INFLUENCE OF PRESUIUGHTER INIECTIONS OF ADRENALIN AND IODOACETATE ON pH, DRIP AND CERTAIN TURKEY MUSCLE PROTEIN CHANGES DURING. FROZEN STORAGE ’ Thesis for the Degreekof Ph’. D. MICHIGAN STATE UNIVERSITY JOSEPH CARTER CRIGLER 1968 . . THESIS LIBRARY Michigan State University This is to certify that the thesis entitled INFLUENCE OF PRESLAUGHTER INJECTIONS OF ADRENALIN AND IODOACETATE ON pH, DRIP AND CERTAIN TURKEY MUSCLE PROTEIN CHANGES DURING FROZEN STORAGE presented by Joseph Carter Crigler has been accepted towards fulfillment of the requirements for Mdegree in. FOOd SCienCe X f é‘wab Major professor Date 18 1968 0-169 mm IIIIAII & 68""? BIIIIK BIIIIIEIII IIIII. IIMRV BINDER ABSTRACT INFLUENCE OF PRESLAUGHTER INJECTIONS OF ADRENALIN AND IODOACETATE ON pH, DRIP AND CERTAIN TURKEY MUSCLE PROTEIN CHANGES DURING FROZEN STORAGE BY Joseph Carter Crigler This study was undertaken to evaluate the effects of a high ultimate pH of turkey muscle, obtained by injec— tion of either adrenalin or iodoacetate, and frozen storage on the percentage drip, changes in certain muscle protein fractions and amino acid changes of drip. The use of gas chromatography for analyses of amino acids from protein hydrolysates was also investigated. Five turkeys received a 1.5 mg subcutaneous injec- tion of adrenalin per kg of body weight and were fasted 20 hours prior to bleeding. A second group of five turkeys received a 200 mg intravenous injection of iodoacetate per kg body weight three minutes prior to bleeding. After processing, light and dark meat was removed from each carcass, ground and formed into slabs prior to freezing. Drip was collected separately from light and dark meat during a thawing period of 36 hours at 5°C after frozen storage periods of 0, 30 and 90 days at -20°C. Joseph Carter Crigler High ultimate pH values for light and dark meat from the adrenalin treated turkeys (6.78 and 7.05, respec- tively) and the dark meat from the iodoacetate treated turkeys (6.97) were obtained. pH values for the control light and dark meat were 5.96 and 6.16, respectively. A high ultimate pH reduced the amount of drip during thawing. Control meat samples yielded 3.3 and 1.2 percent drip from light and dark meat, respectively, before storage. Insuffi— cient drip was obtained from the light and dark meat from the adrenalin treated birds for measurement before storage. Percentage drip from the iodoacetate treated birds before storage, from the light and dark meat, was 10.3 and 1.1 percent, respectively. Light meat had a higher per- centage drip than dark, and the percentage drip from both light and dark meat increased during storage, except for the percentage from light meat from the iodoacetate treated turkeys. Gas chromatography was found to be a satisfactory means of separating and quantitating the amino acids from a mixture of pure amino acids. Results of analyses from a hydrolysate of purified protein (bovine serum albumin) were less satisfactory, with threonine and phenylalanine giving high recoveries and leucine and aspartic acid giving low recoveries. Amino acid analyses of a water soluble protein extract varied considerably from the results of amino acid analyzer. Satisfactory recovery was obtained Joseph Carter Crigler for only six of the 14 amino acids investigated, namely, alanine, valine, proline, glutamic acid, tyrosine and lysine. Sarcoplasmic proteins did not change in solubility as a result of frozen storage up to 90 days. Non—protein nitrogen in the water soluble fraction was higher from light meat than from dark meat, and values remained con— stant during storage. A decrease in myofibrillar proteins with storage was found to occur only in meat samples from the control light meat and dark meat from the iodoacetate treated birds. Similar percentages of myofibrillar nitro— gen were obtained from meat samples with high ultimate pH values and no apparent change occurred with 90 days frozen storage. Percentage denatured protein from the control light and dark meat samples increased with storage, and light meat contained more denatured protein than dark meat. Only slight changes were found in meat samples from the adrenalin treated birds during storage. Denatured protein values from the light meat of the iodoacetate treated birds were considerably higher than from dark meat, and higher than values from both light and dark meat samples from the control and adrenalin treated birds, and increased with storage. Stroma nitrogen values varied between treatments and decreased during storage for all samples. A high Joseph Carter Crigler ultimate pH was found to result in higher percentages of soluble sarcoplasmic and myofibrillar proteins and lower percentages of denatured and stroma proteins. Meat samples with an ultimate pH of 6.2 or higher contained larger amounts of free amino acids than samples with an ultimate pH below 6.2. The amount of amino acids in the drip increased with storage time of the meat and the amino acid content of the drip from light meat was higher than from dark meat from all samples. A dilution effect was observed in the amino acid concentration of drip after 30 days storage but was not apparent in the meat stored for 90 days. The additional storage period allowed proteolysis to continue and resulted in a high concentration of amino acids in the drip. It was concluded that a high ultimate pH of the muscle was of major significance in reducing the amount of fluid exuded during thawing, since there was a direct relationship between amount of drip and total amount of amino acids released from tissues. INFLUENCE OF PRESLAUGHTER INJECTIONS OF ADRENALIN AND IODOACETATE ON pH, DRIP AND CERTAIN TURKEY MUSCLE PROTEIN CHANGES DURING FROZEN STORAGE BY Joseph Carter Crigler A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1968 4,7540% 7] 3-/f‘«é? ACKNOWLEDGMENTS The author deeply appreciates and is sincerely grateful to Dr. L. E. Dawson for his devoted assistance and guidance throughout this course of study. I would also like to extend a special word of thanks to Dr. Dawson for his prompt and critical review of the manuscript, which was of tremendous value in the completion of my course of study in light of my military obligation. The author would like to express his appreciation to Dr. B. S. Schweigert and the faculty and students of the Department of Food Science for their cooperation given throughout my graduate program; to Dr. A. M. Pearson, Dr. R. K. Ringer, Dr. C. M. Stine, and Dr. D. E. Ullrey for their cooperation and prompt and critical review of the manuscript. A special note of thanks to Dr. J. R. Brunner and Ursula Koch for their advice and assistance in amino acid analyses, and to Mrs. Maurice Ritchey for assistance in typing and preparation of the manuscript. To my wife Marilyn, insufficient words are availa- ble to me to express my appreciation for the encouragement, understanding and help given through this trying period. ii TABLE LIST OF TABLES . . . . . LIST OF FIGURES . . . . . INTRODUCTION . . . . . . . LITERATURE REVIEW . . . . Changes in Meat During Freezing and Thawing Amount and Composition of Quantity of Drip . . . Composition of Drip . Gas Chromatography of Ami EXPERIMENTAL PROCEDURE . Experimental Animals . . General Methods Used . . Nitrogen Determinations pH . . Collection of Drip Muscle Protein Fractionation . . Amino Acid Analyses by Gas- ~Liquid Chromatography OF CONTENTS Drip . . . no Acids Preliminary Investigations . Derivative Formation o o n a o Recovery of Amino Acid Derivatives Amino Acid Analyses of Samples from Experimental Animals . Water Soluble fraction Drip . . . . . . . . . RESULTS AND DISCUSSION . . 0 Evaluation of Amino Acid Analysis by Gas— —Liquid Chromatography . . . . Preliminary Investigations . . . Evaluation of Procedure Effects of Adrenalin and Iodoacetate on .pH and Related Changes in Certain Muscle Fractions pH of Muscle . o Page . viii Drip . . . . . . . . . . . . . Nitrogen Released in Drip . . . . . Muscle Protein Fractionation . . Amino Acid Analyses of Ultrafiltrate Amino Acid Analyses of Drip . . . . SUMMARY . . . . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . iv Table l. 10. LIST OF TABLES Page Experimentally derived factors used for quantitation of specific amino acids by GLC . . . . . . . . . . . . . . . . . . . . 47 Percentage recovery of specific amino acids from a standard solution by GLC and amino acid analyzer . . . . . . . . . . . . . . . 48 Percentage recovery of specific amino acids from a solution of bovine serum albumin by GLC and amino acid analyzer . . . . . . . 50 Percentage recovery of specific amino acids from the water soluble fraction of light turkey meat by GLC and amino acid analyzer . . . . . . . . . . . . . . . . . 51 pH values of muscle from adrenalin and iodo— acetate treated turkeys 24 hours after slaughter and after frozen storage . . . . 57 Percentage drip from muscles of adrenalin and iodoacetate treated turkeys after frozen storage . . . . . . . . . . . . . . 60 Nitrogen content in drip from muscles of adrenalin and iodoacetate treated turkeys after frozen storage . . . . . . . . 63 Protein components of light and dark meat from control turkeys after frozen storage . 66 Protein components of light and dark meat from adrenalin treated turkeys after frozen storage . . . . . . . . . . . . . . 67 Protein components of light and dark meat from iodoacetate treated turkeys after frozen storage . . . . . . . . . . . . . . 68 Table 11. 12. I T 13. 14. 15. l6. 17. 18. 19. 20. Amino acid composition of the ultrafiltrate of the water soluble fraction of light meat from control turkeys after frozen storage . . . . . . . . . . . . . . . . . . Amino acid composition of the ultrafiltrate of the water soluble fraction of the dark meat from the control turkeys after frozen storage . . . . . . . . . . . . . . Amino acid composition of the ultrafiltrate of the water soluble fraction of the light meat from the adrenalin treated turkeys after frozen storage . . . . . . . . . . . Amino acid composition of the ultrafiltrate of the water soluble fraction of the dark meat from the adrenalin treated turkeys after frozen storage . . . . . . . . . . . Amino acid composition of the ultrafiltrate of the water soluble fraction of the light meat from the iodoacetate treated turkeys after frozen storage . . . . . . . . . . Amino acid composition of the ultrafiltrate of the water soluble fraction of the dark meat from the iodoacetate treated turkeys after frozen storage . . . . . . . . . . Amino acid composition of drip from the light meat of the control turkeys after frozen storage . . . . . . . Amino acid composition of drip from the dark meat of the control turkeys after frozen storage . . . . . . . . . . . . . . . Amino acid composition of drip from the light meat of the adrenalin treated turkeys after frozen storage . . . . . . . . . . . . . . Amino acid composition of drip from the dark meat of the adrenalin treated turkeys after 90 days frozen storage - . - - . - - - - - vi Page 74 75 76 77 78 79 82 83 84 85 Table 21. 