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'g‘ “memWhJfifi'gi 2w§£ . .‘22'J'12‘2 2‘2‘22 {93" “J“ ‘ 2 22‘2 i.2 'hH 2222 2221\222” 21122222222222?” ([2 222222224 {221} {3“ £32; :I‘éy fin‘ifi‘ f'? $1.2 2-: ng‘j‘i‘ fig "2'! , 2.; 22 """' "' {‘2’ ‘M 2 » 2 2 222 22.2 .2222 222.22.222.22. 22 3 ”or“ ">31 22:“ “ 2H '1" '3” "W' ”2dw22xw2 332 wqu 22$ .2 23 '122'22 '2“222 2’2 «1‘2242' ‘UI‘W, 2‘02?) 2 2’2 22 . 222 22:22:22 . . 222 22‘“ 2 2221‘2222 ‘22 {EH2 "‘“8‘ .22 22W ' 212122 """"2"’2‘22i2 2 ""' 2 I l ‘22 2'3“ 3“"! 2 2.2 22“" 222222 “f.“ 2222" ”2'31"“? "'2222‘2 2""‘2 'W 4' fit"! l , llllllllllllll University This is to certify that the thesis entitled The role of nitrite in preventing Development Of warmed-over flavor in cogéeg mea from different species nlma 5 presented by Mohamad Hassan Fooladi has been accepted towards fulfillment of the requirements for M. S. degree in Food Science Major professor Date November 17. 1977 0-7639 9.“ s'V’ " «r "‘ ‘ V 'V'V ' U 1: 3W THE ROLE.OF NITRITE IN PREVENTING DEVEIDPMENT OF WARMED-OVER FLAVOR IN COOKED MEAT FROM DIFFERENT SPECIES OF ANIMALS By Mohamad Hassan Fooladi 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 1977 AU .II fl ll» 01 pc 86 m' st le to CO re fi SD ABSTRACT THE ROIE OF NITRITE IN PREVENTING DEVEIOPMENT OF WARMED-OVER FLAVOR IN COOKED MEAT FROM DIFFERENT SPECIES OF ANIMAIS by Mohamad Hassan Fooladi The present study was designed to determine the role of nitrite in develOpment of warmed-over flavor in beef. pork and chicken. which was followed by TBA values and sensory panel scores. Samples with and without added nitrite were evaluated both before and after cooking at 0 days and after #8 hours storage at 4°C. The relation- ships between TBA numbers and total lipid and phospholipid levels were also followed to ascertain their significance to development of warmed-over flavor. Added nitrite protected against autoxidation of cooked meat during storage at 4°C for #8 hours. causing a 6-fold reduction in TBA values for pork and a 2-fold reduction in beef and chicken. Sensory panel scores con- firmed the protective effect of added nitrite in all three species. Total lipid levels were not significantly related Mohamad Hassan Fooladi to warmed-over flavor deve10pment, but there was some evidence for involvement of phospholipids. 8f. ACKNOWEEDGMENTS The author is indebted to Dr. A. M. Pearson for his direction and encouragement throughout this study and during the preparation of this thesis. The author also wishes to express his appreciation to the guidance committee, Drs. C. Cress, P. Markakis, and C. M. Stine, for their critical review of this thesis. Appreciation is also expressed to Dr. L. Dugan for his attendance at the oral examination in Dr. Markakis' stead. Finally, the author is particularly grateful to his family, for their encouragement, patience, understanding, and support. ii Ya In. Re TABLE OF CONTENTS IntrOduCtion O O 0 O O O O O O O O O O O O O O 0 Literature Review . . . . . . . . . . . . . . . . Oxidation of Lipids . . . . . . . . . Mechanism of Autoxidation . . . . . . Products of Lipid Oxidation . . . . . Catalysts of Lipid Oxidation in Meat . Role of Phospholipids in Development of Warmed- Over Flavor in Cooked Meat . Influence of Nitrite on Meat Flavor . Improvement of TBA Values on Adding Nitrite Comparison of the Volatiles from Cured and Uncured Meat . . . . . . . . . . . . . . . Thiobarbituric Acid Test . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . . Solvents and Chemicals . . . . . . . . . . . Source of Meat . . . . . . . . . . . . . . . Analytical Methods . . . . . . . . . . . . . Sample Preparation . . . Measurement of lipid Oxidation by the TBA Test . . .. . . Extraction of Total Muscle Lipid . . . Isolation of Phospholipids and Neutral Lipids O O O O O O O I O O O O O I O Sensory Evaluation . . . . . . . . . . Statistical Treatment . . . . . . . . . Results and Discussion . . . . . . . . . . . . . TBA Values for Meat Samples with and without Nitrite . . . . . . . . . . . . . . . . . TBA Values for Chicken . . . . . . . . TBA Values for Pork . . . . . . . . . . TBA Values for Beef . . . . . . . . . . iii Sensory Panel Scores for Nitrite Treated and Non-nitrite Treated Chicken, Pork and Beef . . . . . . . . . . . . . . . . . . Sensory Scores for Chicken . . . . . . . Sensory Scores for Pork . . . . . . . . . Sensory Scores for Beef . . . . . . . . . Total Lipid and Phospholipid Levels in Muscles from Different Species . . . . . . . . . . . Correlation of TBA Values and Sensory Panel scores 0 O O O O O O O O O O O O O O O O 0 Relationship of Total Lipid and Phospholipid Levels to TBA Values . . . . . . . . . . . . Summary and Conclusions . . . . . . . . . . . . . . Bibliogr‘aphy O O O O O O O O O O O O O O O I O O 0 Appendix 0 O O O O O O O O O O O O I O O O O O O 0 iv 38 39 42 an 44 48 50 56 58 65 LIST OF TABLES Table Page 1 Mean Squares for TBA Values of Chicken, Pork and Beef . . . . . . . . . . . . . . . 3O 2 TBA.Ievels from Chicken Muscles Treated with and without Nitrite . . . . . . . . . 32 3 TBA Levels from Pork Muscles Treated with and without Nitrite . . . . . . . . . 35 4 TBA Levels for Beef Muscles with and without Nitrite I I I I I I I I I I I I I I I I I I 37 5 Mean Squares Taste Panel Scores from Chicken. Pork and Beef . . . . . . . . . . 4O 6 Taste Panel Scores for Chicken Samples and the Significance of Mean Differences . . . 41 7 Taste Panel Scores for Pork Samples and Significance of Mean Differences . . . . . 43 8 Taste Panel Scores for Beef Samples and Significance of Mean Difference by LSD . . 45 9 Total Lipid, Neutral Lipid and Phospholipid Levels for Chicken, Pork and Beef Samples . 47 10 The Correlation Coefficients of TBA Values and Taste Panel Scores . . . . . . . . . . 48 ll Correlation Coefficients between TBA Values and Total Lipid Levels . . . . . . . . . . 51 12 The Correlation Coefficient of TBA Values and Phospholipids as Percent of Tissue . . 52 13 The Correlation Coefficient of TBA Values and Phospholipids as Percent of Total Lipids I I I I I I I I I I_ I I I I I I I I 54 V] VI Table II-A II-B II-C III IV V-A V-B VI—A VI-B VI-C LIST OF APPENDIX TABLES TBA Values for Chicken. Beef and Pork . . . Table of Analysis of Variance for Chicken . Table of Analysis of Variance for Pork . . Table of Analysis of Variance for Beef . . Sensory Panel Score Sheet . . . . . . . . . Taste Panel Scores . . . . . . . . . . . . Table of Analysis of Variance for Chicken . Table of Analysis of Variance for Pork . . Table of Analysis of Variance for Beef . . Total Lipid, Neutral Lipid and Phospholipid Levels as a Percentage of Tissue and as a Percentage of Lipid in Chicken . . . . . . Total Lipid, Neutral Lipid and Phospholipid Levels as a Percentage of Tissue and as a Percentage of Lipid in Pork . . . . . . . . Total Lipid, Neutral Lipid and PhOSpholipid as a Percentage of Tissue and as a Percent- age of Lipid in Beef . . . . . . . . . . . vi 74 76 78 ’1) mu in an CO! atz' hai I‘eE sta pho and dur; UDSE to t Cata INTRODUCTION When uncured cooked meat is stored for a relatively short period of time, it develOps an undesirable taste and odor which is commonly referred to as warmed-over flavor (WOF). The objectionable stale rancid odor becomes especially noticeable after the cooked meat is refrigerated and then re- heated again. The problem of warmed-over flavor has assumed much greater significance in recent years due to the rapid increase in fast food service facilities (airlines. vendors. and franchises) requiring the use of large quantities of pre- cooked or partially-cooked meats and meat products. Oxidative rancidity is a major cause of flavor deterior- ation in meat during storage (Turner gt al.. 1954; Timms and watts, 1958). The lipids present in muscle tissue are responsible at least in part for problems related to product stability. Love and Pearson (1971) concluded that the phos- pholipids result in oxidative deterioration of cooked meat and the resulting rancid flavor. which develops rapidly during refrigerated storage. The relatively high content of unsaturated fatty acids in the phospholipid fraction appears to be responsible for development of warmed-over flavor. Heme pigments have traditionally been considered as catalysts of lipid oxidation in meat. Metmyoglobin in raw meat and ferric denatured hemichromes in cooked meat have been implicated as the catalytically active forms of the muscle pigments. Younathan gt gl. (1959) showed that ferric hemochromogen is an active catalyst for oxidation of unsatur- ated fat, whereas, ferrous nitric oxide hemochromogen in cured meat does not catalyze lipid oxidation. Zipser gt al. (1964) reported that TBA numbers during storage of cured pork samples are lower than those for un— cured pork. Liu and watts (1970) and Sato and Hegarty (1971) concluded that the presence of nitrite in the meat products inhibits oxidation of the lipids. while off—flavor in uncured meat is due to oxidation of lipids. Thus, the present study was undertaken to investigate the role of nitrite in prevent- ing develOpment of warmed-over flavor in cooked meat from different species of animals. Th au ac ou PI" W1 LITERATURE REVIEE Oxidation of Lipids The lipid components of foods are readily susceptible to autoxidation, which occurs slowly even at normal temperatures according to Waters (1971). He concluded that the deleteri- ous effects of oxidation are more serious because peroxides produced by lipid oxidation can attack molecules of other types. He further reported that secondary reactions of autoxidation are responsible for undesirable changes in foods. Thus, exposure of food lipids to atmospheric oxygen causes extensive deterioration. As a result of autoxidation, un- pleasant odors may develop in foods. and sometimes even toxic compounds may be produced (Holman. 