D EMULS‘RFYKNG CHARAC‘EER‘STICS SOLUBIUTY AN BEEF MUSCLE PROTHNS OF INTRACELLU LAP. Them {at m 00g!» of Ph. D. MGMGAN STAY! URNERSITY Gera‘d Ray Hegafly i963 'IHESXS This is to certify that the thesis entitled SOLUBILITY AND EMULSIFYING CHARACTERISTICS OF INTRACELLULAR BEEF MUSCLE PROTEINS presented by Gerald Ray Hegarty has been accepted towards fulfillment of the requirements for Ph.D. degree in Food Science ' 0 m f< ~ . 3.. Mi/ g/ i Major professg/u/ Date W— 0-169 LIBRARY Michigan State University F ABSTRACT SOLUBILITY AND EMULSIFYING CHARACTERISTICS OF INTRACELLULAR BEEF MUSCLE PROTEINS By Gerald Ray Hegarty It has been thought for many years that the salt soluble proteins of muscle have been primarily reSponsible for the characteristics of meat re- quired for the manufacture of acceptable sausage products. However, the role of the remaining intracellular proteins and the fundamental reasons why certain protein fractions have superior water-holding and emulsifying properties have not been clarified. Certain age and sex groups of beef animals have been assumed to yield meat which is more acceptable for the production of sausage products, however, there is little scientific basis for these beliefs. It was the object of this research to study the solubility behavior of the intracellular proteins during a seven day period post mortem, and compare the protein alterations occurring during this period between sever- al age and sex classes. In order to do this, a relatively rapid and accur- ate procedure was developed to chemically partition the intracellular proteins of beef muscle. This protein fractionation procedure was also utilized in an attempt to study the relationship of muscle protein solu- bility to tenderness. The emulsifying prOperties of the major intracellu- 1ar proteins were studied in model systems using purified muscle protein preparations. Results indicated that considerable variation in protein composition existed between muscles of the same animal. Muscles considered in this Gerald Ray Hegarty research were the infraspinatus and the longissimus dorsi. The solubility of the sarcoplasmic protein fraction exhibited little variation during the first seven days post mortem. Fibrillar protein solubility varied consider- ably in the same period for the three classes of animals studied. The general trend was a high degree of solubility at 0 time, followed by a sharp decrease to 24 hours and a gradual increase to seven days. The be- havior of bulls in this respect deviated somewhat from heifers and cows. Fibrillar protein solubility, in the case of longissimus dorsi muscles of yearling bulls was found to be highly correlated with tenderness measured by two methods (r - -0.69 for shear and r = 0.59 for panel). water-holding capacity was significantly correlated with tenderness as measured by the shear (r - 0.49). Emulsifying capacity and emulsion stabilizing characteristics of muscle proteins were studied under varying conditions of pH and ionic strength. Emulsifying capacity of all the major intracellular proteins was found to increase as protein concentration decreased. Emulsifying capacity of the proteins studied were ranked from greatest to least as follows: actin (u - 0), myosin, actomyosin, sarcoplasmic, and actin (u I 0.3). In general, myosin and actomyosin produced emulsions with the most desirable stability characteristics, however, at pH 5.5 the sarcoplas- mic fraction produced the most acceptable emulsion from a stability stand- point. Actin produced very undesirable emulsions under all conditions. SOLUBILITY AND EMULSIFYING CHARACTERISTICS OF INTRACELLULAR BEEF MUSCLE PROTEINS By Gerald Ray Hegarty A THESIS Submitted to Rflchigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1963 r! a -‘\) 'Q \\ ('y 5 (In K ACKNOWLEDGMENT The author wishes to eXpress his appreciation to Professor L. J. Bratzler for his guidance throughout this research and for his assistance in the preparation of the manuscript. He also wishes to thank Dr. A. M. Pearson for his advice and en- couragement. The author is grateful to Mrs. Dora Spooner for conducting the taste panels, to Mr. Harold Swaisgood for assistance in the ultracentri- fugal analysis, and to Mrs. Beatrice Eichelberger for typing the manu- script. The author is indebted to the Griffith Laboratories, Chicago, Illinois, for their support furnished him by a fellowship. ii TABLE OF CONTENTS Page INTROD UCT ION O O O O O O O O O O O O O O O O O O O C O O O O O O O 1 REVIEW OF LITERATURE O O O O O O I O O O O O I O O 0 O O O O O O O 3 Structure of Muscle . . . . . . . . . . . . . . . . . . . . . 3 Muscle proteins . . . . . . . . . . . . . . . . . . . . . . . 5 a. Myogen . . . . . . . . . . . . . . . . . . . . . . . 5 b. Actomyosin . . . . . . . . . . . . . . . . . . . . . 6 c. Myosin . . . . . . . . . . . . . . . . . . . . . . . 8 d. Actin . . . . . . . . . . . . . . . . . . . . . . . 11 e. Trapomyosin . . . . . . . . . . . . . . . . . . . . 12 Muscle Contraction and Rigor Mortis . . . . . . . . . . . . . 13 Extractability and Fractionation of Muscle Protein . . . . . 14 Protein Studies Applied to Meat Research . . . . . . . . . . 19 Theory of Emulsions . . . . . . . . . . . . . . . . . . . . . 33 Emulsion Technology Applied to Meat Research . . . . . . . . 38 EXPERIMENTAL METHODS . . . . . . . . . . . . . . . . . . . . . . . 40 General Methods . . . . . . . . . . . . . . . . . . . . . . . 40 Nitrogen analysis . . . . . . . . . . . . . . . . . . . 40 pH measurement . . . . . . . . . . . . . . . . . . . . . 40 Non-protein nitrogen determination . . . . . . . . . . . 40 Reagents . . . . . . . . . . . . . . . . . . . . . . . . 41 Centrifugation . . . . . . . . . . . . . . . . . . . . . 41 Statistical analysis . . . . . . . . . . . . . . . . . . 41 Part I O O O O O O O O O O O O O O O O O O O O O O O O O 0 Experimental animals . . . . . . . . . . . . . . . . . Sampling procedures . . . . . . . . . . . . . . . . . Sample preparation for protein fractionation studies . Fractionation procedure . . . . . . . . . . . . . . . Tenderness measurements . . . . . . . . . . . . . . . Water-holding capacity determinations . . . . . . . . Part II . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation and purification of sarcOplasmic proteins . Isolation and purification of actomyosin . . . . . . . Isolation and purification of actin and myosin . . . . Tests for confirmation and purity of myosin and actin Interfacial tension measurements . . . . . . . . . . . Emulsion preparation and evaluation . . . . . . . . . Depolymerization of proteins by urea . . . . . . . . . pH adjustment of protein solutions . . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . Part I . . . . . . . . . . . . . . . . . . . . . . . . . . Post mortem variation in protein composition . . . . . Tenderness study . . . . . . . . . . . . . . . . . . . Part II . O O O O O O 0 O O O O O O O O O O O O O O O O O O S M AND CONCL US IONS O O C O O O O O O O O O O O O O 0 O O 0 Part I O C O O O C O O O O O C O O O O O O O O O O O O O 0 Part II 0 O O O O O O O O O O O O O O O O O O O O O 0 O O 0 iv Page 41 41 42 42 43 48 48 48 48 49 51 56 59 60 62 62 64 64 64 76 79 95 95 96 Page BIBI‘IOGMHiY O O O O O O O C O O O O O O O O O O O O O O I O O O O 9 9 APPENDIX . O O O O O O O O O I O O O I O I O O O O I O O O O O O O 107 6. 10. 11. 12. 13. LIST OF TABLES Page Protein composition of muscle as reported by several authors 0 O O O O I O O O O O O O I O I O O O O O O I O O O 17 Average nitrogen composition of infrasginatus muscle of three classes of beef animals expressed as percent of total nitrogen and as mg. nitrogen/g. of tissue . . . . . . . . . 66 Average nitrogen composition and percent total nitrogen of longissimus dorsi muscle of 20 yearling bulls . . . . . . . 67 Means and standard errors for amount of sarc0p1asmic protein extracted at three periods post mortem and for three classes of animals (mg. N/g. tissue) . . . . . . . . . . . . . . . 69 Means and standard errors for percent of total fibrillar protein which was soluble at three periods post mortem among three classes of animals . . . . . . . . . . . . . . . . . 70 Means and standard errors for percent of total water released at three periods post mortem and for three classes of animals 0 O I O O O O O O O O O O O O O O O O O C O O O O O 71 Analysis of variance (approximate) of sarcoplasmic protein among three classes of animals and three periods post mortem O O 0 O O O O I O O O O O I O O O O O O O O O I O O 72 Analysis of variance (approximate) of percent of total fibri- llar protein which is soluble at three periods post mortem among three classes of animals . . . . . . . . . . . . . . 72 Analysis of variance (approximate) of percent of total water released among three classes and three periods post mortm O I O O O O O O I O O O l O O O O I O I C O O O O O 73 Means and standard errors of three factors, over three periods post mortem, for three classes of animals . . . . . 74 Means and standard errors of three factors, including 11 animalg for three periods post mortem and showing signi- ficant differences . . . . . . . . . . . . . . . . . . . . 74 Means and standard errors for pH of three classes of beef animals at three different periods post mortem . . . . . . 75 Tenderness, water-holding capacity and nitrogen composition of the longissimus dorsi of 20 bulls . . . . . . . . . . . 78 vi Table Page 14. Correlation coefficients for various factors related to tenderness (tenderness measured by shear and panel) . . . . 79 15. Characteristics of emulsions prepared with actomyosin and sarc0p1asmic protein fractions . . . . . . . . . . . . . . 90 16. Characteristics of emulsions prepared with actin, myosin and crude protein fractions . . . . . . . . . . . . . . . . 91 17. Percent protein nitrogen remaining soluble in the aqueous phase after maximum amount of oil was emulsified . . . . . 93 18. Relative interfacial tension of some protein solution-oil systems determined by the drop volume method . . . . . . . 94 vii LIST OF FIGURES Figure Page 1. Scheme for the quantitative determination of sarcoplasmic protein nitrogen, non-protein nitrogen and total fibrillar protein nitrogen O O O O O O O O I O O O O O I I O O O O O 44 2. Scheme for the quantitative determination of fibrillar protein nitrogen (complement to figure 1) . . . . . . . . 45 3. Scheme for the isolation of actomyosin . . . . . . . . . . 50 4. Scheme for the isolation of myosin . . . . . . . . . . . . 53 5. Scheme for the isolation of actin . . . . . . . . . . . . 55 6. Ultracentrifugal sedimentation diagrams of myosin at 32 ‘minutes and at 64 minutes . . . . . . . . . . . . . . . . 58 7. Emulsifying capacity of purified actomyosin vs. concentration of protein N in aqueous phase (protein in 0.6 M and 0.3 M KCl) 0 O I O O O O O O O O O O O O O O O O O O O O O O O O 82 8. Emulsifying capacity of water soluble sarc0p1asmic protein and g. of oil emulsified vs. concentration of protein in aqueous phase (u u 0) O O O O O O O O I O O O C O O O O O 83 9. Emulsifying capacity of water soluble sarcoplasmic protein and g. of oil emulsified vs. concentration of protein N in aqueous phase (protein in 0.6 M KCl) . . . . . . . . . . 84 10. Emulsifying capacity of actin and myosin vs. concentration of protein N in aqueous phase . . . . . . . . . . . . . . 85 ll. Emulsifying capacity of sarcoplasmic proteins (buffer extracted, u I 0.05, pH - 7.6) before and after dialysis vs. concentration of protein N in aqueous phase . . . . . 86 12. Emulsifying capacity of water soluble sarcoplasmic proteins vs. protein N concentration of aqueous phase as affected by pH and kind Of Oil 0 O O O I O O O O O O O I O I O O O O O 8 7 l3. Emulsifying capacity of purified actomyosin vs. concentra- tion of protein N in aqueous phase as affected by pH (u - 0.65) O C I O O O O I O O O O O O O O O O O I O O O O 88 viii Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix LIST OF APPENDIX TABLES Page Composition of solutions used for the fractionation and isolation of proteins . . . . . . . . . . . . . . . 107 Complete calculated data from infraspinatus muscle of 12 beef animals 0 I O I O I O I O O O O O I O O O O O O 108 Complete calculated data from infraspinatus muscle of 12 beef animals (continued) . . . . . . . . . . . . . . 109 Complete calculated data from infraspinatus muscle of 12 beef animals (continued) . . . . . . . . . . . . . . 110 Complete calculated data from longissimus dorsi muscle Of 20 bu118 O O C O O O O I O I O O O O O C O O O O O I 111 Complete calculated data from longissimus dorsi muscle of 20 bulls (continued) . . . . . . . . . . . . . . . . 112 Complete calculated data from longissimus dorsi muscle of 20 bulls (continued) . . . . . . . . . . . . . . . . 113 Protein concentration of aqueous phase and average amount (g.) of oil emulsified for various trials involving determination of emulsifying capacity . . . . 114 Protein concentration of aqueous phase and average amount (3.) of oil emulsified for various trials involving determination of emulsifying capacity (continued) . . . 115 Protein concentration of aqueous phase and average amount (3.) of oil emulsified for various trials involving determination of emulsifying capacity (continued) . . . 116 ix INTRODUCTION Differences in physical characteristics of meat are due primarily to the variation in amount and character of protein constituents, which in turn vary with Species, age, sex, and muscle. This variation in pro- tein makeup is reflected by differences in tenderness, juiciness, color, freezer drip, storage shrinkage, cooking losses, emulsion forming ability, and water-holding capacity of meat. The proteinsof muscle are classed into three major types: sarco- plasmic proteins which are soluble in water or weak salt solutions, fibrillar proteins which are soluble to various degrees in salt solutions, and the stroma proteins which are insoluble in either salt solutions or water. The literature abounds with information concerning the partition of muscle proteins of guinea pigs, rabbits, rats, embryonic chickens and fish, but such information concerning meat animals is rather scarce and not in complete agreement. It has been thought for many years that the salt soluble portion of muscle proteins is primarily responsible for desirable sausage processing characteristics. However, fundamental rea- sons underlying this belief have not been established. Hansen (1960), Swift st 21. (1961), and Sherman (1961b) have estab- lished that fat incorporated into a sausage product is diSpersed in small droplets and envelOped with a layer of protein material, producing, essentially, an oil in water emulsion, stabilized by muscle proteins. Swift et_§1, (1961) utilized crude muscle protein fractions to study fat emulsifying characteristics in model systems. However, much work remains to be done in this area. -2- For many years the amount of stroma or connective tissue of meat was believed to be the exclusive source of variation in the tenderness of meat. Wierbicki 35 a1. (1954), Kamstra and Saffle (1959), Carpenter 25.3l, (1961) and many others, have shown that the remaining protein con- stituents may be in some cases closely related to tenderness of meat. Research in this area could provide some further insight into the con- troversial subject of meat tenderness. It was the object of this study to: (1) Develop a simple and relatively rapid method for the routine partition of muscle proteins and apply this to a study of post mortem changes in protein solubility, pH, and water-holding capacity of beef from different age groups and sexes. (2) Apply the partition techniques and water holding-capacity data to a tenderness study of a group of beef animals closely controlled in regard to age, grade, sex, and feeding, and which exhibited fairly large variations in tenderness. (3) Study fat emulsifying capacity and relative emulsion stabilizing prOperties of various plflifiEd intracellular muscle proteins in model systems. REVIEW OF LITERATURE Structure of Muscle In order to provide for a full appreciation of the functional and morphological relationships of muscle proteins, it is necessary to pre- sent a brief gross and microscopic picture of muscle structure. Maximow and Bloom (1954) presented a comprehensive outline of muscle structure. They recognized two distinct kinds of muscle in vertebrates: smooth muscle and striated muscle. Generally, smooth muscles contract indepen- dently of voluntary control, while the striated muscles are subject to voluntary control. Cardiac muscle, though striated, contracts indepen- dently. They stated that a muscle fiber is generally considered the functional unit of a muscle. In striated muscle these are large multi- nucleated cells. The thickness of the fiber varies from 10 to 100 microns and depends on the type and age of the animal and the particular muscle. Fibers are relatively long, some of which extend the full length of a muscle. The striated fibers are covered with the sarcolemma, a thin structureless membrane which completely invests the fiber. Muscles are formed of parallel muscle fibers held together by connective tissue. The muscle fibers combine to form the primary bundles, and several primary bundles combine to form secondary bundles. Bailey (1944) provided a more detailed explanation of some important structural characteristics of muscle. He stated that fibers of striated ‘muscles are composed of numerous fibrils one micron in diameter, arranged parallel to each other and to the fiber axis. The fibrils are the ulti- mate morphological units of muscle. In the electron microscOpe the -3- -4- fibril appears to be composed of thinner threads called filaments. The fibril is composed, primarily, of actin, myosin and actomyosin which are the proteins responsible for muscle contraction. The fibrils also con- tain some minor protein constituents, the function of which is not clear. The fibrils are imbedded in the sarc0plasm. The