22. Amino acid composition of drip from the light meat of iodoacetate treated turkeys after frozen storage Amino acid composition of drip from the dark meat of iodoacetate treated turkeys after frozen storage Page 86 87 LIST OF FIGURES Figure Page 1. Outline for the fractionation of turkey muscle proteins . . . . . . . . . . . . . 32 2. Chromatogram of the sequence and temperature of elution for 14 N—trifluoroacetyl n-butyl amino acid derivatives and internal standard . . . . . . . . . . . . . . 45 3. Chromatogram of a water soluble protein hydrolysate from light turkey meat . . . . 53 viii INTRODUCTION For many years, most of the turkeys produced have been fresh—frozen for both the retail and institutional trade. Since most adult turkeys are marketed in late summer or early fall, it is essential to preserve them for consumption throughout the year. There has been a marked increase in use of turkey and chicken in conven- ience foods such as turkey or chicken rolls, roasts and frozen dinners. Further processing of turkey and chicken in Federally inspected plants rose from 105 to 335 million pounds for turkey and 295 to 481 million pounds for chicken between 1961 and 1966 (U.S.D.A., 1967). During freezing ice crystal formation results in expansion of the product. As these ice crystals form, an ice front moves through the product and results in the formation of a concentrated salt solution in the intersti- tial spaces. The severity of the damage resulting from this expansion, the formation of ice crystals and the concentration of salt in solution affects the quality of the frozen product. During thawing, fluid is released from the tissue and collects as drip. The amount of fluid exuded during thawing is affected by the pH of the meat. Hamm (1953, 1958) reported that the amount of drip was closely related to the water-holding capacity and Whitaker (1959) reported that pH affects the water-holding capacity by modifying the charges of the protein. Kuprianoff (1952) found that there may be no drip on defrosting, especially when meat is in the pH range 6.3—6.4. Chicken meat proteins undergo both denaturation and proteolysis during frozen storage. Since beef contained a protease active at freezing temperatures (Balls, 1938), it was concluded that a similar enzyme could possibly be present in chicken muscle (Khan, 33 31., 1963). While results of fractionation data and amount of drip from frozen turkey meat has been reported by a few investigators in the past, little work has been reported on the effect of a high ultimate pH by pretreatment of the birds. Since pH has been shown to affect the amount of drip and water—holding capacity, protein characteristics could be different. The use of the amino acid analyzer has proven to be a satisfactory method for evaluation of amino acid composition. In the past few years amino acid analyses by gas chromatography for biological systems has been investigated. Information concerning the accuracy of this method is limited except for a few purified proteins. The purposes of this study were: 1. to obtain a high ultimate pH in turkey meat by treatment of the birds with either adrenalin or iodoacetate before slaughter; 2. to evaluate the effects of high ultimate pH and storage on the amount of drip, on certain muscle protein fractions, on changes in amino acid content of drip and on changes in the water soluble protein fraction; 3. to evaluate the use of gas chromatography as a means of amino acid quantitation and to compare the results with those obtained by an amino acid analyzer. LITERATURE REVIEW Changes in Meat During Freezing and Thawing Histological structure of frozen poultry was investigated by Koonz and Ramsbottom (1939a). They re— ported that in small pieces of meat which were frozen almost instantaneously, the frozen water appeared within the fibers as minute evenly distributed ice columns, whereas in tissue frozen more slowly there were fewer, larger ice columns with a peripheral distribution. With a further decrease in freezing temperature a single cen- trally located ice column resulted. However, when the freezing process was sufficiently prolonged, a temperature was reached at which water was lost from the fibers; freezing externally to the fibers. The explanation of drip formation during thawing of fish or meat includes more than the rupturing of cells by the expansion of ice crystals (Taylor, 1930). He stated that there were certain physical—chemical changes related to the conditions under which colloidal protein systems were converted from liquid to gel or gel to liquid and form heterogeneous systems that have a role in the forma— tion of drip. Three means by which the cell jelly could be converted to a liquid were suggested by Taylor: 1) the salting out of proteins by strong brine in the frozen cells, 2) the contraction of the jelly and squeezing out of the then dilute liquid, and 3) autolysis of the cell membrane. The cell membrane would have remained undamaged by quick—freezing, and the juice would have been unable to escape through the membrane. The process of autolysis offered a possible explanation since it might account for digestion of the cell membrane or increased membrane permeability. Denaturation of protein in fish muscle and fish muscle juice was reported by Riddell 3E 21. (1937). The extent of denaturation was governed largely by storage temperature and length of the holding period. Later Reay (1933, 1934) emphasized the denaturation phenomenon by investigating the influence of freezing temperature on haddock. He found that muscle protein solubility in salt solutions decreased after the fish were held in frozen storage. The globulin (actomyosin) fraction was most affected and the change was greatest in range —2 to —6°C. He also concluded that the denaturation and deterioration, which occurred during thawing was similar to that which occurred during freezing. Plank (1925) emphasized the importance of two effects of freezing: 1) the damage to the protoplasmic structure by formation of ice crystals, and 2) the consequent dehydration of the colloid. It was pointed out by Callow (1952) that the salts in muscle juice become more concentrated during the formation of ice in muscle. Callow (1955) and Luijpen (1957) found that high concentrations of salt may be capable of denaturing some cellular proteins. Dyer (1951) stated that the formation of ice crystal fragments in frozen fish was attributed to the dehydration of tissue cells. He also reported that the proteins present in the sol form in the fresh tissue were converted to gel form at the eutectic point by denaturation of the salts in the fresh tissue. Smorodintsev and Bystrov (1937) reported that low temperature freezing (—25°C) resulted in no denaturation of muscle proteins as determined by ATP—ase activity or by the solubility and swelling of tissue in salt solution. Rapid freezing and thawing together with storage at low temperatures may be important in denaturation, since these conditions minimize changes due to denaturation of the proteins by strong salt solutions (Finn, 1932). Deatherage and Hamm (1960) found that quick freezing and thawing of beef caused no significant decrease in hydra— tion and no change in the isoelectric point of the meat; however, small but significant changes in acidic and basic groups of muscle proteins were noted. While the overall extent of denaturation depended on the length of storage (Love, 1955) large differences in protein denaturation were caused by different freezing rates of fish. Love (1956) concluded that an important factor which affected denaturation was the mode of ice formation, distribution and the resulting concentration patterns of tissue components rather than actual mechanical damage. Love (1958a), in re-examining the question of tis— sue damage, reported that the appearance of deoxypentose— nucleic acid (DNA) in the expressible fluid from muscle indicated rupture of the sarcolemmas with liberation of nucleic materials. The presence of DNA provided a means of assessing the degree of cellular damage. As fish fillets were frozen more rapidly, the expressible nucleic acid suddenly reached a maximum; this was thought to correspond with the point of formation of intracellular ice crystals. The great damage reported in the ultra—rapid freezing range was thought to be due to a different type of cell damage, an expansion of the interior during cooling, and subsequent cracking of the hard outer shell which had formed earlier. Using different freezing methods (solid CO2 and plate at -78°C) Love (1958a) further examined the action of intercellular ice on muscle cells. He showed that DNA content in the drip corresponded with actual rupture of the sarcolemmas, both from histological evidence (Love, 1958a) and from the presence of cell particles in the expressible fluid (Love, 1958b). A zone of minimum damage was found with a freezing rate of about 115 minutes, (Love, 1958a) the freezing time at which all ice was able to form in the intracellular spaces. At slower freezing rates (200 to 500 minutes) cell damage was found to be at a maxi— mum value and then decreased. With very slow freezing, the strong salt solutions, which were created by the freezing out of water from the weak saline interstitial fluids, exer- cised a considerable solvent effect on the fibers without actually rupturing the cells. An increase in the protein concentration in the interstitial fluid tended to increase during very slow freezing (approximately 750 minutes). Love (1962a) found that denaturation was less when the quantity of ice present at a given temperature was reduced, since a reduction in the amount of ice reduced the concentration of tissue salts. These results further supported the theory (Love, 1958c) that denaturation results directly or indirectly from the action of concentrated salts on the proteins. Chicken muscle proteins undergo both denaturation and proteolysis during frozen storage according to Khan gt 31. (1963). Stepwise denaturation of actomyosin decreased ATP—ase activity and protein solubility. Prote- olysis also occurred in both leg and breast muscle at —l8, —10 and —4°C, and the rate of change depended directly on storage temperature and time. Bandack-Yuri and Rose (1961) reported that muscle proteases have a pH optimum of 4 to 7 and temperature optimum of 37°C. Since beef contains a protease active at freezing temperatures (Balls, 1938), it has been concluded that a similar enzyme system could possibly be present in chicken muscle (Khan gt 21., 1963). Khan and van den Berg (1965), in further investigation of changes occurring during storage, showed that there was a proteolytic enzyme active during frozen storage. This