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Occurence (sic) of a pink color in cooked turkey breast Girard, Benoît 1987

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OCCURENCE OF A PINK COLOR IN COOKED TURKEY BREAST by BENOIT GIRARD B.Sc.(Agr.), Universite Laval, 1984 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Food Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1987 ® Benoit Girard, 1987 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f PfloA •V'Q.^Cg The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 ABSTRACT A peculiar problem involving cooked turkey products is the appearance of a pink color which consumers often associate with undercooking. Measurements of redness over a period of 4 days of storage were taken on 2 mm thick breast slices from 12 or 18 week old turkeys cooked at selected temperatures of 65, 75, 85 and 95 °C. An analysis of variance indicated that, unlike the age effect, temperature, storage time and their interaction were statistically significant at p<0.01 level. Proper cooking, achieved when an end-point temperature of 85 °C was reached, did not only produce a white to golden-brown color. A tinge of redness was detected by Hunter a^ measurement immediately after cooking and disappeared after 3 to 4 days. A simple method developed for the evaluation of pigments in situ, using transmission spectrophotometry, revealed that the residual pink color could be caused by cytochrome c. Visible spectra and electrophoretograms of extracts from cooked breast slices supported the involvement of this pigment. The concentration -2 -2 of cytochrome c in these extracts was 7.9x10 and 5.4x10 mg/g tissue in samples from 12 and 18 week old birds respectively. However the total hemoprotein content in these same breast samples increased from 0.60 to 0.77 mg/g tissue. The method for the evaluation of pigments in situ also was modified in order to investigate the effect of air contact on color immediately after cooking. Additional absorption bands were present when meat slices were kept under anaerobic conditions. The rapid disappearence of these peaks once the meat surface was ii exposed to air indicated the susceptibility of other hemochromes to oxidation and therefore underlined their relative unimportance in the pinkening phenomenon. Since the above hemoproteins and hemochromes involved in the pinkening phenomenon need to be in a reduced form to give the obtained difference spectra, procedures using ultrafiltration and chromatography were established to search for reducing compounds. The reducing capacity of 1 mL fraction during the first minute of contact with a solution of hemoprotein was called hemoprotein initial reducing activity (IRA). Two groups, one above (higher molecular weight compounds or HMW) and one below (lower molecular weight compounds or LMW) 3000 daltons, were separated. The LMW had a low IRA for both myoglobin and cytochrome c. On the other hand, HMW were produced after heating at 85 °C for 10 min and strongly reduced only cytochrome c. It is postulated that the isolated material may be Maillard reaction products. iii TABLE OF CONTENTS ABSTRACT " TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES vii LIST OF APPENDICES x ACKNOWLEDGEMENTS xi I. INTRODUCTION 1 II. LITERATURE REVIEW 4 A. Ultrastructural organization of skeletal muscle 4 B. Meat Color 6 C. Myoglobin 6 1. Myoglobin distribution 6 a. Animal species 7 b. Muscle type 7 c. Genetic and environmental factors 7 d. Post-slaughter treatment 8 2. Physiological role of myoglobin 9 3. Structure of myoglobin 10 4. Electromagnetic properties of myoglobin 14 5. The common myoglobin derivatives 22 a. Deoxymyoglobin 22 b. Oxymyoglobin 24 c. Nitric oxide myoglobin 24 d. Carboxymyoglobin 25 e. Ferrohemochromes 26 f. Metmyoglobin 28 g. Ferrihemochromes 29 D. Cytochrome c 29 1. Structure of cytochrome c 30 2. Physiological function of cytochrome c 33 E. Hemoproteins as part of the meat system 36 III. MATERIALS AND METHODS 41 A. Chromaticness scale ^ measurements 41 B. Identification of pigments in cooked turkey breast 44 1. Transmission spectrophotometry of integral meat slices 44 2. Transmission spectrophotometry of meat extract 45 3. Electrophoresis of meat extract 46 4. Transmission spectrophotometry of soluble hemoprotein complexes 47 iv 5. Determination of total heme proteins and of cytochrome c ... 47 C. Measurement of reducing activity 49 1. Detection of reducing activity using hemoproteins 49 2. Fractionation of reducing compounds 53 a. Gel filtration 53 b. Ultrafiltration 54 IV. RESULTS AND DISCUSSION 56 A. Colorimetric evaluation of red color in cooked turkey breast 56 B. Characteristics of involved pigments 61 1. Transmission spectra of integral meat slices 61 2. Extraction of cytochrome c 67 3. Effect of air contact on color after cooking 70 4. Transmission spectra of soluble heme-protein complexes 73 5. Hemoprotein content in turkey breast meat 76 C. Reducing activity in cooked turkey breast 78 1. Reduction reactions with myoglobin and cytochrome c 80 2. Gel filtration of cooked turkey breast extract 83 3. Combination of ultrafiltration and chromatography 85 CONCLUSION 92 REFERENCES 95 APPENDICES 106 v LIST OF TABLES Table 1. Absorbance (414 nm) of serial dilutions of oxidized and reduced cytochrome c with an initial concentration of 12.5 mg/100 mL 50 Table 2. Analysis of variance on aj_, values of breast meat slices from 12 and 18 weeks old turkeys 57 Table 3. Theoretical wavelengths at maximum absorption (^MAX) °^ s o m e hemoproteins (adapted from Mahler and Cordes, 1971) 75 Table 4. Hemoprotein content of breast meat from 12 and 18 weeks old turkeys 77 vi LIST OF FIGURES Figure 1. Schematic representation of the structure of myofibrils 5 Figure 2. Backbone structure of myoglobin 11 Figure 3. Structural representation of myoglobin 12 Figure 4. Energy level diagrams 16 Figure 5. Energy level diagram for the Fe-C>2 complex 17 Figure 6. The bonding geometry for the Fe-02 complex 18 Figure 7. Absorption spectra of the three most common pigments in fresh muscle tissue 23 Figure 8. Structure of mitochondrial cytochrome c from tuna 31 Figure 9. Absorption spectra of oxidized and reduced horse heart cytochrome c extracted into an aqueous solution 34 Figure 10. Difference spectra of intact rat ascites tumor cells and of mitochondria isolated from such cells 34 Figure 11. Scheme of probable interactions between cytochrome c and mitochondrial membrane 35 Figure 12. Changes in the adenosine triphosphate content and pH of breast muscle during 8 h post-mortem aging at 15 °C 38 Figure 13. Changes in pH of turkey breast muscle during 72 h post-mortem . 39 Figure 14. Coupled microscope glass slides separated with 2.0 mm spacers 42 Figure 15. Linear relationship between percentage of reduced form of cytochrome c with a starting concentration of 12.5 mg/100 g, and absorbance at 414 nm 51 Figure 16. Linear relationship between percentage of reduced form of myoglobin with a starting concentration of 12.5 mg/100 g, and absorbance at 408 nm 52 Figure 17. Surface plot of the relationship between cooking temperature (T), storage time (S), and value of turkey breast meat slices 60 vii Figure 18. Absorbance difference spectra of a turkey breast slice cooked at 65°C for 10 min and stored at 0°C for 0 day (A), 1 day (B), 2 days (C), 3 days (D) and 4 days (E) 63 Figure 19. Absorbance difference spectra of a turkey breast slice cooked at 75°C for 10 min and stored at 0°C for 0 day (A), 1 day (B), 2 days (C), 3 days (D) and 4 days (E) 64 Figure 20. Absorbance difference spectra of a turkey breast slice cooked at 85°C for 10 min and stored at 0°C for 0 day (A), 1 day (B), 2 days (C), 3 days (D) and 4 days (E) 65 Figure 21. Absorbance difference spectra of a turkey breast slice cooked at 95°C for 10 min and stored at 0°C for 0 day (A), 1 day (B), 2 days (C), 3 days (D) and 4 days (E) 66 Figure 22. Absorbance spectra of: (A) a commercial solution of reduced cytochrome c, (B) myogen extracted from turkey slices cooked at 85 °C for 10 min and reduced with sodium dithionite 68 Figure 23. Electrophoretogram of myogen extracted from turkey breast slices cooked at 85°C for 10 min (Myo) and standard solution containing myoglobin and cytochrome c (Std) 69 Figure 24. Absorbance difference spectra of a turkey breast slice cooked at 65 °C for 5 min in a cuvette: (A) covered with a strip of pyrex, (B) uncovered and exposed to air 71 Figure 25. Absorbance difference spectra of a turkey breast slice cooked at 85°C for 5 min in a cuvette: (A) covered with a strip of pyrex, (B) uncovered and exposed to air 72 Figure 26. Absorbance spectra of 5 mg/mL bovine serum albumin added to: (A) 2.5xl0"2 mg hematin/mL and 3.0x10"^  mg imidazole/mL reduced with sodium dithionite; (B) 2.5x10"^  mg hematin/mL reduced with sodium dithionite; (C) 2.5x10"^  mg hematin/mL 74 Figure 27. Influence of myogen extracted from 18 weeks old turkey breast slices cooked at 85 °C on the oxidation state of myoglobin over time 81 Figure 28. Influence of myogen extracted from 18 weeks old turkey breast slices cooked at 85 °C on the oxidation state of cytochrome c over time 82 Figure 29. Hemoprotein initial reducing activity (IRA) of gel filtrate fractions of extract from cooked turkey breast slices 84 viii Figure 30. Myoglobin initial reducing activity (IRA) of gel filtrate fractions of cooked and uncooked turkey breast ultrafiltrate 86 Figure 31. Cytochrome c initial reducing activity (IRA) of gel filtrate fractions of cooked and uncooked turkey breast ultrafiltrate 87 Figure 32. Effect of heating and storage time on the hemoprotein initial reducing activity (IRA) of turkey breast ultrafiltrate: Mb, myoglobin; CYT c, cytochrome c; UNE, uncooked; CKE, cooked at 85 °C for 10 min 89 ix LIST OF APPENDICES Appendix A. Step-by-step instructions concerning the development solutions, times and temperatures used for SDS-PAGE 106 Appendix B-l. Absorbance difference spectra of a turkey breast slice cooked at 75 °C for 5 min in a cuvette: (A) covered with a strip of pyrex, (B) uncovered and exposed to air 1Q7 Appendix B-2. Absorbance difference spectra of a turkey breast slice cooked at 95 °C for 5 min in a cuvette: (A) covered with a strip of pjrrex, (B) uncovered and exposed to air 108 x ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. John Vanderstoep for his patience and knowledgeable advice throughout the course of this research project and review of the thesis. Many thanks are directed to the members of the research committee, Dr. J.F. Richards, Dr. W.D. Powrie, Dr. S. Nakai and Dr. R.C. Fitzsimmons for their helpful and constructive assistance. I am also particularly grateful to Sara Weintraub and my parents, Yolande and Claude, for their inspiration and invaluable encouragement. xi I. INTRODUCTION ln recent years, poultry meat has become more and more important in pur diet. This trend is evidenced by the increasing consumption of poultry meat outside of the home. Fast food restaurants featuring chicken have shown a rapid growth (McDonald's, Kentucky Fried Chicken, etc.). At the same time, other types of fast food restaurants have extended their menus to include chicken sandwiches or sticks made from dark and white chicken meat. Parallel to this increasing use of chicken, there is a continual growth of processing turkey into products other than the "ready-to-cook" carcasses. Today, turkey slaughter-houses are converting increasing quantities of meat into roasts and turkey rolls for institutional use. Acceptable color of poultry products is one of the strongest influences on consumer acceptance. Physiological responses to color, as they relate to appetite, are considered important to consumers. With respect to turkey, the loss of redness of muscle during cooking is an indication of doneness. Consumers often object to variation from the normal white-to-golden brown of cooked turkey. Any trace of muscle pinkness or redness suggests undercooking. Pinkness in adequately cooked turkey breast muscle has been observed for many years. It may, on the one hand, be seen as a uniform pinkish tint after slicing the breast of a cooked whole turkey, or on the other hand, be described as a pink spot or ring in freshly cut surfaces of cooked turkey rolls. 