sarc0plasm is made up of a complex mixture of proteins which correspond to myogen, so named by Von Furth in 1895. Contained in the sarc0plasm is also particulate material, namely the mitochrondia and microsomes common to practically all cells of animal tissue. Bailey (1944) also described the fibers optically, as characterized by striations running throughout their whole width, the isotr0pic I bands alternating with the anisotrOpic A bands. The difference of re- fractive index in the two bands makes the A layer appear dark in ordin- ary light and the I layer bright, while in polarized light the effect is reversed. Ramifying throughout and between the cells (fibers) is a framework of connective tissue fibers which are attached ultimately to the tendon. According to Maximow and Bloom (1954), connective tissue at the periphery of the muscle, called epimysiun, projects into the spaces between the bundles of fibers as perimysium. Thin fibrous networks which enclose the fibers within the primary muscle bundles are called endomysium. Bailey (1944) indicated that this tissue consists primarily of the pro- teins, collagen and elastin, and is termed the extracellular protein. The protein components of the sarc0plasm and the fibril are intracellu- lar. Maximow and Bloom (1954) described smooth muscle cells as short (15 to 500 microns), spindle shaped, and containing a single centrally located mucleus. Smooth muscle cells do not have a distinct membrane corresponding to the sarcolemma of striated muscle. The arrangement of smooth muscle cells in respect to one another varies with their function. Muscle Proteins a. Myogen The sarcoplasmic protein fraction (myogen), which can be extracted with very dilute salt solutions, was divided by weber and Meyer (1933) into an albumin portion and a globulin portion. The globulin portion which precipitated upon dialysis or standing in solution was denoted as globulin X. Bate-Smith (1937) obtained a fraction he called myoalbumin by a technique which he called analytical denaturation. According to Bailey (1944), neither the uniformity nor the native state of these two protein fractions has been proven, nor have biochemical functions been attributed to them. He stated that most probably several native and denatured protein constituents of the myogen group contribute to globu- lin X and myoalbumin. The whole myogen complex is relatively unstable, and on standing becomes turbid and slowly precipitates. According to Mommaerts (1950), myogen, although a relatively con- stant unit in preparative work, is far from homogenous. Myogen consists of an extremely complicated mixture of biologically active proteins among which are found most of the enzymes of the glycolytic cycle along with such proteins as myoglobin. He stated that since none of these, accord- ing to present knowledge, is directly involved in the structural and mechanical changes of muscle tissue, the attention of most muscle re- searchers has been focused on the proteins that form the contractile structure. b. Actomyosin In 1930 Edsall extracted minced muscle with salt solutions of high ionic strength. These extracts had preperties ascribable to solutions of fibrous molecules. Edsall termed this protein extract muscle globu- lin. Muscle globulin was further studied by v. Muralt and Edsall in 1930 and found to produce double refraction of flow (flow birefringence). Considering this pr0perty, they suggested that muscle globulin was com- posed of rod shaped particles. Edsall's protein generally became known as myosin. After several years of research in this area involving studies of the swelling, X ray diffraction, and elastic properties of myosin threads, Weber (1934), Asbury and Dickinson (1935), and various other workers concluded that the A.band of muscle is composed of myosin. In 1941 Needham gt 31. found that ATP diminished both the viscosity and flow birefringence of myosin solutions. Shortly after this, Schramm and Weber (1942) showed for the first time that myosin solutions are polydisPerse, containing a slowly sedimenting component with a low bire- fringence of flow (L-myosin) and several rapidly sedimenting components with high flow birefringence (S-myosins). Straub (1942), considering the findings of previous workers, an- nounced that the myosin of earlier workers was in reality a complex of two fibrous proteins, actin and L-myosin. Bailey (1944) stated that after this discovery, the complex of actin and Ldmyosin was termed acto- myosin which corresponded to the S-myosin of Schramm and Weber. Accord- ing to Bailey (1944), it is now generally accepted that ATP dissociates actomyosin into its two components, resulting in the prOperties observed by Needham 25 El. (1941). In connection with this ATP dissociation, Engelhardt and Ljubimova (1939) discovered that actomyosin preparations possess ATPase activity. Banga (1941) found this pr0perty to belong solely to the myosin part of the molecule. Considering their observation in the ultracentrifuge of several S-myosins (actomyosins), Schramm and weber (1942) indicated that actin and myosin combine step by step to form actomyosins of quite different sedimentation constants and hence, various molecular weights. Bailey (1944) stated that artifidal actomyosin can be prepared by mixing solu- tions of actin and myosin. He also pointed out that artificial actomyo- sins appear to sediment faster in the ultracentrifuge than natural actomyosins, and in the electron microsc0pe the fibers of artificial actomyosin dc not appear as fine as the natural fibers. From electron microscope studies conducted by Ardenne and Weber (1941), the length of natural actomyosin particles was found to vary from a few thousand A. to several microns and in thickness from 50 to 250 A. Bailey (1944) summarized the solubility prOperties of actomyosin. He stated that actomyosin is the least soluble of the muscle proteins; the threshold for salting-in is by far the highest, and for salting-out the lowest. Actomyosin gel is insoluble in water and begins to dissolve at an ionic strength of about 0.3. c. Myosin Myosin is a remarkable protein that possesses several properties that are unique for a protein. Bailey (1954) indicated that myosin possesses the solubility pr0perties of globulins, although the salting in threshold of globulins in general at pH 7 is lower (ionic strength = 0.04 vs. 0.3). Myosin can be prepared in a purified state and appears electrOphoretically homogenous in the pure state. In 0.5 M KCl it gives a water clear solution that shows no birefringence. Bailey (1954) also pointed out that myosin is relatively easily denatured. Freeze drying, dehydration with organic solvents, and mild heat all cause its denaturation. Depolymerizing solvents, such as urea or guanidine-HCl induce a change to a salt insoluble form. Mblecular weight determinations by Portzehl (1950), using both the ultracentrifuge and the osmotic method, showed the value for myosin to be approximately 850,000. Work by Tsao (1953b) indicated that the myo- sin framework is built up of five units of particle weight 165,000. Tsao depolymerized myosin with 6.7 M urea and studied molecular weight of the fragments by osmotic pressure and fluorescence polarization measurements. Bailey (1951) found that the molecule does not possess an identifiable N-terminal residue and thus may be cyclic. According to Weber and Portzehl (1951), all molecular measurements published so far indicate a very long, thin particle with a high axial symmetry. According to ultracentrifuge suidies of Weber (1950), the particles are 22-23 A. wide and 100 times as long. IMommaerts (1951), by measuring the angular dissymmetry of light scattering at two wave lengths, found the myosin molecule to be rod shaped and possessing a molecular length of 1500 A. Recent work by Kielley and Harrington (1960) using equili- brium ultracentrifugation techniques indicated that the molecular weight of the myosin molecule is 619,000. Depolymerization of myosin with guanidine-HCl resulted in the formation of molecules with an average weight of 206,000. These workers proposed a model for the myosin molecule made up of two light components and one heavy component, with the heavy component folded back upon itself. Kielley and Harrington's model has a length of 1650 A and a diameter of 22 A. Engelhardt and Ljubimova in 1939 announced that ATPase, the enzyme that liberates inorganic phOSphate from ATP, could not be separated from myosin and seemed to be identical with it. Bailey (1954) believed there was no doubt that ATPase was either closely associated with myosin, or was part of the molecule itself. Perry (1951) found that the activity is 20-2000 times less than that of other pure enzymes. Bailey (1954) pointed out that the Qp value of an average preparation is only 3000- 6000 (Perry, 1951), compared to a Qp of 100,000 for a purified inorganic perphOSphataSe. He also stated that acidification inactivates the enzyme, as also does any treatment which blocks SH groups. However, loss of solubility does not always indicate the loss of enzymatic acti- vity in the case of myosin ATPase. The effect of ions on ATPase activity is extremely important and at the present time no less confusing. Bailey (1954) stated that there are essentially only two points of agreement, that certain bivalent metals are necessary for the action of ATPase, and that calcium is the most powerful activator at alkaline pH. -10- MOmmaerts and Seraidarian (1947) made a thorough study Of the in-' fluence of ions on ATPase activity. .At pH 7 both myosin and actomyosin split ATP optimally in 0.3 M KCl; in the presence Of 0.001 M CaClz the potassium Optimum is shifted to lower concentration, and the activity is three times as great. In the presence of 0.1 M.KC1 the Optimum is at pH 9 when calcium is the activating ion. The effect Of magnesium is always to suppress the activation by other ions. There is evidence that actin rich myosins are activated by magnesium ions at neutral pH pro- vided potassium ions are absent. Perry (1951), utilizing the intact myofibril, made the following observations Of ion effect on ATPase activity: as noted with myosin and actomyosin, there is an Optimal potassium concentration for activation by calcium ions. The activation by magnesium can be just as great as that by calcium, but it occurs at a much lower concentration. Magnesium is strongly inhibitory above the Optimum concentration. Kielley and Meyerhof (1948) discovered the presence Of another ATPase in the sarc0plasm that is separate from the myosin ATPase and is activated by magnesium. Ions also have a profound influence on other prOperties Of myosin. In fact, according to Szent-Gyorgyi (1951), the colloidal state Of most hydrOphylic colloids is influenced by their ionic environment. The amount Of potassium or sodium absorbed by a protein molecule affects the charge, thereby altering its isoelectric point as evidenced by titration curves presented by Szent-Gyorgyi (1951). According to Szent-Gyorgyi, any substance causing a shift in the isoelectric point will alter the -11- solubility characteristics of a protein. In the absence of salts the isoelectric point Of myosin is at pH 5.4. The addition Of 0.025 M KCl shifts the isoelectric point to pH 7. .Maximal precipitation now occurs at neutral reaction, while the protein becomes soluble at its former isoelectric point, pH 5.4. At the highest potassium concentration studied, 0.8 M, the protein.was again soluble at neutral reaction, in fact, the isoelectric point was shifted down to pH 3.0. According to Szent-Gyorgyi (1951), myosin does not distinquish between potassium and sodium ions. He also stated that the reaction of myosin with other ions is not so clear. Every ion induces new and specific changes in the protein which modify its affinities toward other ions. Secondly, the effect of anions is probably as important as that of the cations with which they are added. d. Actin Prolonged extraction of muscle with salt solutions extracts a mix- ture of myosin, actin and actomyosin. Straub (1942) extracted relatively pure actin by removing the sarc0plasmic proteins and myosin by a treat- ment with a K01 phosphate solution, denaturing the remaining protein ex- cept actin, with butanol and preparing an acetone powder of actin from the residue. Actin obtained in this manner was in the globular (G) form, its viscosity was low and no flow birefringence was shown. Addition of a salt or acid resulted in the rapid formation of fibrous (F) actin. Szent-Gyorgyi (1951) summarized the solubility properties of actin as follows: while actin is soluble in water or low concentrations of neutral salt solutions, these solutions do not extract it readily from -12- the muscle. The isoelectric point of actin is pH 4.7. The fact that actin is present in the muscle in fibrous form may exPlain the diffi- culties encountered in its extraction. If actin is depolymerized by agents like KI, it is readily extracted. The molecular weight of the actin monomer is approximately 70,000 as determined by Tsao (1953a) by osmotic pressure measurements. Actin contains small amounts of phOSphate which in G actin solutions has been found to be associated with ATP by Straub and Feuer (1950). They have suggested that the G --->'F transformation involves the reversible change of ATP to ADP in the actin molecule. weber and Portzehl (1951) pointed out that heavy metal compounds prevent the transformation of G --->-F actin, supposedly by their combination with SH groups which are necessary for polymerization. e. Tropomyosin Tropomyosin, discovered by Bailey (1946), is the most recent addi- tion to proteins which form the muscle fibril. Trapomyosin has many prOperties in common with myosin. Bailey (1954) summarized these pro- perties. The amino acid composition is similar, as are some of the solubility prOperties and the isoelectric point. Most significant of all, is that neither protein possesses appreciable amounts of N-terminal residues. This would indicate that some type of cyclic chain structure is present. Under the electron microscOpe the tropomyosin fibrils appear to be about 3000 A. long and 250 A. broad. True molecular weight is thought to be about 50,000. -13.. Muscle contraction and Rigor Mortis The morphological configuration of the actin and myosin components during contraction is controversial. According to one of the present views presented by COpenhaver and Johnson (1958), there are two types of myofilaments (thick myosin filaments in the A band and thin actin filaments in the I band and part of the A band), and it is thought that contraction is accompanied by a sliding of the thin filaments along the thick filaments. Another viewPOint outlined by these authors is that only one type of myofilament exists (an actin filament throughout a sarcomere accompanied by myosin in the A band). Contraction in this case is accomplished by an altered molecular arrangement which draws the I band material into the A band. COpenhaver and Johnson go on to explain that when the nerve to a 'muscle is stimulated, there is a change in electrolyte balance along the sarcolemma. As a consequence of nerve stimulation, the actomyosin complex absorbs ATP and certain cations (K and Mg). In the process, ATP is converted to ADP with the release of free phOSphate ions to sup- ply energy for the process. The mechanism of rigor mortis is, understandably, closely asso- ciated with the process of contraction. Bate-Smith (1948) summarized the two most popular theories for this phenomenon. The first theory advanced by Szent-Gyorgyi (1947) emphasizesintimate molecular processes. Resting muscle, i.e., before and shortly after death, contains myosin in the globular form and dissociated from actin. Muscle is maintained in this state because the contractile proteins are prevented from combining -14- by potassium ions. When potassium (by diffusion) and ATP (by enzymatic breakdown) are removed from myosin as occurs when muscle dies, actin combines with myosin to form actomyosin which causes the muscle to be extremely inextensible. According to Bate-Smith's theory, rigor mortis involves the asso- ciation of the ultimate filaments of a muscle fiber (this filament re- presents a unit containing a number of actomyosin molecules packed side by side). This association is stabilized by weak cross linkages. These cross linkages which account for the decreased extensibility of muscle are formed as a result of total removal Of ATP. Extractability and Fractionation of Muscle Protein Deuticke (1932) was the first to report that muscles which had been fatigued by stimulation, frozen, and pulverized, imparted less protein to an extracting solution than those freshly extracted. Weber and Meyer (1933) Obtained very similar results and in addition found that the de- crease in protein extractability for muscle stored over twenty four hours resided largely in the myosin fraction and to a smaller extent in the globulin X fraction. Bate-Smith (1934a) studied the effects of a series of extracting solutions. He found that with ammonium and lithium chlorides of ade- quate strength, no differences could be observed between the behavior Of fresh and rigor muscle. As pointed out by Bailey (1954), the most direct eXplanation of this early work was that stimulation and rigor involve a change of state which is reflected in a loss of solubility in some salt solutions but -15- not all; or in the light of recent knowledge, they involve the combina- tion of myosin and actin to give a less soluble complex. In freshly minced relaxed muscle, the ATP acts as a Specific dissociating agent. In rigor or fatigued muscle, extraction is facilitated by salts which depolymerize the complex. Bailey concluded that, considering the large amount of recent work on the theory of contraction and rigor, this is probably an over simplified explanation. From the preceding discussion of rigor, it might be thought that the disappearance of ATP