conclusion was based on an analysis of cooked meat stored at temperatures between -10 and —40°C for two years. They reported that since there was no formation of protein— breakdown products, as in raw meat, the enzymes must have been inactivated by heat. Sawant and Magar (1961) studied the denaturation of proteins in frozen fish and concluded that denaturation was restricted to the actomyosin fraction of protein while the sarcoplasmic fraction remained unchanged. Dyer (1951) observed that the albumin fraction was not denatured during frozen storage, while Seagram (1958, 1959) suggested that the sarcoplasmic fraction was not associated with the formation of drip. Denaturation and autolytic changes in compounds of muscle tissue during cooling and freezing of meat were studied by Pavlovskii (1962). Autolysis of muscle was characterized by a degradation resulting from the activity of ATP-ase and glycolytic conversion and resynthesis of adenasine triphosphate. Since freezing increased NaCl, 10 myosin and ATP-ase concentration, he concluded that denaturation of frozen muscle was characteristic of meat previously autolyzed before freezing. Proteolysis in poultry during frozen storage was indicated by an increase in non—protein and soluble nitro- gen of both light and dark meat (Swanson and Sloan, 1953). They also noted a decrease in amino acid nitrogen which suggested that some metabolic processes were being con- tinued in the frozen state resulting in additional degra— dation of the amino acids formed during proteolysis. The protein components of autolyzed muscle tissue were studied during cooling and freezing of meat by Pavlovskii and Grigor'eva (1963a). The amount of readily extractible protein from the tissues decreased during cool— ing and freezing when compared to that of unfrozen meat. However, during the initial storage of frozen meat the soluble protein increased, but after three months this fraction decreased. Tarr (1947) found that undesirable changes in fish (formation of large ice crystals ruptured the cells during freezing) took place during freezing, with much of the deterioration resulting from a change (denaturation) in the myosin, the most important single protein present. This denaturation rendered the myosin insoluble. Denatura— tion occurred most rapidly between the temperatures of 30°F and 23°F although denaturation occurred slowly at much 11 lower temperatures. Tarr concluded that "the only practical method of retarding protein denaturation is by very rapid freezing and storage at a constant low tempera— ture, preferably below -40°F." Khan (1964) reported that the nonprotein nitrogen fraction of chicken increased noticeably during storage for 45 weeks at temperatures of —5 and —40°C. He found about 80 percent of the increase resulted from an increase in amino acids, mostly acidic, aromatic and sulfur contain— ing amino acids. He also believed that limited proteolysis occurred as a result of the release of enzymes due to freezing. Amount and Composition of Drip Quantity of Drip Cook g3 g1. (1926) reported drip to be the clear, reddish—brown colored fluid which exudes from meat after it has been frozen and thawed. Sair and Cook (1938a) also reported that removal of skin, cutting of meat and particu- larly mincing the meat increased the susceptibility of frozen—defrosted tissues to drip. Reay (1934) reported that the quantity of expressible fluid from frozen fish depended on the number of cuts made in the flesh. However, they reported that the major factor determining the amount of drip in haddock was the time spent in the temperature range of —l to —5°C, either during thawing or freezing. - -———~—I 12 I Koonz and Ramsbottom (1939b) reported that the temperature of freezing affected the amount of drip obtained from frozen—defrosted poultry meat. This was based on the fact that the rate of freezing determined the size, distri— bution and number of ice nuclei formed. They reported that white meat frozen at 8°F was much more suceptible to drip loss than when frozen at —50°F. Callow (1952) and Empey and Howard (1954) similarly reported that slow freezing resulted in more drip. The quantities of drip obtained from thawed beef decreased as freezing temperatures were lowered from 18 to -ll4°F (Hiner gg gl., 1945). Protein reabsorbtion of a large proportion of the water originally frozen out was believed to be the cause of reduced drip since there was increased intrafibrillar freezing and rupturing of the fibers at lowered freezing temperatures. Pearson and Miller (1950) studied steaks which had been frozen at various temperatures. They reported that increased frozen storage time resulted in increased drip, which became apparent after 90 days storage. However, they found that rate of freezing did not alter the amount of expressible fluid. Ramsbottom and Koonz (1940) reported that the amount of drip from steaks was increased by in— creasing the period of time between slaughter and freezing. Time of storage, at any one temperature, had little effect on the amount of drip (Moran and Hale, 1932) but 13 temperature of storage affected the amount of drip. They found no variation in drip due to thawing rate when using 10° for rapid thawing and 1°C for slow thawing. Moran (1932) reported that maximum drip was recorded when frozen tissues were stored 80 days at —2 to —3°C. He also found a denaturation effect on the protein at —l.5°C and at temperatures lower than —3°C. Marion and Stadelman (1958) using different methods of freezing (brine, plate, and moving air) found that the method of freezing exerted no significant effect on the amount of drip from chicken fryers, fowl, turkey fryers and mature tom turkeys. They reported that drip from chicken fryers ranged from 5.3 to 5.8 percent; fowl, 5.2 to 5.6 percent; turkey fryers 4.3 to 5.3 percent; and mature turkeys, 2.3 to 3.0 percent. According to Ramsbottom and Koonz (1939) small rapidly frozen steaks retained more drip on defrosting since intrafiber freezing occurred. Whereas in steaks which were slowly frozen, extrafiber freezing took place and upon defrosting more fluid was lost as drip before it could be reabsorbed by the partially dehydrated fibers. They also reported that irrespective of freezing temperature small rib cuts lost more fluid as drip than large rib cuts due to area of the cut surface in relation to the volume. Moisture loss during the defrosting of turkey was affected significantly by the cooling and freezing treatment 14 (Spencer g2 g1., 1956). They found that losses from air—cooled carcasses averaged lower than losses from car— casses cooled in ice water. They also found a higher weight loss from turkeys frozen at 0°F than from turkeys frozen at lower temperatures. Bouton gg g1. (1958) found a gradual decrease in the amount of drip from pgggg and l. dorsi muscles when the pH increased above 5.8. Maximum drip was obtained from meat in the pH range between 5.4 — 5.8. Howard and Lawrie (1956) found that the amount of drip exuded from 1. dorsi was approximately twice that from pgggg at the same pH. They concluded that different muscle proteins may be affected differently by the same pH during thawing. The quantity of drip from meat frozen at a constant rate was affected by the pH of the tissue and the period of time between slaughter and freezing (Sair and Cook, 1938a). The quantity of drip was determined primarily by the amount of acid contained in the tissue, and increased as pH fell below 6.0. Maximum drip was obtained at pH 5.2 - 5.5, and when freezing rates were faster than 3 days (meat tempera— ture changed from 0°C to -5°C) there was a reduction in the amount of drip obtained. The reduced amount of drip was attributed to the high water—retaining capacity of tissue proteins at pH 6.4 and was not affected by the size or number of ice crystals formed during freezing. A de- crease in water—retaining power of the proteins occurred 15 at pH 5.2, and resulted in increased moisture losses. Rapid freezing reduced these losses due to the production of smaller ice crystals and a more uniform distribution of water during defrosting. The reduced moisture-retaining capacity at pH 5.2 — 5.5 was due to isoelectric conditions rather than to accelerated denaturation. Ramsbottom and Koonz (1940) found that thawed beef with pH values between 6.2 and 6.5 produced only 0.7 percent drip compared with 4.3 percent drip for beef thawed at pH 5.7 — 5.9. Empey (1933) reported that pH was the major factor that influenced the amount of drip, and the lower the pH, the greater the volume of drip. After examining the effects of freezing on the swelling of tissues in a buffered solution, Smorodintsev and Bystrov (1937) concluded that the quantity of fluid secreted from the tissue varied inversely with the degree of swelling. The swelling of frozen tissues increased with acidity of the medium (starting at pH 3.4) and a decrease was noted at pH values higher than 6.0. According to Koonz and Ramsbottom (1939b) less drip was obtained from ground dark chicken meat than from ground white meat regardless of the freezing temperatures (—45.5°C, —26.1°C, —l3.3°C). The pH value for meat 2.3 hours after slaughter was 5.7 for the white meat and 6.1 for dark meat, and after 22 days storage the pH values were 6.2 and 6.3, 16 respectively. The greater reabsorption capacity exhibited by the dark muscle was thought to be due to the higher pH values. Wladyka (1965) reported that larger amounts of drip were obtained from white meat than dark meat and that the amount of drip increased with storage. He found that the percentage of drip obtained from white meat after 30 and 90 days storage was 5.4 and 9.6 percent respectively, while that obtained from dark meat was 4.8 and 7.9 percent, respectively. Kaloyereas (1947) found that rapid freezing was less effective in decreasing drip loss from products with high boundwater content than from products with low bound— water content. The amount of drip obtained is closely related to the water-holding capacity (WHC) of meat, Hamm (1953, 1958). When a high WHC was observed in meat before freezing, com— parable WHC was noted after defrosting. Whitaker (1959) found that pH affects the water-holding capacity of the meat by modifying the charges of the protein. Meat with a pH in the range of its isoelectric point (5.0 — 5.5) had a greater drip loss after freezing and thawing than meat with a higher pH (Kuprianoff, 1952). He also found little or no drip from meat defrosted in the pH range of 6.3 - 6.4. Drip losses from frozen aged meat were much less than those from frozen rigor muscles (Wierbicki g3 g1., 17 1957b; Bouton g2 g1., 1958) since the aging of meat resulted in increased water-holding capacity (Hamm, 1960). When whole animals or quarters of beef were infused with NaCl, the water—holding capacity of the meat was markedly increased so that little or no drip occurred after freezing and thawing (Wierbicki gg g£., 1954). The effect of added cations on meat shrinkage at 70°C was studied by Wierbicki g2 g1. (1957a). They found that the cations sodium, calcium, potassium, and magnesium increased the water—holding capacity of meat and that NaCl, added to the meat prior to freezing, decreased the amount of drip. They also stated that the pH shift toward alka- linity, produced by the addition of NaCl, decreased after freezing and thawing, indicating a possible protein modi— fication due to freezing. Howard and Lawrie (1957) reported that there were two ways to reduce drip from frozen beef muscle on thawing, depending on biochemical factors. Their importance was suggested from fundamental work 13 21339 (Empey, 1933; Bendal and Marsh, 1951; Marsh, 1952a, 1952b). Apparently these factors enhance the capacity of muscle proteins to retain fluid. The factors were: a. Reduction in the rate of ATP breakdown during the onset of rigor mortis. b. Restriction of postmortem production of lactic acid from glycogen which resulted in a high ultimate pH of the muscle tissue. 