1 INTRODUCTION / 2 Along with caramelization of carbohydrates and Maillard-type reactions between sugars and amino groups, the behavior of the sarcoplasmic hemoproteins during cooking determines the change of meat color from red to brown. Normally, heating to 85 °C denatures myoglobin (major meat heme pigment) in pure solution (Fogg and Harrison, 1975). But in situ, part of the myoglobin molecules may begin to coagulate at 65°C, co-precipitating with other muscle proteins (Ledward, 1971). As a result, the generation of pinkness in adequately cooked turkey muscle can not be attributed to the presence of undenatured myoglobin. A variety of causes have been suggested to explain the occurence of this peculiar pinkening phenomenon. They could be regrouped as follow: (1) diet, chill water and processing equipments contributing nitrates and nitrites (Froning et al., 1969a; Mugler et al., 1970; Brant, 1984; Nash et al., 1985), (2) carbon monoxide and nitric oxide contamination from gas-fired ovens or freezing processes (Pool, 1956; Everson, 1984), (3) preslaughter stress (Ngoka et al., 1982; Babji et al., 1982), (4) unusual heat stability of myoglobin and hemoglobin (Fransham and Walters, 1981), (5) presence of cytochrome c (Ngoka and Froning, 1982), and (6) presence of reduced nicotinamide-denatured hemochromes (Cornforth et al., 1986). Even if these conditions may possibly lead to pink color in meat, many cases remain unexplained. As Cornforth and his co-workers stated, the generality of the problem suggests that a more unifying explanation is needed, one in which endogenous constituents of turkey meat form the pink color. The object of the present research was: first, to evaluate the effects of cooking end point temperature and storage time on the redness of cooked turkey breast; INTRODUCTION / 3 secondly, to identify the responsible pigments; and thirdly, to investigate the endogenous requirements for the production of the spectral characteristics of these pigments. II. L I T E R A T U R E R E V I E W A . U L T R A S T R U C T U R A L O R G A N I Z A T I O N O F S K E L E T A L M U S C L E Meat is defined as the flesh of animals used as food. Both fresh and processed meats are derived primarily from muscle tissue. An understanding of meat should be based on an appreciation of the fact that muscles are developped and differentiated for definite physiological purposes in response to various intrinsic and extrinsic stimuli. In the living animal, muscles allow for locomotion as they convert chemical energy into mechanical energy through the process of contraction. Vertebrate muscle that is under voluntary control has a striated appearance when examined under the light microscope. It consists of fibers that are surrounded by an electrical excitable membrane called sarcolemma. A large part of the volume of muscle cells is occupied by their contractile elements, the myofibrils which are arranged in parallel bundles. The alternation of light and dark transverse bands along their length is due to the degree of penetration of the thin and thick filaments along each other (Figure 1). It is recognized that there are two major protein components in the water-insoluble filamentous elements of skeletal muscle, myosin and actin. They make up about 80% of the proteins of the contractile apparatus. In addition, there are at least six other protein components, tropomyosin, troponin, C-protein, M-line protein, and a- and /3-actinin (Lehninger, 1975). The myofibrils in turn are surrounded and bathed by the sarcoplasm, the 4 LITERATURE REVIEW / 5 Axis of contraction yu z A i • 'zone line band band \MM?/.IL., / Sarcomere \ / \ / ^ / \ / \ Abend ,lband, Abend I bend Abend Myofibril M / / Z H M M M 4444M H zone J I Thick I filaments \\ J. Thin filaments (actin) Dashed arrows in color show location of cross sections Cross section Cross section Figure 1. Schematic representation of the structure of myofibrils 1975) LITERATURE REVIEW / 6 intracellular fluid of muscle, which contains mainly glycogen, glycolytic enzymes, ATP, phosphocreatine, myoglobin, and inorganic electrolytes, as well as a number of amino acids and peptides. B. MEAT COLOR The color of fresh meat is mainly due to myoglobin, comprising 80 to 90% of the total pigments; hemoglobin, cytochromes, flavins, peroxidases, and catalases are also present but their contribution is minor. Other factors influence the perception of color in meat. These include the texture of the cut meat surface and the lighting conditions. The acquisition of knowledge pertaining to the key molecules, their structure, and their reactivity is desirable in aiding the prediction and control of the color of the products in which they are found. The remainder of this review will concentrate on two sarcoplasmic hemoproteins: myoglobin and cytochrome c. Because myoglobin is the main pigment of concern to meat scientists and processors, much more emphasis will be given to it. C. MYOGLOBIN 1. Myoglobin distribution Myoglobin (Mb) is localized mainly in the heart and skeletal muscles of vertebrates. The relative concentration of myoglobin depends on the species and age of the animal, the muscle type used, and the way the meat is treated following slaughter. To illustrate several general trends of myoglobin distribution in muscle, the following selected data is presented. LITERATURE REVIEW / 7 a. Animal species Myoglobin concentration varies for different animal species. For example, typical ranges of myoglobin concentration are 2.0-5.0 mg/g wet weight in beef (Hunt and Hedrick, 1977; Rickansrud and Henrickson, 1967); 4-7 mg/g in lamb (Ledward and Shorthose, 1971); 2.5-7.0 mg/g in pork (Topel et al., 1966) ; 3-4 mg/g in dark-meat poultry (Blessing and Muller, 1974); and 0.5-1.0 mg/g in light-meat tuna (Brown, 1962). b. Muscle type The muscle type used is perhaps even more important in determining myoglobin concentration than the animal species. In yellowfin tuna, for example, a typical light-meat section will contain approximately 0.7 mg of myoglobin/g, whereas the dark meat (from the epoxial muscle) can contain greater than 20 mg/g (Brown, 1962). In chicken, the myoglobin content is many times higher in concentration in the gizzard, 3.95 g%, than in other muscles; pectoral muscle contains 0.04 g%; leg muscle, 0.40 g%; and heart, 0.56 g% (Blessing and Muller, 1974). Varying myoglobin concentrations in beef longissimus, gluteus medius, and inner and outer semitendinosus were reported by Hunt and Hedrick (1977). c. Genetic and environmental factors Genetic and certain environmental factors in addition to age, exercise, and diet of the animal, may all affect muscle myoglobin concentration. In general, muscle myoglobin increases with the age of the animal, as shown by Ledward and Shorthose (1971) for lamb and by Nishida (1976) for chicken, where myoglobin concentration of the leg, heart, and gizzard doubled between 6 and 27 weeks LITERATURE REVIEW / 8 after hatching. Myoglobin content of the skeletal muscles has been shown to be lowered by iron deficiency in pig tissues (Kainski et al, 1967), increased in the same animal by vitamin E deficiency (Bender et al.,1959), and by excercise (Hagler et al., 1980; Pattengale and Halloszy, 1967). In an interesting study, Thomas and Judge (1970) showed that myoglobin in porcine skeletal muscle reached higher concentrations in animals reared at constant ambient temperatures vs fluctuating temperature environments, provided that the humidity was moderate to high. It has also been shown that animals acclimatized to high altitudes showed increased muscle myoglobin levels (Vaughan and Pace, 1956). Finally, there are always animal-to-animal variations in muscle myoglobin levels for all species, and genetic factors may play an important role. For example, Hart (1961) found lower myoglobin concentrations in porcine pale, soft, and exudate (PSE) muscle. However, this finding has been disputed by other workers (Briskey and Wismer-Pederson, 1961). d. Post-slaughter treatment Post-slaughter treatment of the carcass has a significant effect on the relative concentrations of myoglobin and hemoglobin. Its effect on the state of myoglobin will be discussed further on. Often, results of myoglobin and hemoglobin analysis on the same species and muscle will differ with the condition of the meat before sampling. For example, Hunt and Hedrick (1977) reported myoglobin accounting for 88.4 and 93.2% of the total pigment for longissimus dorsi and semi-tendinosus beef muscles, LITERATURE REVIEW / 9 respectively; whereas Rickansrud and Henrickson (1967) reported values of 79.91 and 68.21% for the same muscles. Presumably the difference is accounted for by the longer holding time of the carcass (4-5 days) by the former group of workers. Data for beef heart analysed immediately after slaughter, where myoglobin accounted for 58.4% of the total pigment, would tend to support this theory (Livingston and Brown, 1980). The total pigment concentration, along with certain properties affecting light scattering, will determine the color and acceptability of a meat product. Since myoglobin and hemoglobin are found at much higher concentration in skeletal muscle than any other pigment -e.g., myoglobin is found at almost 100-fold greater concentrations than cytochrome c in rat skeletal muscle (Akeson et al., I960)- their combined concentrations will determine the darkness of the cut. Since hemoglobin is drained during handling and storage, while the myoglobin is retained by the muscle's intracellular structure, the color of a meat sample can fade if inital Mb/Hb ratios are low. However, assuming that most of the hemoglobin is lost initially, most color changes in meat are due to reactions of myoglobin with other muscle components. 2. Physiological role of myoglobin The fact that myoglobin combines reversibly with oxygen is of utmost importance physiologically. Theoretical and experimental evidence continues to accumulate in support of the position that the passive reservoir function of myoglobin in muscle fibers is primary only in the case of diving mammals (e.g. seals, whales). In terrestrial mammals, the role of myoglobin is both dynamic facilitation of LITERATURE REVIEW / 10 intracellular diffusion to the interior respiratory sites (i.e., mitochondria) and static stabilization of tension at the points of delivery against fluctuations in demand, with the dynamic role perhaps predominating. Such a role demands, of course, that myoglobin be a freely diffusible cytosolute not located at fixed intracellular sites except for possible transient binding to mitochondria, for example during dissociation of oxygen and regeneration of deoxy-ferrous myoglobin which conceivably could be facilitated by binding. It has even been suggested that the facilitated diffusion role of myoglobin extends to its conveying waste metabolic heat from the source (mitochondria) to the sink (the cell wall) in vivo (Hills, 1973) and fatty acid transport (Gloster and Harris, 1977). 3. Structure of myoglobin Myoglobin is composed of a relatively small globular protein moiety called apomyoglobin, and a prosthetic group called heme (Figure 2). The molecular weight of myoglobin is reported to be in the range of 16850 to 17600 daltons (Wang, 1962). The whale myoglobin molecule has been almost completely described through the X-ray diffraction studies of Kendrew et al. (1960, 1961) and the amino acid sequence studies of Edmundson (1965). The analyses by electrophoresis and isoelectrofocusing show several zones of migration which correspond to the microheterogeneity of myoglobin. Renerre (1977) observed, in the case of beef, two main bands with a pi of 7.3 and 7.6. The prosthetic heme (also known as protoheme) is formed by four pyrole rings LITERATURE REVIEW / 11 Figure 2. Backbone structure of myoglobin. (Lehninger, 1975) LITERATURE REVIEW / 12 GLOBIN Figure 3. Structural representation of myoglobin. (Govindarajan, 1973) LITERATURE REVIEW / 13 connected by methine bridges having an iron atom in the central position. The structure of heme is shown in Figure 3. The prosthetic heme is responsible for the color of myoglobin. The polypeptide chain consists of eight helical segments compactly folded into a space 45x35x25 A (Kendrew, 1960) in such a manner that the interior of the molecule is completely filled with hydrophobic groups and all the polar residues are located on the outer surface. Deep in this hydrophobic interior is a cleft into which heme is fitted. The orientation of the heme group is such that the ordered vinyl groups are buried in the hydrophobic interior and the carboxyl group of propionic acid extends from the interior to form a part of the polar surface. Both propionic acid side chains of heme are hydrogen bonded to amino acid side chains of the globin moiety. The heme iron plays the role of nucleus in the whole molecule. It has the ability to accept six electrons in its outer orbital, four of which come from nitrogen atoms of the pyrole in the porphyrin ring, one from nitrogen of the histidine in the globin moiety, and one from the ligand bound by the heme. It is generally believed that the interaction between heme and globin stabilizes the whole molecule. The existence for the hypothesis comes from the work of Breslow et al. (1967) who showed that both protoporphyrin (heme without iron) and heme affect the alpha helix content of apomyoglobin (globin). LITERATURE REVIEW / 14 4. Electromagnetic properties of myoglobin The structure and chemistry of the iron atom is the key to understanding the reactions and color changes which myoglobin undergoes. The oxidation state and type of ligand bound to the iron center determine the color and reactivity of myoglobin under most reaction conditions. Livingston et al. (1981) wrote an comprehensive review about the subject, and it served as a major reference for the following information. Iron is a third-row transition metal, meaning that it has unfilled energy levels below its valence electron levels. It is the transition of electrons from filled to empty "3d" orbitals which accounts for the visible absorption of light, and thus the color of myoglobin complexes. The iron atom has eight valence electrons. Because of its low electronegativity, it 2 + may lose two valence electrons to form ferrous (Fe ) or three electrons to 3 + form ferric (Fe ) iron cations. These are the forms that are found in almost all myoglobin complexes observed. To understand how these forms will react, attention must be focused on how the ligands around the cation affect the remaining electrons. Crystal field theory demonstrates that for an iron cation surrounded by six ligands, the five electron levels will be divided into a group of higher and a group of lower energy levels. The way the iron d-electrons fill the energy levels depends upon the surrounding ligands. LITERATURE REVIEW / 15 Figure 4 shows some representative d-orbital electronic structures. The net "spin" of these complexes depends upon the number of unpaired Gone) electrons. The high spin derivatives have many unpaired electrons, and are named "paramagnetic". High spin ferric iron is found in metmyoglobin. Molecules with no unpaired electrons are "diamagnetic", e.g., the low-spin ferrous derivative. Only the upper right structure in Figure 4 is diamagnetic. This type of structure is formed in myoglobin complexes with certain types of ligand such as nitric oxide, carbon monoxide, and particularly oxygen. The energy-level diagram and the bonding geometry for the oxygen-myoglobin complex (oxymyoglobin) are shown in Figures 5 and 6. From Figure 5, much information about the properties of the oxymyoglobin complex can be deduced. The