from the muscle is largely responsible for an "in vivo" aggregation Of myosin and actin which retards extractions. According to Bailey (1954), this is incorrect. While ATP hastens the rate of solution, it does not increase the final yield, except when the extracting solution has an ionic strength above 0.5. Crepax (1951) in- dicated that the action of ATP on the extractability of the muscle pro- teins is that of strengthening the dissociating action of the electro- lyte on the binding forces which hold the proteins in place in the muscle. The characteristic decrease in extractability of contracted muscles is not due to the hydrolysis of ATP which accompanies these contractions. Bailey (1954) stated that extractability is not solely determined by solubility. He indicated that this is probably because the dissolu- tion of F actin or F actomyosin threads, several microns long, is seri- ously impeded in a mechanical way by the insoluble components of muscle. The extractability of myosin and actin depends, in part, on the mutual combination of these proteins and the hindrance to diffusion by the -16- surrounding insoluble muscle structures. A relaxed muscle, freshly ‘minced, will yield free myosin, even on coarse grinding, but further comminution and stronger salt solutions will bring out large amounts of actomyosin. Homogenization must be continued to break mechanically not only the surrounding structures but to disperse further the concentrated thixotrOpic actin gel inside. Bailey (1954), considering the above facts, made the following conclusions on muscle protein extractability. At any particular stage of rigor the extractability of the intracellular protein fraction appears to be determined by pH, ionic strength of extracting solution, type of extractant and by adequacy Of grinding. Jacob (1947) studied the effects of pH on the extractability of sarc0plasmic proteins. He found that pH 7.7 was optimal for extraction of all muscle proteins within the phOSphate buffer range. At all pH values a precipitate formed on dialysis. The quantity of precipitate varied considerably above pH 7.8, was least at 7.6 and become more abun- dant as pH fell; however, the precipitate fonmed above pH 7.1 was solu- ble in 0.5 M KCl and showed double refraction of flow. weber and Meyer (1933) and Bate-Smith (1934a) were the first to quantitatively partition the proteins of muscle. However, conflicting results were obtained (table 1). Recent workers have obtained more reliable values. Herrman and Nicholas (1948) fractionated embryonic rat muscle. Muscle protein was divided into three fractions by a single procedure. One fraction was insoluble in 0.5 M KCl at pH 7.5 (stroma). -17- Table 1. Protein composition ofimuscle as reported by several authors* Protein N as % total protein N Sarco- Soluble Residual Total Authors ‘Muscle plasmic fibrillar intracellular .fibrillar Stroma Weber & Meyer Rabbit 44 39 17 Bate-Smith Rabbit 16 54 15 69 15-17 Hasselbach & Schneider Rabbit 28 52 4 56 16 Robinson Chick 33 40 22 62 5 Dyer 2; a1. God 21 70 6 76 3 *From Bailey (1954) A second fraction, most of which was myosin, was soluble in 0.5 M KCl but was precipitated at 0.16 M KCl. The remaining proteins which did not precipitate at 0.16 M KCl were designated as soluble proteins (myogen). Hasselbach and Schneider (1951) were the first to attempt the direct estimation of actin. The muscle was coarsely ground and actin free myo- sin plus the sarcoplasmic proteins were first extracted with 0.6 M KCl containing pyrophosphate (actinlsomewhat similar to ATP) at pH 6.3. The perphOSphate dissociates actin and myosin, and the myosin diffuses out, leaving the actin associated with the stroma protein. The tissue was then homogenized in 0.6 M KCl yielding a turbid, viscous extract of actin. Dyer gtflgl. (1950) utilized the Wering blendor to disperse muscle tissue for extraction by a salt solution. This method diSplaced the pre- vious classical methods of extraction.which consisted of repeated (6-9 times) grinding with sand, shaking and centrifuging. -13- Dyer gt El! also designed a plastic baffle plate to prevent foaming in the blendor and subsequent denaturation. These workers tested the efficiency of many salt solutions for extraction of soluble protein. It was concluded that the most important point in the extraction of protein was sufficiently fine subdivision of the muscle fibrils, and when that was Obtained by use of the blendor, the type of salt used for extraction was not critical. They used a one to twenty ratio of tissue to extract- ant solution. The effect Of temperature of extraction was quite marked in the case of fish that had been frozen with much lower extractions at 25°C than at 5°C. With fresh fish, temperature was unimportant. These workers confirmed Bate-Smith's (1934b) results that a constant fraction of 88% of the myosin would be precipitated by a one to ten dilu- tion. It was found that maximum extraction occurred between pH 7 and 9 and in salt solutions of three to five percent. Robinson (1952) developed a standard method for the extraction and estimation of several protein fractions in embryonic chicks. The sarco- plasmic proteins were extracted from homogenized muscle by dilute salt solutions and the myofibrillar proteins were extracted in strong salt so- lutions and precipitated on dilution. Another fraction insoluble in strong salt solutions but soluble in dilute alkali formed a third fraction and the final extracellular residue a fourth fraction. Seagran (1958b) u/ used a similar procedure to fractionate the muscle of the king crab. However, he extracted the entire myofibrillar fraction with 0.1 M NaOH instead of a strong salt solution. Khan (1962) develoPed a technique for the routine fractionation and estimation of major protein fractions in chicken muscle. He also compared -19- different buffer systems for efficiency of extraction and found that KCl- borate and KCl phosphate buffers of pH 7.3 - 7.5 and ionic strength of 1.0 gave the maximum extractability. In one-year-Old chicken meat, stroma-, myofibrillar-, and sarc0plasmic-protein nitrogen, respectively, contributed 13, 42, and 30% of total nitrogen in breast muscle and 27, 30, and 22% in leg muscle. Fujimaki (1962) carried out chromatographic fractionation of muscle protein preparations on DEAE - cellulose and cellulose - phOSphate. Effluent diagrams for the sarcoplasmic fraction from rabbit muscle showed four main peaks, each Of which showed two or three peaks in ultracentri- fugal analyses. He also fractionated actin solutions prepared from acetone powder. In addition to a purified actin peak, he obtained another major peak thought to contain the so called "inactive actin" and a smaller peak believed to be a prosthetic group of actin (a nucleotide). Protein studies applied to meat research An eXplanation of some of the basic concepts of meat hydration and a comprehensive review of previous work in this area has been presented by Hamm (1960). He stated that the "true hydration water" of muscle is the amount of water that attaches to protein by monomolecular and multi- molecular adsorption. This water is bound directly to polar groups of proteins and makes up about 4 - 5% of the water in muscle. The physical prOperties of this fixed, bound water are different from those of free 'water. This bound water has a lower vapor pressure and a lower dissolving power than normal water. -20- Hamm (1960) further stated that studies of muscle tissue by differ- ent physical methods showed that most Of the water in muscle is, chemically Speaking, free. It seems to be free water mechanically immobilized by the network of the cellular protein membranes and protein filaments. Hamm concluded that the considerable changes of the water-holding capacity of meat caused by changes of protein charges (by pH, ions, etc.) are not due to any changes of true hydration water fixed to the polar groups of meat proteins. He stated that the amount of free water "immobilized" within the tissue is strongly influenced by the spatial structure of muscle. Tight- ening this spatial structure (a network of proteins) decreases immobilized water, and loosening the protein structure has the opposite effect. This so-called "stero effect" (Hamm, 1959) is extensively influenced by changes of protein charges. The presence of certain ions or adjustment to cer- tain pH values greatly affects the Spatial protein arrangement and conse- quently affects water-holding capacity. The water-holding capacity of meat means the ability of meat to hold its own or added water during application of any force (pressing, heating, grinding, etc.). Schon and Stosiek (1958a), (1958b) found an inherent difference in water-holding capacity between different Species, age, and sexes of meat animals. The reasons for these differences have not been elucidated. They utilized the adductor and longissimus dorsi muscles, and found that pork had a greater water-holding capacity than beef. There were no differ- ences between sexes in the case of pigs, however, the water-holding capa- city of cattle increased in the order of steer to heifer to cow. The -21- behavior of bull meat in this resPect was very erratic and could not be placed in the above series. Bendall and Pedersen (1962) carried out an extensive experiment to investigate the causes of soft watery pork, a condition quite commonly associated with pork.musc1e. They prepared fibrils from normal and watery pork and compared the water retention at various ionic strengths. Normal and watery fibrils were also titrated to determine isoelectric points. From the results of this study, they concluded that the fibrillar proteins of the watery fibrils are not denatured or aggregated in the usual sense, but are probably covered by a layer of denatured sarc0plas- mic protein that is firmly bound to the surface of the myofilaments. Swift and Berman (1959) and Swift.gtual. (1960) investigated some of the factors affecting water retention of beef. They confined their observations to variations between muscles of the same and different ani- mals. A direct, highly Significant correlation was found between water retention and zinc content, in contrast to the inverse relation found between water retention and either calcium or magnesium content. The ratio, moisture/protein content, was found to be directly related to water retention, hence, the results showed that the relative ability of the muscles to hold added moisture was predictable on the basis of the ori- ginal prOportions of moisture and protein present. They found that the variation in pH over a relatively narrow range, 5.49 - 5.86, was correlated directly and closely with capacity for water retention (r - 0.95). Variation in pH was negatively related to protein content (r = -0.89). -22- Sherman (l96la)studied factors influencing fluid retention by ground pork. Tetrasodium pyrophosphate and alkaline polyphOSphate were found to be more effective than NaCl in retaining fluid. pH was found to have a positive influence on fluid retention and the degree of solubilization of actomyosin. Fluid retention was said to depend on the degree of ion adsorption. The ionic strength of the solutions employed was important only in so far as it controlled the rate of ion adsorption by the meat. The greater the ionic strength, the greater the adsorption of ions. In Sherman's experiment, fluid retention at 100°C appeared to be re- lated to the concentration of actomyosin that went into solution at 0°C. The greater the concentration of actomyosin in solution, the stronger the gel of denatured protein formed upon heat coagulation. A strong gel ex- tending throughout the meat mass improved moisture retention. Sherman (1961c) showed that stronger coagula are developed with alka- line phosPhates than with NaCl. He stated that more fluid is retained by meat at higher temperatures in the presence of the former additives. Coagulation temperatures, the nature of the coagula, and consequently the rate of release of fluid at temperatures above 60°C, depend on the nature and concentration of the additive employed. Sherman (1961c) concluded that the most important factor in the ex- planation of variations in fluid retention appeared to be solubilization, or swelling of proteins, particularly actomyosin, within the meat prior to heating. This process is influenced by pH, time and temperature of the initial aging period, and in the case of added alkaline phosPhates, some additional factor, possibly the ability to Split the bond between actin and myosin in actomyosin. -23- Results obtained by Swift and Ellis (1956) showed that the effect- iveness of treatments with pyrophOSphates was primarily related to the ionic strength and pH of a solution applied to lean meat. The capacity of pyrophOSphate for buffering was also shown to be important. They showed that pyrophOSphates dissolve proteins, especially actomyosin, to an extent affected by ionic strength and pH. In general, the factors governing the moisture retention of meat treated with phOSphate additives are those which influence solubilization of muscle proteins; namely temp- erature, time, ionic strength and pH of treatments, and those specific effects that can be exerted by ions, such as I' and Mg++ ions. Swift and Ellis (1957) investigated the effects of the ordinary curing agents and certain phOSphates on color and binding of frankfurters and bologna. Shrinkage during smoking and cooking and cohesiveness as indicated by tensile strength measurements were employed as criteria Of 'binding. 0f the ordinary curing ingredients, only the action of NaCl in- creased the development of heat when emulsions were prepared, the reten- tion of moisture during heat processing, and the cohesion of sausage products. The addition of phosphates increased the relative binding of sausage components, and decreased shrinkage of bologna heated to an inter- nal temperature of 160°F. The rate at which the temperature of emulsions increased during comminution was reduced in the presence of certain phos- phates. Fukazawa EE.§£- (1961c) undertook to clarify the role played by the addition of various phOSphates in the binding quality of sausage. Phos- phates studied were perphOSphate (PP), tripolyphOSphate (TPP), and hexa- metaphosphate (HMP). They concluded that the effect of phOSphates was to -24- increase the amount of protein extracted from fibrils by the use of a 0.6 M KCl solution. Phosphates, especially PP, induced dissociation of myosin from natural actomyosin as shown by decrease in viscosity of acto- myosin solution upon the addition of PP. Data from the ultracentrifuge supported this view. The amount of protein extracted from intact fibrils with 0.6 M NaCl was shown to increase from pH 5.6 through pH 6.4 to pH 7.0. Bendall (1954) found that orthoPhOSPhate, Calgon, and metaphOSphate tended to increase swelling and water uptake of meat. This effect was increased by addition of NaCl. The effect of the above phOSphates was small comparedwith that of perphOSphate. The effects of the orthophos- phate, Calgon, and metaphOSphate were probably due to ionic strength in- crease alone, while the pyroPhOSPhate, in addition, facilitated the extraction of actin and myosin from the tissue because of its ability to Split actomyosin into its components. Fukazawa gt 3;. (1961b) studied the influence Of fibrillar proteins on the quality of experimental sausages. The binding quality of sausage prepared from intact fibrils, actin poor fibrils, synthetic fibrils and purified myosin was estimated by elasticity determinations. It was con- cluded that myosin present in fibrils exerted a great influence on the binding quality of sausage. Actin and tropomyosin did not greatly influ- ence the binding quality of sausage. It could not be shown that the amount of nitrogen soluble in 0.6 M NaCl, pH, or water-holding capacity directly influenced the binding quality of experimental sausages. Another study by these Japanese workers, Fukazawa g; 3;. (1961a), dealt with the preparation of subcellular muscle preparations from muscle -25- fibrils free of water soluble proteins and also fibrils relatively free of actomyosin called "ghost fibrils" by the authors. It was found that sausage made from the actomyosin poor fibrils showed a considerable de- crease in binding quality, whereas sausage made from water-soluble-protein- free muscle fibrils showed little change in binding quality. Actin- trOpomyosin poor fibrils were also prepared. These fibrils were only slightly inferior in binding quality to sausage made from whole muscle. Sair and Cook (1938) were among the first to investigate the effect of freezing, length of storage, and pH of tissues on exudation of drip during thawing. Beef, pork and mutton were SUJdied. pH was adjusted by injecting lactic acid or NaOH. Observations made during the course of these experiments indicated that mutton behaves like pork, in that the pH is subject to considerable variation between animals. Results sug- gested that the quantity of drip is related to the pH of the tissue at the time of freezing, and that the maximum total drip is associated with low pH. It was concluded that these three kinds of meat will drip to the same extent after freezing and thawing, provided they have the same pH, and that the same freezing rate was used. Sair and Cook also stated that the pH of beef is relatively constant and close to the value at which maximum drip occurs, whereas pork and mutton vary in pH from carcass to carcass, and are generally more alkaline. They claimed that this fact readily explains the small amount of drip from pork or mutton. These authors also concluded that a protein extraction procedure is not suited to a study of the nature of changes in the proteins affecting drip. Dyer (1951) extracted soluble proteins in 5% NaCl, and followed this with an estimation of the actomyosin of the solution. The amount of acto- -26- myosin solubilized was used as an estimate