1115' 7' T 18 Radouco—Thomas g3 g1. (1959) reported on the injection of epinephrine on skeletal muscles of rat, rabbit, sheep and pigs. They found that epinephrine pre— vented the autolytic process from setting in and prevented an increase in build up of lactic acid, pH drop, and re— duced exudation. Anglemier g5 g1. (1961), deFremery (1963), deFremery and Pool (1963) found similar results with pork and poultry respectively. Bendall and Lawrie (1962) reported the effects of ante-mortem injections of various drugs on post-mortem changes in muscle. Injection of adrenalin 4 hours before slaughter significantly depleted muscle glycogen in rabbits and raised ultimate muscle pH. Hedrick g3 g1. (1957) re- ported that injection of epinephrine 24 and 12 hours prior to slaughter significantly elevated the ultimate pH of bovine muscle. pH of the control animals ranged from 5.45 to 5.7 and for the adrenalin treated animals, pH ranged from 6.0 to 6.15. The accepted explanation for this effect is that adrenalin causes activation of phosphorylase so that glycolysis proceeds more rapidly along the anaerobic pathway to lactic acid (Cori and Illingworth, 1956). Iodoacetate was shown by Padieu and Mommaerts (1960) to inhibit the action of the enzyme phosphoglyceraldehyde dehydrogenase and therefore eliminate glycolysis. deFremery (1963) and deFremery and Pool (1963) investigating factors affecting poultry tenderness, found a high ultimate pH 19 after an injection of-iodoacetate and a reduction in the amount of fluid exuded during defrosting. deFremery and Pool (1960) found similar results using sodium monobromo- acetate which inhibited glycolysis. Partmann (1963) reported that when muscle tissue was frozen immediately after death, the ATP present at that moment persisted. When the temperature increased suffi- ciently after thawing, the ATP was split quickly by con— traction of the tissue and produced thaw rigor. The muscle contracted during thawing to about 50 percent of its former length and was accompanied by a high drip loss. Marsh and Thompson (1957) suggested that this was the result of cell membrane rupture. Love (1962b) reported that pre-rigor frozen fragments had about 60 percent lower soluble-protein values after thawing, and suggested that the exudation resulted from a change in the proteins which reduced their ability to bind water. Composition of Drip Godeaux (1957) stated that the fluid which escaped from muscle during thawing, due to cell membrane destruc- tion, contained large amounts of proteins (myogen and globulin x). Pearson gg g1. (1959) reported that the amino acid content of drip from thawed pork indicated losses ranging from 7.15 percent for tryptophane to 11.08 percent for isoleucine. They also stated that the losses did not 20 appear to be associated with the water solubility of the individual amino acids which indicated that the losses in drip may have been due in part to the leaching of more complex substances. The concentration of essential amino acids in the drip from dark and light chicken meat increased as the time of frozen storage increased (Wladyka, 1965). Larger quan— tities of essential amino acids were detected in drip from frozen white meat than from frozen dark meat after both 30 and 90 days of storage. When reported as the percentage of total protein contained in the drip, arginine values ranged from 2.80 to 6.05 percent, and histidine from 4.70 to 12.83 percent after 30 and 90 days storage, respectively. Arginine and histidine content of drip from light meat varied from 3.63 to 7.26 percent, respectively after 30 days storage, whereas after 90 days storage, the values varied from 8.25 to 16.45 percent for isoleucine and histidine, respectively. Khan and Lentz (1965) reported on the influence of pre—rigor, rigor, and post-rigor freezing on drip losses and protein changes in chicken meat. They found an average of 0.8 m1 drip released per 100 grams carcass and 5.9 mg total nitrogen released per 100 grams of carcass in post— rigor chicken muscle. Nonprotein nitrogen accounted for 35 percent and protein nitrogen, 65 percent of the total nitrogen. 21 According to Wladyka (1965) the drip from the white meat contained larger quantities of protein than drip from dark meat, after storage periods of 30 or 90 days. He reported that the drip from white meat held for 90 days contained 10.92 percent protein nitrogen and that stored 30 days, 9.17 percent. Similarly, the protein nitrogen content of drip from dark meat stored for 90 days (6.26 percent) was larger than the protein content of drip from dark meat stored for 30 days (5.16 percent). Love (1958a) investigated cell damage in fish muscle during slow freezing and reported solids and ash content. Solid matter varied from 7.29 to 8.47 grams per 100 grams of expressible fluid with freezing rates of 2 and 47 minutes, respectively. Ash content in the expressi— ble fluid did not vary greatly regardless of the freezing rate (1.27 to 1.34 grams per 100 grams of expressible fluid with freezing rates of 2 and 42 minutes, respectively). Love (1955) reported the solids and nitrogen content of expressible fluid from unfrozen fillets used as controls. Total solids and nitrogen were 8.20 and 1.15 grams per 100 ml of expressible fluid, respectively. Proteolytic changes in meat during storage and during defrosting were studied by Pavlovskii and Grigor'eva (1963b). Electrophoretic patterns were similar for the extractible proteins of meat and drip. Loss of protein in the drip was 15—22 percent greater for meat subjected to 22 preliminary autolysis before freezing than for quick-frozen meat. The proteins of the myoplasmic tissue increased in the drip during the initial storage period. After three months of storage, no increase in volume of drip was ob— served, and the amount of myoplasmic proteins in the drip decreased. Seagram (1958) reported that drip in fish was due, at least in part, to the denaturation of proteins which normally hold the water in the muscle. Using dilution techniques and electrophoresis, he showed a definite simi— larity between the protein composition of drip and extracts of low ionic strength from fish muscle. He found the con- tractile protein, actomyosin, absent in drip from frozen and thawed rockfish. He concluded that the sarcoplasmic fraction of fish muscle was not closely associated with the origin of drip, and suggested that drip formation and texture changes, as a result of freezing and thawing, were due in part to actomyosin denaturation by a dehydration process. Howard gg g1. (1960) found a higher concentration of both hemoglobin and myoglobin in drip than in whole muscle. Higher hemoglobin presumably came from the residual fluid in the vascular system. This hemoglobin containing fluid moved more readily through the sample than fluid con— tained within the cells and interstitial spaces. A similar increase in concentration of myoglobin was expected when 1 .T. 23 the pigment was confined to a portion of the cell fluid which moved more readily than that associated with the fibrillar protein. Paper electrophoresis and ultracentrifugation results indicated that drip has patterns similar to those from low ionic strength extracts of muscle (Howard gg gl., 1960). Since freezing and thawing modified the solids content of the drip, they did not modify the composition of these solids. Modification brought about by freezing were largely restricted to structural proteins which were insoluble at the ionic strength of the drip fluids. Gas Chromatography of Amino Acids A great number of gas chromatographic methods and techniques have been investigated to determine amino acid composition of products, both qualitatively and quantita- tively. Hunter g3 g1. (1956) established the beginning of extensive gas chromatographic analyses of amino acids by using oxidation with ninhydrin to prepare volatile aldehydes from several aliphatic amino acids. Later the procedure was developed (Zlatkis, 1960) into a fully automated method for the analysis of seven amino acids. Numerous other approaches have been evaluated, including decarboxylation to give volatile amines (Bier and Teitelbaum, 1959), and conversion to methyl—chloro esters (Melamed and Renard, 1960). Bayer gg g1. (1957) reported that methyl esters of 24 a number of aliphatic amino acids, prepared by the Fischer method, could be separated by gas chromatography. Success- ful chromatography of the ester hydrochlorides of 14 amino acids, by adding ammonia to the carrier gas, was reported by Saroff gg g1. (1962). Nicholls g2 g1. (1963) explored the possibility of chromatographing the acid salts of amino acid methyl esters by operating the flash heater at a sufficiently high temperature to cause disassociation of the free esters. Youngs (1959) was one of the first to recognize the advantages of N—substituted esters of amino acids for gas chromatographic analysis. He reported that chromatographic peaks had been obtained for the n—butyl N-acetyl esters of glycine, alanine, valine, leucine, isoleucine, and proline. Johnson g3 g1. (1961) investigated the preparation and gas chromatographic separation of the n—amyl N—acetyl esters of amino acids. Chromatographic peaks were obtained for the derivatives of 36 amino acids: however, difficulty was experienced with tryptophan, histidine, cystine, and arginine. An important part of the investigation was the use of columns containing small percentages of liquid phase at relatively low temperatures, to give fair separation. Graff gg g1. (1963) considered the n-propyl N—acetyl esters of the amino acids to be the best derivatives for chromatography because of their ease of separation. How— ever, after examination of the properties of a number of the “T“T‘j 25 derivatives, Blau and Darbre (1963) found that the n-amyl N-trifluoroacetyl esters were the most satisfactory. It was reported by Zomzely gg g1. (1962) that the n—butyl N-trifluoroacetyl esters of 22 naturally occurring amino acids could be prepared and that they could be chromato- graphed on a single column by means of temperature programming. Separation of the derivatives was fairly complete, however, no quantitative evaluation of the pro— cedure to prepare the derivatives was made. Pisano gg g1. (1963) and Landowne and Lipsky (1963) investigated the gas chromatographic behavior of the phenylthiohydrantions and methyl 2,4-dinitropheny1 esters; the latter derivatives were investigated using an electron capture detector. Trimethyl N-trimethylsilyl amino acid derivatives were separated by Rfihlmann and Giesecke (1961) but the derivatives were not sufficiently stable for quantitative analysis. Shahrokhi and Gehrke (1967 and 1968) used trimethyl esters to analyze for sulfur and iodine containing amino acids and Ruyle g3 g1. (1967) quantatively analyzed for purine and pyrimidine bases as the trimethylsilyl derivatives. An ultramicro— method for analysis of amino acids with a minimum detectable limit of 5 x 10'12 g injected was also developed by Ertingshauser (1967). He used the N-diethyl phosphate amino acid methyl esters of the amino acids. 