ferrous ion is in a high-spin, paramagnetic configuration, but a different one 2 + than seen previously for the Fe species with six ligands. This is because 2 + deoxymyoglobin (which is represented by the Fe energy levels) has five ligands around the iron, which alters its electronic distribution. The oxygen molecule, with its two unpaired electrons (paramagnetic), bonds to the paramagnetic ferrous species to form a complex which is clearly diamagnetic. The overall distribution of electrons in the complex is at a lower energy than in either of the starting species. This is the main reason the iron-oxygen complex is stable. The symbol "A" in Figure 5 represents an energy gap between the highest field and the lowest unfilled energy levels. Visible light can excite electrons over this small energy difference in oxymyoglobin. In fact, the exact wavelenghts observed -and thus the visible wavelenghts which are transmitted-L I T E R A T U R E REVIEW / 16 + + -K- + -r- -H- -H- -U-high spin, ferrous low spin, ferrous + + + high spin, ferric low spin, ferric Figure 4. Energy level diagrams. (Livingston et al., 1981) LITERATURE REVIEW / 17 Fed) Fe-0 2 02(t) Figure 5. Energy level diagram for the Fe-0 2 complex (Livingston et al., 1981) L I T E R A T U R E REVIEW / 18 v- Bond Figure 6. The bonding geometry for the F e - 0 2 complex (Livingston et al., 1981) LITERATURE REVIEW / 19 account for the red color which the complex takes on. Any other myoglobin complex with a similar bonding pattern, e.g., nitric oxide myoglobin (NOMb) and carboxymyoglobin (COMb), and thus an energy gap (A), will have a similar color to the eye. That does not mean to imply that the color of the oxymyoglobin derivatives observed is due solely to the electronic transition shown by A in Figure 5 -it is, in fact, predominantly due to the porphyrin-iron ir—A 2 transitions (Makinen et al., 1978). However, these transitions are also similar in heme complexes with ligands having similar bonding patterns. Figure 6 shows the geometry of the bonding orbitals just described. The drawing does not show, however, that the iron-oxygen bond is actually bent at 135° to the heme plane. Nitric oxide (NO) also bonds in a bent mode, whereas the Fe-CO bond in the carbon monoxide complex is close to 180° (Antonini and Brunori, 1971). Two types of bonding may be recognized in Figure 6. The a bond is formed by donation of electrons of the ligand (here, oxygen) to the iron cation. Most of the ligands which bind to myoglobin have this type of bonding, i.e., an electronegative atom (N or O) donates electrons to the metal. The other type of bonding shown is ir bonding, sometimes called "back-bonding", in which the iron donates electrons back to the ligand via the ligand's ir or ir* orbitals. This type of bonding is very important in oxymyoglobin, since oxygen is a rather weak a -electron donor. The iron complex in oxymyoglobin has a histidine ligand opposite to the oxygen. The histidine residue, bonded through a nitrogen atom, feeds extra electron density into the iron and subsequently the oxygen molecule and further LITERATURE REVIEW / 20 strengthens the back-bonding and stability of the complex. However, for back-bonding to occur, the metal must have sufficient electron density for donation to its ligand. Ferrous iron meets this criterion. The relatively low charge on the nucleus leads to expanded d-orbitals. This, along with its relatively many d-electrons, enables 7T-bonding with suitable ligands. In contrast, ferrimyoglobin is unable to form an oxygen adduct because the ferric heme iron is a poorer 7T donor than is its ferrous counterpart. This is compensated for in ferrimyoglobin complexes with six ligands (CN , NO and Ng ) that are superior donors. As such, they form low-spin complexes with electronic properties and, therefore, optical spectra and appearance similar to those of low spin ferrous complexes such as oxymyoglobin. Since oxymyoglobin, along with similar complexes of high field (i.e., ligands which induce pairing of electrons), has the desirable cherry-red color of fresh meat, it follows that to retain a desirable color, myoglobin must remain in the ferrous form. One should keep in mind that oxidation of oxymyoglobin to metmyoglobin and the reverse process (reduction) are always occuring in living muscle and for a significant time postmortem; thus the conditions for tipping the balance to the reduced form must be maintained. Several more concepts in the coordination chemistry of myoglobin help in predicting the behavior of all the important forms of myoglobin: A. Both ferrous and ferric forms of myoglobin usually have six ligands bound to the iron cation. The exception to the rule is native deoxymyoglobin, LITERATURE REVIEW / 21 which has five ligands. Only the native myoglobin conformation can stabilize this pentavalent geometry. If denaturation occurs, the resulting myoglobin derivatives must pick up an extra ligand. Four of the six coordination positions are occupied by the heme pyrole nitrogens, which form such a stable chelate -the association 30 constant is probably on the order of 10 (Buchler, 1975)- that displacement of the iron is unlikely. In the native myoglobin derivatives, the fifth position (also called the axial or proximal position) is occupied by histidine, which is considered a moderately strong ligand. This leaves the sixth position open for substitution by oxygen or other ligands. B. For a ligand to coordinate to the iron center in native myoglobin, there must be access to it via the cleft in the protein structure. Since the cleft is small (Takano, 1977b), there will be significant steric hindrance for a large ligand. Therefore, only small ligands (e.g., O2, CO, NO) can bind to the iron center in native myoglobin. However, if the protein becomes denatured (by heat or low pH, for example), the heme becomes exposed, and coordination by larger ligands can occur. C. Deoxymyoglobin contains a high-spin (paramagnetic) ferrous iron center. This derivative can take on a sixth ligand which will preferentially be paramagnetic and a ff-acid, i.e., a ligand which can accept back-bonding from the metal's d-electrons. Thus, it is perfectly designed for oxygen-binding, but other ligands may also meet these criteria. If the resulting myoglobin-ligand complex is diamagnetic, it will have less tendency to react with a paramagnetic species. D. High-field ligand (i.e., ligands which can back-bond and induce electron-pairing in iron) can stabilize ferrous iron against oxidation. This is desirable from the point of view of color, since the low-spin ferrous complex LITERATURE REVIEW / 22 (MbOg, MbCO and MbNO) cause the cherry-red color of fresh meat. However, these same ligands display another interesting effect. They labilize the ligand opposite to them to substitution. This so-called "trans effect" means that if a complex involving a high field ligand has accessibility to other ligands, one can expect rapid exchange of the opposite ligand. This exchange probably continues until a second high-field ligand is bound. 5. The common myoglobin derivatives a. Deoxymyoglobin Deoxymyoglobin which lacks a ligand in the sixth position, is the ferrous myoglobin derivative found under low oxygen tension (pOg) conditions. These conditions are found in muscle and meat, where intracellular oxygen tensions may go below 0.1 mm Hg (Whalen and Nair, 1967). This form of myoglobin is purplish-red and is characterized by an absorption spectrum with a maximum at 555 nm in the green portion of the spectrum. The soret band in the near ultraviolet region shows a maximum at 435 nm (Figure 7). Deoxymyoglobin, with its open sixth position, can bind high-field ligands such as oxygen, nitric oxide and carbon monoxide by ir back-bonding. However, because of the low charge on the iron (as explained previously), ligands such as HgO or Cl cannot bind, since these ligands bind through a interactions only. In addition, many high-field ligands such as pyridine derivatives do not bind, since they are unable to get through the heme cleft to the iron. LITERATURE REVIEW / 23 Figure 7. Absorption spectra of the three most common pigments in fresh muscle tissue. (Francis and Clydesdale, 1975) LITERATURE REVIEW / 24 b. Oxymyoglobin Oxymyoglobin, the diamagnetic ferrous form, is a stable derivative under high-oxygen conditions. It is bright red in color and is responsible for the familiar "bloom" of fresh meats. The visible spectrum is characterized by maxima at 538 to 542 nm and 575 to 588 nm (Figure 7). The soret band has a maximum around 418 nm. If oxygen tension becomes low enough for partial deoxygenation to deoxymyoglobin (4 mm O2), the stability of the oxy-deoxymyoglobin mixture to oxidation is greatly diminished. It is important to remember that native ferrous myoglobin is required for oxygen to be bound in a stable manner, and that once oxidation has occured, metmyoglobin will permanently replace red oxymyoglobin unless reducing conditions are present. c. Nitric oxide myoglobin Nitric oxide myoglobin and carboxymyoglobin are used to preserve Mb in the ferrous form. Nitric oxide myoglobin (NOMb) can be generated by the reaction of deoxymyoglobin with nitrite under anaerobic conditions (Koizumi and Brown, 1971). Alternatively, it is formed by the reaction of metmyoglobin with NaNOg in the presence of ascorbate (Watts and Lehmann, 1952), sulfhydryl compounds (e.g., cysteine), hydroquinone (Fox and Ackerman, 1968), or nucleotide reductants, such as NADH, if flavins are present (Koizumi and Brown, 1971). Cytochromes have an important indirect effect on meat color. The rate at which cytochromes utilize oxygen determines the depth to which oxygen is found in meat. In absence of oxygen, cytochromes may reduce myoglobin derivatives (Swatland, 1984). LITERATURE REVIEW / 25 NOMb is a bright red derivative with a visible spectrum similar to that of oxymyoglobin. The bonding to iron is via both ir and a bonds. Although NO dissociates from myoglobin much more slowly than does oxygen (about one million times slower, according to Gibson and Roughton, 1957), NOMb is still considered unstable in situ. This is because oxygen can be present in much higher concentration and will replace NO once the latter dissociates. In addition, oxygen and other oxidants react with dissociated NO to form nitrate, which cannot bind to Mb. NOMb is the only common ferrous paramagnetic derivative, and, as we would expect, it reacts with other paramagnetic molecules. For example, NOMb is rapidly oxidized by ferricyanide, a paramagnetic iron complex (Antonini and Brunori, 1971). Native NOMb is thus unstable in situ; therefore, the method used for stabilization is to denature the protein. The resulting derivatives will be discussed shortly. d. Carboxymyoglobin Carbon monoxide binds tightly to myoglobin, forming ferrous carboxymyoglobin (COMb). This derivative is diamagnetic, has a visible spectrum similar to that of oxymyoglobin and NOMb, but is different from the ligands discussed so far in that the bonding is more a in character. COMb has unique features for providing a stable red color in meat, one of which is that the CO complex is stable even when denaturation of the protein occurs, because the a bond does not depend upon maintening the iron-histidine linkage "trans" to the CO. CO dissociates slowly from ferrous myoglobin (about 1000 times more slowly than oxygen), and since CO, unlike NO, is a stable molecule, dissociated CO will likely rebind to myoglobin. The high-field CO ligand imparts stability to the LITERATURE REVIEW / 26 ferrous oxidation state, and the resulting diamagnetic complex is rather inert to reactions with paramagnetic molecules. However, COMb is extremely labile to photodissociation (Brunori et al., 1973). e. Ferrohemochromes The remaining ferrous myoglobin complexes of importance to food scientists are the denatured-protein derivatives. These are classified under the name "ferrohemochromes", which signifies a ferrous heme derivative with a non-oxygen sixth ligand. In fact, ferrous cytochrome c, which has a methionine ligand opposite a histidine, may be classified as a ferrohemochrome. When myoglobin is denatured, several types of amino acid side-chains can coordinate to the heme iron, including histidine or carboxyl groups (Tarladgis, 1962a). These amino acids may in fact be part of other proteins in proximity to myoglobin (Ledward, 1971). Since the exposed heme can readily exchange ligands, different hemochromes are constantly interconverting. Ferrohemochromes have visible spectra similar to that of oxymyoglobin; i.e., they are red (Akoyunoglou et al., 1963). However, if the ligand can dissociate, these derivatives will oxidize quickly, causing browning or color fading to occur (Brown, 1973). Thus, the nature of the ligand determines the stability of the pigment. It has already been noted that COMb is stable when the protein is denatured, whereas NOMb is not, as a result of its paramagnetism and a weakening of the trans iron-histidine bond (Perutz et al., 1976). The NO ligand imparts a "trans" effect to the NOMb complex, and upon denaturation the histidine ligand is labilized and is replaced with a second NO molecule (Tarladgis, 1962b) or a LITERATURE REVIEW / 27 nitrogen-base ligand (Rifkind, 1973). The di-NO-heme complex is diamagnetic, has two high-field ligands, and is quite stable to oxidation and ligand exchange, as are the nitrogen-base hemochromes. For this reason, the curing process provides a stabilizing effect on the color. These pigments are sensitive to photooxidaion under retail lighting conditions if the product is exposed to oxygen (Fox, 1966; Hornsey, 1957). Therefore, appropriate packaging is required for protection of cured products from light and air. The attempts at finding nitrite substitutes have been wrought with difficulties that can be now explained (Brown, 1973). First, a ligand with 7r-acid properties is needed to prevent dissociation and to stabilize the