of denaturation. It was found that the decrease in actomyosin solubility paralleled and anticipated taste panel ratings and provided a quantitative measure of the quality of frozen fish. According to Seagran (1958a), recent work shows that drip in fish is due, at least in part, to denaturation of protein which normally tends to hold the water of muscle. He found the contractile protein, actomyosin, absent in drip from frozen and thawed rockfish. By electrOphoresis and dilution techniques, he showed a definite Similarity between the protein composition of drip and extracts of low ionic strength from fish muscle. From the results of this study, it was concluded that the sarc0plasmic fraction of fish muscle is not intimately associated with the origin of drip. It was suggested that drip formation and texture change resulting from freezing and thawing must be due in part to actomyosin denaturation by a dehydration process. Hunt and Matheson (1958) used three criteria to investigate denatur- ation of actomyosin in freeze dehydrated beef and cod muscle. These were loss of solubility in salt solutions, loss of contractility of muscle fibers in the presence of ATP, and loss of ATPase activity. On dehydra- tion, cod actomyosin became insoluble and the muscle fibers may or may not lose their power to contract and about one-half Of the ATPase activity was lost. iMuscle fibers of beef were always contractile after rehydration and they also retained about half Of their ATPase activity. In connection with studies of protein denaturation during freezing, Connell (1962b) extracted reasonably pure myosin from cod muscle by ex- haustively extracting finely minced muscle with a slightly acid salt -27- solution containing pyroPhosphate ions. He indicated that pyroPhOSphate dissociated the actin-myosin bonds existing in the muscle and inhibited the formation of such bonds during extraction. From this study it ap- peared that 70-80 percent of the myosin became non-extractable at a rate similar to that at which the total myofibrillar protein of flesh became non-extractable. The remainder of the myosin became non-extractable at a much Slower rate. He cautioned the use of the term denaturation in connection with changes in extractability Of the actomyosin fraction in frozen fish. He suggested that part of the myosin and actin interact very strongly during freezing in a manner similar to the interaction occurring during rigor, and that this interaction may partially account for the inextractability of myofibrillar proteins after frozen storage. Hashimoto gt EL. (1959) carried out an experiment to determine whether or not denaturation of actomyosin in muscle during storage pro- ceeded in a manner similar to that of the isolated protein. These work- ers followed changes in nitrogen extractability, in weber Edsall solution, of rabbit muscle stored at 20°C. They found extractability at a high value at 0 time, dropping to a minimum at 12 hours, and in 24 hours rising to a point above that of 0 time. pH changes and water-holding capacity followed much the same pattern. ATPase activity of myosin ex- tracted from muscle at 20°C was found to be inactivated very quickly. The course of denaturation of actomyosin in stored meat coincided com- pletely with denaturation of isolated actomyosin. Seagran (1956) reported on a study of changes in properties Of the actomyosin fraction of fish muscle that had been subjected to frozen -23- storage. The properties of chemical activity, asymmetry, and solubility of actomyosin were investigated. He concluded that the freezing process produced some subtle change in the structure of actomyosin present in post-rigor muscle, yielding a more symmetrical molecule. A significant structural change was not indicated as the increase in -SH groups was verytiight. He suggested a possible correlation between cold storage stability and a lipid complexed with actomyosin of fish. King gtflal. (1962) investigated the effect of free fatty acids on denaturation of cod actomyosin. They found that small concentrations (0.025 ml./350 m1. of solution containing 0.45 mg. of total soluble pro- tein nitrogen/ml.) rapidly reduced the solubility of actomyosin. The extent of insolubilization of actomyosin depended on the fatty acid used, its concentration and the length of storage of the treated solution, thus supporting the hypothesis that the accumulation of free fatty acids in frozen fish muscle causes actomyosin to become inextractable. Connell (1962a) stated that there is good evidence that sarcoplasmic proteins survive freeze-drying virtually intact. The electrOphoretic and ultracentrifuge properties of extracts of this group of proteins prepared from freeze-dried beef and cod are very similar to those of'frozen con- trols. The amounts of sarcoplasmic proteins extractable from these Spec- ies are also unchanged. Toughness developing during freeze-drying appears to be associated with a loss of the true water-holding capacity of the muscle. He indicated that it is evident that changes in actin and myosin, or their association with one another, are prime causes of textural deter- ioration following freeze-drying. Practically no actomyosin can be -29- extracted from freeze-dried cod, however, as much actin is extractable from freeze-dried cod as from a frozen control, and about half of the myo- sin also remains extractable after freeze-drying. Connell related the toughening during freeze-drying and subsequent storage to an increase in the number of bonds between the myofibrillar proteins. He stated that there are probably two types Of bonds involved in this association: (1) actindmyosin bonds and (2) bonds between denatured myosin molecules. Cole (1962) studied the effect of oxygen plus elevated temperature on freeze-dried beef by determining changes in solubility of actomyosin and sarc0plasmic proteins. The solubility data indicated that the sarco- plasmic proteins are much less adversely affected than those of the contractile group. Fujimaki gt El: (1961) Studied the effects of gamma irradiation on the chemical properties of actin and actomyosin during aging of meat. Meats were irradiated at three stages, i.e., at slaughter, at maximum rigor (2 days) and at "rigor off" (7 days). Changes in actin and acto- myosin were determined by extracting these components from the irradiated and control meats and determining sulfhydryl groups, amino acid composi- tion, viscosity, ATPase activity and ATP sensitivity. It was found that actin was only slightly sensitive to irradiation and that actomyosin was very sensitive. Actomyosin becomes more sensitive to irradiation as the stage of rigor advances. Hamm and Deatherage (1960b) summarized the steps Of meat denaturation upon heating. In the range 20°C to 30°C, no change in hydration, rigidity, buffer capacity, solubility or changes was observed. From 30°C to 40°C, -30- changes in muscle proteins include two steps: an unfolding of peptide chains and the formation of new electrostatic and/or hydrogen cross link- ages. In this temperature range changes in muscle proteins influence hydration, rigidity and solubility of meat. Some of these changes may be due to an increased availability of charged groups made possible by the unfolding Of the peptide chains. The strongest denaturation occurs in the range of 40°C to 80°C as shown by a decrease in muscle hydration, increase Of rigidity and decrease of protein solubility. Wierbicki 2; EL- (1954) were among the first to directly approach the study of quality attributes of meat by protein fractionation. These workers attempted to determine the amount of actomyosin in meat and re- late this to tenderness. Their extracting solution was designed to dissolve actin and myosin and other proteins but not actomyosin. This solution was a citric acid buffer of pH 5.6, ionic strength of 0.48 and 0.22 M KCl. These workers also determined connective tissue by the hy- droxyproline method and an alkali insoluble method. No changes were observed in content of connective tissue during aging of beef, however, tenderness did increase significantly. In a group of 48 beef animals, hydroxyproline values showed no relation to tenderness, however, a very good correlation of extractable nitrogen with tenderness was obtained (correlation coefficient was 0.507 for 46 degrees of freedom). These workers also suggested on the basis of their study that in- crease in tenderness with post mortem age may be related to: (a) the dissociation of actomyosin or some similar protein changes which increase protein extractability, and (b) redistribution of ions within muscle, thus causing increased hydration and tenderness. -31- In a subsequent paper, Wierbicki ggnal. (1956) indicated that pro- tein-protein interactions or ion-protein interactions may be the cause of post mortem tenderization rather than classical proteolysis or disso- ciation of actomyosin. In their study both a buffer extract and a water extract were employed to study changes in nitrogen solubility. Both ex- tracts were found to follow the same basic pattern, i.e., they were high at slaughter, decreased to a minimum in 12 to 24 hours and then gradually increased to five days, where they leveled Off. Kronman and Winterbottom (1960) indicated that aging beef for seven days renders the previously soluble protein less liable to extraction from the tissue, which agrees with'Wierbicki.gE.§l. (1956). Using this solubility as a criterion.of denaturation, the former authors concluded that from 10 to 30% of the water soluble protein may be denatured during the seven days of aging. They also found that freezing led to a decrease in extractability of water soluble protein. They stated that extracta- bility or solubility of proteins does not provide a sufficiently sensitive criterion for protein alteration. weinberg and Rose (1960) attempted to study post mortem tenderization of chicken muscle by observing changes in the extractability of the con- tractile proteins from pre and post rigor chicken breast muscle. The amount of nitrogen extracted increased when the carcasses were held for 24 hours at 4°C, and this increase was entirely accounted for by an in- crease in the actomyosin fraction. They suggested that more actin was extracted from post rigor meat and that this actin combined with myosin in the BXtraCt- Faom.the results of the study, they suggested that ten- -32- derization is not merely random autolysis, but results from a Specific cleavage of an actin association responsible for the maintenance of the muscle‘matrix. Arnold g; 2;. (1956) studied the interactions of cations and pro- teins of beef during post mortem aging. They found that the total cationic shift was a movement of cations onto the meat proteins, result- ing in an increased charge on the meat proteins, allowing greater hydra- tion and improved tenderness. The absolute amounts of each cation in the meat, in the water extracts, and in the juice had no significant correlation with tenderness. Following this work, Wierbicki 2531;. (1957a) studied the effects of added cations on meat shrinkage at 70°C. The cations, sodium, potassium, magnesium, and calcium were found to increase the water-holding capacity Of meat, with magnesium showing the most pronounced effect. Sodium chloride, added to meat prior to freezing, reduced the amount Of drip encountered on thawing. Kamstra and Saffle (1959) attempted to evaluate the extent to which rigor contributes to toughness of meat. The normal sequence Of reactions during rigor was interrupted by the infusion of a chelating agent, sodium hexametaphOSphate, into hot hams. The hot hams infused with sodium hexametaphOSphate showed an immediate massive contraction and then re- laxation. Results showed a highly significant increase in tenderness of all hams infused with sodium hexametaphOSphate. Carpenter 2; El- (1961) designed an eXperiment suggested by the work of Kamstra and Saffle (1959) on paired hams. They attempted to interrupt the normal sequence of rigor by infusing sodium hexametaphOSphate into -33- hot beef rounds. Previous basic studies on the contractile proteins indicated that calcium, potassium and magnesium ions influenced the for- mation of actomyosin. The injection of a chelating agent would supposed- ly bind these ions, thereby interferring with the formation of actomyosin. The over all results of taste panel and shear showed a decided in- crease in tenderness for treated rounds over control rounds, however, the treated rounds were dark and flabby. The darkness was somewhat overcome by the infusion of lactic acid. Theory of Emulsions Becher (1957) described an emulsion as follows: "a heterogenous system, consisting of at least one immiscible liquid intimately diapersed in another in the form of droplets, whose diameter, in general, exceeds 0.1 micron. Such systems possess a minimal stability, which may be ac- centuated by such additives as surface-active agents, finely divided solids, etc." According to Holmes (1934), two mutually insoluble liquids may be emulsified by mechanical agitation, but they soon separate into two layers of the original liquid. Stable emulsions of two pure liquids can not be made. A third substance, usually colloidal, is necessary to stabilize emulsions. This is often present as an unsusPected impurity. This third substance is called an emulsifying agent and is concentrated at the interface between the two liquid phases. Soaps and proteins are among the most common emulsifiers. Clayton (1928) stated that emulsions of oil in water (O/W) and water in oil (W/O) are common both in the laboratory and in technical practice. -34- Some Oil/water emulsifiers are casein, albumin, agar, starch, gums, hemo- gLobin, pepsin, peptones, dextrin, lecithin, soaps and alkalis which may form soaps with the free fatty acids found in some fats. Finely divided solids in certain instances may promote emulsification of oil in water or water in Oil. Holmes (1934) stated that emulsions may be broken by: (1) adding an equivalent amount of emulsifying agent of the opposite type; (2) con- version Of the emulsifying agent into some other compound; (3) addition of an excess of dispersed phase with violent agitation; (4) certain types of violent agitation alone; (5) electro-deposition; (6) freezing; (7) centrifuging; (8) certain types of filtration; and (9) a hot dip of metal into the emulsion. Clayton (1928) described the preparation of an emulsion using only pure oil and water. Such a process required relatively violent agitation over very long periods of time. The oil globules in pure oil in-water emulsions have diameters of the order of 10'5 cm. compared with 10"3 cm. for colloidal suSpensions. Without the presence of an emulsifying agent, emulsions of oil in water may be made up to a maximun concentration Of 2 percent, however, they are usually much more dilute. The most note- worthy feature in all cases is that the purer the materials, the less stable are the emulsions. With pure Oil and water, only stable emulsions of the oil in water type are known. Such emulsions are of interest theoretically, but have no practical importance. Clayton (1928) stated that, theoretically, if oil globules were considered as rigid Spheres of equal diameter, it would be possible to -35- pack them together in such a way that each Sphere would touch twelve others. This maximum packing occurs when about 75 percent Of the total available Space is occupied by the spheres. Hence, such an emulsion would be 75 percent oil and 25 percent water. Practically, Oil globules are deformable Spheres and occur in a large variety of sizes in an emul- sion. Therefore, the preceding theoretical explanation is incorrect as experiments have shown that stable emulsions containing 99 percent dis- perse phase may be obtained. He also stated that the Oil globules of emulsions of Oil in water carry a negative charge as proven by cataphore- sis tests. The origin Of this charge is open to question as is the more general case of the electric charge of all colloids. Clayton (1932) stated that there can be no doubt that a protein, acting as an emulsifying agent, concentrates at the dineric interface and sometimes the act of adsorption leads to a change in the physical character of the emulsifying agent, this being "precipitated" as a fibrous or membrano-fibrous solid,ru> longer soluble in its original solvent. IMost of the recent research on protein denaturation at interfaces has been carried out at air-water interfaces. Bull (1947) stated that it is one of the remarkable facts of nature that when soluble, highly organized native protein molecules are placed on an aqueous surface, they promptly Spread on the surface to form insoluble films whose thickness correSponds to one peptide chain, irresPective of the dimensions of the original, native protein. In order to Spread on a water surface and form a stable film, a substance must possess hydrOphilic and hydrOphobic groups. The water side of a Spread film Of protein must be predominantly hydrOphil- ic, while the side directed toward air or Oil, as thecase may be, must be -36- predominantly hydrOphobic. Otherwise, a protein monolayer would not be stable at a water-air or water-Oil interface. He showed that the air side of a protein monolayer is predominantly hydrophobic. According to MacRitchie and Alexander (1961b), protein concentra- tion, nature of the interface, pH, ionic strength, and temperature, all influence the rate of build-up of a denatured layer, although at a parti- cular interface for sufficient protein concentration, pH is probably the major factor. The adsorption-pH curves show a maximum near the isoelectric point, falling away steeply on either Side. They stated that it is gen- erally accepted that the process of surface denaturation occurs by adsorp- tion in the globular form followed by an unfolding caused by the asym- metric surface forces. It is also widely held that the surface denatur- ation step is