1960; Lundberg, 1962). Kummerow (1962) and Matsuo (1962) have pointed out that per- oxides of unsaturated fatty acids are toxic to animals. In lipids containing unsaturated double bonds oxygen attacks at or near the unsaturated center. while in-sat- urated fats breakdown may occur anywhere along the hydro— carbon chain. predominantly at the beta position (Ingold. 1962). ha: an. ti 4 Mechanism of Autoxidation The generally accepted mechanism of lipid oxidation has been reviewed by Dugan (1961). Labuza (1971), and Sato and Herring (1973) and involves a free radical chain reac- tion. They indicated that the reaction proceeds in three stages: (1) (2) Initiation. This step involves the formation of a free radical species (unpaired electron) from an unsaturated fatty acid as shown below: (initiators) heat, light, metals . , R1H > 31 + H (unsaturated fat) (free radical) Propagation. Free radicals combine with molecular oxygen (autoxidation) to form peroxide free radicals, which upon reaction with fatty acids. yield hydrOper- oxides and other free radicals. The free radicals are then available to continue the chain reaction in the following manner: s> (peroxide 3’3i + 02 ,7 R100‘ free radical) 32100 + {:2}; 9 aloon + H'z I (hydrOperoxide) 89 mo am 861 be: (3) Termination. Deactivation of the radical results in stable end products as illustrated below: R' + a' > HP. *1 + 200° > ROOF. 300° + 200' 9 {:00}: + 02 '3' - A I -\ + '21 7 Ale Free radical inhibitors (R1) also include antioxidants. The development of off flavors results from hydrOper- oxide degradation (Kaunitz, 1962; Lundberg, 1962). Although hydroperoxides are odorless, they are degraded through a series of scission and dismutation reactions to yield low molecular weight carbonyl compounds (aldehydes and ketones) and short chain fatty acids, which possess extremely low sensory threshold values (Keeney, 1962; Lea. 1962: Lund- berg, 1962). Factors which effect the rate of off-flavor development include the fatty acid composition of the lipids, tempera- ture, light, metal catalysts, inhibitory compounds. and the availability of oxygen (Lea, 1962; labuza. 1971). Ackman (1976) simplistically emphasized two major points in dis- cussing lipid stability of foods. He first indicated the need to begin with high quality raw materials, and secondly, the need to optimize all handling and storage procedures. 'C .hw. m1 Ali fre one such tissr Products of Lipid Oxidation Hydroperoxides are the primary products of the reaction of oxygen with unsaturated lipids (Farmer gt gl.. 1942: 1943). Decomposition of these primary products produces alcohols. aldehydes. ketones, acids, lactones. and unsaturated hydrocarbons, which are known as secondary degradation pro- ducts (lundberg. 1962). These compounds are highly suscep- tible to further oxidation (Keeney. 1962; Lundberg, 1962; Sherwin, 1972). 'According to Keeney (1962) and Lea (1962), the primary products (hydroperoxides and peroxideS) are odor- less. whereas. the rancid odors in oxidized fat are chiefly due to aldehydes, ketones and acids that are formed from the primary products. Aldehydes are notoriously unstable compounds, and are susceptible to polymerization and condensation reactions. They may be oxidized by active oxygen in the autoxidative system to form carboxylic acids (Sato and Herring. 1973). Although the free aldehyde level in oxidizing fat is low, free aldehydes still cause major flavor problems because some of them (2-4-decadiena1) have flavor thresholds of less than one part per billion (Keeney, 1962). Catalysts of Lipid Oxidation in Meat It has been generally accepted that hematin compounds. such as hemoglobin, myoglobin, and cytochromes in animal tissues, are catalysts for unsaturated fat oxidation (Lew fa (1 Cc ta and Tappel. 1956). Fox (1966) has reviewed the chemistry of meat pigments. He stated that myoglobin in fresh meat exists in three inter- convertible forms. namely, oxymyoglobin, reduced myoglobin, and metmyoglobin. He concluded that oxymyoglobin imparts the desirable bright red color to meat. whereas. reduced myoglobin is purplish-red in color. and metmyoglobin is responsible for the undesirable brown to black discoloration occurring in fresh meat. Watts gt g1. (1966) stated that the balance between the different pigment forms is affected by the activity of enzymatic reducing systems in the meat and the oxygen concentration of the surrounding atmosphere. During cooking, the pigments are irreversibly converted to denatured ferric hemichromogens (Fox, 1966). Brown 23 gl. (1963) reported that ferric hemes are more active catalysts of lipid oxidation than ferrous hemes. The rapid oxidation of lipids in cooked meat has been attributed to catalysis by denatured ferric hemichromes (Younathan and Watts. 1959: Liu and Watts, 1970). The mechanism of the reaction is incompletely understood. Banks (1944) suggested that the active catalyst results from the combination of a fatty peroxide with an iron porphyrin. Maier and Tappel (1959) proposed that catalytically active hemes form unstable compounds with fat peroxides, which then decompose to give two free radicals. each of which in turn is capable of ini— tiating autoxidation. Tarladgis (1961) attributed the catalytic activity of fe‘ uni fi ti 0): of Tl' ve ll ferric hem0proteins to the paramagnetic character of the por- phyrin bound iron. He suggested that the presence of five unpaired electrons in metmyoglobin produces a strong magnetic field that would favor the initiation of free radical forma- tion. He further reported that the decomposition of hydroper- oxides by a ferric porphyrin was mediated through the donation of an electron from their cloud of the porphyrin ring. According to Timms g3 g1. (1958), oxidative rancidity in stored cooked meat, is higher in uncured than in cured meat. They suggested that differences in the heme pigments of cured versus uncured meat might be responsible for the differences in their oxidative behavior. Waters (1971) stated that the nitric oxide complex of the heme containing compounds is structurally similar to organic nitrioxide free radicals (inhibitors of autoxidation) and have an unpaired electron which is more closely associated with the NO groups than with the iron. They suggested that in this way stereochemical and functional blocking of cata- lytic reactivity by heme containing systems may occur. Youna- than and Watts (1959) hypothesized that the cured meat pig- ment, in which the 5th and 6th coordination position on the iron molecule are occupied by denatured globin and nitric oxide. respectively, would not be expected to react with a fat peroxide in the manner postulated for hematin or hemoglobin. Even though heme pigments have traditionally been implicated as the major prooxidants in meat, there is eV 3.0! 1.11 He; J evidence that non—heme iron may play an important role in accelerating oxidation of muscle lipids (Moskovits and (ielsmeier. 1960; MacLean and Castell, 1964; Sato and Hegarty, 1971). Moskovits and Kielsmeier (1960) demonstrated that contaminating iron fractions act as prooxidants in sau- sage. MacLean and Castell (1964) found that trace amounts of iron added to cod muscle produced a rancid odor. Sato and Hegarty (1971) showed that non—heme iron accelerated the oxidation of lipids in water extracted cooked meat. They also reported that myoglobin and hemOglobin failed to act as prooxidants in cooked meat. The principles of metal ion catalysis in lipid oxida- tion have been reviewed by Ingold (1962; 1968) and Waters (1971). Lipids contain heavy metals originating from either metal activated enzymes (Ingold, 1962) or from direct con- tamination by contact with metals during processing (Patron, 1968). These authors concluded that heavy metals, notably iron and copper, which exist in several valency states. generally increase the rate of the oxidative reaction. Metals can also affect the rates of initiation and proPagation and hydr0peroxide degradation (Ingold, 1962). Role of Phospholipids in Develogment of Marmed-Ove;_?lavor in Cooked Meat Watts (1954; 1961) suggested that lipid oxidation of adipose tissue was responsible for meat rancidity. Timms and Watts (1958) noted that there is little, if any. correlation I< ll U1 3C bc be 19 10 between flavor changes occurring in cooked meat and the oxidation of neutral lipids. Younathan and Watts (1960) concluded that the phospholipids in cooked pork are more susceptible to oxidation than the neutral lipid fraction. Campbell and Turkki (1967) reported that during the cooking of meat the neutral lipids are lost more readily than the phospholipids. Thus, the ratio of phospholipids to total lipids increases during cooking. Similar results were obtained by Hornstein g3 g1. (1961), who concluded that off-flavor development in cooked meat is greater than in raw meat because of a higher phospholipid ratio. Horn- stein gt g1. (1961) indicated that polyunsaturated fatty acids (C18, 020 and C22) with two to six double bonds are found in animal tissues and are mainly responsible for development of warmed-over flavor (Lea, 1962). Acosta g§,gl. (1966) showed that the phospholipid fraction is implicated in the early stages of autoxidation in turkey meat. A number of investigators (Watts, 1954; Lea, 1962; love and Pearson, 1971) have concluded that the lability of the phospholipid fraction is a result of their high unsat- urated fatty acid content. For example, 19% of the fatty acids in beef muscle phospholipids have four or more double bonds, while only 0.1% of the triglyceride fatty acids from beef show this degree of unsaturation (Hornstein g1 g1., 1961). Oxidation of polyunsaturated fatty acids is (J on f1 ac! ni- cor Wit fra of . 