26 Methyl N-trifluoracetyl esters were investigated by Weygand gg g1. (1960), Saroff and Karmen (1960) and Hagen and Black (1964). All of the common protein amino acids and ornithine were chromatographed by Cruickshank and Sheehan (1964) after conversion to their methyl N—trifluoroacetyl esters. They did not, however, study the quantitative aspect of the derivative procedure. Analyses of amino acids as their N—trifluoroacetyl n—butyl esters by Lamkin and Gehrke (1965) and later by Gehrke and Stalling (1967) has led to a most important improvement and advancement in the analyses of amino acids by gas chromatography. They were able to quantatively analyze the 20 common amino acids with fair separation on 0.5% neopental glycol sebacate. However, Stafanovic and Walker (1967), using 0.35% ethylene glycol adipate were able to obtain better separation for 17 amino acids and by using an OV—17 column in conjunction, they were able to obtain good separation, as well as good resolution. Satterlee and Lillard (1967) reported on GLC analysis of free amino acids in meats using the method of Lamkin and Gehrke (1965). They found the procedure satisfactory for the analyses of amino acids after they had removed all the carbohydrates, proteins and 98 percent of the inorganic salts. Consistancy in their work was acceptable, however there was no comparison made to the accepted method of the amino acid analyzer. Another application of GLC analysis 27 y for protein was made (Ward gg gl., 1967) using the n—amyl N—acetyl derivatives for analysis of the amino acids in actinomycin hydrolysates. They reported that the minimal detectable limit was at least 10_9mole. Ikehawa g2 g1. (1966) reported on a method for the separation and estimation of DNP derivatives for 13 amino acids of serum by GLC. However, no information was given as to the accuracy of the method. EXPERIMENTAL PROCEDURE Experimental Animals Broad Breasted White turkeys, 20 weeks of age, and raised on the same ration at the Michigan State University Poultry Farm were used in this study. One group of eight birds received no treatment prior to slaughter and served as the controls. Two other groups of five birds each were treated with either adrenalin (as the hydrochloride; Parke, Davis and Company) or iodoacetate (as the sodium salt; Eastman Kodak) prior to bleeding to minimize post-mortem glycolysis (deFremery, 1963). Each bird in one group received a 1.5 mg subcutaneous injection of adrenalin per kg of body weight and was fasted for 20 hours prior to bleeding. Each bird in the other group received a 200 mg intravenous injection of iodoacetate per kg body weight, prior to bleeding. All turkeys were subjected to an electric shock prior to bleeding to prevent excessive activity, bled, scalded in a Rotomatic Scalder, and defeathered in an automatic rubber fingered picker. All the birds were then eviscerated, washed, and chilled in slush ice for 24 hours. After the chilling period the Pectoralis major and minor 28 29 muscles and the thigh muscles were carefully removed from the birds to insure a minimum of connective tissue (tendons) in the samples. After removal of the meat from each bird it was pooled into samples of all breast and all thigh meat and ground with an auger type meat grinder through a 64mm (1/4 inch) plate. The breast and thigh meat samples were formed into slabs approximately 15 x 20 x 4 cm (6 x 8 x 1 1/2 inch) and vacuum packed separately in Cryovac bags. The samples were frozen and stored at —20°C for further analyses. Samples were held in storage for periods of 0, 30 and 90 days to evaluate the effect of storage on protein change. General Methods Used Nitrogen Determinations A11 nitrogen values were determined using the micro-Kjeldahl method outlined by the American Instrument Company (1961). Nitrogen contents were reported as mg of nitrogen per m1 of solution or per g of tissue. All nitro- gen determinations were run in duplicate. PE All pH measurements were made with a Corning Model 10, Expanded Scale pH Meter and read to the nearest one-hundredth unit. A 5g sample of meat was blended with 30 40 ml of deionized water for 1 minute and the pH of the sample read. Collection of Drip The method of drip collection was similar to that reported by Pearson gg gi. (1959), Wladyka (1965) and Crigler (1968). The collection apparatus consisted of a V—shaped trough lined with aluminum foil, which served as a funnel for the collection of the drip in a beaker. A wire grid was placed over the funnel to hold the meat above the aluminum foil and allow the drip to be exuded. The entire device was then placed in a disconnected freezer chest which had a layer of water on the bottom to provide a saturated atmosphere. The previously frozen white and dark meat samples were placed on the grids for thawing after the packaging material had been removed and the samples were weighed. After the thawing period of 36 hours at 5°C, each meat sample was reweighed and the amount of drip measured to the nearest ml. The drip was then frozen and held for further analyses. Muscle Protein Fractionation After thawing, the meat samples were again throughly mixed. A modified extraction procedure of Fischer (1963) and Maier and Fischer (1966) was used to fractionate the 31 muscle proteins of turkey meat (Figure 1). Five 9 samples of meat were weighed and blended at high speed with a Model 1042, two speed Delux Waring-Blendor. Blending was conducted in a semi-micro waring blendor jar with approxi- mately 40 ml of deionized water. A 50 m1 volumetric flask was placed in the blendor jar to prevent the formation of a vortex, which leads to excessive protein denaturation. The blended samples were then transferred to a 250 ml cen- trifuge bottle and the blendor jar washed twice with approximately 30 ml of deionized water to make a final volume of approximately 100 ml. The centrifuge bottles were placed in a refrigerated Model RC2—B Sorvall centri- fuge and centrifuged in a Type GSA head for 10 minutes at 10,000 rpm. After centrifugation, the supernate was decanted into a 100 ml graduated cylinder and read to the nearest 1.0 ml. The residue from the water soluble frac- tion was placed in a semi-micro Waring Blendor jar and approximately 40 m1 of KCl—PO4 buffer (ionic strength of 1.00, pH 7.50, 0.01 Mole KF/liter) was added and the sample blended for 3 - 5 seconds at low speed. The blendor jar was washed several times with the buffer solution and the washings were added to the blended portion of the residue in a 250 ml centrifuge bottle to give a final volume of approximately 200 ml. After standing for 4 hours at 1°C the samples were centrifuged at 3,000 rpm for 10 minutes. The supernate was decanted off, measured to the nearest _59 TISSUE SAMPLE TISSUE WATER HOMOGENIZED CENTRIIFUGED j SUPEIRNATE PRECIPITATE | 1 50m. for PRECIPITATED ULTRAFILTRATE in 5% TCA CENTRIFUGED ph 7.5 KCI-PO4 BUFFER MIXED THROUGHLY CENTRIFUGED NPN PROTEIN N (TCA Soluble) (Sarcoplasmic Proteins) I PRECIPITATE SUPERNATE (Myosin,Actin, Actomyosm) O.IN NoOH MIXED THROUGHLY CENTRIFUGED PRECIPITATE SUPERNATE I (Stroma) (Denatured I Proteins) I Figure 1. Outline for the fractionation of turkey muscle I proteins. I 33 1.0 m1, and the residue suspended in approximately 100 ml of 0.1 N NaOH. The NaOH soluble fraction was separated from the NaOH insoluble fraction, after standing 4 hours, by centrifugation at 3,000 rpm for 10 min. The volume of supernate was read to the nearest 1.0 ml. As a means of quantitation of each fraction, and to determine the difference between treatments, total nitro- gen was determined on each fraction and each meat sample. In an attempt to follow the change in non—protein nitrogen (NPN) during storage, and to evaluate treatment differences, NPN was determined on the water soluble fraction. NPN was determined by pipetting out 10 ml of the water soluble fraction and adding 10 ml of 10 percent trichloracetic acid (TCA) to give a final concentration of 5 percent TCA. After mixing, the above mixture was centrifuged at 3,000 rpm in a Type SS-34 Sorvall centri— fuge head in 50 ml centrifuge tubes for 10 min. To determine how proteolytic activity (which occurres during storage) was affected by treatment, an ultrafiltrate of the water soluble fraction was obtained. The ultrafiltrate was obtained by pipetting 50 ml of the water soluble fraction into a 4.76 cm (1 7/8 inch) dialysis tube and the dialysis tube was placed in a 250 ml polyeth- ylene screw cap centrifuge bottle with the tapered part of the bottle removed. To increase the surface area and the speed of diffusion of the free amino acids and peptides 34 through the tube, approximately 4 cm of 6 mm glass beads were placed in the bottom of the centrifuge bottle. After placing the filled dialysis tube in the centrifuge bottle the sample was centrifuged at 3,000 rpm in a Model CS International Centrifuge for approximately 5 hr or until a volume of 10 — 15 ml was collected. The samples were centrifuged (in a walk-in cooler) at 1°C. After collection of the ultrafiltrate, it was placed in sample containers and stored at -20°C for later amino acid analysis by GLC. Amino Acid Analyses by Gas-Liquid Chromatography Preliminary Investigations An F & M, model 810, gas chromatograph (GLC) equipped with dual hydrogen flame detectors was used for the amino acid analysis by GLC in this study. Temperature data, program rate, and column packing material parameters were obtained from Gehrke and Stalling (1967). From the parameters obtained from their work the following optimum conditions for gas