practically instantaneous. MacRitchie and Alexander (1961a), in exPeriments with protein stab- ilized foams, found that the general effects of sucrose were an overall increase in foam stability and an increase in the ease of formation up to a point beyond which higher concentrations depress the foaminess. Interfacial tension has been one of the most widely studied physical prOperties of emulsions. According to Becher (1957), the phenomenon of surface or interfacial tension may be explained on a molecular basis by the fact that the Van der waals field of force acting on a molecule at the surface of a liquid is different from the forces acting on a similar molecule in the bulk of the liquid, where the forces are balanced out because their environment is the same on all sides. The value of inter- facial tension will usually lie between the individual surface tensions of the two liquids involved. -37- Limited research has been done involving the Spreading of the con- tractile muscle proteins on airawater interfaces. Lajtha and Rideal (1951) found that the spreading of the muscle proteins, myosin and acto- myosin, depends both on the salt solution in which the proteins are dis- solved and on the solution on which they are Spread. The viscosity of these proteins in the monomolecular fiim state was compared with other proteins. Myosin was found to possess an unusually high viscosity in this state. Various phOSphates were found to affect the Spreading of myosin and actomyosin at interfaces. It was noted that phOSphates were more effective in this reSpect than the other salts studied. The order of effectiveness of the phosphate compounds was ATP >-ADP >-hexaphOSphate > triphosPhate >-perphosphate >'AMP. The Spreading of actin was unaff- ected by the addition of salts. Cheesman and Davies (1954) stated that ATP accelerates the spreading of actomyosin but not myosin. In fact, spreading of myosin was retarded by ATP. They explained these findings by assuming that the unfolding of both actin and myosin is retarded by the combination of the two proteins. When the complex is Split by ATP, the actin will be free to spread, while the unfolding of the myosin may then be delayed by the combination with ATP. This, they state is probably a manifestation of the common finding that substrates tend to stabilize their enzymes against inactivation. According to the same authors, the tensile strength of monolayers of myosin and actomyosin Spread on‘M KCl at pH 7.0, is greatly reduced in the presence of ATP. Although the fully unfolded myosin protein found at an interface has no ATPase activity, ATP continues to exert a plasti- cizing effect on the protein as evidenced by the reduction in talsile strength. -33- Emulsion Technology Applied to Meat Research Swift 25 El- (1961) undertook a series of experiments to investigate the factors affecting the capacity of meat to stabilize emulsions. They prepared emulsions with meat slurries and with crude salt and water ex- tracts of meat. The fat globules present in an emulsion prepared from a meat slurry were examined microscOpically. They had a mean diameter of 18 microns and possessed a non-uniform polyhydral fonm. Histological preparations treated with a protein stain exhibited fat globules outlined by protein membranes. The rate of fat addition and the temperature of the process were found to significantly affect the amount of fat emulsi- fied. Increased rate of fat addition, up to l ml./sec., caused an increase in fat emulsified while an increase in temperature from 18-46°C caused a decrease in fat emulsified from 180 ml. to 130 ml. Data from emulsions prepared from meat extracts showed that salt soluble proteins were more efficient emulsifying agents than water soluble proteins. They also found that water soluble proteins had no marked capacity as stabilizers in the absence of salt. Hansen (1960) made an extensive microscopic study of comminuted meats, diluted comminuted meat batter, and emulsions prepared with pro- tein solutions. Stained preparations of diluted batter and emulsions prepared from protein solutions indicated that a fat globule membrane is formed in a weiner batter. This membrane was Observed only in emulsions prepared with the salt soluble extract. Results of this study also indi- cated that if excessive temperature rise occurs during chOpping, the pro- tein matrix may be partially denatured and broken, giving rise to an unprotected fat diSpersion. -39- Sherman (1961b) attempted to determine if phOSphates play a role of emulsion stabilization in sausages through the formation of soaps with free fatty acids. Soap formation was promoted when inedible hog fat (high in free fatty acid content) was used for the diSpersed phase. If NaCl was present, fat emulsification was obstructed as any soap that was formed would be salted out. He concluded that soap formation in sausage did not occur because of the low content Of free fatty acids in edible fat and the presence of salt normally used in a sausage formulation. EXPERIMENTAL METHODS This research was composed Of two parts. Part I included two exper- iments, both of which involved fractionation and solubility studies of beef muscle proteins. The first of these experiments was designed to study changes in extractability, or solubility, of certain proteins dur- ing post mortem aging of carcasses from three different sex and age groups. The second experiment was designed to investigate the relation- ship of intracellular muscle proteins to meat tenderness. Part II was composed of an experiment designed to study the fat emulsifying prOper- ties Of semi-purified intracellular muscle proteins. General Methods Nitrogen analysis. All nitrogen analyses were performed by the micro-Kjeldahl method as outlined by The American Instrument Co. (1961), unless otherwise Spec- ified. All nitrogen contents were reported as mg. Of protein nitrogen or non-protein nitrogen per ml. of solution, or per g. of tissue. Nitro- gen analyses were made in duplicate. 1H meas urement . A11 pH measurements were made with a Beckman MOdel G pH meter. The electrodes were placed directly into the ground sample or protein solution and the Observed values recorded to the nearest one tenth unit. Nongprotein nitrogen determination. Non-protein nitrogen was determined by mixing 15 m1. Of protein sol- ution.with 5 m1. of 10% trichloroacetic acid. After 15 minutes this -40- -41- material was filtered through Whatman NO. 1 filter paper. The filtrate was analyzed for nitrogen. This value was multiplied by 1.33 (necessary because of the TCA dilution) to give non-protein nitrogen per ml. of ori- ginal solution. Reagents. Reagent grade chemicals and deionized distilled water were used throughout the experiment. Detailed composition of all solutions is contained in appendix A. Centrifugation. A model PR—2 refrigerated International Centrifuge was used through- out the experiment. Centrifuging was done at 2500 rpm (1400 X gravity) with the exception of water-holding capacity determinations. Statistical analysis. Simple correlation coefficients, standard errors, standard devia- tions and analyses of variance were calculated as outlined by Snedecor (1956). Part I Experimental animals. All meat samples were obtained from carcasses of cattle fed on the Michigan State University farms and slaughtered in the Michigan State University meat laboratory. The twelve animals used in the initial fractionation study were of three different age and sex groups. Three of these were cows of varying ages and grades, five were Standard and Good grade yearling heifers and four were Standard and Good grade year- ling bulls. -42- Twenty yearling bulls, sired by bulls selected for variation in ten- derness and muscling, were utilized for the tenderness study. These bulls, of the Standard and Good grades, had received the same management and feeding treatment and were very nearly the same age. Samplingrprocedures. The infraspinatus muscle was selected for the initial fractionation study because of its convenient size and accessibility. Samples to be fractionated were taken at three periods post mortem: 0 time, generally taken within 20 minutes after death, at 24 hours, and at 7 days. Only that part of the muscle used for a particular sample (about 500 g.) was removed from the carcass. The remainder Of the muscle was left on the carcass until used. The sample at 0 time was taken from the middle of the muscle, at 24 hours from the posterior portion and at 7 days from the anterior portion. The extreme ends Of the muscle were not used be- cause Of increased connective tissue at the muscle attachments. The longissimus dorsi muscle of a two inch steak taken five inches from the anterior of the short loin, was used for protein fractionation in conjunction with tenderness studies. These steaks were vacuum pack- aged in Cryovac bags, immediately frozen at -30°C. and stored at this temperature for approximately one month. Sample. preparation for protein fractionation studies. All separable fat and connective tissue were removed from the muscle samples. The sample was ground twice through a 1 cm. plate and twice through a 2 mm. plate. The grinder head and plates were prechilled in all cases to prevent heat denaturation of the sample, which was ground -43- into a beaker and immediately covered with aluminum foil to prevent evaporation. A portion of this (about 50 g.) was frozen and analyzed later for total nitrogen and moisture. Nitrogen content (Fn) of these samples was determined by the micro-Kjeldahl method as outlined by the A.O.A.C. (1960). Fat and moisture were also determined according to the A.O.A.C. Methods. The only exception to the above procedure occurred in the case of steaks utilized for protein fractionation in the tenderness study. They were partially thawed by removing from the freezer and storing at 4°C overnight. No drip occurred during this period. The longissimus dorsi muscle was removed from the steak and trimmed of fat and connective tissue. After being cut into small cubes, the partially frozen meat was ground and held as described above. Fractionation procedure. The fractionation procedure was adapted from that of Seagran (1958b) and of Turner and Olson (1959). The principal changes made in adapting these procedures were an increase in the volume of extracting solutions relative to sample size, increase in sample size, and length of time in- volved in each extraction. All fractionation procedures were carried out in duplicate at 4°C unless otherwise stated. The design outlined in figure 1 was utilized for the quantitative determination Of sarcoplasmic protein nitrogen, non- protein nitrogen, and total fibrillar protein nitrogen. Additional data Obtained from the design outlined in figure 2 were necessary for the determination of fibrillar protein solubility. Details of these proce- dures are outlined below. residue residue, C -44- 5 g. sample liendorized in 70 m1. of P04 buffer + a 30 m1. rinse with same buffer after one hour centrifuged for 15 minutes SUpernatant agded 100 ml. P04 buffer, mixed, after one hour centrifuged 15 minutes supernatant added 200 ml. 0.1 NaOH, after 4 hours filtered through nitrogen solution, A, gauze extracted at low ionic strength residue filtrate 15 ml. aliquot of A treated with TCA for V NPN determination alkali insoluble material, connective tissue (discarded) Figure 1. V' solution, D, containing total fibrillar protein nitrogen precipitate fildiate, B, (discarded) containing NPN Scheme for the quantitative determination Of sarc0plasmic pro- tein nitrogen, non-protein nitrogen and total fibrillar protein nitrogen. -45- 5 g. sample blendorized in 70 ml. weber Edsall solution + a 30 ml. rinse with same solution after one hour centrifuged for 15*minutes residue supernatant added 100 ml. Weber Edsall solution, mixed, after one hour centrifuged for 15 minutes residue supernatant nitrogen extracted at high ionic Strength, solution E (digcarded) Scheme for the quantitative determination of fibrillar protein Figure 2. nitrogen solubility (complement to figure 1) -46- For scheme1(figure l), a five g. sample was weighed into a 250 ml. centrifuge tube. Seventy ml. of a phOSphate buffer (pH 7.6, ionic strength 0.05) was added to the tube and the entire contents of the tube were transferred to a microblendor jar. An attempt was made to approxi- mate the degree of comminution generally achieved in a normal bologna emulsion and at the same time to avoid protein denaturation by excess- ive foaming. This was achieved by blendorizing for one minute at a blendor speed of 8000 rpm (adjusted with a Powerstat transformer setting of 40). It was extremely important to control blendor speed and time, as variations in these factors caused differences in extractability. After blendorizing, the material was transferred back to its original tube. The blendor jar was rinsed with 30 m1. of extracting solution, and this too, was added to the centrifuge tube. After one hour the mater- ial was centrifuged for 15 minutes, the supernatant decanted and its volume recorded. One hundred ml. of extracting solution was again added to each tube. The tube was stOppered and Shaken until complete disper- sion of the tissue was Obtained (about 20 seconds). After one hour the material was centrifuged, the supernatant decanted and the volume recorded as before. The two solutions obtained were combined and filtered through eight layers of gauze to remove fat and other particulate material not removed by centrifugation. This combined solution was designated as A (protein solution extracted at low ionic strength). A 15 ml. aliquot was taken for nitrogen analysis, and a 15 ml. aliquot was used for the deter- mination Of non-protein nitrogen. The filtrate resulting from the TCA precipitation was designated as B. The residue, C, remaining from the -47- extraction with phosphate buffer was extracted with 200 ml. of 0.1 M NaOH for four hours at room temperature. The volume of the tube contents was measured and then filtered through gauze. A very small amount of residue (alkali insoluble material, i.e., connective tissue) was retained in the gauze. An aliquot of the filtrate, D, was taken for nitrogen analysis and the remainder discarded. The procedure in figure 2 is ex- actly the same as the first two steps outlined in figure 1, except that the extracting solution was Weber Edsall solution (NaCl carbonate buffer, pH 9.0, ionic strength 0.67). The solution extracted by this scheme was designated E. Solutions A, B, D, and E were analyzed for nitrogen and results designated as An, Bn, etc. These symbols (nitrogen contents) represent the following fractions: B = non-protein nitrogen 51> :3 ll nitrogen extractable at low ionic strength U :8 ll total fibrillar protein nitrogen ['11 D I nitrogen extractable at high ionic strength A - Bn = sarcoplasmic protein nitrogen :11 :1 I An - soluble fibrillar protein nitrogen Fn (Dn +.An) = connective tissue protein nitrogen The above values were averages of duplicate analyses recorded to the second decimal place. Variation between duplicates for An, D“, and En was normally from 0 - .02 mg, with an extreme range in one or two cases of .05 mg. Variation in the second decimal place was seldom Observed in the case of En. -43- Tenderness measurements. Tenderness determinations were made on the first three 1 1/2 inch steaks from the anterior end of short loins removed from carcasses which had aged for seven days. Steaks were cooked in deep fat at 141°C to an internal temperature Of 63°C. They were allowed to cool 24 hours and one inch cores were submitted to a 12 member taste panel for tenderness evaluation on a nine point hedonic scale. The scores were rated from 1 (extremely tough) to 9 (extremely tender). One-half inch cores were 'measured for tenderness with the Warner-Bratzler shear. Water-holding capacity determinations. Water-holding capacity was determined according to the centrifugal method of Wierbicki et-al: (1957b). Ground samples of 25 g. were heated for 30 minutes at 70°C and centrifuged at 1000 rpm. (250 X gravity) for 10 minutes. Triplicate determinations were made. Part II Isolation and purification 2f sarcoplasmic proteins. SarcOplasmic proteins, S, for emulsion preparation were isolated from frozen longissimus dorsi muscles of Good or Choice grade steers. The muscle was allowed to partially thaw by holding overnight at 4°C and then ground through a 2 mm plate. One hundred g. of ground muscle were mixed with 400 m1. of water or phosPhate buffer (pH 7.6, ionic strength 0.05). The mixture was macerated for 20 seconds in a Waring blendor. After one hour the material was centrifuged for 20 minutes, and the supernatant collected. The nitrogen content of the buffer extracted -49- solution was about 0.25 mg./m1. greater than the water extracted solution, which contained about 2.25 mg. nitrogen/m1. When the phosphate buffer was used for extraction, the protein solution contained the globulin X fraction of the sarcoplasmic proteins. The phOSphate buffer extract was dialyzed against water for 36 hours to precipitate globulin X. This fraction was then partially redissolved in a buffer similar to that used for the original extraction. The globulin X fraction was rather unstable as evidenced by partial insolubility of the precipitate. Isolation and purification 9f actomyosin. Actomyosin was prepared according to Szent-Gyorgyi (1951) with cer- tain modifications found necessary in applying this procedure to frozen beef muscle. Actomyosin was prepared as outlined in figure 3, from the same type of tissue as the sarcoplasmic fraction. One hundred g. of muscle ground through a 2 mm. plate, were mixed with 400 ml. of Weber Edsall solution and stirred mechanically until the mixture attained a jellylike consistency (about two hours with previously frozen tissue). The solution was then diluted with enough Weber Edsall solution to faci- litate separation of fibrous material by centrifuging for 