ferrous complex. Second, the complex should be diamagnetic and thus more unreactive to paramagnetic species: however, many of these desired compounds will labilize their trans ligands to substitution, which may lead to oxidation by oxygen or other oxidants. In addition, under denaturing conditions the heme periphery becomes exposed, and oxidation of the pigment may now occur via attack on the porphyrin edge (which is protected by the native protein). Much effort has been spent studying the formation of ferrohemochromes (Howard et al., 1973; Akoyunoglou et al., 1963; Fox et al., 1974; Kemp, 1974) from various compounds, most notably pyridine and nicotinic acid derivatives. However, the instability of these compounds to oxidation in situ, causing fading of color when exposed to air, leaves one still in search of practical nitrite substitutes. LITERATURE REVIEW / 28 f. Metmyoglobin Deoxymyoglobin can be oxidized by one- or two- electron oxidants to metmyoglobin. The sixth ligand in metmyoglobin is HgO (or OH at basic pH), 3 + which bonds to Fe by a donation. This is a paramanetic complex which can act as a catalyst in free-radical reactions such as lipid oxidation by including breakdown of hydroperoxides (Hirano and Olcott, 1971; Brown et al.,1963). Metmyoglobin spectra are characterized by a sharp peak at 505 nm and a minor peak at 627 nm (Figure 7). The soret band has a maximum at 409 nm. This pigment is brown and is the myoglobin derivative responsible for discoloration of meat held under normal (i.e., no heating) conditions. Since metmyoglobin is unable to bind oxygen, the muscle has developed a means for reducing it back to reduced myoglobin. This system remains functional in situ for varying amounts of time, depending upon storage conditions. Of the high-field ligands discussed earlier, only nitric oxide can bind to metmyoglobin. This is likely a major intermediate in the curing process. Nitrosylmetmyoglobin can be formed (as opposed to an oxygen metmyoglobin derivative) because of the superior a-donor character of nitric oxide. However, the resulting paramagnetic complex should be quite reactive, and in fact is unstable in air since it reacts with oxygen (Fox, 1966). This complex is formed in the presence of reductants (e.g., cysteine or ascorbic acid) from nitrous acid or nitric oxide and metmyoglobin (Fox, 1966). However, in the presence of the same reductants, the resulting nitrosylmetmyoglobin is reduced to nitrosylmyoglobin. There is also evidence that this reduction can be autocatalytic (Chien, 1969). It has already been noted that the NOMb formed must be denatured to prevent color fading in the cured product. LITERATURE REVIEW / 29 g. Ferrihemochromes The remaining ferric derivatives may be classified as ferrihemochromes. They contain a sixth ligand which is a good a-donor for bond formation with the ferric cation. All ferrihemochromes are labile to ligand exchange, and so form a heterogeneous class of molecules. Their formation may result from denaturation of metmyoglobin, with a subsequent replacement of the by another sixth ligand. Alternatively, ferrihemochromes may be formed by oxidation of ferrohemochromes, which is a facile process. Many ligands, such as nicotinate and nicotinamide derivatives proposed as nitrite substitutes, can form both ferro- and ferrihemochromes (Akoyunoglou et al., 1963). The oxidized form of hemochrome is however the stable one under aerobic conditions; many ferrohemochromes are paramagnetic and labile to substitution and oxidation by oxygen. D. C Y T O C H R O M E C Cytochrome c was probably originally isolated by MacMunn in 1885, but Keilin in 1930 was the first to recognize its chemical uniqueness and its biological role. He showed that cytochrome c could act as a necessary cofactor for the oxidation of various substances by molecular oxygen via the membrane-bound oxidase of mitochondria. This hemoprotein is, in fact, part of the terminal oxidation chain, which completes the breakdown of foods to C O 2 and ^ O , storing the liberated energy in molecules of ATP. LITERATURE REVIEW / 30 1. Structure of cytochrome c Cytochrome c has been studied in many organisms and its structure determined in over one hundred species. It can be found across the entire spectrum of animals, plants, and aerobic microorganisms except bacteria, making it an admirable tool for studying the process of molecular evolution. The three-dimensional structure of mitochondrial cytochrome c appears to have been essentially fixed at the time of emergence of the first eucaryotes and is usually considered to be one of the most evolutionarily conservative proteins (Dickerson and Timkovich, 1976). A schematic representation of a mitochondrial cytochrome c from tuna is shown in Figure 8. The heart of all cytochrome c molecules is the heme group, a porphyrin ring surrounding a central iron atom. The outer electrons of the atoms of the porphyrin skeleton are delocalized, that is, they are free to wander from one atomic center to another; the iron atom itself is a part of this delocalized-electron system. An electron flowing into the heme from one edge can move freely to the central iron atom. Conversely, if an electron is lost at one edge, the vacancy can migrate inward until it reaches the iron, making it into a 3 + 2 + ferric iron atom (Fe ) rather than a ferrous atom (Fe ) as in the reduced heme iron. This is now thought to be the way electrons migrate into and out of cytochrome c: through one exposed edge of the heme. Current evidence indicates that the heme iron remains low-spin and in the plane of the heme in both its 5 6 single oxidized (Fe d ) and its uncharged reduced states (Fe d ). Figure 8. Structure of mitochondrial cytochrome c from tuna. (Dickerson, 1980) LITERATURE REVIEW / 32 The heme is held rigidly within the protein framework by four covalent bonds. Two bonds connect edges of the heme to sulphur atoms in the side chains of two amino acids (cysteines) in the protein; two more connect the iron itself to a sulfur atom (in a methionine side chain) and to a nitrogen atom (in a histidine side chain) on each side of the planar heme. The two cysteines are separated by two other amino acids along the protein chain, and the histidine immediately follows the second cysteine. This sequence, cysteine-X-Y-cysteine-histidine, is one of the distinguishing characteristics of a cytochrome of the c type. The same heme is also present in myoglobin, but there it is held within the protein framework in a different way. The covalent attachments to cysteines are missing, and only the histidine ligand is present. A heme has two polar propionic acid side chains (-CH^-CH^-COOH) attached to one edge. In myoglobin these side chains stick out into the aqueous surroundings. Instead the heme is rotated 90 degrees, so that one propionic acid group is just under the surface of the molecule of cytochrome c and the other group deeply buried. Although the overall environment of the heme crevice is hydrophobic due to the packing of aromatic and aliphatic amino acid side chains with the heme, tyrosine and tryptophan (amino acids no. 48 and no. 59 in the sequence of cytochrome c) compensate by forming hydrogen bonds with the buried propionic acid. These two hydrogen-bonded side chains are absolutely constant among all eukaryotes -animals, plants, fungi and protists (Dickerson, 1980). Cytochrome c is the only mammalian cytochrome that is soluble in water. All the others are bound to cell membranes and cannot be released without LITERATURE REVIEW / 33 disrupting those membranes. Cytochrome c can be removed from minced muscle simply by washing it with. a salt-containing medium. The molecule is small (M.W. 12600), containing 104 amino acid residues. One of the striking features of cytochrome c is its high lysine content; this amino acid is not uniformly distributed along the chain, but is clustered. The result is a basic protein, with an isoelectric point near pH 10 (Racker, 1970). Figure 9 shows that the reduced form of cytochrome c possesses four characteristic peaks at 550, 521, 415, and 315 nm identified by the letters a, /?, 7, and 6. It also shows that the difference between the reduced (pink) and oxidized (orange) forms of the cytochrome can account for the peaks at 550 and 521 nm in the spectra of both intact particles and intact cells of Figure 10. 2. Physiological function of cytochrome c In the inner membrane of mitochondria, soluble high-potential cytochromes c generally function as isopotential electron carriers between membrane-bound cytochrome b and the most oxidizing protein electron acceptor, cytochrome a. The probable nature of the interactions made between the cytochromes c and their membrane-bound oxidases (OX) and reductases (RED) is schematically represented by Figure 11. Cytochrome c is peripherally bound on the membrane surface by complementary charge interactions formed between a ring of positively charged membrane surface and/or membrane-bound oxidoreductase. The ionic interaction formed between the cytochrome c molecule and the membrane is relatively weak, giving the cytochrome c two-dimensional mobility (arrows) such that it may reversibly interact with its physiological oxidoreductases. The LITERATURE REVIEW / 34 Figure 9. Absorption spectra of oxidized and reduced horse heart cytochrome c extracted into an aqueous solution. (Nicholls, 1984) Figure 10. Difference spectra of intact rat ascites tumor cells and of mitochondria isolated from such cells (in each case the spectrum is that of an anaerobic substrate reduced system minus that of a complete oxidized preparation. (Nicholls, 1984) LITERATURE REVIEW / 3 5 Figure 11. Scheme of probable interactions between cytochrome c and mitochondrial membrane. (Salemme, 1977) LITERATURE REVIEW / 36 complementary ionic interactions serve to orient the cytochrome c molecule so that it is oxidized (shown) or reduced by a mechanism involving direct interaction of the cytochrome c heme and prosthetic groups of its oxidoreductases. E. HEMOPROTEINS AS PART OF T H E M E A T SYSTEM Several changes take place in muscle following the death of an animal, that have a profound effect on meat pigment and hence on the meat color. When a bird is slaughtered, the continuous oxygen supply is cut off and the muscle system resorts to anaerobic glycolysis for energy production. As pointed out by Pearson (1970), three sources of energy available for glycolysis would include ATP, creatine phosphate, and glycogen. Of these, only glycogen is present in considerable amounts in the muscle and, therefore, serves as the principal energy source. The anaerobic postmortem glycolysis causes a net gain of three molecules of adenosine triphosphate for each 6-carbon unit of the glycogen that is converted to lactic acid. There is a decline in pH from an initial value of about 6.8 to a value of 5.7 to 5.8 accompanied by a considerable drop in temperature from 36-39°C to 2-5°C. This drop of pH may induce myoglobin oxidation since the oxidation of myoglobin is reported to be faster at lower pH values. The changes in pH also have a profound effect on the water binding capacity of meat proteins, which in turn affects the gross morphology of the muscle. When the pH is much above the isoelectric point of actomyosin, more water is bound, resulting in a subsequent decrease in the fluid phase of the muscle. Typical curves obtained for glycolysis and for dephosphorylation of ATP in LITERATURE REVIEW / 37 poultry meat of high post-slaughter pH (restrained struggling) are shown in Figure 12. A decrease in pH to a value of 6.2-6.3 coincides with the dephosphorylation-phase during which the ATP level of the muscle tissue fell to below 40-50% of its initial level. During this period, the shear force value of meat is generally low and remains more or less constant. The rapid stiffening of muscular tissue does not begin until the ATP content of the muscle has declined to about 30% of its initial concentration. Thereafter, the ATP content of the meat decreases to a minimum value and toughening occurs (de Fremery and Pool, 1960). Holding samples at 30 or 37 °C during postmortem glycolysis and dephosphorylation of high energy phosphates causes toughness, while holding at 10, 15 or 25°C does not affect tenderness. Lowering temperature from 37-40°C to 15°C before the pH has dropped to a value of about 6.2-6.3 appeared to minimize the loss of tenderness (Khan, 1971). The changes in pH of turkey breast muscle from anesthetized and non restricted struggling birds, are shown in Figure 13. The pH of the muscle from control birds dropped rapidly during the first 15 min. postmortem, then declined slowly through 72 h except for a slight increase from 1/4-1/2 h. The pH of muscle from anesthetized birds behaved quite differently. It dropped very little through the first 6 h postmortem, and was significantly higher than the pH of muscle from control birds through 1/4-6 h postmortem (Landes et al., 1971). The pH decline observed in the anesthetized birds indicated that the rate of postmortem glycolysis was decreased when compared with the control birds. LITERATURE REVIEW / 38 0 1 2 3 4 5 6 7 8 POST-MORTEM AGING TIME, HRS. Figure 12. Changes in the adenosine triphosphate content (•) and pH (°) of breast muscle during 8 h post-mortem aging at 15°C. (Khan, 1971) LITERATURE REVIEW / 39 Figure 13. Changes in pH of turkey breast muscle during 72 h post-mortem. Solid line represents average pH for anesthetized turkeys; dashed line for control turkeys. Hatched area shows significant difference between control and treatment. (Landes et al., 1971). LITERATURE REVIEW / 40 The most important agents which have been proven to be stressors to fowl are temperature extremes, handling, shaking, and food and/or water deprivation (Freeman, 1971). In the initial "alarm phase", which occurs after exposure to a stressor, adrenalin or epinephrine is released from the adrenal medulla causing the passage of potassium from the