chromatography were determined and are reported below: Column: 3—mm I.D. x 6 ft coiled borosilicated glass Column packing: 0.5% neopentyglycol sebacate (Analabs) on 80/100 mesh Chromosorb G (Johns- Manville) 35 Conditions: Column temperature:' Initial 70°C, ' final 220°C Program rate: 2°C/min Injection port temperature: 230°C Detector temperature: 250°C Carrier gas and flow rate: Helium, 40 ml/min Hydrogen flow rate: 50 ml/min Air flow rate: 550 ml/min Sensitivity: 10/4 Chart speed: 0.63 cm/min Commercially prepared N-trifluoroacetyl n—butyl esters (Regis Chemical Company) of the 14 amino acids to be investigated in this study were used to determine the elution sequence. The elution sequence was determined by injecting one amino acid and then recording the position of elution. Amino acids were injected in the order of expected elution by the work of Gehrke and Stalling (1967). A second amino acid was then added to the first and mixed, after which the position of the second peak was recorded. This procedure was continued until the position of each amino acid in the sequence was determined. Calibration of the detector and recorder systems used for quantitative analysis was necessary before evalua— tion of the procedure could be conducted. This was done by calculating factors which related peak height per weight of internal standard to the peak height per weight of amino acid to be analyzed. The commercially prepared N—trifluoroacetyl n—butyl derivatives of the amino acids which contained a known concentration (0.05M) were mixed with the internal standard, butyl stearate (1 mg/ml 36 n—butanol), and analyzed by GLC. An appropriate factor for each amino acid was calculated from the resulting peak heights of the N—trifluoroacetyl n-butyl derivatives by the following equations: Factor _ wt. of amino acid x peak height of internal standard _ peak height of amino acid x wt. of internal standard To quantitate, the equation was rearranged as shown below: peak height wt. of factor x of amino acid x internal standard Wt' Of amino ac1d = peak height of internal standard Derivative Formation The N-trifluoroacetyl n-butyl derivatives of the amino acids were prepared by the procedures of Lamkin and Gehrke (1965) and Gehrke and Stalling (1967). The deriva- tization of amino acids involves the following steps: 1. Esterification of the amino acids to form methyl ester hydrochlorides. RCHCOOH+CH OH —E9l€> RCHCOOCH 3 25°C 0.5 hr + _ NH2 NH3 Cl 3 2. Interesterification of the methyl ester hydro— chlorides to form n—butyl ester hydrochlorides. RCHCOOCH +n-C 3 4 H +CH OH H90H -3§EL> RCHcooc4 9 3 100°C _ 2.5 hr NH +c1 + 3 NH3 Cl 37 3. Acylation of n-butyl ester hydrochlorides with trifluoroacetic anhydride to form N—trifluoro- acetyl n—butyl esters. (CF3CO)20 + RCHCOOC4H9 IEEZ?‘ RCHCOOC4H9 5 min + _ NH3 Cl NHCOCF3 Methyl ester formation was necessary to achieve solubility of lysine in n-butanol. The following procedure was used to form deriva— tives: l. Esterification of the amino acids with methanol. a. To a dried amino acid sample 10 m1 of 1.25N HCl in methanol (Regis Chemical Company) was added for each 60 mg of amino acids. b. A 24/40 standard tapered erlenmeyer flask was stoppered and stirred with a magnetic stirrer for 30 minutes at room temperature. c. The methanol HCl was removed with a rotary evaporator at 60°C. 2. Interesterification of methyl ester hydrochlorides. a. 10 m1 of 1.25N HCl in n—butanol (Regis Chemical Company). b. Add an exact amount of internal standard (butyl stearate). c. The flask was placed in 100°C oil bath with a cold water condensor positioned in the flask and a CaSO4 drying tube in the end exposed to the air. The temperature was maintained and the sample agitated for 2 1/2 hours by a Fisher Thermomix magnetic hot plate. d. The butanol HCl was removed with a rotary evaporator at 60°C. 3. Acylation of n-butyl ester hydrochlorides. a. 4 ml of 25% by volume of trifluoroacetic anhydride in methylene chloride (Regis Chemical Company) were added to the flask. b. Sample was stirred at room temperature for 15 minutes. 38 c. One ml of the solution was transferred to a high-temperature acylation tube and heated for 5 minutes at 150°C in an oil bath (immersed only to the sample liquid level). d. After cooling to room temperature the sample was ready for analysis by GLC. Recovery of Amino Acid Derivatives An amino acid standard solution and a protein hydrolysate of each bovine serum albumin and a water soluble protein extract of turkey meat were used to quan- titatively evaluate the derivative formation as follows: 1. Amino acid standard solution The accuracy of the method was evaluated by first preparing a standard solution of amino acids (Nutritional Biochemicals Corporation) with a known concentration of 0.5 uM per m1. Five ml of the standard solution were pipetted into a 125 ml standard tapered erlenmeyer flask and lyophilized. The accuracy of the method was determined for the amino acid standard solution using the factors obtained from the commercially prepared N—trifluoroacetyl n—butyl derivatives and the procedure outlined previously. In addition, the amino acid standard solution was analyzed by the amino acid analyzer and the percentages of amino acids recovered by each method were determined. A Beckman/Spinco, Model 120, Amino Acid Analyzer was used to obtain the amino acid ion—exchange values in this study. Samples were prepared according to the method 39 of Spackman (1960). Protein hydrolysates containing 3 to 4 mg protein were transferred quantitatively to a 25 ml pear—shaped flask fitted with a ground glass joint. Nor- leucine was added to the hydrolysate before transferring in order to check for transfer losses. The hydrolysate was evaporated to dryness on a rotary evaporator. The residue was taken up in a small amount of deionized water and redried. Five ml of citrate-HCl buffer (pH 2.2) was added to the dried hydrolysate. When all the soluble material had dissolved the sample was ready for analysis on the amino acid analyzer. Standard amino acid mixtures were analyzed with the same ninhydrin solution within four or five days. 2. Purified protein The amino acid content of the purified protein was determined by the GLC procedure, and values obtained were compared with ion—exchange data from similar samples. A 100 ml solution containing 4 mg of bovine serum albumin (Nutritional Biochemicals Corporation) per ml of water was prepared to evaluate the method. Two ml of the solution were pipetted into a 20 ml ampoule with enough concentrated HCl added to give a final concentration of 6N HCl. After making to 6N with HCl the ampoule was evacuated and sealed under vacuum. The sample was hydrolyzed in a forced air oven at 110°C for 20 hours. Before analysis by GLC, the sample was analytically transferred from the ampoule to a 40 125 ml standard tapered 24/40 erlenmeyer flask. The HCl was removed on a rotary evaporator using a 60°C water bath. Derivative formation of the amino acids was conducted on the entire 2 ml sample in the same manner as for the amino acid standard solution. A short Dowex 50 column was used to determine if the recovery of the amino acids from bovine serum albumin could be improved by removing interferring substances. The use of a Dowex 50 column for biological systems was recommended by Gehrke and Regis Chemical Company (1967). 3. Water soluble fraction of light turkey meat After evaluation of the GLC method for the purified protein, it was applied to the water extractible protein fraction from light turkey meat. A 125 g sample of breast meat from turkey was blended in a Waring Blendor with 1500 ml of water. After blending, the suspension was cen- trifuged in a Model RC2—B Sorvall Automatic Refrigerated Centrifuge using a Type GSA head at 10,000 rpm. The super— nate was decanted off, frozen and held at —20°C for further analysis. The water extracted protein was analyzed by the amino acid analyzer to determine the concentration of the various amino acids. Amino acid analysis by GLC was then conducted on 3 ml samples of the water soluble protein, which were hydrolyzed with 6N HCl in an evacuated ampoule for 20 hours at 110°C. HCl was removed from the sample by 41 a rotary evaporator at 60°C and analyzed by GLC after derivative formation. Since there were numerous inter— ferring substances which resulted in extraneous peaks and poor recovery of the amino acids, a purification procedure was required. In an attempt to clean up the samples prior to GLC analyses, and obtain better quantitative results, the following procedures were tried: 1. a. Ether extraction of protein hydrolysate b. Ether extracted protein hydrolysate placed on a short Dowex 50W-X12 (H+ form) column to remove acids, sugars and other neutral con- taminants by absorbing the amino acids on the column and eluting with an aqueous ammonium hydroxide. 2. The effluent from 1 was placed on a short Dowex 2-X8 (OH—form) column and eluted with HCl. 3. Steam was bubbled through the hydrolyzed samples to remove amines and other volatiles. 4. Samples were mixed with activated charcoal and filtered through Whatman No. 2 filter paper. The above purification procedures did not result in satisfactory recovery of all amino acids when compared to the results obtained from the amino acid analyzer. However, since the recovery data obtained by the use of activated charcoal was comparable to other purification procedures, and required less sample preparation time, it was used in this study. The entire hydrolyzed sample was filtered through the activated charcoal and dried with the aid of a rotary evaporator at 60°C to remove the water and HCl. Analysis 42 was then carried out on the protein'hydrolysate using the same procedure as for the amino acid standard solution. Amino Acid Analyses of Samples from Experimental Animals Water Soluble Fraction Ten ml of the ultrafiltrate were pipetted into a 20 ml ampoule and hydrolyzed in 6N HCl at 110°C for 20 hours. The hydrolysate was then mixed with activated charcoal and filtered into a 125 ml standard tapered erlenmeyer flask. To insure complete transfer of the hydrolysate to the flask, the ampoule and charcoal were washed with 5 separate 10 ml washings of deionized water which were collected in the flask. The sample was dried using a rotary evaporator and water bath at 60°C. After drying, the sample was analyzed by GLC to quantitate the amino acids as outlined in the derivative formation section. Five ml of the drip were pipetted into a 20 ml ampoule and hydrolized in 6N HCl at 110°C for 20 hours. The hydrolysate was then mixed with activated charcoal and filtered into a 100 ml volumetric flask and made up to volume after 5 separate 10 m1 washings with deionized water. A 3 ml aliquot from the 100 ml was dried and ana— lyzed by GLC using the procedure for derivative formation previously outlined. RESULTS AND DISCUSSION Evaluation of Amino Acid Analysis by Gas-Liquid Chromatography Preliminary Investigations The elution sequence of the amino acids and internal standard on a column packed with Chromosorb G and coated with 0.5 percent neopentylglycol sebacate is shown in Figure 2. Good separation and resolution were obtained for all the amino acids investigated, except for the tendency of glycine and isoleucine to overlap with increased use of the column. Good separation was obtained by Stafanovic and Walker (1967) using a dual type column system consisting of one column packed with 0.325 percent ethylene glycol adipate on Chromosorb G and a second column containing 2.5 percent OV-17 on Chromosorb G. Samples injected on the first column were programmed at 4°C per minute to 210°C and then the temperature reduced to 125°C and another portion of the sample injected on the second column and programmed to 215°C at 6°C per minute. This procedure, although it required a two column system, has been shown to have substantial advantages over the other systems investigated for complete amino acid analysis. 