11 accompanied by the destruction of both fat-soluble and water-soluble vitamins (Holman, 1960; Kummerow, 1962; Lundberg, 1962). The products of oxidation of polyunsaturated fatty acids and their subsequent degrada- tion products impart objectionable flavors and odors to (foods (Holman, 1960; Keeney, 1962; Lundberg, 1962). Influence of Nitrite on Meat Flavor Bailey and Swain (1973) stated that nitrite serves several purposes during meat curing, including color fixa- tion and as an antibacterial agent, but perhaps the most important feature of nitrite is its influence on flavor. The relationship of nitrite to flavor was first des- cribed by Brooks ggflgl. (1940) in studying the use of nitrite in curing bacon and ham. Although they presented no taste panel data, these authors stated that the panel showed a preference for meat cured with nitrite. Barnett‘gt‘gl. (1965) reported an extensive study on the factors affecting cured ham flavor. They reported that nitrite improved flavor of cured ham, but the level did not greatly influence acceptability. Cho and Bratzler (1970) studied the effect of nitrite and smoke on the flavor of cured pork roasts, and concluded that the flavor was improved in the roasts cured with nitrite. Wasserman and Talley (1972) found that frankfurter flavor became less desirable upon elimination of sodium nitrite from the cure. Thus, results demonstrated 12 that cooked, uncured frankfurters have an unappetizing V flavor. . Swain (1972) studied the effect of nitrite on the flavor of hams and found that a taste panel rated smoked cured hams, unsmoked cured hams, smoked uncured hams and unsmoked uncured hams in order of intensity for cured flavor. Simon gt gt. (1972) also investigated the flavor of frankfurters produced with either beef and pork or from beef alone while using different levels of sodium nitrite (0, 39, 78, 156 ppm). In frankfurters containing both beef and pork, taste panel results indicated that both nitrite level and storage time were associated with taste acceptance. In both vacuum packaged and bulk packaged frankfurters taste acceptance decreased with time, but was not associated with the nitrite level. In frankfurters produced from beef alone the addition of nitrite improved the flavor. Although taste panel scores slowly decreased with storage time, they were not related to the nitrite level. Simon gt_g;. (1972) reported that the flavor of uncured frankfurters can be improved by incorporation of antioxidant (0.01% by weight of BHA or BHT), but the taste acceptance is still not equal to that of frankfurters cured with sodium nitrite. r< his 11' at de e) TE wt 11‘. (1 0X pr de in at Va th du: 801 of Th; 13 Igprovement of TBA Values on Adding Nitrite Bulk stored frankfurters produced from beef, pork and mechanically deboned chicken with a spice extract of rosemary as a substitute for nitrite were studied by MacNeil and Mast (1973). TBA values were used as being indicative of oxidative changes during 16 days storage at 45°F. Results indicated that there was a significant decrease in TBA values when either nitrite or the spice extract was included in the frankfurter formation. The TBA values support results obtained by panel flavor scores, which show that extracts of rosemary as well as nitrite inhibit oxidation (Chipault gt_g;., 1956). Zipser gt gt. (1964) found a high correlation between TBA values and oxidative off flavor in cooked meat. Thus, the TBA procedure has been used routinely to measure off flavor development in cooked meat and fish. Swain (1972) used TBA values to measure the changes in oxidative products after cooking and following storage at 7°C for hams cured with and without nitrite. The TBA values of the nitrite-treated hams were initially lower than comparable samples without nitrite and remained lower during storage up to 2 weeks. Younathan and Watts (1959) reported that nitrite and sodium chloride acted synergistically to retard oxidation of lipids in cooked meat stored at refrigerated temperatures. They concluded that the difference in flavor between nitrite 14 cured and non—nitrite cured meat soon after cooking is probably due to development of warmed~over flavor caused by rapid oxidation of unsaturated fatty acids. Comparison of the Volatiles from Cured and Uncured heat The volatile compounds from cooked meat or meat extracts were found to comprise carbonyl compounds, organic acids, alcohols, sulfur compounds and ammonia (Lillard and Ayres, 1969). The carbonyls and the sulfur—containing substances are believed to be the predominant contributors to meat flavor (Bernstein gt gt., 1960; Kramlich and Pearson, 1960; Bender and Ballance, 1961). Cas chromatographic examination of the volatiles from cured and uncured ham by Cross and Ziegler (1965) showed that hexanal and valeraldehyde were present in appreciable quantities in the uncured product, but were barely detectable in the volatiles of the cured meat. They assumed that these volatiles were derived by oxidative clea- vage of unsaturated fatty acid residues, probably from linoleate. Although butyraldehyde, propionaldehyde and acetaldehyde were also more prevalent in uncured hams, the differences between the cured and uncured meat were less pronounced. The results were essentially the same on com- paring cured and uncured beef or chicken. The branched chain aldehydes (isobutyraldehyde, isovaleraldehyde, and 2-methylbutyra1dehyde) occurred to the same extent in both cured and uncured meat. Sulfur compounds in both cured and 15 uncured meat were H23 and methanethiol. Cross and Ziegler (1965) concluded that curing with nitrite does not seem to contribute any volatile compounds other than nitrogen oxides that are not present in cooked uncured meat. Thus, different aromas of the cured and uncured meat depend on the spectra of carbonyl compounds derived by oxidation of fats. They stated that nitrite prevents the oxidation of unsaturated lipids by deactivating the hematin cata- lysts. Swain (1972) also compared volatiles from cured and un- cured hams, and reported that isobutanal and higher molecular weight aldehydes were more concentrated in the uncured than the cured samples. There was an even greater difference in the level of these compounds when the hams were stored for 8 hours at 4°C prior to evaluation. They suggested that oxida- tion of unsaturated fatty acids in uncured hams was respon- sible for formation of high molecular weight aldehydes. These results supported the findings of Cross and Ziegler (1965) showing that nitrite retarded oxidation of unsat- urated fatty acids. Piotrowski gt al. (1970) concluded that there is a difference in the flavor of cured and uncured meat, which is associated with variation in the lipid phase. l6 Thiobarbituric Acid Test Sherwin (1968) reviewed the methods for determining the stability of fats and oils in foods. Current methodology available for evaluating the stability of lipids in foods has also been reviewed by Erickson and Bowers (1976). These workers concluded that the methods for determining lipid stability are based upon either measuring oxygen uptake, peroxide formation, or peroxide decomposition products. The thiobarbituric acid test (TBA) was classified as measuring the final reaction products of peroxide decomposi- tion. This method has been used for determining the extent of lipid oxidation in foods under a variety of test condi- tions (Turner gt g;., 1954). The method is based on the development and quantitation of a red pigment formed by the condensation of one molecule of malonaldehyde and two mole-- cules of 2—thiobarbituric acid (Sinnhuber gt gl., 1958). The condensation occurs as shown on the following page. The chemistry of the pigment has been studied by a number of investigators (Sinnhuber g; g;,, 1958; Tarladgis g3 gl., 1962; Yu and Sinnhuber, 1962; Marcuse and Johansson, 1973). Maximum absorbance of the red pigment has been shown to occur at 530 to 535 nm (Sinnhuber and Yu, 1958). The prOposed mechanism of malonaldehyde formation is by dismutation and scission of aldehydes generated during hydr0peroxide degradation (Day, 1966). The reaction proposed by Day (1966) is shown on Page 18. l7 Immm§ o~I~+ I» Ate: 25:53 <95 :0 z\ finfid/If r2 . OI /2.\=om .00; .23 obxcoEmcoIms. I I I I / \ so..%uo + ‘ o oIo Eo< ?, while the chelated form (III) domin- ates at pHI< 3. Therefore, Kwon and Watts (1964) indicated that maximum volatilization of free, preformed malonaldehyde occurs at pH‘<'3. The acid is added in order to free the malonaldehyde from possible combinations in food constituents. H O C H \ / II C II //C - Ii C\\ CI-I —-—¥ \c «4—— .\c/ \o 12 I 9. 7' l! ,3 /” ”\ ”\ O // ‘\\H C// H O// (I) (II) (111) According to Tarladgis g§,gl. (1960), malonaldehyde itself does not contribute to typical rancid odors, although a high correlation between malonaldehyde content and rancid odor has been noted (Zipser ggpgl., 1964). The relationship may be limited to moist foods, specially to animal tissues (Kwon and Watts, 1964). Pearson (1968) pointed out that the TBA test apparently measures the deterioration in both the extractable and non- extractable lipids. However, he further reported that relatively high TBA values may be found in some fresh samples, and yet in advanced stages of rancidity the TBA values may actually fall to zero or remain constant after reaching a maximum value. »2 +3 Trace amounts of Be. or Te" have been reported to increase TBA values (Mills, 1964). Ascorbic acid has also been alluded to as a cause of high TBA values (Wills, 1966). Hougham and Watts (1958) reported that the presence of 200 ppm of nitrite decreased the TBA value by 20-30%, but a concentration of less than 100 ppm did not interfere with the TBA test. Zipser and Watts (1962) stated that small amounts of nitrite ion are capable of significantly reducing TBA numbers in rancid meat, with the reduction increasing linearly with nitrite concentration. Nitrite interference with the TBA test takes place during the distillation step and is believed to be due to nitrosation of malonaldehyde. Diazonium salt formation with sulfanilamide is util- ized to bind the nitrite before beginning the TBA test (Zipser and Watts, 1962). The reaction is shown below. “Hg ' p 5 1‘4“” c1 + Nach + 212101 —-> + H20 + NaCl V V SCZNH2 SCZNHZ Sulfanilamide Diazonium salt 21 An advantage of the TBA test is that the fat does not ‘need to be extracted from the rest of the muscle tissue (Tarladgis gt gl., 1960). Therefore, the TBA test would be expected to measure malonaldehyde produced from autoxi- dation occurring in all of the lipid fractions. Off-flavor threshold values have been reported for TBA numbers in the range of 0.5 to 1.0 (Tarladgis gt g1., 1960; Watts, 1962). However, this range has not been firmly established. Jantawat and Dawson (personal communi- cation) have reported threshold values for cooked chicken to be 4.0 mg malonaldehyde per 1,000 g of meat. Yu gt_gl. (1969) stated that fish samples with a TBA value of 2.4 was judged to be acceptable, whereas, the samples with TBA values of 3.1 or greater were very rancid and unacceptable. Younathan and Watts (1959) have reported that TBA values for pork ranging from 0.46 to 0.60 are indicative of tissue rancidity. Patton (1974) stated that the TBA test is highly sensi— tive and useful in monitoring lipid oxidation. However, Dugan (1976) stated that all objective methods available for determining lipid stability have their limitations. Therefore, sensory methods are necessary for absolute confirmation. MATERIALS AND METHODS Solvents and Chemicals All solvents, chemicals and reagents were of analytical grade. Distilled deionized water was used throughout the study. Source of Meat Four 6 1b samples from each of three different species (chicken, beef and pork) were obtained for this study. The samples were used to determine the effect of nitrite on develOpment of warmed—over flavor. Chicken breast and thigh muscles were obtained 24 hrs postmortem. Pork semitendinosis and biceps femoris muscles were obtained at 24 hrs postmortem and were pooled and used for this study. Beef flank steak and hanging tender muscles were obtained at 24 hrs postmortem and were mixed together in the same prOportion as they occur in the beef carcass. All samples were trimmed of excess connective tissue and of subcutaneous adipose tissue. The chicken skin was removed from all chicken muscle samples. 22 23 Analytical Methods Sample Preparation The meat samples were chOpped with a Hobart Silent Cutter, Model #841810, for five minutes. During the chOpping proce— dure, 5 g of sugar and 12 g of salt were dissolved in 200 ml of water, which was then mixed into the meat sample. After chopping, the sample was divided into two equal por- tions. One portion was mixed with 75 ml of water and chopped for an additional 5 minutes in the silent cutter. This was used as the control. The remaining 3 lb sample was mixed with 156 ppm of nitrite ion (as sodium nitrite) and the same volume of water and was chopped for an additional five minutes. Each of the nitrite treated and control samples were divided into two portions. One portion was used raw and the other portion was cooked prior to chemical and sensory analyses. Samples to be cooked were packaged in Cry-O-Vac bags and sealed. After weighing, the nitrite treated and control samples were placed in a boiling water bath separately and cooked until the meat reached an internal temperature of 70°C. After cooling at room temperature for 20 minutes, the samples were again weighed. Drip was then obtained by difference, by subtracting the weight of the cooked meat from the weight of the raw sample. Zero-day samples from the raw and cooked meat with and 22+ without added nitrite were analyzed immediately for TBA values, taste panel (raw samples were tested for aroma), and lipid analyses. The remaining raw and cooked samples from the nitrite and control treatments were refrigerated for 48 hrs at 4°C. After storage, these samples were then analyzed for develOp- ment of warmed-over flavor by the TBA test, taste panel and lipid analyses. Measurement of Lipid Oxidatign by the ‘BA Test The distillation method of Tarladgis g3 a1. (1960) was used for measuring TBA numbers. The distillation.apparatus consisted of a 250 m1 round bottom flask, which was attached to a Friedrich Condensor with a three-way connecting tube, and it was placed in an electric heating mantle. A d3pli-~ cats 10 g sample of meat was homOgenized with 50 m1 of distilled deionized water for 2 minutes in a Virtis homogen- izer at low speed. The homogenate was transferred quanti- tatively into a 250 m1 round bottom flask by washing with 47.5 ml of distilled deionized water. The pH of the meat slurry was adjusted to 1.5 by the addition of 2.5 ml of 4 N HCl. Boiling chips were added and a small amount of Dow antifoam was sprayed into the flask to prevent foaming. The slurry was steam distilled using the highest setting on a power stat (the Superior Electric Company, Bristol, Connecticut) until 50 ml of the distillate were collected. The distil~ late was mixed and 5 ml were transferred to a 50 ml test 25 tube. Then, 5 m1 of TBA reagent (0.02 m 2-thiobarbituric acid in 90% glacial acetic acid) were added. The tubes were stoppered and the contents mixed. The tubes were heated in a boiling water bath for 35 minutes. After cooling in cold water for 10 minutes, absorbance was read on a Beckman DU spectrOphotometer at 538 nm against a blank containing distilled deionized water and TBA rea- gent. Absorbance readings were multiplied by a factor of 7.8 (Tarladgis gt gl., 1960). TBA values are expressed as mg malonaldehyde per 1,000 g of sample. According to studies by Younathan and Watts (1959), Hougham and Watts (1958), and Zipser and watts (1962), nitrite interferes with the distillation step by nitro- sation of malonaldehyde. Thus, for the nitrite treated samples a modified TBA test (Zipser and Watts, 1962) was sed to bind the nitrite by formation of diazonium salt with sulfanilamide. Ten g of nitrite treated meat was blended with 49 ml distilled deionized water and 1 ml of sulfanilamide reagent (0.5% sulfanilamide in 20% H01 ~ v/v) using a Virtis homogenizer at low speed for 2 minutes. The mixture was quantitatively transferred to a 250 m1 round bottom flask by washing with 48 ml of distilled deionized water. Then, 2 ml of 4 N HCl was added to bring the pH to 1.5. The remainder of the procedure for TBA analysis was carried out as described by Tarladgis 33 a1. (1960). 26 Extraction of Total Kuscle Lipid The procedure of Folch gt_a1. (1957) as modified by Igene (1976) was used to extract the total lipids from the muscle tissue. A 100 g sample was homogenized in a Waring blender and extracted three times with 500 ml of a chloro- form—methanol mixture (2:1 - v/v). The extract and tissue residue were then transferred to a medium grade sintered glass funnel and filtered under vacuum. The homogenizer and the residue in the funnel were washed with an additional volume of chloroform-methanol and fil- tered. The extract was quantitatively transferred into a 1,000 ml separatory funnel and 10% by volume of distilled water was added and thoroughly mixed. The mixture was al- lowed to separate into two phases until the interface was clear. The lower phase was transferred to a 500 ml volum- etric flask and evaporated in a vacuum Rotavapor-R (Buchi, Switzerland) at 20-300C. When the volume of the total lipid extract was reduced to 10-20 ml, the extract was quan- titatively transferred to a previously tared 100 ml volumetric flask by washing with an additional quantity of chloroform- methanol. The final extract was further evaporated under a stream of nitrogen until it reached a constant weight. The weight of the lipids was then obtained by difference. Isolation of Phospholipids and Neutral Lipids Separation of the phospholipids from the total lipids was accomplished using the method of Choudhury g: 31. (1960). 27 This method involves separation on activated silicic acid, in which neutral lipids are preferentially removed by washing with chloroform, followed by solubilization of the phospholipids with methanol. A weighed amount of silicic acid (20-25 g) was activated for 16 hrs by drying in a 100°C oven. The lipid sample was then quantitatively transferred to a 125 ml Erlenmeyer flask containing the activated silicic acid. The contents were shaken and allowed to settle for 6 hrs. The mixture was then thoroughly stirred and filtered through a sin- tered glass funnel under vacuum. The silicic acid was washed six times with 50 ml portions of chloroform. The filtrate and washings were combined and evaporated using the Pota- vapor-? as described previously. The phospholipid fraction was determined by washing the silicic acid residue with six 50 ml portions of methanol. The filtrate and washings were combined and evap- orated to a constant weight using the Rotavapor-B. The percent total lipids, neutral lipids and phospho- lipids in the raw and cooked meat samples were calculated. SensorypEvaluatigg To determine flavor changes, the samples were evaluated at 0 day and after 48 hrs storage at 4°C by 3 trained panel- ists. At each evaluation time, the panelists were presented with four different coded samples (raw without nitrite, cooked without nitrite, raw with nitrite, cooked with nitrite). The stored cooked samples were reheated in Cry-O-Vac bags in a boiling water bath for 20 minutes prior to evaluation. The raw samples were scored only for aroma by panelists. Score sheets were designed so that the samples were scored from 1 to 5 (l = very pronounced warmed- over flavor and 5 = no warmed-over flavor). The score sheet used is given in Appendix Table III. Statistical Treatment Analysis of variance for TBA values and taste panel scores was calculated using a Michigan State University computer package program identified as MSU Stat System and run on a Control Data Corporation (CDC) 6500 computer. Correlation coefficients were calculated using a Texas Instruments programmable calculator, Model SR52. Factors analyzed were TBA values, taste panel scores, total lipids, phospholipids as percent of