30 minutes. The supernatant was decanted, volume measured, diluted with nine volunes of cold water, and allowed to set overnight. The actomyosin formed a precipitate which slowly settled to the bottom of the container. The clear solution above the actomyosin layer was siphoned Off. The actomyo- sin containing layer was centrifuged for 45 minutes. The precipitate, which had a jelly-like consistency, was transferred to a 250 ml. graduate cylinder. A calculated amount of 1.2 M KCl was added to redissolve the -50- 100 g. ground muscle + 400 ml. weber Edsall solution, stirred 2 hours centrifuged 30 minutes residue supernatant (digcarded) diluted with 9 volumes water, stored over- night, centrifuged 45 minutes residue supernatant redissolved by addition of 1.2 M KCl to an ionic strength of 0.6, filtered through gauze (discarded) and diluted with 9 volumes water, after precipitation, centrifuged 45 minutes precipitate sppernatant 'V dissolved in 1.2 M KCl to give final (discarded) actomyosin solution, N, with ionic strength of 0.6 Figure 3. Scheme for the isolation of actomyosin. -51- actomyosin and adjust the ionic strength of the solution to 0.6. The solution was filtered through gauze and again diluted with nine volumes of cold water. After a length of time necessary for precipitation of the actomyosin (the time varied from 4-12 hours in this step), the preci- pitate was isolated as in the preceding step. The actomyosin precipitate was again dissolved by adding a sufficient amount of 1.2 M KCl to give an actomyosin solution N, of ionic strength 0.6. This preparation gen- erally contained between 0.7 and 0.8 mg. of protein nitrogen/ml. Non- protein nitrogen analysis revealed that only protein nitrogen was present. The presence of actomyosin was verified with an ATP sensitivity test. This involved the observed drop in viscosity upon the addition of two drops of 0.014 M ATP to 5 ml. of actomyosin solution containing 0.5 mg./ml. of protein nitrogen. In all cases the time required for a 2 ml. slow delivery pipette to deliver 2 m1. of actomyosin solution was reduced by approximately one half upon addition of the ATP solution. Isolation and purification_gf actin and_myosin. Preparation of myosin was performed according to the method of Mom- maerts and Parrish (1951), and actin was prepared as outlined by Tsao and Bailey (1953). Certain modifications were necessary to adapt these pro- cedures to bovine muscle. Actin and myosin were prepared from the same muscle sample, i.e., the longissimus dorsi muscles of young calves. An attempt was made to extract these proteins from the neck muscles of year- ling steers, but this source proved to be unsatisfactory for the isolation of myosin. It was concluded that too much unseparable fat was contained in the neck muscles and when this lipid material was carried through the -52- procedure it caused a large amount of surface denaturation of the myosin preparation. In fact, a very creamy emulsion was Observed on the surface of the myosin preparation at several stages during the extraction. Longissimus dorsi muscle samples were generally Obtained from three ‘months Old calves within three minutes after death. All separable fat and connective tissue were removed from the sample which was ground through a 2 mm. plate. The entire myosin extraction procedure was carried out at 4°C as outlined in figure 4. Four hundred g. of ground muscle were extracted with approximately three volumes of cold KCl-phosphate buffer (pH 6.8, ionic strength 0.57). This mixture was stirred with a glass rod for 10 minutes, strained through several layers of gauze and the residue, H, pressed as dry as possible and retained for the extract- ion Of actin. Myosin was precipitated by a 10 fold reduction of ionic strength of the original extract, the volume of which was about 900 m1. This was achieved by dialyzing without stirring against nine volumes of water. The dialysis was carried out for 20 hours in one inch dialysis tubing. The contents of the tubing were centrifuged for 20 minutes and the result- ing supernatant discarded. Assuming that 900 ml. of original extract were Obtained, the precipitate was dissolved in 60 ml. of 2 M KCl and 60 ml. of 0.5 M KCl-phosphate buffer (extracting solution). The pH was in the range 6.7-6.9. This preparation was centrifuged for 20 minutes to remove any particulate material. After centrifugation, the preparation was diluted to four liters by slowly adding water over an interval of about 15 minutes with rapid stirring to promote the formation Of crystalloid -53- 400 g. ground muscle stirred 10 minutes with 3 volumes cold KCl phosphate buffer filtered through gauze residueLpH filtrate dialyzed against V 9 volumes water retained for actin for 20 hours, extraction centrifuged for 20 minutes precipitate sppernatant (discarded) dissolved in 60 ml. 2 M KCl and 60 ml. Of 0.5 M KCl P04 buffer, added water to volume of 300 ml., centri- fuged for 20 minutes, diluted to 4 liters, after precipitation, centrifuged precipitate Sppernatant dissolved in 60 ml. of 2 M.KCl, diluted to 4 liters, after (discarded) precipitation, centrifuged precipitate supernatant dissolved in 60 ml. of 2 M KCl, v added water to final volume of (discarded) 125 m1. myos n solution, J, in l M KCl Figure 4. Scheme for the isolation of myosin. -54- needles. When the myosin had settled sufficiently (4-24 hours) the super- natant liquid was siphoned Off and the remainder of the material centri- fuged for one hour. The precipitate was then dissolved in 60 m1. of 2 M KCl and precipitated by diluting as before. Stirring was not necessary in this step and separation of the precipitate was accomplished as in the preceding step. The final precipitate was dissolved in 60 m1. of 2 M KCl and water added to a final 125 ml. volume. The ionic strength of the final preparation, J, was about 1. Protein nitrogen concentration of this solution was about 1.5 mg./ml. MOmmaerts and Parrish (1951) stated that preparations of rabbit myosin prepared essentially according to the above procedure were stable for about one week. However, the prepara- tions of calf myosin obtained in this manner began to show evidence of de- naturation £124 to 48 hours as evidenced by the appearance of insoluble shreds in the solution. It was found that the myosin preparation was more stable when stored in a salt solution of high ionic strength, i.e., l M KCl. Preparation Of actin acetone powder, K, was performed at room temp- erature according to the scheme in figure 5. The residue, H, from the myosin extraction was washed with four volumes of 0.05 M NaHCO3 to adjust the pH to 7.0. This was followed by washing with 10-15 volumes of water. Each washing was carried out for 20 minutes under constant stirring with a magnetic stirrer. The washed residue was separated from the washing solution by straining through several layers of gauze. After washing with water, the residue was pressed as dry as possible and then macerated for one minute in a Wering blendor at full Speed with 5 volumes of n- residue -55- Residue, H, from figure 4, washed 20 minutes with 4 volumes of 0.05 M NaHCO3 filtered through gauze washed with 10-15 volumes water, filtered through gauze resi ue macerated in blendor for one ‘minute with 5 volumes n-butanol, after 112 hour, filtered through gauze residue residme \/ residue washed with 3 volumes cold acetone, treated for 20 seconds in blendor, filtered through gauze washed with 3 volumes acetone for 20 minutes, filtered through gauze washed with 3 volumes acetone for 20 minutes, filtered through gauze filtrate (discarded) filtrate ‘V (discarded) filtrate i (discarded) filtiate (discarded) filtrate (dis arded) residue filtjate ‘ldried under exhaust hood for 12 hours (disdhrded) actin acetone powder, extracted with 25-30 volumes cold water for 30 minutes, vacuum filtered residue filt te (discarded) actin soluj:on,M Figure 5. Scheme for the isolation of actin. -56- butanol. The butanol was precooled so the temperature in the blendor did not rise above 20°C. The butanol treatment dissolved the contaminat- ing lipid material and denatured most of the remaining sarcoplasmic, myosin, and stroma proteins. After one-half hour, the butanol was strained through gauze and the residue washed with three volumes of cold acetone. The lumps of debris observed at this step were disintegrated by treating for a few seconds in the Waring blendor. The acetone was strained through gauze and was followed by two more washings of 20 min- utes each in three volumes of cold acetone. The preparation was stirred occasionally with a glass rod during these washings. After the final acetone wash was strained, the residue was placed in a shallow layer in an Open container and dried 12 hours under an exhauSt hood. Actin was extracted from the resulting powder, K, by stirring 30 minutes at 4°C with about 25 - 30 volumes of cold water. A higher yield could be ob- tained if each 100 ml. of water used to extract actin contained about 20 mg. ATP. The actin solution was separated from the residue by vacuum filtration through Whatman No. 41H filter paper. The protein nitrogen concentration of the solution Obtained in this manner was about 0.25 mg./ ml. This was concentrated to the necessary nitrogen content by pervapor- ation. Tests for confirmation 311i purity 9.13. mygsin and actin. An ATP sensitivity test for myosin purity was the same as that pre- viously described for actomyosin. A.pure solution of myosin would not exhibit a change in viscosity upon the addition of ATP. Therefore, any reduction in viscosity caused by ATP would indicate actomyosin contamin- ation of a myosin preparation. All of the myosin preparations obtained -57- Showed a slight drop in viscosity upon the addition of ATP, indicating a slight contamination with actomyosin. Equal volumes of actin and myosin were mixed to observe the forma- tion of "artificial" actomyosin. All actin and myosin preparations were tested in this manner. In all cases, a gel-like precipitate of actomyo- sin was observed upon the reduction of the ionic strength of the mixture to 0.3. Actin extracted by the method of Tsao and Bailey (1953) is in the G form. G actin can be transformed to the viscous F form by adjusting the pH to 7 and adding 0.1 M KCl. All actin preparations responded to this test with a great increase in viscosity. In most cases, the G actin preparation, which was only slightly more viscous than water, was trans- fOrmed to a semi-solid gel of F actin. One sample of calf myosin in 1‘M KCl was subjected to ultracentrifu- gation in a Model L Spinco ultracentrifuge at 59,780 rpm at 0°C. The protein concentration of the sample was approximately one percent. Sev- eral small fast peaks were observed which were concluded to be different sized actin-myosin polymers (figure 6). Similar small fast peaks were also evident in ultracentrifuge patterns of myosin presented by Johnson and Rowe (1960) and Kominz EEHEA° (1959). A large, very sharp myosin peak, indicated that the preparation was predominately myosin with a degree of purity necessary for the preparation of emulsions and inter- facial tension studies. The 520 of the myosin peak, 3.14, was slightly higher than the $20 of myosin for this concentration determined by the extrapolation of the values reported by Johnson and Rowe (1960). l -58- Figure.6.Ultracentrifugal sedimentation diagrams of myosin at 32 minutes and at 64 minutes (in l M KCl at 59,780 rpm.) -59- To determine the amount of active actin, i.e., actin that will react with myosin to produce actomyosin, the following procedure was employed. The myosin in 2 ml. of solution, J, (ionic strength reduced to 0.5), was precipitated by the addition of 18‘ml. of water. This material was cen- trifuged, analyzed for nitrogen and the total nitrogen in the supernatant calculated (this value was denoted as X). In all cases, this supernatant contained no nitrogen. Next, 2 ml. of the above myosin solution, J, was mixed with an equal amount of actin solution, M (ionic strength - 0), and diluted to 16 ml. The preparation was centrifuged to remove the resulting actomyosin. The total amount of nitrogen in the supernatant from the actin-myosin mixture was determined (this value was denoted as Y). By subtracting X from Y, the amount of inactive actin and other impurities in 2 m1. of the actin preparation was determined. The amount of active actin was determined from the difference between nitrogen content of in- active actin/ml. and nitrogen content of actin solution,M,/m1. Actin con- tained eight percent and myosin preparationscontained no measurable non- protein nitrogen as determined by the TCA method. Interfacial tension measurements. Clayton (1928) described a drOp volume method for determining rela- tive interfacial tension of emulsion systems. A similar apparatus was constructed to determine the relative interfacial tension between soybean oil and the previously described protein solutions, S, N, M, and J. A 10 ml. buret was solidly mounted in a vertical position on a ring stand. A five inch section of one-quarter inch glass tubing was formed in the shape of the letter J. The short end of the J tube was drawn to -60- a capillary diameter and the long end was connected by means Of Tygon tubing to the delivery end of the 10 ml. buret. The J tube and the buret were filled with soybean oil by immersing the J tube in Oil and applying a vacuum to the Open and of the buret. For the determination of drOp volume, the J tube was wiped completely free of all oil and immersed in 25 ml. of the protein solution (concentration, 0.25 mg. nitrogen/m1.) to be studied. The protein solution was contained in a water bath main- tained at 25°C. The stOpcock on the buret was fully Opened and the drOps of oil that were formed and released from the end of the capillary were counted. The oil was allowed to flow until approximately 1 m1. had been used. The volume of oil used and the number Of draps correSponding to this volume of oil were recorded. From these data, volume of oil/drOp could be calculated. Relative interfacial tension was calculated as fol- lows: the oil droppvolume in the protein solution. the Oil drOp volume in the protein solvent. A low relative interfacial tension should have indicated good emulsifying prOperties of the protein in question. Emulsion preparation and evaluation. Emulsions were prepared in a manner similar to that described by Swift‘gg El. (1961). The principle difference between the two methods was the stirrer Speed. Swift 2; EL. used a stirrer Speed of 13,000 rpm., while the stirrer Speed used in these experiments was 1750 rpm. A Light- nin Model L stirrer equipped with a three bladed prOpellor with a diameter of 5 cm. was used. In most cases, the emulsions were prepared in one pint Mason jars, as they proved to be more durable than glass beakers. Oil -61- was delivered into the emulsion at an average rate of 0.9 m1./sec. with a glass tube connected by Tygon tubing to a 300 ml. separatory funnel. All emulsions were prepared at room temperature. To prepare an emulsion, 25 ml. of protein solution were initially placed in a jar, and the weight recorded to the nearest gram. The pro- tein concentration of this solution was varied for studying the emulsify- ing capacity of each protein. After the jar and contents were weighed, stirring was begun and the oil flow started. As oil was added to the emulsion, viscosity increased to a maximum, and at a point slightly past the maximum viscosity, a sudden drop in viscosity was easily Observed. This was the point at which the emulsion broke. At this point, the Oil flow was shut off, stirring was discontinued, and the amount of oil added was determined by weight difference. Variation between duplicate deter- 'minations was generally within 10 g., however, in a few cases it was as high as 20 g. Emulsifying capacity was expressed as g. Of Oil emulsified/ ‘mg. of protein nitrogen in the solution. After the broken emulsion was weighed, it was centrifuged for 30 minutes. This treatment separated the broken emulsion into three distinct layers: a tOp layer of clear soybean oil, a bottom layer Of water con- taining varying amounts of protein, and an intermediate layer of varying thickness consisting largely of denatured protein plus some water and oil. The bottom layer was assumed to contain protein that was not involved in the formation of an interface, as Bull (1947) and Clayton (1928) stated that proteins are denatured at a surface, i.e., an interface, and lose their solubility in the usual solvents. In other words, the proteins -62- which retained their solubility were not utilized in forming the emulsion. A sample of the protein solution at the bottom of the centrifuge tube was recovered and analyzed for protein nitrogen. This value was divided by the amount of protein nitrogen originally present and the resulting per- centage value reported as a supplementary measure of emulsifying capacity. Relative stability of emulsions prepared from the various protein preparations was determined by storing at room temperature and noting the degree of fat separation and loss of white color as the emulsions aged. A white color indicates small drOplet size or a high degree of diSpersion (Clayton, 1928). As an emulsion begins to acquire the color of its fat component, this is an indication that drOplet size has increased and the emulsion stability has decreased. Emulsions for stability tests were prepared with 25 ml. of protein solution (concentration 0.5 mg. of pro- tein nitrogen/m1.) and 200 g. of Oil. When the protein in question would not emulsify 200 g. of Oil, the amount of Oil was reduced to a suitable level. Depolymerization 9: proteins 21 papa. The protein solution to be treated was dialyzed against three volumes of 81M urea for seven days at room temperature. At equilibrium the pro- tein was in a 6'M urea solution. ‘pH adjustment 2; protein solutions. Emulsion stability and emulsifying capacity of proteins