muscle to the blood and the breakdown of liver and muscle glycogen to glucose and lactic acid (Lawrie, 1966). If the effect of the stressor is severe enough, one would expect alterations in postmortem muscle due to glycogen depletion. Results obtained by injecting broilers with epinephrine preslaughter verify this effect (Wood and Richards, 1975). Froning et al. (1978) observed that breast muscles from heat stressed and free struggle turkeys had increased a^ values, lower muscle pH, lower water holding capacity (WHC), and higher shear values than anesthetized and cold stressed turkeys. More recently, Ngoka et al. (1982) observed substantially greater redness and increased myoglobin concentration in breast from birds subjected to normal excitement prior to slaughter and allowed to struggle freely during slaughter. III. M A T E R I A L S A N D M E T H O D S A . C H R O M A T I C N E S S S C A L E A ^ M E A S U R E M E N T S Three 12 week old and three 18 week old male turkeys of the Nicholas strain were processed by a local packing house. The paired breasts were excised and, prior to freezing at -25°C, the pH of each muscle was taken. Two g of meat were placed in a 50 mL centrifuge tube with 20 mL of deionized distilled water and homogenized with a Polytron model PT10/35 (Brinkmann Instrument, Westbury, NY) at speed 6.5 for 15 sec. All pH values, which were measured using a Fisher model 420 (Fisher Scientific Co., Pittsburgh, PA), varied from 5.7 to 5.9. As needed, the frozen breast muscles were cut 2.0 mm thick using a Hobart model-410 meat sheer (The Hobart Manufacturing Co. Ltd., Don Mills, Ont.) and trimmed in order to fit between two juxtaposed microscope glass slides separated by 2.0 mm spacers (Figure 14). Two paper clips held the pieces together. Each mounted sample was inserted in a laminated retortable pouch and vacuum-sealed at 40.5 kPa. A preliminary study was done to find out the time-temperature programs required when cooking in a water-bath. Temperatures at the center of the meat samples were probed by copper-constantan thermocouples and recorded on a Kaye Ramp II scanner/processor (Kaye Instrument Inc., Bedford, MA). It was found that 10 min of complete immersion was sufficient to reach the target temperatures of 65, 75, 85 and, 95°C in the pre-equilibrated water-bath. 41 MATERIALS AND METHODS / 42 Figure 14. Coupled microscope glass slides separated with 2.0 mm spacers. MATERIALS AND METHODS / 43 A model D-25-D Hunter Color and Color Difference Meter (Hunter Associates Laboratory Inc., Fairfax, VA) equipped with a 5 cm circular port was used to obtain the color parameter a^, measuring red when plus, and green when minus. The instrument was standardized using a white ceramic tile with specifications of L=93.10, a^—0.6, bj^=+0.4. Four cooked meat units, each 5 x 5 cm, were placed side by side in a plastic petri dish, overlaid with a white background. The color was measured five times over a period of four days of storage at 0°C. Samples were rotated four times through 90°, to evaluate the effects of meat heterogeneity. The data were collected according to a split-plot in time model, in a randomized block design with fixed effects (Steel and Torrie, 1980). The complete mathematical description is represented by the following equation: Y.„. = n + a. + 7- + co, + p\, + T J . . , + d. ykl l IJ k lk ljk 1 + X a + + p H + o i k l + e i j k, + A..,. (1) yklm where i = 1 to 2 ages of turkeys j = 1 to 3 turkeys or blocks k = 1 to 4 temperartures or whole-unit treatments 1 = 1 to 5 storage days or subunit treatments m = 1 to 4 rotations or replications Computations were performed for analysis of variance and contrasts by UBC MFAV (Le, 1978), and for general linear model by SAS (SAS User's guide: MATERIALS AND METHODS / 44 Statistics, 1985). Both program packages were available on the UBC Amdahl 470 V/8 computer. B. IDENTIFICATION OF PIGMENTS IN COOKED TURKEY BREAST 1. Transmission spectrophotometry of integral meat slices The procedure detailled in this section was derived from the work of Fransham and Walters (1981). Meat units from Pectoralis major and minor were prepared and cooked at either 65, 75, 85, or 95°C for 10 min as described in Chapter A. Immediately after cooking, the samples were cut in order to fit the interior side of 10 mm spectrophotometer cells. Difference spectra were recorded intermittently with a Cary 210 automatic recording spectrophotometer (Varian Associates Inc., Palo Alto, CA) over a period of four days. The following operating conditions were used: Chart display : 20 nm/cm Scan rate : 0.2 nm/sec Period : 1.0 sec Spectral band width : 3.5 nm Similar meat slices were bleached by immersion in hydrogen peroxide (20%) at room temperature for approximately 1 h and tissues treated in this manner were used as reference samples. In a similar manner, another phase of this study investigated the effect of immediate air contact on cooked turkey meat. Fresh 2 mm slices were cut and MATERIALS AND METHODS / 45 put directly on one side of the cuvettes. Then, in order to keep the meat in an anaerobic environment immediately after cooking, 10 x 50 x 1 mm pyrex strips were deposited on top of the meat. The resulting cuvettes were vacuum-packaged at 40.5 kPa, and cooked for 5 min at 65, 75, 85, and 95°C. After cooling to room temperature, the samples were removed from the pouches and scanned with and without the pyrex strips. 2. Transmission spectrophotometry of meat extract The extraction of pigments in turkey breast slices cooked at 85 °C for 10 min was done using a low ionic strenght salt solution. Each step is outlined in the following: 5 g of cooked meat and 20 mL of 0.04M sodium phosphate buffer, pH 6.8 (Gomori, 1955), were placed in a 50 mL centrifuged tube and homogenized with a Polytron at speed 6.5 for 15 sec. The slurry was allowed to stand for 1 h at 4°C before centrifuging at 750 x g for 15 min. The supernatant was filtered through coarse fluted no. 1 filter paper. The filtrate was centrifuged at 20000 x g for 15 min and the supernatant was considered as the crude extract. In a cuvette, 30 mg of sodium dithionite was added to 3 mL of extract. After 10 sec of gentle mixing, the solution was then scanned in the visible range. MATERIALS AND METHODS / 46 3. Electrophoresis of meat extract Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried TM out on a PhastSystem using PhastGel Gradient 8-25 and PhastGel SDS buffer strips (Pharmacia, Uppsala, Sweden). Gels had a 13 mm stacking gel zone and a 32 mm continuous 8 to 25% gradient gel zone with 2% crosslinking. The buffer system in the gels was 0.112 M acetate (leading ion) and 0.112 M Tris, pH 6.4. The buffer system in PhastGel SDS buffer strips was 0.20 M tricine (trailing ion), 0.20 M Tris and 0.55% SDS (analytical grade), pH 7.5. The buffer strips were made of 2% agarose. SDS to 2.5% and /J-mercaptoethanol to 5%t was combined to 4 mL of crude extract from turkey breast cooked at 85°C for 10 min. After heating at 100°C for 5 min, bromophenol blue to 0.01% was added. A standard solution of 20 mg/100 mL of each of the proteins, myoglobin and cytochrome c, was handled the same way. 0.5 mL of prepared samples were applied to the gel and the separation carried out using the following recommended optimized conditions: limiting voltage: 250 V limiting current: 10.0 mA limiting power: 3.0 W temperature of the separation bed: 10 °C duration of separation step: 60 Vh t Concentration in the final solution. MATERIALS AND METHODS / 47 The gel was then silver stained using a development technique derived from Heukeshoven and Dernick (1985). The step-by-step instructions concerning the development solutions, times and temperatures are outlined in Appendix A. 4. Transmission spectrophotometry of soluble hemoprotein complexes The preparation of a soluble heme-protein complex similar to denatured meat hemoproteins was done according to Ledward (1971). Bovine serum albumin (BSA) (Cohn fraction V, Sigma Chemical Co., St-Louis, MO) and hematin (from bovine blood, Aldrich Chemical Co., Milwaukee, WI) were used without further -2 purification. 20 mL of a 4 day old mixture of 10 mg BSA/mL and 5.5x10 mg hematin/mL in 5.5 M urea at pH 6.0 was dialysed against 0.05M sodium phosphate buffer, pH 6.0 (Gomori, 1955). The dialysis (MW cut-ofT = 12000-14000) was performed over 4 days at 4°C using 4 changes of 8 litres of phosphate buffer. Different visible spectra were obtained from the solution of BSA-hematin itself as well as combined with imidazole and/or dithionite. 5. Determination of total heme proteins and of cytochrome c In order to determine quantitatively the total heme pigments, an extraction according to Warriss (1979) and a chemical conversion according to Drabkin (1950) was performed. 4 g of raw turkey breast slices and 20 mL of 0.04M phosphate buffer, pH 6.8 (Gomori, 1955), were taken through the three first steps described in section B.2. Next, 25 *xL of 2.5% K gFe(CN) 6 and 25 uL of 5.0% KCN were added to 10 mL of supernatant. After mixing, the solution was centrifuged at 20000 x g for 15 min. The absorbance of the supernatant was then measured at 540 nm and the concentration of total pigments was calculated MATERIALS AND METHODS / 48 from the Beer's law formula: A c = (MW x D) (2) e x 1 where c = concentration of total heme pigments (mg/g wet tissue) A = absorbance at 540 nm e = absorptivity of cyanmetmyoglobin (11300 mL/mmol-cm) 1 = thickness of cell (1 cm) MW = molecular weight expressed in terms of myoglobin (18200 mg/mmol) D = dilution factor (20 mL/4.0 g) Cytochrome c was extracted and reduced as detailed stepwise in section B.2. The absorbance at 550 nm was determined and the appropriate terms of the equation 2 therefore were: c = concentration of cytochrome c (mg/g wet tissue) A = absorbance at 550 nm e = absorptivity of reduced cytochrome c (27700 mL/mmol-cm) 1 = thickness of cell (1 cm) MW = molecular weight of cytochrome c (12500 mg/mmol) D = dilution factor (20 mL/5.0 g) MATERIALS AND METHODS / 49 C. MEASUREMENT OF REDUCING ACTIVITY 1. Detection of reducing activity using hemoproteins Standard curves for myoglobin and cytochrome c (both from horse heart, Sigma Chemical Co., St-Louis, MO) had to be generated in order to determine the proportions of reduced and oxidized forms in solutions of a certain concentration. Two series of dilutions, starting with an original concentration of 12.5 mg of hemoprotein per 100 mL of 0.04M phosphate buffer, pH 6.8, were elaborated: one series oxidized with 0.2 mL of 2.5% KoFe(CN)„/100 mL and another o 6 reduced with 1 g of sodium dithionite/100 mL. Table 1 summarizes the results of this procedure for cytochrome c. As demonstrated by Figure 15, a linear relationship between the proportion of reduced cytochrome c and the absorbance at 414 nm was found. In Figure 16, a similar equation was established for the relationship between proportion of reduced myoglobin and the absorbance at 408 nm, using a starting concentration of 12.5 mg/100 mL. In the latter case, the first series of dilution was done directly with the commercial myoglobin since it is provided in the oxidized form. The reducing effect of aqueous turkey breast extract was measured as follows. 2.5 mL of hemoproteins at 15 mg/100 mL phosphate buffer and 0.5 mL of turkey extract were mixed. The absorbance at 408 nm and 414 nm were recorded every minute for one hour and then converted into % myoglobin and cytochrome c with the respective equations. MATERIALS AND METHODS / 50 Table 1. Absorbance (414 nm) of serial dilutions of oxidized and reduced cytochrome c at an initial concentration of 12.5 mg/100 ml. Cyt. c^ + Absorbance Cyt. c ^ + Absorbance Total Abs. (%) (414 nm) (%) (414 nm) (414 nm) 10 0.037 90 1.269 1.306 20 0.130 80 1.154 1.284 30 0.220 70 1.027 1.247 40 0.309 60 0.895 1.204 50 0.411 50 0.756 1.167 60 0.503 40 0.635 1.138 70 0.609 30 0.512 1.121 80 0.694 20 0.383 1.077 90 0.794 10 0.242 1.036 100 0.893 0 0.115 1.008 MATERIALS AND METHODS / 51 Absorbance, 414 nm Figure 15. Linear relationship between percentage of reduced form of cytochrome c with a starting concentration of 12.5 mg/100 g, and absorbance at 414 nm. MATERIALS AND METHODS / 52 Absorbance, 408 nm Figure 16. Linear relationship between percentage of reduced form of myoglobin with a starting concentration of 12.5 mg/100 g, and absorbance at 408 nm. MATERIALS AND METHODS / 53 2. Fract ionat ion of reducing compounds a. Gel filtration Extracts of turkey breast slices cooked at 85°C for 10 min were obtained by the procedure described in Section B.2 using a 0.05 M tris hydrochloride buffer, pH 8.5, containing 0.002% chlorhexidine acetate. However, an additional step was done to minimize oxidation: immediately after homogenization, samples were degassed under vacuum. Fine grade Sephadex G-25 (Pharmacia, Uppsala, Sweden) was swollen in a boiling water bath for 1 h, cooled down to room temperature, degassed and packed in a column of dimensions 1.27 cm x 33 cm. Tris buffer previously degassed was poured in a Mariotte flask. Nitrogen gas slowly purged the buffer from the time of bed equilibration to the end of sample elution. The total volume of the packed bed (V ) was approximately 38 mL and the void volume (V ), determined using Blue dextran 2000, was 16 mL. 2 mL of extract was applied to the column and a peristaltic pump used to maintain the flow rate at approximately 10.7 mL/cm-h. Fractions (2 mL) were collected with an Ultrorac type 7000 (LKB-Produkter AB, Stockholm-Bromma 1, Sweden) and were analysed for their reducing activity during the first minute of contact with the respective standard solutions of hemoprotein (see Section C. 1). MATERIALS AND METHODS / 54 b. Ultrafiltration An Amicon Ultrafiltration apparatus Model 8050 (Amicon Corp., Lexington, MA) consists of a batch-type system having a cell designed to accomodate a circular membrane of 4.5 cm diameter. Two of these cells were attached to a one liter capacity reservoir which in turn was