tissue, and phospho- lipids as a percent total lipid. The significance of the computed correlation coefficients was determined by using the distribution of "r" table given by Snedecor and Cochran (1973). RESULTS AND DISCUSSION TBA Values for Meat Samples_With and Without Nitrite Raw and cooked samples of chicken, pork and beef with and without nitrite were analyzed for TBA numbers initially (0 days) and again after #8 hrs storage at #00. Table 1 presents the mean squares of TBA numbers of the different species. Appendix Table I contains the raw data, and Appendix Table II contains the analysis of variance for the TBA values. Table 1 indicates that with the exception of pork. lipid oxidation in muscles from different animals of the same species behaved differently. This agrees with the results of Fitzgerald and Nickerson (1939), who found that the keeping quality of chicken fat varied between individual birds. The difference in behavior of the meat from differb ent animals found in the current study may be due to the variation in the environment or to the previous history of the individual animals. Table 1 also indicates that in chicken, pork and beef, there is a significant difference (P < .01) between TBA values for the nitrite treated and non-nitrite treated samples. Cooking treatment as well as storage time were also shown to be factors which significantly influenced 29 30 .Hm>mH no.0 map pm pcmoHeHcmHm * .Hm>mH Ho.o map pm pcaOHchsHm ** Hm Hapoe mH.o om.o mm.o Hm soups HmseHmmw *am.o *amH.HH 0H.H H mmapopm x mconoo x mpanHz $*mm.s agmo.sH **:N.a H mmmpopm x econoo aamm.m **mo.mH am.m H mmmpopm x mHm>mH mpprHz **um.oH aaoe.mH **oH.wn H sszooo x mHm>wH mpHesz *eo.H *gsm.mm ms.o H empopm .m> sweep *tmm.m *adm.wa *mm.m H cmxooo .m> 3mm **me.m **mm.oa **Hm.so H mpanHc 0: .m> mpprHz **~N.~ a:.m **ms.n m mHmeHce Mmom xnom smefino .mouooum ho mosmwpm> we mopsom mmhmzvm smog newsman AepHupHg eases escasz cam 59H; mmHasmmv mowm use xnom .cmxowgo we mosam> ¢ma sch mouwswm smog .H mamas 31 the TBA values of the samples. Table 1 shows that there is a significant interaction between nitrite levels (0 and 156 ppm) and cooking treatment for all three species. There was a significant interaction between nitrite levels and storage time (0 day and #8 hrs) for pork and beef samples but the interaction was not significant for chicken, while all three species have significant interactions between cooking and storage. The interaction between nitrite x cooking x storage is not significant in chicken, but it is significant for pork and beef samples. TBA Values for Chicken Inspection of the values in Table 2 indicates that the TBA values of raw chicken with and without nitrite initially were not statistically different. However, the effect is in the same direction as the other effect and is probably real even though not significant. In fresh cooked samples there was a significant difference (P < .01) between TBA values of the nitrite treated and control (non-nitrite treated) samples. The nitrite dramatically inhibited the effect of storage and cooking on the develOpment of warmed-over flavor by prevent- ing oxidation as shown by much lower TBA values. The TBA values for fresh raw nitrite treated chicken were only about half that of the samples without nitrite. Although the small number of samples prevented the differ- ence from being statistically significant, the magnitude of the mean difference suggests that the addition of .QmH.%p Hm>ma Ho.o 0:» pm psmonHsmHm a: on .umnopmp psmnmahwc : Bosh mmmsm>w one mH mSHs> scam ADV .maomse Pmmmsn cam :mwna Amv 32 **mm.m mo.m mm.e as we amass ewxooo **mo.: Nd.H Nm.m A: m: HmPMd 3mm *smm.m oo.H mm.m see 0 pm cmxooo eH.H om.H mm.m see 0 pm a m museumemHa mpHupHa :pHn mpanHz psonga . wHasmm on smog mmz N mpHesz psogsH; use :pHa empmmse mmHoms: :mgoHno gone mHm>mH ame .N mHnee Adv 33 nitrite protected against autoxidation. Upon cooking at 0 days, the samples without nitrite showed a considerable increase in TBA values, but the nitrite treated chicken did not change appreciably. In fact, the nitrite treated sample had a slightly lower TBA value after cooking. Slight changes in TBA values occurred in raw nitrite treated meat during #8 hrs storage at 4°C, whereas, nitrite free raw samples consistently increased in TBA values, in- creasing from 2.52 (0 day) to 5.52 (48 hrs at 4°C). The high rate of oxidation in raw chicken may be due to the grind- ing process. since Sato and Hegarty (1971) reported that warmed-over flavor occurs in raw ground meat with about the same rapidity and to the same extent as in cooked meat. Thus, they postulated that any process that disrupts the muscle membrane system, such as cooking or grinding, will result in exposure of the highly labile lipid components to oxygen and other reaction catalysts, thus, accelerating autoxidation. In both nitrite treated and in non-nitrite treated cooked chicken, there was a marked increase in TBA numbers during storage at “OC for an additional #8 hrs. In spite of the relatively large increase in the TBA values for the samples with nitrite, it was still below the threshold level. Dawson (personal communication) stated that the threshold value for TBA numbers in chicken is approximately four. The data in Table 2 clearly show that nitrite protects 34 against autoxidation, as shown by the considerably higher value for the nitrite free sample (6.98) as compared to the sample with nitrite (3-05)- TBA Values for Pork Table 3 compares lipid oxidation for pork with and without added nitrite. The TBA value of the raw sample without nitrite before storage (0 day) was 1.52, which was about twice as high as that of the sample containing nitrite. The TBA value increased slightly on cooking the samples without nitrite, whereas, the mean value for similarly treated samples containing nitrite decreased slightly. How- ever, changes in the TBA values of the porkzsamples with added nitrite were negligible upon cooking. After storage for #8 hrs at u°c, raw pork had a mean TBA value of 2.48, which is well above the threshold level of one to two reported by Watts (1962). Similar samples with added nitrite had a TBA value of only 1.42, which is lower than the samples without nitrite but still approaches the threshold leVel. Table 3 shows that cooked pork with added nitrite is much more resistant to lipid oxidation during storage as compared to the stability of nitrite free cooked pork under the same conditions. Control cooked pork (withOut nitrite) increased about five-fold.in TBA numbers during storage, with a mean value of 7.85. On the other hand, storage and cooking resulted in a negligible change in the TBA value 35 .amH an Hm>oH Ho.o was we p:AOHaH:uHm ** on .nawaflcw psosom%flo 2 some ommam>m ens ma msam> nomm Apv .mHouSE mfisosmw mnmcfln use mamofiaqupflEom Amy aeHm.o :m.H mm.a as a: eases swgooo mo.H ms.H m:.m a: a: Amped saw HH.H ms.o mw.H ass 0 as emsooo no.0 mw.c mm.H has c as awn mocmnmmaHo mpHesz :pHa mpanHz psonp.s . mHaemm ADV Cmmfi mm: m . mpwupfim pzonpfls ecu spas awesome moaomsa show Eon“ mam>mq cma .m magma Amy for I‘Gdl and on of car fre 83‘. di ad tr 11 of mu: Va: Sig abc par 36 for pork containing added nitrite, with a TBA value of 1.64. Table 3 indicates that although addition of nitrite reduced TBA values for both raw and cooked pork initially and during storage, the greatest inhibitory effect of nitrite on development of warmed-over flavor occurred during storage of the cooked samples. In this case, there was a signifi- cant (P <:.01) difference between TBA values for nitrite free (7.85) and pork with added nitrite (1.64). TBA Values for Beef The oxidative process is not limited to meats containing a relatively high percentage of unsaturated fatty acids (chicken and pork), but also occurs in beef which contains a lower prOportion of polyunsaturated fatty acids (Timms and Watts, 1958). Table 4 indicates there was a significant difference between the TBA values of beef with and without added nitrite both in the raw and cooked state. This was true both initially and after 48 hrs storage at 4°C. Addition of nitrite significantly (P < .01) reduced lipid oxidation in cooked beef during storage (TBA values of 2.06 vs. 4.12). However, the variance estimate for beef is much smaller than that of chicken and pork. Thus, the smaller variance resulted in lesser differences in TBA values being significant. The TBA value for nitrite treated beef is just above the threshold level. Thus, results indicate that nitrite partially retarded lipid oxidation due to cooking and storage. The data in Tables 2, 3 and 4 indicate that rate of 37 .mmH an H0>0H H0.0 pm PcsOHeHcmHm as “00 .omH an H0>0H no.0 #0 psmoHeHcmHm a A00 .mawafisw Psmam%wwv : Sosa mmsnm>m one ma msam> comm ADV .mmaomss houses mswwsmn 0cm xmmvm MCUHH we oHQEwm opfimomEoo Amv .[ Ill. ssoo.m wc.m 7 NH.: a: me am coxooo samo.o ma.~ :m.a a: m: pm 3mm *amm.o mm.o no.H zmu 0 pm Uexoou *0N.0 00.0 00.0 aa0 0 00 3mm HovmocmmeeHm mpprHa 09H: mpHuPHz pzocsz . 0Hasam H00000H0> <00 Amvmpwhpflz 930:9“; and Spa; mmaoasm %mmm new mHm>mH 0H no.0 one so somOHchsHm * .H0>0H H0.o 0:0 pm pcmoHeHomHm ** Hr Hmpoe Hm.0 mm.0 00.0 Hm house Hmsunmom no.0 0n.0 on.H H memeopm x sconoo x mpHusz mNH.0 mm.0 0H.0 H mmMQOPm x mwaooo H0.0 Ho.0 0H.0 H omwnopm x opanHz 03.0 05.0 ** m.m H mcwxooo x mpwppfiz *mm.m *0N.H *emm.: H concem .m> nwmhm ##mm.oa *mm.a 00.0 H vmxooo_.m> 30m *mm.H *mm.H 0N.0 H mpnppns on .w> mpwmpwz 05.0 H0.0 mm.0 m mHMEmc¢ mmum, xnom sexowmo .nnc mosmnhm> no mopsom mmsmsvm Gama “means“: 0000s enonpns 020 pp“; wmamemmv 0000 0:0 xsom .smxowno scan mopoom Hmswm mpmme mommsvm coma .m canoe .009 an Hm>mH no.0 pm mossonhficunm mmpmonccH “*V .mHmEnsm psmsmmmfio : Bonn mwwpm>w 0:9 ma msam> comm Amy 41 *nm.H H0.» 