were studied in the acid, alkaline and neutral pH ranges. The pH of the protein solu- tions was adjusted by the addition of solid reagents. The pH of all pro- tein solutions as extracted, was near neutrality, except the sarc0plasmic -63- fraction, which was approximately pH 5.5. An increase in pH from neutral- ity was achieved by the addition of Na2C03 or NaH003 and a decrease was attained by the addition of KH2P04. The pH of the sarcoplasmic protein solution was adjusted to neutrality from 5.5 by the addition of K2HPO4 and KH2P04 in a predetenmined ratio. Na2003 was used to adjust the sar- c0plasmic protein solution to an alkaline pH. Calculated amounts of the above materials were added to produce an increase in ionic strength of 0.05. The ionic strength of the solutions which were not changed in re- gard to pH, i.e., those which were studied as extracted, was increased by 0.05 with KCl in order to remove the variable of ionic strength from the pH experiments. RESULTS AND DISCUSSION Part I Post mortem variation ig_protein composition. Bate-Smith (1934a) develoPed a long procedure to partition the pro- teins of beef muscle and applied this to only a few animals. This proce- dure utilized repeated extractions with a salt solution to quantitatively recover the fibrillar proteins. Such a procedure usually furnishes rather variable results. Since he reported the results of the above experiment, very little information has been reported on the complete protein composi- tion of beef muscle, except in connection with studies of changes during carcass aging or freezing, and in these cases usually only changes in solubility were studied. In such studies it would be beneficial if total amounts of the major nitrogen containing components were determined. If these were determined, changes in protein solubility could be reported as a percentage of the total protein fraction. Values reported on this basis would be more meaningful because the present study has shown considerable differences exist between animals in regard to the total amounts of the major protein components, esPecially the fibrillar fraction, in which so much interest has been directed in connection with aging and freezing stu- dies. The fractionation procedure outlined in the previous section could facilitate a relatively rapid and reasonably accurate determination of nitrogen components in muscle. The procedure outlined could be carried out in duplicate in one day. In order to make a complete analysis, some type of connective tissue analysis would be desirable, as connective tissue -64- -65- was determined by difference in this procedure and such a determination reflects all the errors involved in all the separate determinations of the other components. Ritchie ggugl, (1963) reported values of collagen determined by the hydroxyproline method of approximately 1.3 g. collagen nitrogen/100 g. total nitrogen for beef longissimus dorsi muscles (1.3 percent). This differs considerably from the values for the same muscle obtained in this study (0.5 percent). Accurate determinations of such small amounts of connective tissue would be practically impossible to determine by difference. ‘Muscle samples of 28 animals involving three different classes of beef cattle were fractionated. Twelve of these involved the infrappinatus muscle, which is a muscle of the shoulder and of intermediate connective tissue content and 20 involved the longissimus dorsi muscle, which is of low connective tissue content. Both longissimus dorsi and infraSpinatus muscles were fractionated from four of these animals which explains the discrepancy in total number of muscles and animals studied. Data Obtained from these studies are outlined in tables 2 and 3. From these data there appears to be little difference between classes of animals (table 2) in regard to nitrogen composition, at least for the infraspinatus muscle. Noticeable differences occurred in the case of the fibrillar protein fraction and stroma fraction. It appears that bulls possibly have a high- er percentage of fibrillar protein, which may in some way account for the supposed superiority of bull meat for the manufacture of sausage products, as, according to Hamm (1960), it has been fairly well established that the fibrillar fraction is primarily reSponsible for the water-holding capacity of meat. Not only do bulls have a higher percentage of nitrogen as fibril- -66- lar protein nitrogen, but they also have a greater amount of total nitro- gen per unit of tissue weight by virtue of their lower fat content. The combination of these two factors results in a considerable increase in total fibrillar protein per unit of muscle tissue. Table 2. Average nitrogen composition of infraspinatus muscle of three classes of beef animals expressed as percent of total nitrogen and as mg. nitrogenlg. of tissue. Nitrogen containing fraction Class of animal SarcOplasmic Fibrillar NPN Stroma Total Percent cows (3) 16 66 10 8 heifers (5) 18 64 9.5 8.5 bulls (4) 18 68.5 9.0 4.5 all animals (12) 17.5 66 9.5 7.0 Mg. N/g. of tissue cows 5.10 20.97 3.23 2.37 31.67 heifers 5.95 20.74 3.10 2.81 32.60 bulls 6.01 22.85 3.05 1.38 33.35 all animals 5.76 21.50 3.11 2.20 32.57 Std. dev. 0.75 1.76 0.49 Std. error 0.22 0.51 0.14 The four bulls from which the infraspinatus muscle was fractionated were from the group of 20 from which the longissimus dorsi muscle was fractionated. This furnished a good comparison between muscles of the same animals. There seemed to be little similarity between the nitrogen -67- Table 3. Average nitrogen composition and percent total nitrogen of longissimus dorsi muscle of 20 yearling bulls. Type of compound Total Sarcoplasmic Fibrillar NPN Stroma nitrogen Mean (% of total nitrogen) 31 62 6.5 '0.5 Mean (mg. of N/g.) 10.70 21.22 2.22 0.16 34.30 Std. dev. 0.56 0.80 0.10 0.80 Std. error 0.12 0.18 0.02 0.18 composition Of these two muscles from the same animals. The longissimus 'dggsi muscle contained almost twice as much sarcoplasmic protein and al- most 1 mg. more total nitrogen/g. Of tissue than the infraspinatus. It was possible that the value for sarc0plasmic proteins was somewhat higher than the actual sarc0plasmic protein content, due to the fact that the longissimus dorsi muscles were frozen for a short time before being frac- tionated. Mechanical damage to the intimate muscle structure by freezing could possibly have allowed a greater dissolution Of actin and myosin in the 0.05 ionic strength buffer, ultimately resulting in a slight overesti- mation of the sarc0plasmic proteins. There were considerably less of the fibrillar protein fraction and the non-protein nitrogen compounds in the longissimus dorsi than in the infraSpinatus. Standard deviations and standard errors were much lower in the case of the longissimus dorsi from bulls than from the infraspinatus muscles of the mixed group. This would be expected since this was more or less a homogenous group of bulls as contrasted with a heterogenous group Of different beef animals. Standard errors and standard deviations were not ~68- calculated for each separate class of animals for the study involving the infraspinatus. Because of the small numbers involved, such data would be rather unreliable. An analysis of variance for each of three factors was made for each class of animals, i.e., cows, bulls, and heifers, to determine if signi- ficant differences existed between samples of the infraSpinatus muscle at three periods post mortem. The three factors studied were amount of sarc0plasmic protein extracted, percent Of the total fibrillar protein extracted, and percent of the total water released (water-holding capacity). Data from only 11 of the original 12 animals were included in this study as all of the data were not available from one animal. The only significant difference shown in table 4 was the smaller amount of sarc0plasmic protein extracted at 0 time from the bulls. This was followed at 24 hours and seven days by an increase in.amount of extract- able sarcoplasmic protein to levels greater than either cows or heifers at 24-hours and seven days. This was unexpected as such differences have not been previously reported for any species. The variation in amount of sarc0plasmic protein is probably due to a small amount of actin and myo- sin being extracted with the sarcoplasmic fraction. There undoubtedly was a amall amount of myosin and actin dissolving in the 0.05 ionic strength buffer used for the extraction of sarcoplasmic proteins, however, it would seem that the amounts of actin and myosin dissolving in this buffer would be greatest at slaughter before any appreciable amount of these proteins combined to form actomyosin. NO significant differences in regard to amount of sarc0plasmic protein were noted between individual -69- Table 4. Means and standard errors for amount of sarc0plasmic protein extracted at three periods post mortem and for three classes Of animals (mg. ng. tissue)p(l) Period Standard Class 0 24 hours 7 days error cows 4.73 5.47 5.50 0.17 heifers 5.75 6.16 6.62 0.53 bulls 4.70* 7.10 7.70 0.53 (1) The means underlined with the same line do not differ significantly. * P < 0.05 animals in each class. In each class the trend was an increase in extract- ability Of sarcoplasmic proteins as the carcass aged up to seven days, however, the increase was not significant except in the case of bulls from 0 time to 24 hours. Changes in fibrillar protein solubility are presented in table 5. Periods were not listed in chronological order because Duncan's Multiple Range Test (1955) requires that the means be listed in order of increasing size. The changes Observed in this table are of particular importance because of the relation this protein fraction has to water-holding capa- city of meat. In the case of cows and heifers, fibrillar protein solu- bility was greatest at slaughter, decreased sharply to 24 hours and then increased slightly to seven days. Bulls again proved to be the exception. At 0 time fibrillar protein extractability was at its highest level, followed by a sharp decrease to a low at 24 hours. The solubility then rose at seven days to a level almost as high as at slaughter. It is also interesting to note that the solubility of the fibrillar proteins was higher for bulls than the other two classes at all periods studied. With- -70- Out exception, fibrillar protein solubility was highest at slaughter and was followed by a sharp decrease with the onset of rigor mortis. A simi- lar sequence of events also occurs in other species, according to Bailey (1954). Table 5. Means and standard errors for percent of total fibrillar protein which was soluble at three periods post mortem among three classes of animals (1) Period Standard Class 24 hours 7 days 0 error cows 20.90 22.87 49.70** 2.58 heifers 19.24 27.52 54.98** 3.13 bulls 24.87** 51.73 57.40 3.79 (l) The means underlined with the same line do not differ significantly. **P < 0.01 Several workers (Turner and Olson, 1959; Hamm, 1960) have related water-holding capacity to fibrillar protein solubility. In the present research, there seemed to be very good agreement in this reSpect (compar- ing data in tables 5 and 6). For cows and heifers, water-holding capacity (which is inversely related to percent water released, which is reported in the present study) followed a similar pattern to fibrillar protein solubility, i.e., it was highest at slaughter, dropped to a low at 24 hours and gradually increased to seven days but never reached the capacity observed immediately after slaughter. The water-holding capacity for cows and heifers at slaughter was significantly higher than at the other two periods. No significant differences in reSpect to water-holding capacity were observed between periods post mortem in the case of bulls, even -71- though a decrease was noted at 24 hours. For bulls, the water-holding capacity followed the pattern for fibrillar protein solubility very close- ly; water-holding capacity was high at slaughter, decreased to 24 hours and recovered in seven days to its original value at slaughter. Table 6. Means and standard errors for percent of total water released at three periods post mortem and for three classes of animals (1) Period Standard Class 0 7 days 24 hours error cows 45.67* 47.20 49.60 0.61 heifers 45.76* 47.62 48.90 0.83 bulls 46.57 46.57 48.03 0.82 (l) The means underlined with the same line do not differ significantly. *P < 0.05 Information Obtained on differences between classes of beef animals in respect to the above three factors would be of interest. As the numbers of animals in all classes were not equal, an exact analysis of variance would have involved a series of rather difficult and time consuming calculations. According to Snedecor (1946), an approximate analysis of variance would provide a reasonably accurate test providing interaction was non-significant. Interaction was judged by inSpection to be non-significant, and the approximate analysis was performed. The non-significance of interaction was confinmed by the small F values Ob- tained from the approximate analysis of variance presented in tables 7, 8 and 9. -72- Table 7. Analysis of variance (approximate) of sarcoplasmic protein among three classes of animals and threepperiods post mortem. Sums of Degrees of Mean Source squares freedom square F value clauses 2.7 2 1.35 3.65* periods 3.9 2 1.95 5.27* interaction 2.0 4 0.5 1.35 between 8.6 8 within 30.7 24 1.28 x .29(1) - 0.37 total 39.3 32 0.29 is the factor used to correct the error term for the F test. (1) * P < 0.05 Table 8. Analysis of variance (approximate) of percent of total fibril- lar protein which is soluble at three periods post mortem among three classes of animals. Sums of Degrees of Mean Source squares freedom square F value classes 305 2 152.5 2.09 periods 1600 2 800.0 10.98** interaction 223 4 55.7 0.76 between 2128 8 within 6038 24 251.4 x .29“) -= 72.9 total 8162 32 (1) correction factor **P < 0.01 -73- Table 9. Analysis Of variance (approximate) of percent of total water released amongpthree classes and three periods post mortem. Sums of Degrees of Mean Source squares freedom sguare F value classes 0.5 2 0.25 0.15 periods 12.3 2 6.15 3.72* interaction 2.1 4 0.42 0.25 between 14.9 8 within 136.8 24 5.70 x .290) - 1.65 total 151.7 32 (l) correction factor * P<0.05 The only difference between classes of beef animals (tables 7, 8 and 9) was in regard to the amount of sarcoplasmic protein. In the analysis of variance, (table 7), this was barely significant at the 0.05 level, however, when Duncan's Multiple Range Test (1955) was applied to the means (table 10), the difference was not significant. The discrepancy could no doubt be traced to the approximate analysis of variance. The approximate analysis of variance also furnished a test for over- all differences between periods post mortem for percent water released, amount of sarcoplasmic protein and percent soluble fibrillar protein (table 11). The means listed in tables 10 and 11 for these factors were not true means, .but each was merely the average of three means (one mean for each class of animal). Differences between periods for percent water released and amount of sarcoplasmic protein were barely significant at the 0.05 level. These differences were not considered important because -74- an exact analysis was not performed. In the case of soluble fibrillar protein, there is little doubt that a definite difference does exist and that solubility is greatest immediately after death. Table 10. Means and standard errors of three factors, over three periods ppost mortemppfor three classes of animals. (1) Class of animals Standard Factor Cows Heifers Bulls error % water released 47.49 47.47 47.00 0.74 mg./g. sarc0plasmic protein nitrogen 5.57 6.17 6.50 0.35 % fibrillar protein soluble 31.16 33.91 44.67 4.95 (1) The means underlined by the same line do not differ significantly. Table 11. Means and standard errors of three factors, including 11 animals, for three periods post mortem and showing significant differences. (1) Periods Standard Factor 0 7 days 24 hours error % water released 46.00 47.06 48.84* 0.74 mg./g. sarcoplasmic protein nitrogen 5.05* 6.60 6.24 0.35 % fibrillar protein soluble 54.03** 34.04 21.67 4.95 (l) The means underlined by the same line do not differ significantly. * P<0.05 ** P‘< 0.01 There is the possibility that the changes in muscle protein behavior Observed at different periods post mortem in this study are not exactly those that would occur in the uncut infraspinatus muscle under similar conditions. Paul and Bratzler (1955) reported that the tenderness of a -75- muscle could be altered by cutting the muscle before rigor had occurred. Locker (1960) reported that merely loosening a muscle from either its origin or insertion resulted in a decrease in meat tenderness. Consi- dering these facts, the normal changes occurring during and after rigor may have been altered after the section of the infraspinatus was removed for analysis immediately after slaughter. Table 12 contains the mean muscle pH and standard error at three periods post mortem for each class Of animals. The pH behavior was simi- lar for all classes and followed a definite pattern, i.e., pH was highest at slaughter (near neutrality), drOpped to a low at 24 hours (pH 5.6) and remained at this pH for considerable time. This pH pattern was in agree- ment with normal post mortem pH changes in beef muscle as outlined by Bate-Smith (1948). In this study the bulls did appear to attain a slight- ly lower ultimate pH than the other two classes. Table 12. Means and standard errors for pH of three classes of beef ani- mals at three different periods post mortem. Period Class 0 24 hours 7 days cows (3) A i 6.56 5.87 5.83 32 (1) 0.13 0.10 0.06 heifers (5) x 1 6.50 5.72 5.82 s,2 ( ) 0.06 0.05 0.05 bulls (3) x (1) 6.60 5.53 5.52 SR 0.07 0.02 0.07 (1) Standard error -76- Tenderness study This work was originally designed to