43 .Uumpsmum Hmsnmusfl can mw>flpw>fluwp pflom osfiEm amusnnc vamomonODHMHnulz ea How soapsam mo OHDDmuomEmp psm.oosw5wom map mo Eonmoumfiouso .N onsqflm N mnsmflm Avovmmnh._ t mz_o>40 w 33:55 _>.:mv omqozflrw 4._. m. mz_n_<> v o.o< 0.24.550 v. wz_z<..< m 0.04. O_hmIz< oFmozmrn_ N. sz>40m _ in 39N08838 83080038 46 Quantitation factors for the amino acids were obtained using the commercially prepared N-trifluoroacetyl n-butyl derivatives of the amino acids. These factors are reported in Table l. The use of these factors for quanti- tation should give repeatable results since an extremely small standard deviation was obtained among replicate samples for all amino acid derivatives except leucine, threonine and methionine. Relatively low standard devia- tion among replicate samples were obtained for these latter amino acids. Evaluation of Procedure The accuracy and precision of the method for quan- titating amino acids were evaluated using a standard amino acid solution containing 0.5 uM of each amino acid per m1 of solution. The amino acid analyzer was used to determine the actual concentration by an established and accepted procedure. Results of the amino acid concentrations by both GLC and the amino acid analyzer are shown in Table 2. Both procedures gave good recoveries of each amino acid when values were compared to the actual amount added. Variation among replicate samples was small when analyzed by GLC, as shown by the small standard deviation obtained for each amino acid. The least variation among replicates was found for glycine and largest variation for isoleucine. 47 Table l. Experimentally derived factors used for quantitation of specific amino acids by GLC 1/ Standard Amino acid Factor deviation Alanine 1.010 .010 Valine .934 .010 Isoleucine 2.059 .231 Glycine .505 .052 Leucine 1.034 .390 Threonine .921 .271 Proline 1.272 .064 Serine .920 .089 Methionine 1.587 .307 Phenylalanine 1.088 .104 Aspartic acid .713 .094 Glutamic acid 1.006 .058 Tyrosine 1.046 .034 Lysine 1.227 .106 1/ Average of eight independent determinations. 48 Table 2. Percentage recovery of specific amino acids from a standard solution by GLC and amino acid analyzer Amino acid Amino acid recovered recovered by 191 GLC ion exchange Amino mgi/ g/ é/ acid added Av. mg Percent Av. mg Percent Alanine 44.5 44.2 i .055 99.3 43.6 98.0 Valine 58.6 58.8 i .089 100.3 57.3 97.8 Isoleucine 65.6 67.1 i .780 102.3 65.3 99.5 Glycine 37.5 36.8 i .013 98.1 35.9 95.7 Leucine 65.6 64.3 1 .092 98.0 65.0 99.1 Threonine 59.6 56.0 i .074 94.0 54.8 91.9 Proline 57.6 52.8 i .195 91.7 54.5 94.6 Serine 52.5 54.0 i .113 102.9 54.1 103.1 Methionine 74.6 71.4 i .169 95.7 72.2 96.8 Phenylalanine 82.6 83.7 i .147 101.3 82.9 100.4 Aspartic acid 66.6 65.4 i .135 98.2 63.6 95.5 Glutamic acid 73.6 63.4 i .059 86.1 72.5 98.5 Tyrosine 90.6 90.8 i .065 100.2 87.6 96.7 Lysine 58.53/ 55.8 i .101 95.4 58.8 100.5 l/ 0.5 uM per ml solution of standard amino acids. 2/ Average of seven replicates. 3/ Average of two replicates. g/ Corrected value since Lysine was added as L—Lysine monohydrochloride. 49 Recovery and quantitation of the amino acids from bovine serum albumin were more variable between GLC and amino acid analyzer procedures (Table 3) than results obtained from the standard amino acid solution. The re— covery of threonine and phenylalanine (neutral and aromatic, respectively) were not accurate since values were 147.2 and 243.7 percent, respectively. Recovery of leucine (62.7 per- cent) and aspartic acid (50.0 percent); neutral and dicar— boxlyic, respectively, on the other hand, were incomplete with low values obtained. The reasons for the high re— coveries of threonine and phenylalanine, or the low values for leucine or aspartic acid are not known since the amino acids were from hydrolysates of a purified protein, hydro- lized under the same conditions for both methods of analysis. These results differ from results of Gehrke and Stalling (1967) based on comparisons between ion exchange and GLC values. Their GLC values were within 1 percent absolute of the analyzer results for bovine serum albumin, kappa casein and purified soy-protein. Amino acid values obtained from the water soluble fraction of light turkey meat by GLC and the amino acid analyzer (Table 4) varied considerably. Figure 3 shows a Chromatogram of the separation and resolution of the amino acids from the water soluble protein extract. Only 6 of the 14 amino acids were recovered with satisfactory results. These 6 amino acids were alanine, valine, proline, glutamic 50 Table 3. Percentage recovery of specific amino acids from a solution of bovine serum albumin by GLC and amino acid analyzer _1_/£/ Av. mg by 1/;/ y Amino amino acid Av. mg by Percent acid analyzer GLC recovery Alanine 26.45 21.11 .264 79.8 Valine 24.84 23.72 .137 95.5 Isoleucine 8.99 9.16 .280 101.9 Glycine 6.27 4.60 .151 73.4 Leucine 41.35 25.91 .192 62.7 Threonine 18.61 27.40 .377 147.2 Proline 26.05 21.32 .308 81.8 Serine 13.77 10.40 .257 75.5 Methionine 1.31 1.16 .05 88.5 Phenylalanine 23.54 57.37 .165 243.7 Aspartic acid 36.20 18.11 .186 50.0 Glutamic acid 54.55 51.93 .168 95.2 Tyrosine 19.03 19.44 .281 102.2 Lysine 44.70 46.45 .358 103.9 l/ 4mg/ml of bovine serum albumin. 2/ Average of two independent analyses. 3/ Average of five independent analyses. 4/ GLC values as a percentage of amino acid analyzer values. Table 4. Percentage recovery of specific amino acids from the water soluble fraction of light turkey meat by GLC and amino acid analyzer i/g/ - 5/ AV. mg by 3/4/ Percent Amino amino acid Av. mg recovery acids analyzer GLC by GLC Alanine 18.34 14.91 i .166 81.3 Valine 19.00 15.66 i .198 82.4 Isoleucine 15.26 33.75 i .505 222.6 Glycine 20.53 5.14 i .069 25.0 Leucine 24.60 12.67 i .188 51.5 Proline 12.67 11.58 i .108 91.4 Serine 13.17 6.09 i .115 46.2 Methionine 2.90 4.54 i .115 156.5 Phenylalanine 13.16 32.02 i .318 243.3 Aspartic acid 29.93 6.46 i .092 21.6 Glutamic acid 35.10 29.29 i .261 83.4 Tyrosine 7.76 6.53 i .077 84.1 Lysine 22.19 18.82 i .360 84.8 1/ Average of two samples. 2/ mg/100 ml of water soluble fraction. 3/ Average of 10 independent samples. 4/ mg/100 ml of water soluble fraction. 5/ GLC values as a percentage of amino acid analyzer value. .umoE hoxusu usufla Eoum GunmmHonphg samuoum mHQDHOm Hmnms m mo Emnmopmfionno .m musmflm 53 wow mm— 09 m musmfim Gov mmaquwdEMF mm m# Oh IJI 0. mm. v.2 mm. mm. Om. NE VB 0N. _ _ _ 1 _ _ _ 1 n. mz_m>3 »_ 181°.owzm .szsmv om._. 0.04 02240 m_Z_ODm._om_ m23<> mz_z<4< mo_mo>Iz< O_HmodomODIEEH kzm>n_om 9: —NK)VIDKDI\€D05 35‘N085‘38 83080338 54 acid, tyrosine and lysine with recoveries of 81.3, 82.4, 91.4, 83.5, 84.2 and 84.8 percent, respectively, when compared with values for the amino acid analyzer. To improve the percentage recovery by GLC in com- parison to the values obtained by the amino acid analyzer, several techniques were tried. Ether extraction, as sug— gested by Gehrke and Regis Chemical Company (1967) in conjunction with a short Dowex 50W—12X (H+ form) column to remove acids, sugars and other neutral contaminants, was not effective. This procedure did not reduce the high recovery values for isoleucine, methionine or phenylalanine. In addition, this procedure did not remove substances which may have ifiterferred with esterification or acylation, since the low percentage recoveries for these amino acids were not improved. When elution of tHe amino acids from a Dowex 2W—X8 (OH_ form) or bubbling live steam through the protein hydrolysates were used, no improvement in the per- centage recovery could be detected. This indicated that neither amines nor volatiles were causing a significant effect on the recoveries of the amino acids. Filtration through activated charcoal was used to clarify the samples to determine if amino acids from a protein hydrolysate could be more effectively recovered. This method proved to be as satisfactory as any of the methods tried and required less time, thus making it the most desirable. Since the percentage recovery for threonine 55 was approximately 500—600 percent, no further attempt was made to quantitate or collect data for this amino acid from biological systems. The chemical classification of the amino acids had little effect on the percentage recoveries. Recoveries of aliphatic, aromatic, sulfur—containing, secondary, dicar— boxylic and basic amino acids were similar. At least one amino acid from each classification (except sulfur- containing) gave satisfactory results. However, it should be noted that there was only a small amount of methionine present in the water soluble fraction and this could have affected actual quantitation and resulted in high values. These results have shown that amino acids in standard solutions and hydrolysates of purified protein extracts can be satisfactorily recovered and quantitated using the GLC procedure. However, when attempting to separate and recover these amino acids from a complex meat hydrolysate, the results were quite variable and in most cases, not closely related to the values obtained from the amino acid analyzer. Since the use of an amino acid ana— lyzer has been an accepted procedure, it is assumed to be more accurate than these procedures for separating and quantitating the amino acids in complex meat hydrolysates. However, bearing in mind the consistancy of the values obtained, the GLC procedure can still provide a valuable means in detecting differences or changes in samples. 