0H.n a: as hopes omxooo mn.0 no.0 0H.0 awe 0 p0 omxooo WH.0| ua.: mm.: a: m: pmpmm 300 0 05.: 35.: has 0 #0 30m mocoeonnHo .llw mpHepHa 00H: opHssz escapHm 0Hassm coma monoom Hmsmm opmme A00 mmocmnownwm smog no cosmoflnnsmfim one 0:0 meQEmm meonno non monocm Hmcwm mpmme .0 manna 42 samples after storage at 4°C for 48 hrs, there was a signifi- cant difference in the values for the untreated and nitrite treated chicken. Off-flavor development in the nitrite free sample was in the moderate warmed-over flavor range and was accompanied by high TBA values (Table 3), while nitrite treated chicken was scored in the slight to no warmed-over flavor range and was accompanied by lower TBA numbers. Sensory Scores for Pork The data in Table 7 demonstrate that raw pork did not have any off odor at 0 day, regardless of whether or not nitrite was added. Furthermore, pork did not produce distinguishable off odor during storage and both nitrite treated and control (without nitrite) samples were scored as having no warmed-over flavor. Even after cooking, there was no significant difference between the flavor/aroma of samples with or without nitrite, since both were judged as having slight to no warmed-over flavor. Nitrite free cooked pork produced moderate Warmed—over flavor during 48 hrs storage at 4°C, whereas, the flavor of similar samples with added nitrite did not change upon storage and were scored as having very slight to no warmed-over flavor. Thus, cooked pork with added nitrite Was signficantly (P\< .05) preferred over the similarly treated samples without nitrite. This Suggests that cooked pork is Very susceptible to development of Warmed-over flavor, while the addition of nitrite definitely retarded development of off flavor. 43 .H0>0H no.0 0:9 pm pcmoHanmHm Hav $00.0 on.o sm.n e: no omens omxooo 0.0 on.0 mm.0 see 0 pm ooxooo 0 wn.0 on.: a: we popes 20m 0H.0 nu.0 00.: see 0 as sex 000mmMnnH0 opanHz epHa oprpaz psoana . 11. 0H0smm a mmho 0m qumxw memB and an moosmsmnmflm 2003 no cosmoflnncwnm 0cm mmamemm xmom pom mmhoom Hmcmm opmwe .n manna 44 Sensory Scores for Beef Data from Table 8 also indicate that there was no significant difference in the sensory scores of raw beef in the presence or absence of sodium nitrite, as neither sample had any off odor at 0 day. When the nitrite free beef was cooked, there was a decline in the sensory panel scores, and it was judged as having moderate warmed-over flavor. 0n the other hand, similar samples treated with added nitrite were scored as having slight warmed-over flavor. Raw samples of beef, both with and without nitrite after 48 hrs storage at 4°C, were judged as having very slight off odor. After storage, there was a significant difference between flavor of nitrite treated and non-nitrite treated cooked beef; however, nitrite did not greatly re- tard the development of warmed-over flavor. In this case. the non—nitrite treated beef was scored as having moderate warmed-over flavor, while the nitrite treated samples were judged as having moderate to slight warmed-over flavor. Total Lipid and Phospholipid Levels In Muscles from Different Species Extraction of total lipids was carried out using a chloroform and methanol extraction procedure modified from the technique described by Folch gt filo (1957). Separation of the phospholipids from the total lipids involved frac- tionation on a silicic acid column. The neutral lipids Were “5 .000 so H0>0H H0.0 pm pcAOHanme A**0 .004 an H0>0H no.0 pm pamOHanme H* 0 .mHMEwsm pampmmwflc 3 Beam ommhm>m ma whoom comm Amv **oo.0 no.0 00.N an 00 nouns owxooo **00.H 00.: 00.0 000 0 pm ooxooo 00.0: H0.0 03.: a: 0: means 30m 0H.0- 00.: H0.0 000 0 pm 300 monouomnHo opHusz :sz mpngHz psoanz 0ngmm smog me00 mg 0 mm A00 m H 0 p a 0mg an mosmnmmnnn :002 no cosmoanQMnm 02m mmamsmm Hoom pom monoom Hmsmm opmme .w mapme 46 preferentially removed by washing with chloroform followed by subsequent removal of the phospholipids with methanol. Table 9 gives the mean values for total lipids and for neutral lipids as a percentage of tissue and for phospho- lipids, both as a percentage of total lipid and as a per- centage of tissue. Appendix Table VI presents the raw data. Table 9 shows the percentage of total lipids in chicken (2.85) and pork (3.51) were fairly similar; however, the total lipid level in beef (7.22%) was much higher. The percentage of neutral lipids in the tissues were very similar for chicken (2.14) and pork (2.77), while beef contained over two-fold more neutral lipids. The data in Table 9 also show that the percentage of phospholipid to total lipid increases as the percentage of lipid decreases. For example. beef, which contained 7.22% total lipid expressed as per- cent of tissue, contained only 10.40% phospholipid when expressed as a percentage of total lipid. This can be compared to corresponding values for chicken of 2.85 and 23.57%, respectively. This is in general agreement with the premise of Dugan (1971) that the percentage of phospho- lipid to total lipid increases from less than 10% to nearly 70% as total lipid decreases from 5 to 1%. Table 9 also reports the average levels of phospho- lipid as a percentage of tissue for the three species. The mean phospholipid levels were 0.66% for chicken and pork and 0.73% for beef. These values are within the range of 0.5 to 1.0% phospholipid as a percentage of muscle, which u? .mmaomss nousop msfimsmn 0cm xmmpm Maman mo oamemm 000000800 A00 .0000800 @0009 0:0 £903 00x08 mm; maomse mamaS0csmPHEmm one on .umnpmmop 00x08 one: mmaomse anSp 0:0 Pmmmpm ADV .Amnm m: and 000 00 many ommnopm no 000000: 00 cosmmmpm 0o mwmacnwmms .mamEasm #:0000000 0 000 zHCO mmamswm 300 no mwmse>m HHmhm>o cm ma msam> Scam Amv 00.0 0:.0H 00.0 00.0 A000000 00.0 00.0H 00.0 Hn.m “000000 00.0 0n.nm 0H.m n0.~ H000000000 “000000 00 Hofion1av AosmmH0 av A0000H0 av mHaemm oHaaHoemmoam oflaHHoaamoam 00000H Hmcpsoz moflaHg H0000 Amv moamsmm 000m 0:0 0000 .cmonao 000 0H0>0H onHHongmoa0 one 0000H Hmcpsmz .0000; H0000 .0 0H000 48 was reported earlier by Dugan (1971). Correlation of TBA Values and Sensory Panel chres The correlation coefficients between TBA values and sensory scores for chicken, pork and beef were determined as described by Snedecor and Cochran (1973). The TBA values utilized in the calculations are shown in Appendix Table I, while the sensory panel scores are found in Appen- dix Table IV. Table 10 presents the calculated correlation coefficients. Table 10. The Correlation Coefficients of TBA Values and Taste Panel Scores No. of Sample Samples "r” Value Chicken 32 -0.71** Pork 32 '0-57** Beef 32 -0.36* ** Significant at the<: 0.01 level. * Significant at the<: 0.05 level. 49 All three species showed a significant negative corre- lation between TBA values and sensory panel scores. This suggests that in chicken, pork and beef, regardless of treatment, the samples with high TBA values tended to be scored lower than those with low TBA values. This further confirms the existence of a relationship between warmed-over flavor and sensory panel scores. In general, these results agree with those of Zipser gt a1. (1964), who found a high correlation between TBA values and develOpment of oxidative off flavor in cooked meats. The significance of the correlation coefficients between TBA numbers and sensory evaluation scores were tested for significance using the Z-test as described by Snedecor and Cochran (1973). It was found that the correlation coefficients for chicken and beef were significantly different (P < .05), whereas, the "r" values for chicken and pork and for pork and beef were not significantly (P < .05) different. The coefficient of determination for chicken was 50.4%, which indicated that over 50% of the variation in panel scores could be accounted for by a corres- ponding change in TBA values. For beef, on the other hand, only 12.9% of the variation in panel scores could be accounted for by a similar change in TBA numbers. Thus, results verify the greater degree of oxidative deterioration in chicken as compared to beef, while pork was intermediate between the other two. 50 Relationship of W The level of lipids in samples with and without nitrite (Appendix Table VI) were correlated separately against their corresponding TBA values (Appendix Table I). The lipid measurements subjected to correlation analysis with TBA values were total lipid and phospholipid as a percentage of tissue and phospholipid as a percentage of total lipid. Table 11 presents the correlation coefficients between the TBA values and total lipids for samples with and without nitrite. The relationships between total lipid levels and TBA numbers were not statistically significant (P < .05) for any of the three species (i.e., beef, pork and chicken). These results suggest that total lipid level is not an important contributor to TBA values. Thus, total lipids do not appear to be involved in develOpment of warmed-over flavor. Con- versely, Wilson (1974) reported that both porcine red and white muscle showed a significant positive correlation (P < 0.10) between TBA numbers and total lipid levels. Table 12 shows that the TBA values and phospholipid levels as a percentage of muscle tissue were significantly (P <:.05) related for pork samples without nitrite. This relationship suggests that high levels of phospholipids are major determinants for warmed-over flavor development in pork. However, addition of nitrite retarded oxidation of these lipids in pork muscle. 