connected to a nitrogen gas tank. An extract was prepared from thawed turkey breast muscle using deionized distilled water (0.002% chlorhexidine) according to the method in Section B.2. The cells were filled with 70 mL of aqueous turkey extract solution and deionized distilled water containing 0.002% chlorhexidine was poured into the reservoir. An integral magnetic stirrer helped to dissipate the retained solutes which otherwise form a cake on the membrane surface and lower the flux. This phenomenon, also known as "concentration polarization", was minimized by ajusting the stirring speed to obtain a vortex equivalent to 1/3 of the height of the solution. Nitrogen pressure (40 psi) served to pump water through the cell that progressively removed from the solution, compounds of molecular weights below 10000 daltons, the cut-off point of the membranes. A total ultrafiltrate volume three times that of the cell, i.e. 3 x 70 mL, was collected from each of the two cells. Ultrafiltrates were then frozen, freeze-dried, and stored at -25°C in a dessicator. The samples were reconstituted, taking into account the extraction (4 g/20 mL), MATERIALS AND METHODS / 55 moisture in meat (70%) and ultrafiltrate recovery (85%): 70 mL x 4/20 x 70/100 x 85/100 = 8.33 mL This means that the freeze-dried samples were rehydrated with 8.33 mL of water. Cooked (85 °C for 10 min) and uncooked ultrafiltrate samples were subjected to gel filtration and their initial reducing activity was measured as described in Subsection C.2.a. The reducing activity of these samples was also tested over time. IV. RESULTS AND DISCUSSION A. COLORIMETRIC EVALUATION OF RED COLOR IN COOKED TURKEY BREAST The Hunter Color and Color Difference Meter is basically a chosen light source, filter and detector designed to provide a measurement which correlates meaningfully with visual judgement. The L, a^ and b^ coordinates obtained from this instrument define the color of an object and situate it at a precise point in a color solid. The positive and negative ranges of the a^ and bj^ scales represent different hues while the L scale establishes the degree of lightness. Since redness was the "color" of prime concern, data were accumulated for the a^ scale only. All measurements were made on slices of meat 2 mm thick. Besides eliminating temperature gradients during cooking and long cooking times inherent to thick samples, additional spectrophotometric information, as will be seen later, could be obtained from the same specimen. Part of a study done by Howe et al. (1982) showed that a^ values did not change when the thickness of cooked pork decreased from 20 mm to 2 mm, using a white tile as background. A preliminary experiment carried out on one sample of turkey breast meat cooked at an internal temperature of 65 °C verified this fact. Table 2 shows the results of an analysis of variance on the redness measurements from turkey breast meat. Normal distribution and homogeneity of variance among the data were assumed as well as the other underlying 56 RESULTS AND DISCUSSION / 57 Table 2 . Analysis of variance on aj_, values of breast meat slices from 12 and 18 weeks old turkeys. Source of variationf df SS MS F Age Error (a) Temperature T L T Q Deviation Age x Temp. Error (b) Storage time S L SQ Residual Age x Storage Error (c) Temp, x Storage TL * s L T L x SQ T L * S C TQ x S L TC * S L Residuals Age x Temp, x Stor. Error (d) Sampling error Total 1 4 3 1 1 1 3 12 4 1 1 2 4 16 12 12 48 360 479 4.76 32.6984 916.28 822.61 89.542 4.1300 2.4308 54.332 498.48 437.12 57.983 3.3770 6.0104 15.136 16.042 3.6365 7.3046 1.2988 0.91613 2.4824 0.40357 1.5907 3.6754 5.6697 393.53 4.76 8.1744 305.43 822.61 89.542 4.1300 0.81024 4.5276 124.62 437.12 57.983 1.6885 1.5026 0.94600 1.3369 3.6365 7.3046 1.2988 0.91613 2.4824 5.7652 0.13173 7.6568xl0'2 1.5749xl0-2 0.58 1.81 67.46** 181.69** 19.78** 0.91 0.18 131.74** 462.08** 61.29** 1.78 1.59 17.46** 47.49** 95.40** 16.96** 11.96** 32.42** 0.75 1.72 4.86** t Subscripts used are: L = linear; Q = quadratic; C = cubic. ** Significant at 1% level. RESULTS AND DISCUSSION / 58 assumptions. First, it seemed logical to begin by examining the experimental error. This term, represented by error (d), was significant at P<0.01. In other words, the rotation variation or internal heterogeneity of each meat unit revealed a contribution which was relatively small compared to the experimental error. According to the literature review, differences in meat within a muscle, and for the same muscle type between animals, could be seen and accounted for by many factors. For this reason, the birds were taken as block effects and meat slices from each turkey underwent all treatments. Apparently this precaution was not necessary since the variation between birds within each age of 12 and 18 weeks, or error (a), was not significant. This term was used to test the age effect and it was found to be not significant. As the response means of both ages were similar, all the interactions of the treatments and age were also non-significant. These results clearly indicate that redness of turkey breast upon cooking and storage was the same regardless of the age of the birds. For fixed effects such as the chosen model of this design, the MS value was tested using error (b) for temperature, error (c) for storage time, and error (d) for the interaction. Temperature, storage time, and their interaction were significant at 0.01 level. It is apparent from these results that the differences among a T values for the meat slices cooked at the temperatures chosen in this XJ experiment did not behave consistently during the days of storage. In order to graphically picture this interpretation, the equally spaced treatments were partioned into contrasts. Only the F ratios associated with the significant terms have been compiled in Table 2. The predicted coefficients obtained from these RESULTS AND DISCUSSION / 59 contrasts were assembled to form the following orthogonal polynomial: a L= 42.317 - 8.8193x10"^ + 4.935xl0"3T2 + 9.4881S - S.lSgSxlO^S2 - 3.8594xlO"1TS + 1.0440TS2 - 6.4352xlO"4TS3 + 4.7192xl0"3T2S - 2.09xlO"5T3S (3) r 2= 0.9948 s =0.177 y-x The surface response shown in Figure 17 was then drawn from equation 3. In this three dimensional illustration, the predicted redness values were interconnected and the presence of a sheet articulated by smaller structural frames can be visualized. The sheet posseses a certain distortion created by variations in the rates at which redness decreases as the thermal and temporal treatments proceeded. The measurements of redness fluctuated between 0.0 and 6.0. When the readings were taken immediately after cooking (0 day of storage), the degree of redness sharply decreased over the temperature interval of 65 to 85 °C, and levelled as temperature was increased from 85 to 95 °C. This follows the pattern of myoglobin denaturation; a temperature of 65 °C allows oxymyoglobin to display the highest a^ values, while 85 °C corresponds to the stage where all myoglobin molecules are denatured (Bernofsky et al., 1959). Concerning the storage effect, every day brought a significant decrease in redness. Refrigerated meat slices were probably in conditions conducive to oxidation. Metalloproteins such as myoglobin may loose an electron and become oxidized. They may further degrade by the opening and decomposition of the porphyrin rings. However, one of the major RESULTS AND DISCUSSION / 60 Figure 17. Surface plot of the relationship between cooking temperature (T), storage time (S), and aj_, value of turkey breast meat slices. RESULTS AND DISCUSSION / 61 points evident in this Figure is the fact that, upon cooking at 85 or 95°C, the a^ values were just over 3.0 and three to four days of storage were necessary to bring the redness down to zero. Visually it was found that the observation or detection of this remaining pinkness seemed easier when recent cooked slices were compared to a stored sample. These results support the idea that color in cooked turkey breast is not only driven by myoglobin. Pinkening likely involves other pigments which are susceptible to deterioration as well. Refrigerated storage has marked effects on uncured cooked meat and oxidation is certainly one mechanism, among others, to be explored. B. CHARACTERISTICS OF INVOLVED PIGMENTS 1. Transmission spectra of integral meat slices Many attempts have been made in the past to characterize and quantify the heme pigments in a meat product by a variety of procedures. Reflectance spectrophotometry has been employed successfully, for instance, in cases where all pigments present were converted into one form, e.g., metmyoglobin or deoxymyoglobin (Wolfe et al., 1978). However reflectance spectra are poorly defined and restrict the evaluation of complex mixtures of heme proteins. Consequently, a method has been adapted for the study of pigments in situ, using transmission spectrophotometry. In determining the dimensions of slices to be used, two major factors were considered. As the thinnest samples got thicker, RESULTS AND DISCUSSION / 62 the absorbances increased until further increments resulted in a surcharge of the multiplier phototube trying to compensate for low level of transmitted light through thicker samples. Similar to the findings of Fransham and Walters (1981), a thickness of 2.0 mm was adopted as an optimum compromise between intensity of response and amount of light transmitted through the section. The series of Figures 18, 19, 20 and 21 brings more insight to how meat pigment from turkey breast behaves upon subjection to cooking and storage. All these difference spectra of cooked meat sections had a peak between 410 and 420 nm. The absorption in this range of wavelenghts required a scale twice as high as the rest of the visible range. Such difference in extinction is one of the characteristic features of hemoproteins. In Figure 18, curve A which represents meat slices cooked at 65°C exhibited peaks at 414, 550 and 575 nm, and shoulders at 520 and 545 nm. The use of the first derivative feature of the spectrophotometer greatly helped in locating the wavelenghts at maximum absorption. Oxymyoglobin is known to absorb at 417, 543 and 575 nm. The disappearance of the two last bands at 75 °C (Figure 19, curve A) suggests that the denaturation of myoglobin took place. Furthermore, in curve A of Figures 19 and 20, a strong maximum at 414 nm, relative to two others at 520 and 550 nm, remained. In terms of proportions and positions of the absorptions, these three-banded spectra correspond to the respective y, |3 and a bands typical to cytochrome c, in a low-spin ferrous state. Cytochrome c remains stable at heat treatments that myoglobin cannot withstand and is therefore thought to be responsible for the residual pink color RESULTS AND DISCUSSION / 63 0.02 j—1 1 1 1 1 1 1 i 1 i 400 450 500 550 600 650 W a v e l e n g t h (nm) Figure 18. Absorbance difference spectra of a turkey breast sbce cooked at 65 °C for 10 min and stored at 0°C for 0 day(A), 1 day(B), 2 days(C), 3 days(D) and 4 days(E). RESULTS AND DISCUSSION / 64 Figure 19. Absorbance difference spectra of a turkey breast slice cooked at 75 °C for 10 min and stored at 0°C for 0 day(A), 1 day(B), 2 days(C), 3 days(D) and 4 days(E). RESULTS AND DISCUSSION / 65 Figure 2 0 . Absorbance difference spectra of a turkey breast slice cooked at 85 °C for 10 min and stored at 0°C for 0 day(A), 1 day(B), 2 days(C), 3 days(D) and 4 days(E). RESULTS AND DISCUSSION / 66 Figure 21. Absorbance difference spectra of a turkey breast slice cooked at 95°C for 10 min and stored at 0°C for 0 day(A), 1 day(B), 2 days(C), 3 days(D) and 4 days(E). previously observed. RESULTS AND DISCUSSION / 67 When the cooking temperature reached 95°C (Figure 21), the relative height of the a, 0 and 7 bands decreased. This is also observed for all spectra as storage time progressed. Moreover, the 7 peaks or Soret bands were shifted slightly toward lower wavelengths. The oxidation and decomposition of the mentioned heme proteins, as is demonstrated by all the declining and more diffuse curves B, C, D and E, could explain the additional decreases of redness over time noted in Figure 17. 2. Extraction of cytochrome c Additional work was carried out to provide additional support for the postulation concerning the involvement of cytochrome c in the pink color. Because cytochrome c is a water soluble pigment, an extraction with 5 volumes of 0.04 M phosphate buffer, pH 6.8, was performed on slices of turkey breast cooked at 85°C for 10 min. The extract was reduced with a few grains of sodium dithionite and scanned. Curve B of Figure 22 is an example of the resulting spectrum and demonstrates a definite resemblance to a commercial solution of reduced cytochrome c (curve A). When this extract was subjected to SDS-PAGE (Figure 23), a band at approximately 12500 daltons adjacent to a standard of cytochrome c was present. As expected no band corresponding to myoglobin appeared since the meat had received a heat treatment at 85 °C which was high enough to denature the protein. RESULTS AND DISCUSSION / 68 0.02 l 1 i • i • i • i 1 400 450 500 550 600 650 Wavelength (nm) Figure 22. Absorbance spectra of: (A) a commercial solution of reduced cytochrome c, (B) myogen extracted from turkey slices cooked at 85 °C for 10 min and reduced with sodium dithionite. RESULTS AND DISCUSSION / 69 Figure 23. Electrophoretogram of myogen extracted from turkey breast slices cooked at 85° C for 10 min (Myo) and standard solution containing myoglobin and cytochrome c (Std). RESULTS AND DISCUSSION / 70 3. Effect of a i r contact on color after cooking Figures 24 and 25 represent other absorbance difference spectra of turkey breast meat, measured over the visible wavelength range. In these instances, pieces of pyrex covering the meat slices prevented contact with air after heat treatment and allowed spectrophotometric scanning under stable anaerobic conditions. The sample in Figure 24 was cooked at 65°C for 5 min. The peak at 550 nm of curve A can be largely accounted for by myoglobin in the deoxygenated native form. But a shoulder at 520 nm also suggests the presence of cytochrome c. When the rectangular piece of pyrex was removed, the redness quickly increased in intensity, which was also noticed visually. The Soret band shifted to a lower wavelenght and peaks appeared at 545 and 575 nm; this corresponds to the fingerprints of oxymyoglobin. Figure 25 shows the spectra of a sample cooked at 85 °C for 5 min. Compared to curve B, curve A had different spectral characteristics, particularly in the region 500-600 nm. It showed maxima at 423, 532, 552 and 558 nm. Once exposed to air, curve B indicates that the pigments readily oxidized leaving the stable cytochrome c to display its distinctive features. Many heme related proteins, for instance, cytochrome b and c^, may explain such oxidative behavior. Similar results were obtained for the cooking temperatures of 75 and 95 °C. These respective difference spectra can be found at the Appendices B-l and B-2. Moreover, heat denaturation forces the structure of myoglobin to open. The heme may then bind to new exposed sites of denatured apomyoglobin (Anson and Mirsky, 1928) or be transfered to any of several proteins found in meat (Ledward, 1971). The heme, electrophilic in nature, could participate in coordinate RESULTS AND DISCUSSION / 71 0.02 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 Wavelength (nm) Figure 24. Absorbance difference spectra of a turkey breast slice cooked at 65 °C for 5 min in a cuvette: (A) covered with a strip of pyrex, (B) uncovered and exposed to air. RESULTS AND DISCUSSION / 72 0 . 