56 Effects of Adrenalin and Iodoacetate on pH and Related Changes in Certain Muscle Fractions pH of Muscle Turkeys were given several different treatments before slaughter to alter the ultimate pH of the muscle after processing. The pH values for the tissue from the control turkeys and those treated with adrenalin and iodo- acetate are shown in Table 5. pH of the light and dark meat samples from control birds 24 hours after slaughter and after 0, 30, and 90 days storage are within the ex- pected range (Wladyka, 1965). The pH of the meat from adrenalin treated birds was also as expected since the pH values of the dark and light meat were 7.05 and 6.87, re— spectively, 24 hours after slaughter. These results from the light meat agree with those reported by deFremery (1963), who found that the pH of the light meat was 6.94, however, no comparable data were found for dark meat. pH values of 6.04 and 7.03 for light and dark meat, respec— tively, were obtained for meat from the iodoacetate treated turkeys. The pH values obtained for dark meat were as expected, however, the results for light meat differ from those reported by deFremery (1963), who found, using the same treatment, a pH of 6.76 for the light meat. A possible explanation for the low pH of the light meat from the birds treated with iodoacetate is the time required for complete mixing of the blood in the bird. 57 Table 5. pH values of muscle from adrenalin and iodoacetate treated turkeys 24 hours after slaughter and after frozen storage 24 hours after Days of Storage Treatment slaughter 30 90 pH values 1/ 2/ _ CL 5.95 i .10 5.96 i .09 5.95 i .07 5.90 i .12 CD 6.25 1 .13 6.16 i .08 6.12 i .12 6.14 i .09 4 / AL 6.87 i .16_ 6.78 i .12 6.80 i .15 6.81 i .11 AD 7.05 i .19 7.05 t .11 6.91 i .15 6.97 i .09 IL 6.04 1 .21 6.01 i .19 5.96 i .11 5.87 1 .15 ID 7.03 i .22 6.97 1 .11 6.90 1 .15 6.99 1 .24 1/ CL — Control—Light AD — Adrenalin-Dark CD — Control—Dark AL - Adrenalin-Light IL - Iodoacetate—Light ID — Iodoacetate—Dark CL and CD - Average of individual determinations on muscle samples from eight birds. Average of three independent analyses on each pooled sample. AL, AD, IL, and ID - Average of individual determina— tions on muscle samples from five birds. 58 Pino gg gi, (1951) reported that birds required from 2 to 3 minutes for complete mixing of the blood. Blood mixing time could have affected results since the birds were slaughtered 3 minutes after injection to prevent death. Further confirmation of insufficient mixing time was ob— tained from George and Berger (1966). They reported that each white fiber, of which light meat of the domestic fowl mainly consist, receives considerably less blood per unit area than red fibers which makes up the larger portion of the dark meat. Insufficient time for the iodoacetate to be distributed within the light tissue may have resulted. As a result sufficient iodoacetate may not have been pres— ent to inhibit the action of the phosphoglyceraldehyde dehydrogenase, which was reported by Padieu and Mammaerts (1960) to be the limiting enzyme in anaerobic glycolysis within the muscle complex. These factors could have a direct effect on the results, since birds in this study were slaughtered 3 minutes after injection of the iodo— acetate, whereas deFremery (1963) slaughtered 3 — 6 minutes after injection. In addition, there is a higher concentration of glycogen in white than red muscle fibers, and anaerobic glycolysis is predominant in the white meat (white muscle fibers) according to George and Berger (1966). Thus, it would seem that a higher concentration of 59 phosphoglyceraldehyde dehydrogenase would exist requiring more iodoacetate for inactivation. The pH values obtained showed that treatment had a marked effect on the ultimate pH values of the tissue. The light and dark meat from the adrenalin treated, and the dark meat from the iodoacetate treated birds showed marked changes in ultimate pH, and were sticky and gummy. BLiB The effects of the various treatments and storage times on the percentages of drip obtained are shown in Table 6. Percentages of drip from the control light meat samples at 0, 30 and 90 days of storage were 3.3, 5.8 and 9.1 percents, respectively. Control dark meat samples had 1.2, 7.9 and 9.4 percents of drip as storage time increased. The percentages of drip from the dark meat after 30 and 90 days of storage were higher than from light meat, which according to previous literature would not be expected. Kuprianoff (1952) reported that at the isoelectric point of meat (pH 5.0 to 5.5) maximum amount of fluid would be exuded. Since the pH of the dark meat was higher than that of the light meat, it would be expected that maximum drip would be obtained from the light meat. Percentage drip from the light and dark meat of the adrenalin treated turkeys and from control samples increased with storage time. Insufficient drip 60 Table 6. Percentage drip from muscles of adrenalin and iodoacetate treated turkeys after frozen storage Days of Storage Treatment 0 30 90 Percent _1_/ CL 3.3 5.8 9.1 CD 1.2 7.9 9.4 3/ AL 0.0 2.0 2.9 3/ AD 0.0 0.5 1.1 IL 10.3 8.4 8.4 ID 1.1 1.3 2.0 1/ CL — Control—Light AD — Adrenalin-Dark CD — Control-Dark IL — Iodoacetate—Light AL - Adrenalin — Light ID - Iodoacetate—Dark 2/ Insufficient drip for measurement. 61 (less than 1 ml) was obtained at 0 time to determine percent loss of drip. Percentages of drip obtained from the dark meat were 0.0, 0.5 and 1.1 percent and from the light meat 0.0, 2.0 and 2.9 percent for samples evaluated after 0, 30 and 90 days storage, respectively. Dark meat samples from iodoacetate treated turkeys had an increase in the percentage drip released with in— creased storage. Percentage drip from dark meat after 0, 30 and 90 days of storage was 1.1, 1.3 and 2.0 percent, respectively. The percentage drip from light meat did not follow the same pattern with the highest percent obtained before storage (10.3 percent). No effect of storage could be determined since 8.4 percent drip was obtained from samples stored for 30 and 90 days. The large amount of initial drip from the light meat can be explained as follows: The iodoacetate treat— ment caused the birds to go into rigor immediately (within 2 minutes after bleeding), as determined by the stiffness of the carcass, and poor bleeding occurred. This resulted in high fluid retention by the tissue. Also, since the iodoacetate treatment did not affect the pH of the light meat, optimum pH conditions for maximum fluid release or minimum water—holding capacity were approached. No evidence is available to explain why percentage drip released did not increase with longer storage periods, as it did from the other samples. One possibility is that a physical and/or 62 biochemical phenomenon occurred in the frozen samples during storage due to the presence of iodoacetate in the white muscle fibers. A direct relationship between pH values and per- centage drip was found in this study and has been confirmed by a number of workers. Empey (1933) demonstrated that a high ultimate pH was associated with reduced drip loss from beef muscles. Maximum amount of drip was obtained at about pH 5.2 and as the pH increased the amount of drip decreased to zero at about pH 6.4 (Sair and Cook, 1938b). Meat with a pH near its isoelectric point (5.0 to 5.5) has a larger drip loss after freezing and thawing than meat at a higher pH (6.3 to 6.4) according to Kuprianoff (1952). Nitrogen Released in Drip The mg of nitrogen released per 100 g of tissue was greater from light meat than dark meat control samples and increased with storage for both light and dark meat (Table 7). Mg of nitrogen released per 100 g of light and dark meat were 16.7 and 4.2, respectively, before storage. Effects of storage times of 30 and 90 days on percentages of drip from meat samples of the adrenalin treated birds are shown in Table 6. Insufficient drip was obtained for analysis from both light and dark meat before storage. As with the control samples, the mg 63 Table 7. Nitrogen content in drip from muscles of adrenalin and iodoacetate treated turkeys after frozen storage Days of Storage Treatment 0 30 90 Grams l/ 3/ CL 16.74 27.98 43.58 CD 4.19 25.25 40.81 3/ AL 0.00 10.89 14.58 3/ AD 0.00 1.94 4.20 IL 56.90 45.24 38.42 ID 4.05 4.88 6.66 1/ CL — Control—Light AD - Adrenalin-Dark CD - Control—Dark AL — Adrenalin—Light IL - Iodoacetate-Light ID — Iodoacetate—Dark Mg of N released in drip per 100 grams of tissue. Insufficient drip for determination obtained). (less than 1 m1 64 nitrogen released per 100 9 tissue was greater from the light meat than dark meat and both showed an increase with storage. The amount of nitrogen released per 100 g of tissue for light meat was 10.9 and 14.6 mg for 30 and 90 days storage, respectively, and from the dark meat 1.9 and 4.2 mg, respectively. Results of 0, 30 and 90 days storage on the mg nitrogen released per 100 g of tissue from the iodoacetate treated turkeys are shown in Table 7. Mg nitrogen released per 100 g of tissue for the light meat is considerably higher than from the dark meat. Values of 56.9, 45.2 and 38.4 mg nitrogen per 100 g light meat and 4.1, 4.9 and 6.7 mg nitrogen per 100 g dark meat, were obtained for 0, 30 and 90 days storage, respectively. The higher nitrogen values obtained for light meat at 0 and 30 days storage are the direct result of a higher percentage drip obtained from these samples. The amount of nitrogen released after 30 and 90 days of storage is of interest. Percentage drip released after 30 and 90 days of storage was approximately the same (Table 6), however, the amount of nitrogen de— creased with storage. This could indicate that the protein in solution (drip) for this treatment (iodoacetate) was being retained by the meat, since dilution was not account— ing for the decreased nitrogen release. Based on these observations some unknown relationship may be responsible 65 for the greater amount of drip and nitrogen released at 0 time. Wladyka (1965) reported that the protein nitrogen increase, which occurred in drip from dark and light meat, although of limited proportion, does indicate the possibil- ity of denaturation and proteolysis in the chicken meat during frozen storage. According to Khan g2 g1. (1963) denaturation and proteolysis occurred in chicken muscle proteins during frozen storage. Swanson and Sloan (1953) concluded that proteolysis occurred in poultry meat during frozen storage as evidenced by an increase in soluble nitrogen and non—protein nitrogen of both dark and light meat. Muscle Protein Fractionation Muscle protein fractionation data of dark and light meat proteins from the control, adrenalin and iodo- acetate treated birds are shown in Tables 8 — 10. The water soluble protein extract (sarcoplasmic proteins) of light and dark meat from the control birds remained rela— tively constant during storage, however, more sarcoplasmic protein was extracted from light than dark meat. The amount of sarcoplasmic protein of tissue from adrenalin and iodoacetate treated birds was also constant during storage. These results are in agreement with those re— ported by Sawant and Magar (1961) and Khan g2 g1. 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