'All other correlation 51 .H0>0H no.0 v 000 00 0000H0H00Hm a. 00.0 0H 00.0 0H 0000 00.0 NH 00.0 0H 0000 00.0 0H nm.0 0H 0000000 mesam> =0: mamsmm mo .03 mmus> =0: magfism 00 .oz maasam 00000Hz 00H; 00Hassm 00H0002 psospHs 00Hmemm mam>mq 0090A Hmwoe 0cm mmzam> 0H m0.0 0:9 #0 0oswofihfiswwm 0000000GH Aav 0m.0 0H 0H.0 ma 000m 0H.0 NH am0.0 0H #000 00.0: 0H H:.0 3H 20x00£0 003H0> :0: 0Hmemm he .02 003H0> =0: 0H0§0m Mo .02 0Hasmm 0000000 0003 00H000m 00H00Hz psoanz 00H0000 000000 Mo vs0000m 00 muwmfiflonmmomm 0cm 005H0> «m9 00 02000000000 £0000H00000 059 .NH manna 53 coefficients were not statistically significant. In con- trast to these results. Wilson (1974) found a significant negative correlation (P < .05) between TBA values and phospholipids as a percentage of muscle tissue. This was true in both red and white muscle from pork and suggested that there was an inverse relationship between TBA values and phospholipid levels as a percentage of tissue. Table 13 shows the correlation coefficients between TBA values and phospholipids as a percentage of lipid. The data show that the TBA numbers and the phospholipid levels were significantly (P < .05) correlated for the nitrite treated chicken, but the relationship was negative. Thus. results suggest that the TBA values increased in the nitrite treated chicken as the level of phospholipids (as percentage of lipids) declined. All other correlations were not signifi- cant. In contrast, Wilson (1974) found that the TBA values and the levels of phospholipid as a percentage of total lipid were significantly (P < .05) correlated for red muscles from.pork, although the relationship was negative. Dugan (1971) reported that the composition of phospho- lipids varies between animals and for different carcass locations. Lea (1957) stated that the phosphatidylethano- lamine fraction accounts for very small percentage of the total phospholipids, whereas, phosphatidylcholine com- prises the bulk of the phospholipids. He found that oxida- tion of phosphatidylcholine was only of the order of one hundredth of phosphatidylethanolamine. He further reported 54 mn.0 0 .H0>0H no.0 000 00 000000000Hm A00 HN.0 ma 0000 00.0- NH 00.0 0H 0000 000.0- ‘ 0H 00.0 0H 0000000 m0sHm> :0: 0H080m 0o .02 00:00> =0: magsmm Mo .02 0Haswm 00H00Hz 0003 00Hgs0m no vcmonmm 00 mnwmwaonmmonm 020 0000002 0000000 00H0000 00H00q H0000 000H00 000 00 00000000000 00000H00000 000 .mH 0H000 55 that the cephalin ("Kephalin") content of commercial lecithin has antioxidant effects based upon the inactivation of traces of catalytically active metals. such as Fe or Cu. Thus, in the present study the lower rate of oxidation at higher levels of phospholipid as a percentage of total lipid in chicken is postulated to be due to a change in phospholipid composition, more specifically, to a decline in the proportion of phosphatidylethanolamine and a corres- ponding increase in the pr0portion of the more oxidatively stable phospholipids such as phosphatidylecholine. SUMMARY AND CONCLUSIONS The studies reported herein were designed to ascertain the role of nitrite in development of warmed-over flavor in chicken, pork and beef. Samples with and without added nitrite were evaluated by TBA numbers and panel aroma/flavor scores before and following cooking at 0 days and after #8 hours storage at 4°C. In addition. the relationship between TBA numbers and total lipid and phospholipid levels were determined in an effort to ascertain their relative contri- butions to warmed-over flavor develoPment. The most dramatic change in TBA values for all three species occurred during refrigerated storage (48 hours at 4°C) of the nitrite-free cooked meat. Cooked meat without added nitrite had a 5-fold higher TBA value for pork and was 2-fold higher for beef and chicken than similarly treated samples containing nitrite. Thus, the addition of nitrite protected against oxidative changes during the storage of cooked meat. Sensory panel scores confirmed the protective influence of the addition of nitrite upon deve10pment of warmed-over flavor in meat from all three species. However, the higher coefficient of determination verifies the greater magnitude of warmed-over flavor development in chicken as compared to 56 5? beef. whereas. pork was intermediate between chicken and beef. Total lipid levels were not significantly correlated with TBA values for any of the three species. which suggests that total lipids are not major contributors to warmed-over flavor development. However, the correlation coefficient between TBA numbers and phospholipids as a percentage of tissue was significant (P <:.05) in nitrite free pork. but was not significant in pork containing nitrite. Thus, re- sults suggest that nitrite blocks the autoxidation of the phospholipids. A significant negative relationship (P < .05) was ob- tained between TBA values and phospholipids as a percentage of total lipid in chicken with added nitrite. It is postu- lated that the lower rate of autoxidation at higher levels of phospholipids may be due to an increase in the relative amounts of the more oxidatively stable phospholipids. such as phosphotidylcholine. BIBLIOGRAPHY BIBLIOGRAPHY Ackman, R. G. 1976. Prediction of fat stability. In "Proceedings of a symposium: Objective methods for food evaluation". Nat'l Acad. Sci.. Washington. D.C.. p. 272. Acosta, S. 0.. Marion. W. w., and Forsythe, R. H. 1966. Total lipids and phospholipids in turkey tissue. Poultry Sci. 45:16 9. Bailey, M. E. and Swain. J. w. 1973. Influence of nitrite on meat flavor. Proc. Meat Ind. Res. Conf. p. 29. Banks. A. 1944. A method for studying the effect of antiox- idants on the oxidation of aqueous suspensions of un- gatgrated fatty acids. J. Soc. Chem. Ind. (London) 38 o Barnett. H. W.. Nordin, H. R., Bird, H. D. and Rubin. L. J. 1965. A study of factors affecting the flavor of cured ham. 11th European Meeting of Meat Research Workers. Cited by Wasserman. A. E. and Talley. F. J. Food Sci. 37:536 (1972). Bender, A. E. and Balance. P. E. 1961. A preliminary exaggnation of the flavor of meat. J. Sci. Food Agr. 123 30 Brooks. J.. Haines. R. B., Moran, T.. and Pace. J. 1940. The function of nitrate. nitrite and bacteria in the curing of bacon and hams. Dept. Sci. and Indust. Res.. Food Invest. Spec. Report No. 49 (Great Britain). Brown, w. D.. Harris, L. S., and Olcott. H. S. 1963. Catalysis of unsaturated lipid oxidation by iron protoiorphyrin derivatives. Arch. Biochem. BiOphys. Campbell, A. M. and Turkki, P. R. 1967. Lipids of raw and cooked ground beef and pork. J. Food Sci. 32:143. Chipault. J. R.. Mizuna. G. B.. and Lundberg. W. 0. 1956. Antioxidant prOperties of spices in foods. Food Technol. 10:209. 58 59 Cho. L. C. and Bratzler. L. J. 1970. Effect of sodium nitrite on flavor of cured pork. J. Food Sci. 35:668. Cross, C. K. and Ziegler, P. 1965. A comparison of the volatile fraction from cured and uncured meat. J. Food Sci. 30:610. Choudhury. R.. Roy, B.. and Arnold, L. K. 1960. The determination of the neutral oil content of crude vegetable oil. J. Amer. Oil Chem. Soc. 37:87. Day, E. A. 1966. Autoxidation of milk lipids. J. Dairy 801 I “‘3 3 1360 o Dugan, L. R.. Jr. 1961. Development and inhibition of oxidative rancidity in foods. Food Technol. 15(4):10. Dugan, L.R..Jr. 1971. Fats. In "The science of meat and meat products". Price. J. F. and Schweigert, B. S. (eds.). W. H. Freeman and Co.. San Francisco. Calif.. p. 133. Dugan. L. R.. Jr. 1976. Prediction of fat stability. In "Proceedings of a symposium: Objective methods for food evaluation". Nat'l Acad. Sci.. Washington. D.C.. p. 265. Erickson. D. R. and Bowers, R. H. 1976. Objective deter- mination of fat stability in prepared foods. In "Proceedings of a symposium: Objective methods for food evaluation". Nat'l Acad. Sci.. Washington. D.C., P- 133. Farmer, E. H. and Sundralingam, A. 1942. The course of autoxidation reactions in polyiSOpresence and allied compounds. I. The structure and reactive tendencies of the peroxides of simple olefins. J. Chem. Soc. p. 121. Farmer, E. H. and Sutton, D. A. 1943. The course of autoxidation reaction in polyisopresence and allied compounds. IV. The isolation and constitution of photochemically formed methylaleate peroxides. J. Chem. Soc..p. 119. Fitzgerald, G. A. and Nicherson. J. T. R. 1939. Problems arising during holding of poultry prior to eviscera- tion and freezing. Proc. 7th World's Poultry Congress. Cleveland, Ohio, p. 505. Folch. J.. Lees. M., and Stanley, G. H. S. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 49?. 60 Fox. J. B.. Jr. 1966. The chemistry of meat pigments. J. Agr. Food Chem. 14:207. Holman. R. T. 1960. Enzymes and polyunsaturated fatty acids. In "Food Enzymes". H. W. Schultz (ed.). Avi Publ. Co.. Westport. Conn.. p. 75. Hornstein, I. and Crowe, P. F. 1960. Flavor studies on beef and pork. J. Agr. Food Chem. 8:494. Hornstein, 1.. Crowe, P. F., and Heimburg. M. J. 1961. Fittg acid composition of meat lipids. J. Food Sci. 2351. Hougham, D. and Watts, B. M. 1958. Effect of variations in curing salts on oxidative changes in radiation sterilized pork. Food Technol. 12:681. Igene, J. 0. 1976. Effects of dietary fat and vitamin E upon the stability of meat in frozen storage. M.S. Thesis. Michigan State University, East Lansing, Michigan. Ingold. K. U. 1962. Metal catalysis. In "Symposium on foods: Lipids and their oxidations". H. W. Schultz. E. A. Day and R. C. Sinnhuber (eds.). Avi Publ. Co.. Westport. Conn., p. 93. Ingold, K. U. 1968. Principals of metal catalyzed lipid oxidation. In "Metal catalyzed lipid oxidation". R. Marcuse (ed.). SIK, Goteberg, Sweden. SIK— Rapport nr 240, p. 11. Kaunitz, H. 1962. Biological effects of autoxidized lipids. In "Lipids and their oxidation". H. W. Schultz, E. A. Day and R. C. Sinnhuber (eds.). Avi Publ. Co.. Westport, Conn., p. 269. Keeney, M. 1962. Secondary degradation products. In "Lipids and their oxidation". H. W. Schultz. E. A. Day and R. C. Sinnhuber (eds.). Avi Publ. Co.. Westport. Conn.. p. 79- V Kramlich. W. E. and Pearson. A. M. 1960. Separation .. and identification of cooked beef flavor components. Food Res. 25:712. Kummerow, F. A. 1962. Toxicity of heated fats. In "Lipids. and their oxidation". H. W. Schultz. E. A. Day and R. G. Sinnhuber (eds.). Avi Publ. Co.. Westport,Conn.. p. 294. 61 Kwon, T. W. and Watts. B. M. 1963. Determination of mal- onaldehyde by ultraviolet spectrophotometry. J. Food 8010 288 6270 Kwon, T. W. and Watts. B. M. 1964. Malonaldehyde in aqueous solution and its role as a measure of lipid oxidation in foods. J. Food Sci. 29:294. Labuza. T. P. 1971. Kinetics of lipid oxidation in foods. Crit. Rev. Food Technol. 