0 2 i 1 1 1 1 1 I ' l 1 r— 400 450 500 550 600 650 Wavelength (nm) Figure 25. Absorbance difference spectra of a turkey breast slice cooked at 85 °C for 5 min in a cuvette: (A) covered with a strip of pyrex, (B) uncovered and exposed to air. RESULTS AND DISCUSSION / 73 covalent bonds with nitrogenous ligands such as histidine, lysine, and terminal alpha amino group of proteins (Keilin, 1960). Cornforth and his co-workers (1986) came to the conclusion that the high content of nicotinamide in turkey breast muscle favors the formation of nicotinamide-denatured globin hemochromes. 4. Transmission spectra of soluble heme-protein complexes In order to accumulate some spectral data about such hemochromes, hematin was incorporated into bovine serum albumin (BSA) according to the method of Ledward (1971). Curve C of Figure 26 shows the spectrum of the soluble complex obtained. It absorbed at 414 and 531 nm and is analogous to a difference spectrum of slices cooked at 95°C. There is considerable evidence that histidine, but not primatry amino groups in the albumin are involved in the binding (Little and Neilands, 1960; Maehly, 1961). The reduction of the solution with sodium dithionite caused a shift to the right of the Soret band and new absorptions at 530 and 558 nm (curve B). When an aromatic heterocyclic nitrogenous compound such as imidazole was added, the absorbances remained at the same wavelenghts (curve A). However the intensity of the peaks was much greater indicating increased chemical involvements through a same type of heme-nitrogen interaction. According to Falk (1964), the sharpening of the spectrum is doubtless due to the change from the dimeric to the monomeric speciest. Such heme-nitrogenous base complexes are likely to be formed during cooking but, due to their instability, they would oxidize immediately upon contact with air. In order to summarize this part of the work, Table 3 lists the t Dimerization of ferriporphyrins is due to electrostatic forces between the dissociated carboxyl groups in one molecule and the residual positive charge on the iron atom in another (Lemberg and Legge, 1949). RESULTS AND DISCUSSION / 74 Figure 26. Absorbance spectra of 5 mg/mL bovine serum albumin added to: (A) 2.5x10"^  m g hematin/mL and 3.0x10"^  mg imidazole/mL reduced with sodium dithionite; (B) 2.5xl0"2 mg hematin/mL reduced with sodium dithionite; (C) 2.5x10"^  mg hematin/mL. RESULTS AND DISCUSSION / 75 Table 3. Theoretical wavelengths at maximum absorption (^MAX) or* some hemoproteins (adapted from Mahler and Cordes, 1971). HEMOPROTEINS SPECTRA X M AX ( N M ) a 0 SORET BAND Hemoglobin/Myoglobin 555 - 439 Oxymyoglobin 581 543 417 Oxyhemoglobin 577 542 415 Methemoglobin/Metmyoglobin 630 500 406 Cytochrome b 563 532 429 Cytochrome c 550 521 415 Cytochrome cj 554 524 418 Reduced hemochrome (BSA-Hematin) 558 530 422 Oxidized hemochrome (BSA-Hematin) - 531 411 RESULTS AND DISCUSSION / 76 wavelenghts at maximum absorption of some hemoproteins believed to be involved in meat color under various conditions. 5. Hemoprotein content in turkey breast meat The evaluation of meat color would be incomplete without the determination of hemoprotein concentrations in the samples. Table 4 presents the contents of total heme pigments for turkey breast. On a wet basis, these mean content of myoglobin for 12 week and 18 week old turkeys were respectively 0.60 and 0.77 mg/g of tissue. These results appeared a little higher than values given by Froning et al. (1968) but were very close to a more recent investigation by Pikul et al. (1986). As the last paper stated, freezing meat samples before comminuting and using phosphate buffer instead of water are conditions which significantly improved the extractability of the heme pigments. It is also evident that the amount of hemoproteins in turkey significantly increased as age of the bird increased. This is in agreement with work reported by Froning et al. (1968) and SafTle et al. (1973). The second half of Table 4 pertains to the level of cytochrome c found in -2 turkey breast. The average content of cytochrome c decreased from 7.9x10 _2 mg/g for 12 week old birds to 5.4x10 mg/g for 18 week old birds. Contrary to total heme pigments, cytochrome c concentration was inversely proportional to age. Cytochrome c is an integral part of mitochondria and therefore such results should be related to the distribution of mitochondria during the development of muscles. As a muscle fiber accumulates contractile proteins during growth, it also increases its volume of sarcoplasm and the number of its mitochondria. RESULTS AND DISCUSSION / 77 Table 4. Hemoprotein content of breast meat from 12 and 18 weeks old turkeys. Age Hemoprotein content on wet basis *f (weeks) Total heme pigments Cytochrome c (xlO"1 mg/g) (xlO"2 mg/g) 12 6.0 ± 0.9 b 7.9 ± 0.5 a 18 7.7 ± 0.5 a 5.4 ± 0.4 b * Mean and standard deviation of three duplicates. "f Means with the same letter within a column are not significantly different according to a Student-Newman-Keuls multiple range test. RESULTS AND DISCUSSION / 78 Mitochondria are very abundant in the sarcoplasm of muscle fibers of young animals. They appear to proliferate by fission after each mitochondria has been internally subdivided by the formation of a septum (Duncan and Greenway, 1981). However the proliferation of mitochondria may lag behind the increase in sarcoplasmic volume that occurs with fiber growth. According to Swatland (1984), this is most evident in white muscle. Little information has been published concerning cytochrome c content of turkey muscle. Comparison of the results in Table 4 for 18 week old turkeys and those from Pikul et al. (1986) for 17 week old turkeys, shows a 4 fold difference. The method of this experiment used a somewhat crude extract and an overestimation is possible. But a large difference such as this should be accounted for by other factors. The extraction with low ionic strength buffer and reduction with sodium dithionite was done on meat previously cooked at 85 °C. Heat treatments are known to alter the integrity of cellular constituents. Following this logic, the degradation of mitochondria by cooking could have facilitated the release of cytochrome c from the inner membranes and therefore improved the extractability. Further investigation is needed to confirm this. C. REDUCING ACTIVITY IN COOKED TURKEY BREAST In general, one would think that heat treatment coupled with the presence of some oxygen, fat and certain metallic ions such as copper, iron, zinc, and aluminum, would promote oxidation of hemoproteins in meat. However myoglobin can react with oxygen to produce the "blooming" phenomenon until a cooking temperaure is reached high enough to destabilize its native structure. Beyond this RESULTS AND DISCUSSION / 79 point cytochrome c remains and produces a pink tinge. The alpha and beta absorption bands in the visible spectra of the latter hemoproteins are due to low-spin ("covalent") complexes (Falk, 1964, pp. 56-57) in which the iron d-electrons have been forced to pair. The iron in the porphyrin ring has to be in the ferrous form and this implies that reducing conditions prevail in the meat environment. Giddings (1974) wrote an extensive review of the reduction of ferrimyoglobin in fresh meat. According to this review, ferrimyoglobin reduction in post-rigor meat is no doubt primarily enzymatic in nature; it may involve mitochondrial and/or submitochondrial particles as a generator of reducing equivalents. Most sources of reducing equivalents used in various hemoprotein-reducing systems are either the reduced forms of nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH). Although in vitro studies indicate the possibilitiy of NAD-mediated reduction of metmyoglobin, the loss of NAD in post-mortem muscle is quite rapid. Severin et al. (1963) reproted that several enzymes, such as nucleosidase, come into contact with NAD upon maturation of tissues and degrade the nucleotide. During and after cooking, these systems are likely not to be available, and therefore other reducing pathways should be examined. Probable mechanisms through non enzymatic reduction can be classified into two types. The action of ascorbic acid on metmyoglobin is essentially a two-electron reduction. It proceeds under aerobic and anaerobic conditions and nitrogenous bases serve as catalysts (Fox et al., 1975). However it is not as active as RESULTS AND DISCUSSION / 80 one-electron paramagnetic reductants. High rates of metmyoglobin reduction by dithionite (Olivas et al., 1977), chromium II (Huth et al., 1976), and hydroxyl and alcohol radicals (Van Leeuwen et al., 1979) illustrate the activity of paramagnetic reductants. 1. Reduction reactions with myoglobin and cytochrome c In order to detect any reduction reaction in turkey breast cooked at 85 °C, myogen was extracted and added to an oxidized solution of hemoproteins. Respective absorbances were monitored for 60 min and percentage of reduction in solutions of 15 mg of hemoprotein per 100 mL of phosphate buffer were extrapolated from standard curves. Figure 27 shows that the percentage of reduced myoglobin increased quickly in a linear fashion for the first five minutes, slowed down to level off at approximately 18 min and then declined. Reduction of metmyoglobin occured but no red color appeared as anticipated. The method of purification used by Sigma probably affected the properties of the myoglobin. Upon contact with oxygen, the molecule oxidized instead of getting oxygenated. Therefore the results ma}' not reflect absolutely the way native myoglobin would behave. Cytochrome c was also reduced by cooked turkey extract (Figure 28). Although not as abruptly as for myoglobin, the percentage of reduced cytochrome c increased at a decreasing rate over time. No apparent re-oxidation response was detected for cytochrome c indicating its stability upon reduction by cooked turkey extract. RESULTS A N D DISCUSSION / 81 Figure 27. Influence of myogen extracted from 18 weeks old turkey breast slices cooked at 85 °C on the oxidation state of myoglobin over time. RESULTS AND DISCUSSION / 82 20 1 1 1 1 1 1 f 0 10 20 30 40 50 60 Time, min Figure 28. Influence of myogen extracted from 18 weeks old turkey breast slices cooked at 85 °C on the oxidation state of cytochrome c over time. RESULTS AND DISCUSSION / 83 Presence of a reducing environment in cooked turkey rolls was recently evaluated by Cornforth et al. (1986). In fact they found that the variable most affecting the appearance of pink color was oxidation-reduction potential. The normal commercial turkey rolls had a higher redox potential (-374 mV) than the sample with pink defect (-417 mV) but turned pink when the redox potential was lowered with dithionite. These observations lead to the conclusion that compounds of low redox potential are produced during cooking. However, the extent of their formation depends on certain conditions. If highly favorable, these conditions would accentuate reduction of ferrihemoproteins and hemichromes, and cause pinker products. Two main questions seem to arise at this point: what are these compounds and which conditions favor their formation. The next few experiments were conducted in order to collect more information about the nature of these reducing compounds. 2. Gel filtration of cooked turkey breast extract Hemoproteins such as myoglobin and cytochrome c have their heme buried inside their globin portion. This limits access to the porphyrin ring. Compounds that would be able to reach the interior are likely of small molecular weight. Sephadex G-25 was therefore chosen since its fractionation boundaries range from 1000 to 5000 daltons. Gel filtration of cooked (85 °C) turkey breast extract was carried out and the collected fractions were measured for their initial reducing activity (IRA) on myoglobin and cytochrome c. Figure 29 presents a typical elution pattern under these circumstances. Two main groups were segregated: one above and one below 3000 daltons. The higher molecular weight compounds RESULTS AND DISCUSSION / 84 80.0 60.0-• = myoglobin • = cytochrome c 40.0-20.0-0.0 10 15 20 25 30 35 Elution volume, mL 40 Figure 29. Hemoprotein initial reducing activity (IRA) of gel filtrate fractions of extract from cooked turkey breast slices. RESULTS AND DISCUSSION / 85 (HMW) reduced only cytochrome c while the lower molecular weight compounds (LWM) reduced both hemoproteins. The visible spectra (not shown) of the early fraction revealed absorption bands at 414, 520, and 550 nm denoting the presence of cytochrome c. This fact raised some doubts as to whether or not the cytochrome c IRA contained in HMW was solely due to absorption by cytochrome c from turkey breast extract previously reduced by LMW produced during cooking. 