2:355. Lea. C. H. 1957. Deteriorative reactions involving phos- pholipids and lipoproteins. J. Sci. Food Agr. 8:1. Lea, C. H. 1962. The oxidation deterioration of food lipids. In "Symposium on foods: Lipids and their oxi- dation". H. W. Schultz, E. A. Day and R. C. Sinnhuber (eds.). Avi Publ. Co.. Westport, Conn., p. 3. Iew, Y. T. and Tappel. A. L. 1956. Antioxidant and synergist inhibition of hematin-catalyzed oxidative fat rancidity. Food Technol. 10:285. Lillard. D. A. and Ayres. J. C. 1969. Flavor compounds in country cured hams. Food Technol. 23:251. Liu. H. P. and Watts, B. M. 1970. Catalysts of lipid peroxidation in meat. 3. Catalysts of oxidative rancidity in meats. J. Food Sci. 35:596. Love, J. D. and Pearson, A. M. 1971. Lipid oxidation in meat and meat products. A review. J. Amer. Oil Chem. Soc. 48:547. Lundberg. 0. 1962. Mechanisms of lipid oxidation. In "L1pids and their oxidation". H. W. Schultz. E. A. Day and R. C. Sinnhuber (eds.). Avi Publ. Co.. Westport, Conn.. p. 31. MacLean, J. and Castell. C. H. 1964. Rancidity in lean fish muscle. I. A proposed accelerated copper-catalyzed method for evaluating the tendency of fish muscle to become rancid. J. Can. Fisheries Res. Board 21:1345. MacNeil, J. H. and Mast. M. G. 1973. Frankfurters without nitrates or nitrites. Food Prod. Devel. 7:36. Maier, V. P. and Tappel. A. L. 1959. Products of un- saturated fatty acid oxidation catalyzed by hematin compounds. J. Amer. Oil Chem. Soc. 36:12. 62 Marcuse. R. and Johansson. L. 1973. Studies on the TBA test for rancidity grading. 11. TBA reactivity of diffgrent aldehyde classes. J. Amer. Oil Chem. Soc. 50:3 7- Matsuo, N. 1962. Nutritional effects of oxidized and thermally polymerized fish oils. In "Lipids and their oxidations". H. W. Schultz. E. A. Day and R. C. Sinnhuber (eds.). Avi Publ. Co.. Westport, Conn., p. 321. Moskovits, V. G. and Kielsmeier, E. W. 1960. An effect of iron and sodium chloride on flavor in sausage. Proc. 12th Res. Conf., Amer. Meat Institute Found.. Univ- ersity of Chicago, Illinois. p.121. Patron, A. 1968. Oxidation through metal catalysis and flavor stability of foods containing animal fats. In "Metal catalyzed lipid oxidation". R. Marcuse (ed.)i SIK, Goteberg, Sweden. SIK-Rappert nr. 240, p. 17 . Patton, S. 1974. Malonaldehyde, lipid oxidation. and the thiobarbituric acid test. J. Amer. Oil Chem. SOC. 51(3):1140 Pearson, D. 1968. Application of chemical method for the assessment of beef quality. 111. Methods related to spoilage. J. Sci. Food Agric. 19:553. Piotrowski, E. G., Zaika, L. L.. and Wasserman, A. E. 1970. Studies on aroma of cured ham. J. Food Sci. 35:321. Sato, K. and Hegarty. G. R. 1971. Warmed over flavor in cooked meats. J. Food Sci. 36:1098. Sato, K. and Herring, H. K. .1973. The chemistry of warmed over flavor in cooked meats. In "Proc. 26th Ann. Recip. Meat Conf." Amer. Meat Sci. Assn.. p. 64. Sherwin, E. R. 1968. Methods for stability and antioxi- dant measurement. J. Amer. Oil Chem. Soc. 45:632A. Sherwin, E. R. 1972. Antioxidants for food fats and oils. J. Amer. Oil Chem. Soc. 49:468. 63 Simon. 8.. Ellis. E. D.. MacDonald, B. D.. Miller, D. G.. Waldman, R. C..and Westberg, D. O. 1972. Influence of nitrite on quality of packaged frankfurters. 18th Ann. Meeting EurOpean Meat Research Workers, University of Guelph. Guelph, Ontario, Canada. Cited by Bailey. M. E. and Swain, J. W.. Proc. Meat Ind. Res. Conf. (1973). Sinnhuber, R. 0.. Yu. R. C.. and Yui. T. C. 1958. Char- acterization of the red pigment formed in the 2-thio- barbituric acid determination of oxidative rancidity. Food Res. 23:626. Snedecor, G. W. and Cochran, W. G. 1973. Statistical methods. 6th ed.. The Iowa State University Press. Ames. Iowa. Swain. J. W. 1972. Volatile flavor constituents of pork cured with and without nitrite. Ph.D. Thesis. University of Missouri, Columbia. Mo. Tarladgis, B. 0., Watts. B. M.. Younathan, M. T.. and 1960. A distillation method for the Dugan. L. R. quantitative determination of malonaldehyde in rancid J. Amer. Oil Chem. Soc. 37:44. Tarladgis. B. G. 1961. A hypothesis for the mechanism of the heme-catalyzed lipid oxidation in animal J. Amer. Oil Chem. Soc. 38:479. foods. tissues. Tarladgis. B. G., Pearson. A. M.. and Dugan, L. R.. Jr. The chemistry of the 2-thiobarbituric acid 1962. test for the determination of oxidative rancidity in foods. I. Some important side reactions. J. Amer. Oil Chem. Soc. 39:34. Timms, M. I. and Watts. B. M. 1958. The protection of Food Technol. 12:240. cooked meats with phosphates. Turner, E. W.. Paynter, W. D.. Montie, E. G.. Bessert, M. W.. Struck. G. M.. and Olson. F. C. 1954. Use of the 2-thiobarbituric acid reagent to measure rancidity in frozen pork. Food Technol. 8:326. 1972. The effect of Wasserman, A. E. and Talley, F. J. sodium nitrite on the flavor of frankfurters. Food Sci. 37:536. The kinetics and mechanism of metal- Waters. W. A. 1971. Jo mere Oil Chem. SOC. 48: catalyzed autoxidation. 427. Watts, B. M. 1954. Oxidative rancidity and discoloration in meat. Adv. Food Res. 5:1. 64 Watts. B. M. 1961. The role of lipid oxidation in lean tissues in flavor deterioration of meat and fish. In "Proceedings flavor chemistry symposium". Campbell Soup Co.. Camden. New Jersey. p. 83. Watts. B. M. 1962. Meat products. In "Symposium on Foods: Lipids and their oxidation“. H. W. Schultz. E. A. Day and R. C. Sinnhuber (eds.). Avi Publ. Co.. Westport. Conn.. p. 202. Watts. B. M.. Kendrick. J.. Zipser. M.. Hutchins. B.. and Saleb. B. 1966. Enzymatic reducing pathways in meat. J. Food Sci. 31:855. Wills. E. D. 1964. The effect of inorganic iron on the thiobarbituric acid method for the determination of lipid peroxides. Biochem. Biophys. Acta 84:475. Wills, E. D. 1966. Mechanism of lipid peroxide formation in animal tissues. Biochem. J. 99:667. Wilson. B. R. 1974. Effect of total lipids and phospho- lipids on warmed-over flavor measured by TBA analysis in muscle from several species. M.S. Thesis. Michigan State University. East Lansing. Mich. Witte. V. C.. Krause. G. F. and Bailey. M. E. 1970. A new extraction method for determining 2-thiobarbituric acid values of pork and beef during storage.. J. Food Sci. 35:582. Younathan. M. T. and Watts. B. M. 1959. Relation of meat pigments to lipid oxidation. Food Res. 24:728. Younathan. M. T. and Watts. B. M. 1960. Oxidation of lipids in cooked pork. Food Res. 25:538. Yu. T. C. and Sinnhuber. R. O. 1962. Removal of inter- ferring pigments in determining malonaldehyde by the 2-thiobarbituric acid reaction. Food Technol. 16:115. Yu. T. C.. Ianders. M. L.. and Sinnhuber. R. O. 1969. Storage life extension of frozen silver salmon steaks. Food Technol. 23:1602. Zipser. W. M. and Watts. B. M. 1962. A modified 2-thio- barbituric acid (TBA) method for the determination of malonaldehyde in cured meats. Food Technol. 16(7):102. Zipser. W. M.. Kwon. T. W..and Watts. B. M. 1964. Oxida- tive changes in cured and uncured frozen cooked pork. Jo Agr. FOOd Chemo 12:105. APPENDIX 65 APPENDIX TABLE I TBA Values for Chicken. Beef and Pork Species Chicken Beef Pork Sample ~ No. 0 Day 48 Hr 0 Day 48 Hr 0 Day 48 Hr Raw 1 1.75 3.78 1.38 2.41 0.80 3.14 Without 2 2.89 6.84 1.13 2.46 2.04 2.66 NaNO2 3 3.21 7.59 0.15 0.39 1.34 2.00 4 2.24 3.79 1.04 2.12 1.91 2.12 mean 2.51 5.50 0.92 1.84 1.52 2.48 Raw 1 1.36 0.87 0.92 1.68 0.21 1.26 With 2 0.70 0.70 1.15 1.42 1.9 2.02 NaNO2 3 2.15 3.01 0.10 0.44 0.2 0.73 1.24 1.32 0.48 1.16 1.01 1.69 mean 1.36 1.47 0.66 1.17 0.85 1.42 Cooked 1 4.10 8.13 1.92 4.96 0.81 5.61 Without 2 2.89 5.68 1.34 3.84 2.23 6.23 NaNO2 3 4.23 8.89 0.18 3.43 1.64 9.99 4 3.12 5.23 0.86 4.25 2.66 9.60 mean 3.58 6.98 1.07 4.12 1.84 7.85 Cooked 1 1.09 1.92 1.18 1.74 0.28 0.24 With 2 0.65 4.36 1.27 1.97 1.41 1.73 NaNO2 3 1.62 4.23 0.14 1.74 0.35 1.74 4 0.91 1.72 0.41 2.81 0.87 2.83 mean 1.06 3.07 0.75 2.06 0.73 1.64 j- .E _...I-|_I .llIa-ll 66 .00>00 00.0 0:0 00 00000000w0m *. .00>00 00.0 000 00 0:000m0cw0m * Hm Hma.ooa Hmpoa mmm.o Hm dém.ma pound Hmscwmmm m-.0 ~00.0 0 «00.0 0m00000 x mc00000 x 0000002 0sm.~ 000.~ 0 000.~ 0w00000 x 000np0z *tmon.oa n:m.m H m:m.m ammuopm x mcwxooo :om.o¢ mwa.om a nma.on m20xooo x mao>oa 0900902 ttmzm.o 0mm.o H wmm.o emanovm A: m: m> amp 0 0000.m mmm.s 0 mmm.s 000000 0> 300 *awmo.nm $00.50 A mam.nw mp0090: on m> op0npwz *a:mo.o mm:.m m oom.wa mamswc< -owvaPmpm m mumsvm c002 Eocooum mohmsdw mocmfihm> Mo mohsom Ho mmohwon Ho 85m 0:00> #:0020000 .smonno 000 mocmwum> mo m0mha¢2¢ no manna 00 00.0 0:0 00 0:000000w0m 0* .00>00 no.0 0:» #0 0200000cw0m 0 Hm Hm.ona Hmpoa wam.o Hm wwm.wa gouge Hmzcwmom. 0.000.~0 000.00 0 000.00 0w00000 x mz00000 x 000up0z 0*m0~.00 000.00 0 000.00 0w00000 x wc00000 $0000.00 mmo.m0 0 mm0.m0 0mmuopm x 000000: ##mom.mm m:m.mm H m:m.mm ammuovm 0: m: m> mac 0 0*Am0.00 000.00 0 000.00 mc00000 x 000>00 000up0z *tmow.ma mam.wa a mam.ma coxooo m> 3mm *tomm.m¢ mmm.o: H mma.o: opwnpwc oz m> ovwnp0z m0a.~ 00:.m n mum.s 00ma0a< 009m0pmam m oumzdm cams aocmmum monmsvm 0020000> mo oonaom Ho mmmpwon mo 85m mIHH mumda NHszmmd 0:00> 020000009 .xnom now oosm00m> mo mHthms< Ho manna .Hm>ma H0.0 ms? 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IIII IIII IIII H 30¢ 00.00 00.00 H5.0 05.0 50.0 00.0 0H.0 0H.0 .0 00.H0 00.H0 00.0 00.0 0H.0 .00.0 00.0 00.0 .0 00000Hz 50.0H 00.00 00.0 05.0 50.0 00.0 00.0 00.0 0 0000003 00.0H 05.0H 05.0 05.0 50.0 00.0 55.0 0H.0 H :00 00 00 000 0 00 00 000 0 00 00 000 0 nm 00 000 0 .0z 0Hna0m A0000H 00 00 0HnHH0000000 A000000 00 00 0H00H0000000 A000000 00 00 00000 H000smz £00m :H chdH no mm0PCmonmm 0 m0 000 0 00 0H0>00 0000H0000000 000 000H0 H0u0smz .00000 H0000 m|H> mnm00 00:0H0000000 0:0 00000 H00000z .00000 H0000 UIH> mnm<9 NHszmm< 79 APPENDIX TABLE VI-C (continued) % Drip Lost During Cooking Without With Animal Nitrite Nitrite Beef 1 20.00 20.00 Beef 2 28.00 28.00 Beef 3 9.89 6.20 Beef 4 30.00 14.00 HICHIGRN STRTE UNIV. LIBRRRIES \llilWHlllllll‘HIWHIWIMIHIIIHIINHIINNIHWHI 31293102003708