3. Combination of ultrafiltration and chromatography In another experiment an attempt was made to recreate a meat system in which all large molecules would be excluded. Constituents of fresh turkey breast extract below 10000 daltons were separated by ultrafiltration and freeze-dried. Deionized distilled water was added to the solids in order to reconstitute myogen from breast meat at an approximated moisture level of 70% wet basis. Unheated as well as heated (85 °C for 10 min) solutions were subjected to gel filtration. Figure 30 illustrates the results for myoglobin IRA. The reducing activity -8 -7 increased to higher levels (from 10 to 10 ). This provided somewhat more accurate data since previously the method was at its limit of sensitivity. However, when a dilution factor is considered, low reducing activity very similar to what is shown in Figure 29 was found for myoglobin. This "background" activity may be caused by molecules such as ascorbic acid, cysteine, and glutathione which are known for their reducing power. Furthermore the heat treatment had almost no effect as shown by the two similar curves. The results for cytochrome c are expressed in Figure 31. The unheated solutions had small cytochrome c reducing activity. But heating to 85 °C allowed the formation of R E S U L T S A N D DISCUSSION / 86 25.0 .5 20.0 15.0-10.0-• = unheated • = 85'C, 10 min 20 25 30 35 40 Elution volume, mL 45 Figure 30. Myoglobin initial reducing activity (IRA) of gel filtrate fractions of cooked and uncooked turkey breast ultrafiltrate. RESULTS AND DISCUSSION / 87 6 0 . 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 Elution volume, mL Figure 31. Cytochrome c initial reducing activity QRA) of gel filtrate fractions of cooked and uncooked turkey breast ultrafiltrate. RESULTS AND DISCUSSION / 88 high molecular weight substances which had a powerful reducing activity. After mixing, the solution of cytochrome c turned from oxidized orange to reduced pink in a matter of seconds. The fraction containing this material absorded very strongly in the UV range. Although myoglobin and cytochrome c are relatively similar hemoproteins, only cytochrome c was susceptible to the action of these new heat dependent substances. In order to explain this specificity, steric confinement of the hemes within the globins, molecular complementarity of functional groups between heme and reducing agent, and mode of electron transfer are all important factors to consider. It must also be restated that commercial myoglobin used in this study was somewhat altered and therefore, conclusions extrapolated to the native form are limited. The effect of heating on the hemoprotein IRA of a different but similarly treated ultrafiltrate was followed over time. The results obtained are shown in Figure 32. Both curves related to myoglobin remained near one another and had a low profile. The reduction of cytochrome c by the uncooked extract was this time higher if compared to Figure 31. Prior to heating, the freeze-dried samples were stored at -25°C in a desiccator to optimize their stabilit3'. Despite of these precautions, one can not rule out possible creation of new entities that could have reducing activity. Nevertheless heating significantly increased the cytochrome c IRA. Decreasing activity was registered over time. After 30 h of storage, all -6 curves had reached a plateau below 10x10 mol/mL«min. RESULTS AND DISCUSSION / 89 Figure 32. Effect of heating and storage time on the hemoprotein initial reducing activity (IRA) of turkey breast ultrafiltrate: Mb, myoglobin; CYT c, cytochrome c; UNE, uncooked; CKE, cooked at 85 °C for 10 min. RESULTS AND DISCUSSION / 90 During and after the process of cooking turkey meat, a lot of reactants could be involved in redox reactions with ferrihemoproteins and hemichromes. In this speculative labyrinth, even gases such as SHg from decomposition of sulfur containing amino acids are a possibility that should be considered. However, these previous experiments were restricted to soluble material subjected to ultrafiltration and/or gel filtration. Reducing compounds can be viewed as antioxidant substances, ln a study conducted by Einerson and Reineccius (1977), evidence about the inhibition of warmed-over flavor in retorted turkey breast was presented which suggested the production of antioxidants during processing. A year later in an other publication, the same workers (Einerson and Reineccius, 1978) further characterized the antioxidant material. Antioxidant property was measured by oxygen uptake in model systems. Fractionation on Sephadex G-50 indicated the active material to have a molecular weight of between 200 and 500. It was not volatile, and exhibited strong reducing properties similar to those found in reductones, which are known intermediates in the browning reaction. The Maillard reaction between reducing sugars and amino acids involves condensation, dehydration and polymerization. As a result of this complex reaction, a variety of by-products, intermediates and brown pigments (melanoidins) are produced, which may contribute to the flavor, antioxidative activity and color of food. It is widely accepted that the non-enzymatic browning reaction produces strong RESULTS AND DISCUSSION / 91 reducing substances, whose reducing power is responsible for their antioxidative activity. Franky and Iwainsky (1954) first reported the effect of browning reaction products on the oxidative stability of margarine. This antioxidative property has been successfully used in foods, such as frozen sausage, cookies, milk powder, and minced herring (Beckel et al., 1985). Other workers also found, as in this experiment, compounds of high molecular weight and strong reducing activity formed during heating. Yamaguchi et al. (1981) investigated Maillard reaction products (MRP) obtained in a model system by heating 2 M glycine and 2 M xylose at 100°C for 2 hours. When fractionating by gel filtration and thin layer chromatography, they discovered a strong antioxidative effect in fractions containing melanoidins with a molecular weight of about 4500. The antioxidative effect of MRP from glycine-xylose was reported to be comparable to that of BHA, higher than that of tocopherols, but lower than that of BHT, when compared on a weight basis. It is worthwhile to mention that a considerable number of factors may influence the formation of MRP in the sarcoplasm of turkey breast during cooking. Composition, molar ratio of constituents, time-temperature program, pH and are among the important contributors which ultimately determine the antioxidative properties of MRP. CONCLUSION This study initially dealt with the assessment of red color in cooked turkey breast. Slices of meat from birds of two different ages underwent heat treatments at specific temperatures and were measured for their redness over several days of storage. The Hunter a^ value, used to evaluate this trait, was not different in either 12 or 18 week old turkey samples. On the other hand, cooking temperature, storage time and their interaction were found to be significant sources of variation. Among the studied conditions, the highest red color occured immediately after cooking at 65 °C. The responsible hemoproteins, mainly myoglobin, theoretically denature when an end-point temperature of 85 °C is reached. Although this stage is associated with adequate cooking, it did not only produce an expected white to golden-brown color. A pink tinge was exhibited and disappeared after 3 to 4 days. A simple method was developed for the evaluation of meat pigments in situ, consisting of scanning cooked thin slices by transmission spectrophotometry. Absorption bands at 414, 520 and 550 nm of the obtained difference spectra lead to conclude that the residual pink color was caused by cytochrome c. Additional spectra and electrophoretograms of extracts supported this assertion. Extraction with low ionic strength phosphate buffer was also useful to determine the concentration of hemoproteins in both 12 and 18 week old turkey breasts. As the birds got older, the total heme pigment content increased from 0.60 to 0.77 mg/g tissue and the cytochrome c content respectively decreased from -2 -2 7.9x10 to 5.4x10 mg/g tissue. The trend of these results finds favorable 92 / 93 explanation in the relation between the physiological growth of the breast muscle volume and the slower rate of multiplication of mitochondria. Meat slices were further studied for their behavior upon air contact immediately after cooking. Normally a heat treatment at 65°C does not suffice to affect the structure of myoglobin. The difference spectra effectively showed the appearence of the characteristic bands at 545 and 575 nm as reduced myoglobin interacted with oxygen to produce the known "blooming" phenomenon. When preventing exposure to an aerobic environment, meat slices cooked at 85 °C demonstrated the presence of several other shoulders of absorption. They however vanished upon exposure to air. Possible candidates are cytochrome b and c^ and various denatured globin hemochromes. These pigments may at first contribute to the turkey breast pinkening but spontaneously oxidize as soon as the meat surface is exposed to air. All the forementioned ferrohemoproteins and hemochromes required low redox potential in the meat interior to display the difference spectra illustrated earlier. Procedures involving ultrafiltration and gel filtration were performed to search for compounds responsible for the establishment of a reducing environment. The capacity of 1 mL fraction to reduce myoglobin and cytochrome c for a period of 1 min was expressed as the hemoprotein initial reducing activity (IRA). Two groups of soluble materials were separated: a group of compounds below (LMW) and another above (HMW) the molecular weight of 3000 daltons. LMW had low IRA for both hemoproteins compared to the HMW which were produced by a heat treatment at 85 °C. The latter strongly reduced cytochrome c but not / 94 myoglobin. A possible identity of these compounds could be Maillard reaction products (MRP). Non-enzymatic browning offers reaction pathways for the production of substances whose strong reducing power accounts for their known antioxidative properties. Myogen of turkey breast naturally contains many amino acids, peptides as well as reducing sugars. These materials are the starting reactants necessary for the complex Maillard reaction. Pink color after cooking is not restricted to turkey breast from whole carcasses but occurs in other products such as turkey rolls. During the production of such items, ingredients including salt, sugars, and starch are currently added. These modifications create entirely different molecular environments which in turn bring more variation to the formation of MRP. Certain formulations could favor stronger reducing activity than others. Since the pinkening phenomenon is directly linked to the reduction of heme related compounds, changes previous to processing, e.g., diets of the birds and stress before slaughter, and changes during processing, e.g., alteration of formulation and cooking procedure, could provide a wide range of results. Enumerating all these avenues might help to explain the sporadic reports of pink color in cooked turkey breast products. R E F E R E N C E S Akeson, A., Ehrenstein, G.V., Hevesy, G., and Theorell, H. 1960. Life span of myoglobin. Arch. Biochem. Biophys. 91:310. Akoyunoglou, J.-H.A., Olcott, H.S., and Brown, W.D. 1963. Ferrihemochrome and ferrohemochrome formation with amino acids, amino acid esters, pyridine derivatives and related compounds. Biochemistry, 2:1033. 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Whalen, W.J., and Nair, P. 1967. Intracellular p0 2 and its regulation in skeletal muscle of the guinea pig. Circ. Res. 21:251. Wolfe, S., Watts, D., Duane Brown, W. 1978. Analysis of myoglobin derivatives in meat or fish samples using absorption spectrophotometry. J. Agric. Food Chem. 26:217. Wood, D.F., and Richards, J.F. 1975. Effect of some antemortem stressors on postmortem aspects of chicken broiler Pectoralis muscle. Poultry Sci. 54:528. Yamaguchi, N., Koyama, Y., and Fujimaki, M. 1981. Fractionation and oxidative activity of browning reaction products between D-xylose and glycine. Prog. Fd Nutr. Sci., Pergamon Press Ltd. vol.5, p.429. APPENDICES Appendix A. Step-by-step instructions concerning the development solutions, times and temperatures used for SDS-PAGE. Step number Solution^ Time Temperature (min) CC) 1 50% ethanol, 10% HAc 2.0 50 2 10% ethanol, 5% HAc 2.0 50 3 10% ethanol, 5% HAc 4.0 50 4 8.3% gluteraldehyde 6.0 50 5 10% ethanol, 5% HAc 3.0 50 6 10% ethanol, 5% HAc 5.0 50 7 Deionized distilled water 2.0 50 8 Deionized distilled water 2.0 50 9 0.25% silver nitrate 13.0 40 10 Deionized distilled water 0.5 30 11 Deionized distilled water 0.5 30 12 Developer 0.5 30 13 Developer 4.0 30 14 5% HAc 2.0 50 15 10% HAc, 5% glycerol 3.0 50 f Abbreviation used is: HAc = acetic acid; Developer = 0.04% formaldehyde in 2.5% sodium carbonate 106 / 107 Appendix B-l. Absorbance difference spectra of a turkey breast slice cooked at 75°C for 5 min in a cuvette: (A) covered with a strip of pyrex, (B) uncovered and exposed to air. / 108 Appendix B-2. Absorbance difference spectra of a turkey breast slice cooked at 95 °C for 5 min in a cuvette: (A) covered with a strip of pyrex, (B) uncovered and exposed to air. 


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