investigate muscle protein char- acteristics in relation to their utilization in sausage products, however, during the course of the research an excellent Opportunity was presented to study the relationship of intracellular protein characteristics with tenderness. It was decided to use the previously described fractionation techniques to study protein characteristics in relation to tenderness. ‘Wierbicki 33 El- (1954) were among the first to carry out an experi- ment to investigate the role of intracellular muscle protein in tenderness variation. These workers reported post mortem changes in protein extract- ability that appeared to be related to tenderness. Kamstra and Saffle (1959) produced definite differences in tenderness of paired hams by a1- tering the normal course of rigor and thereby changing some protein solu- bility characteristics. The American Meat Institute Foundation (1960) stated that the ratio of sarCOplasm to myofibrils is apparently directly prOportionate to the amount of work the muscle is required to do. From the present study, it appeared that this ratio was inversely related to the amount of work a muscle performs. The corresponding ratios Obtained in this research were 18/68.5 for the infraspinatus and 31/62 for the longissimus dorsi. It is commonly accepted that the muscles of the shoul- der, of which the infraspinatus is a good example, perform more work than the muscles Of the back, e.g., longissimus dorsi. If a relationship be- tween this ratio and the work performed by a muscle actually existed, be it direct or inverse, there was the possibility that this ratio could be related to tenderness. Consequently, this aspect was investigated. -77- Table 13 shows the tenderness measurements, water-holding capacity and amounts of the more important nitrogen containing fractions of the longissimus gggga of the 20 bulls involved in the tenderness study. The only large variations between animals in this table were the values for tenderness and fibrillar protein solubility. Total nitrogen, sarc0plas- mic protein, fibrillar protein, water-holding capacity, and non-protein nitrogen were remarkably constant from animal to animal as shown by small standard deviations and standard errors. Non-protein nitrogen for indivi- dual animals was not shown in table 13. The mean (2.22), standard deviation and standard error are shown in table .3. Simple correlation coefficients were calculated between tenderness (measured by shear and panel) and sarc0plasmic protein nitrogen/total fibrillar protein nitrogen, soluble fibrillar protein nitrogen/total fibrillar protein nitrogen, and percent of total moisture released (table 14). Tenderness as measured by both shear and panel, was highly corre- lated with fibrillar protein solubility (r = -0.69 and 0.59 reSpectively). Correlations of the other factors with tenderness were all very close to the 0.05 level of significance, however, only one r value was signifi- cant at this level. This was the correlation between water-holding capacity and tenderness as measured by the shear (r = 0.49). Hamm (1960) reported that a correlation existed between water-holding capacity and tenderness, but that the relation appeared only if the differences of water-holding capacity of meat were relatively great. In other words, a high correlation between water-holding capacity and tenderness will usually be found only in extreme cases. Tenderness, water-holding capacity and nitrogen composition of the longissimus dorsi of 20 bulls. Table 13. Nitrogen composition mg. N/g. Water-holding Total nitrogen Soluble fibrillar Tenderness Bull NO. capacity Fibrillar SarcOplasmic released Panel ° Shear 34.5 8.20 10.60 22.2 10.1 47.6 5.4 4.1 10.60 247 34.3 21.6 10.4 46.5 12.30 23 34.1 13.40 15.0 20.4 10.2 45.3 7.3 9.05 9.27 12.50 33.5 20.9 10.1 45.4 7.4 4.7 37 8.2 35.3 14.6 21.3 10.7 46.3 32 «JQ'NIQ'WS 20.7 11.3 46.6 6.0 9.68 9.82 7.33 10.64 15 d‘Ut 8.0 17.4 20.7 21.3 11.1 11.9 43.9 42.1 6.8 6.9 25 50 20 700 361 35. 34. 11.2 20.6 10.2 43.6 5.4 7.7 11.6 19.6 10.8 46.3 7.41 8.29 8.37 7.78 .40 32.3 34.1 12.4 20.9 10.1 44.3 7.6 14.2 20.5 10.1 44.1 6.3 22 34.1 33.3 15.0 9.6 18.2 23.3 21.5 10.7 10.7 43.3 45.6 6.8 5.8 21 672 O‘OICDQ' 34.8 33. 12.2 21.3 21.4 11.5 10.2 45.0 46.2 7.3 5.8 7.30 9.58 9.38 8.83 10.91 47 35. 35. 10.2 22.2 11.2 46.7 6.5 720 14.0 21.0 11.8 44.7 6.5 29 42 8.2 11.4 21.6 10.4 47.1 3.5 33.8 21.4 10.4 47.1 7.0 8.57 12 34.3 12.2 21.2 10.7 45.4 6.24 1.16 9.35 1. Mean 0.80 3.04 0.80 1.48 0.56 51 Std. dev. Std. 0.18 0.68 0.18 0.12 0.33 0.26 0.34 error -79- Table 14. Correlation coefficients for various factors related to ten- derness (tenderness measured by shear and panel) Factor Tenderness Shear Panel Sarc0p1asmic N - Total fibrillar N 0-43 0.41 Soluble fibrillar N .. *7? *1): total fibrillar N 0°69 0.59 percent of total moisture released 0,49* -0.40 Part II The effect of ionic strength on emulsifying capacity of actomyosin is presented graphically in figure 7. The similarity of the curves for protein solutions in 0.3 M K01 and 0.6 M K01 indicated that ionic strength, at least in this range, had no apparent influence on the emulsifying cap- acity of actomyosin. An ionic strength of 0.3 or 0.35 was used in many instances throughout this experiment because this approximated the ionic strength of the aqueous phase of a normal sausage batter. The shape of the above curves was characteristic of emulsifying capacity curves for meat proteins which were studied in this experiment. Without exception, the proteins studied exhibited a greater emulsifying capacity as the protein concentration was reduced. This observation may be of practical significance to the sausage industry as the increased efficiency for stabilizing emulsions at reduced concentrations may par- tially compensate for the post mortem reduction in protein solubility shown in part I of this research. -30- The emulsifying capacity curves (figures 8 and 9) indicated that the sarc0plasmic fraction had a higher emulsifying capacity in the ab- sence of salt. When protein concentration was plotted against total grams of oil emulsified, a marked difference was observed. The curve in case of the water solution was practically linear while a sigmoid type curve was obtained in case of the salt containing protein solution. In the presence of 0.6 M.KCl the total grams of oil emulsified remained constant for a range in protein concentration from 0.3 mg. nitrogen/ml. to 1.0 mg. nitrogen/m1. In this range approximately 210 grams of oil were emulsified, regardless of protein concentration. Swift gt 31. (1961) reported that the water soluble protein fraction in the absence of salt was incapable of stabilizing an emulsion and Han- sen (1960), using histological techniques, found that the water soluble protein fraction was not observed at the oil-water interface of a sausage emulsion. The data in figure 8 tended to contradict these findings, however, in stability studies discussed later, it was found that emul- sions stabilized with the water soluble proteins in salt solution were considerably more stable at normal pH's than emulsions stabilized with this protein fraction in the absence of salt. The behavior of myosin and actomyosin could not be studied at low ionic strength or in pure water as they were insoluble under these condi- tions. However, actin once extracted from muscle tissue was completely soluble in water. Curves representing the emulsifying capacity of myosin in 0.3 M K01 and actin in both 0.3 M K01 and water are presented in figure 10. Obvious differences occurred in the emulsifying capacitflas -31- of these fractions. The presence of salt greatly depressed the effect- iveness of actin. The emulsifying capacity of myosin was about midway between the above mentioned actin solutions. This relationship was al- ways observed regardless of protein concentration. About seven percent of the nitrogen in muscle is TCA soluble, i.e., non-protein nitrogen. Swift gtflél, (1961) were of the opinion that non- protein nitrogen compounds had no role in the formation or stabilization of emulsions. In the present study the phosphate buffer extracted sar- c0plasmic fraction was subjected to dialysis which removed practically all of the non-protein nitrogen and caused the precipitation of the globulin X fraction. Curves of emulsifying capacity of this fraction before and after dialysis arepresented in figure 11. From these curves it appeared that neither globulin X nor the non-protein components was involved in emulsion formation. A noticeable difference was observed only at the lowest concentration, where the emulsifying capacity of the non-dialyzed fraction was the highest. A water extracted sarcoplasmic fraction was also dialyzed to remove non-protein nitrogen. A complete curve was not determined in this case, but approximately identical emul- sifying capacities for dialyzed and non-dialyzed protein solutions at several protein concentrations indicated that non-protein compounds had no effect in forming emulsions. The effect of pH on emulsifying capacity was studied with the acto- myosin and sarc0plasmic fractions (figures 12 and 13). The pH effect in this regard seemed to be quite small in that the only noticeable differ- ence due to pH occurred in the case of actomyosin (figure 13), in which -82- . AHUM S m.o cam Z c.o a“ :Hmuoumv mmmcd moooovm CH 2 samuOMQ we GOMumuucmocoo .m> awm0%EOuom vmflwwwsa wo xufiomamo mafiszmHJEm .m wpnwwm A.a8\z .wEV cowuwuucmucou z awouOHm To 4.0 To Nd no . . q . om Q 10m 3 )w 000 . T. S OT: 1)./M3 / OT: “4. .31.". .100 / m3 Q08 .d 9 N3 (T: «J. rA .nom 8m 2 m.oll|ll.lx HUz 2 0.0 o -83.. .m> pofimeHDEo Awe mo OCH 0 O O m N .—« O Lfi N Total g. of oil emulsified O O ("1 0mm Ao n :V wmmnm wsowsvm aw Camacho we cowumuucmocou .w was :Hououa owEmmaaoopMm MHLDHOm Houmz mo zuMUMQMO wc«%MMmH:Em AHHo\z .wEv newumuucoucoo z :Hoqum md 06 md qd md Nd ad 1 _ 11 4‘ I _ . wowmmeDEw ago we .m Houou .m> z awououm llllll o muwomamo w:w%MfimH:Eo .m> z chuOMQ o 0H ON on oq cm 00 (N 'Sm/Iro ;o '8) Kuroedeo BUTKJISIan .m muowfim -34- .Aquz z c.o CM :Hoooual owns; maoosvn CH : awesoun mo :oflumwucoocoo .m> woflwflmfisao HMO mo .w vcm seamene UMEwmaeoonmm mHQDHOw swam: mo xuflownmo w:MAWHm~:Em .m A.HE\z .mEV scammpucmocoo z :waOHQ N.le ampllllbwwlilbmow mwo n.o 0.0 m.o q.o m.o w.o H.s n OMHW ~ 4 . q . 4 a i d l coagwmasao HMO .w HMoOu .m> z :Hououe.| In I..lx OOH f. xUflommmo wcfixmflmHDEo .m> z Camacho o #w 0 . Ina owa .0H d o e m l . L... . \ n n n 1... , \ 8.. m. m o I. e . A \ .JMH 1... CON I \ quO m. .l _ / \ TIL?) O / f _ \ g m 3 o 8 e _ \ \ H mm. m . :1 l I. v: I I. x I: g l \ . u . m m. 1 \ a CNN \ .. A m \ mm T \ \ \ u cm x OQN 10¢ 9 shaman .ommcm mucus—um 5 2 2580.3 00 cowumuucoocoo .m> cflmoze was :Huow mo zuwommmo wcTCMmHDEm A.HE\z .wEV cOHuwuucooaoo z afimuowm m.0 «.0 m0 «.0 H0 . 1., my I. 1.1 .a I. 4 -85- 30M, 2 m.8 camozfi lilo Aaox 2 m.00 swoon IIIII x AcoquHOm Houmzv .5qu Ill 0 ON 0 0 0 on O \1’ Ln (N “gm/11° J0 '8) Auroedeo Burfigrsmmg O \C .\. w . 0H 93w?“ -86- .mmmsa wsomovm :0 z chuoud mo coHumpucoocoo .m> mwmxamww Hmuwm 0cm oMOwon A0.m u :0 .mo. n 5 hwouompuxw pmmmonv mswmuOMQ oHEmmHaoopmw wo xuwomdmo wcwameJEm AH mpnwwm A.HE\Z .wEV :oHumnucoocoo z :«muopm we 1-- r 0.0 «no To io 4 i J] 1.1 A q a o 50H 1 mm as )m 8 n . T. L we 0 w. T31. ,A o 1. In 1.8 / m o E 393230 umuwm I llll-.. «0 N w (I. mflmszwv ouowon o ,M 1 ow L 00 .Hfio mo vcflx wcm in An kuomwwm mm wmmaa mnowovm mo COflumpucmocoo z ckuOHa .m> mcwquMQ EmeQOUHMm mHLDHOm pmumz mo zuwomamo wcfizmme35m .NH wnnwflm A.HE\Z .wEv coHumHucwocou Camacho 0.H 0.0 0.0 5.0 0.0 m.0 N.0 m.0 N.0 H.0 0 . . HHo cmmn>0m — l 7 RV . A 05 ma III 0.5 mm IIIIII «o voom n.m ma lacuuocr 0H ON on 0c 0m 00 On (N 'Bm/Iro go '8) AJIaedeo EurfigrsInmg Am0.0 u :v :0 x0 kuowwwm mm omega moomsvm GM 2 cfimuoua mo cofiumuucmucoo .m> cho>EOuom uoflmwhna mo huwummmo w:H%MflmH:Em .MH ouswflm A.HE\z .wEV cofiumuucmuaou z saquHm m.o To 90 To 4.9 TP who fio o . . . q q q 1 q o Ilnwt’lnflllonll O // IX/ /0 [I / / l/ O I / )3 / // mom a . o // um ofi . 4 JH / OrA / 1.1.. & o // mm% _ o ,x .3 . e d / Na (3 T.- I m w.m ma IIIII Ix / a .8 53 mallullo , N; me o / o .3 mm -89- case the emulsifying capacity was lowered slightly at low pH. At pH 5.8 actomyosin is approaching its isoelectric point which probably has some effect on its ability to form at an interface. Data obtained from pH studies involving two kinds of oil (soybean and cottonseed) are presented in figure 12. At lower protein concentra- tions apparent differences between the two oils were observed, however, the relative effect of pH for each oil did not differ, indicating that res1lts obtained from one oil could be applied to another oil. Information on stability of emulsions prepared from the various protein fractions at various pH's and ionic strengths is presented in tables 15 and 16. Actomyosin and myosin were studied at only one ionic strength, 0.35. The low emulsifying capacity of actin, myosin and acto- myosin at low pH necessitated a reduction in the amount of added oil. These proteins produced very unstable, short lived emulsions at low pH. Actomyosin and myosin at near neutral pH's produced very similar emul- sions which were superior to emulsions stabilized by any other proteins at any pH or ionic strength. Myosin and actomyosin also produced fairly stable emulsions at alkaline pH's, although these were not as stable as those near neutrality. Actin produced emulsions with extremely low stability at all pH's and ionic strengths studied. The possibility remains that actin in the fresh muscle may act differently than the actin utilized in this experi- ment. The preparation of an acetone powder constitutes a rather harsh treatment for protein extraction, however, more than one third of the actin obtained in this manner was of the active type, i.e., it combined chemically with myosin. suooEm moONM z 5H0.0 .4900 m sea suo> soafiox sagas“ mNH Hoe z om.o o.oH owEmmHaouumm mmmw 0H ouwawmeuouca ouwna nuooam 00m moommz.z_nao.0 0.0 owammHnooumm euooam somNmM z mmoo.o mans N 303 soda.» 4344.0 oom sommNM z undo.o o.x anmmaaooumm «cause 2 mmoo.o song—NM z 385 mason NH 30H onHmh hcqmuw 00H HUM E 00.0 5.0 oHEmmHmoonm Mm mxmms N 33 30.39» 5085 00m 00M 2 00.0 0.0 owemmamooumm - mhmv 0 30a zoaamz nuooam 00m Hmum3, 0.0 oHEmenooumm moomuz z mo.o mamas N smug sum> «“403 04008. com Hue z m.o “.02 :Hmomaouu< moommz z.no.o 4x043 m.A ems: sum> snags auoosm osH Hoe z m.o o.m a44042040< mxmoB m.A swan muo> ouwna nuooam 00m 00M E 00.0 m.0 nanomeouod «ENE 2 mod mamv n oumfiwmaumuafi 300000 mmuwoo 00H HOM_Z 0.0 0.0 afimohaouo< manmum mafia NuHmOOmH> uoHoo ouauxma Hwo ammnNOm uao>aom .. ma. addflfiz .mfl n.al .udduv moaumauouomumno doamasam mo .0 dwmuonn mo mama .maoquomum awmuoun owEmmANOOHMm 0am chomaouom £uw3 0mumadun.mao«mHDEm mo mogumfiumuomumso .ma «Hams -91- moomsz z 35 moommz 2 89¢ muses qm oumwwmeumucw 30HH00 SpooEm 000 002 2 00.0 0.5 nomuuxm manuo .4839 2 mod 832 z 85 .8002 2 Sad musoa 00 mumwmeumucH soHHmh snooEm 000 002 2.00.0 5.0 nomuuxm mvsuo moommz 2 mod 3.43 N am: 333 foes... com 8M z 85 ed 38.x: mxmm3 ¢_A 2003 mum> oufisz suooem 000 H02 2 00.0 0.0 awmoa2 «ENE 2 mod mmmv 0 muMfiwmaumuaH aoaamh wmumoo 00H H02 2 00.0 0.0 awmoz2 002 2 00.0 .. _. .. .. com moommz 2 85 ed 33¢ : : : : OON MOUde z no.0 O.w GHUU¢ : : z : OON HUM z mmoo N.5 fiduo< : : = : 000 Hmum3 0.5 awuo¢ auooem 8:5: 8 v 33 302% 08> o2 48.99 2 mod fin 53¢ manmum maHH hwwmooma> Hoaoo musuxme ~00 :mmnNOm udm>aom ma. m.HB\zqu 0.0l.oaoov moHumHHmuomumno aowmasam mo .0 awmuoua mo 0008 .maoHuomnm :Hmuoum musuo 0cm a0m008 .awuom nuwa 0mumawua maowmasew mo mowumwumuomumau .0H manna ~92- A great deal of variation was observed in the stability behavior of emulsions prepared from the sarcoplasmic proteins. At low pH, i.e., 5.5, (approximately the pH of post rigor fresh meat) and in the presence of 0.35 M KCl, the sarcoplasmic fraction was superior to any other fraction in regard to emulsion stability. If no salt was present at pH 5.5, the emulsion broke in about two or three days which was very similar to those emulsions prepared with sarcoplasmic protein solutions at pH 7.0 and at ionic strength 0.05. With these protein solutions at pH 9.0 and ionic strength 0.05, emulsions had more desirable qualities and were stable for about 10 days. Proteins at alkaline pH's and ionic strength's of 0.35, produced very unstable emul- sions which broke in a few hours. Emulsions prepared with crude protein extracts (containing primarily sarcoplasmic and actomyosin proteins in appro- ximately equal prOportions) were typical of emulsions prepared with sarcoplas- mic proteins in regard to stability characteristics. Actomyosin and myosin in their native states are probably completely utilized in the interface of oil in water emulsions. The protein nitrogen in the aqueous phase recovered from a broken emulsion that was stabilized by actomyosin or myosin, probably consisted of previously denatured actomyosin and myosin and contaminating protein of sarc0plasmic origin. As the emulsions were prepared at room temperature some of the myosin and actomyosin, which are extremely heat labile, were undoubtedly partially denatured before emulsification began and their interfacial activity thereby reduced. The amount of protein nitrogen which retained its solubility in the aqueous phase after maximum emulsifying capacity was attained furnished addi- tional information on the activity of the muscle proteins in.amulsion systems (table 17). The pH effect in this regard was investigated in the case of -93- Table 17. Percent protein nitrogen remaining soluble in the aqueous phase after maximum amount of oil was emulsified. Ionic % Nitrogen remaining Type of protein pH strength soluble in the squeous phase Actomyosin 5.8 0.65 8.5 " 7.2 0.65 11.3 " 10.7 0.65 13.7 Actin 7.6 0.30 48.4 " 7.6 0.0 59.0 Myosin 6.8 0.30 10.0 Sarcoplasmic 5.7 0.0 40.2 " 7.0 0.05 72.5 " 9.0 0.05 78.4 actomyosin and the sarcoplasmic fractions. There appeared to be a definite pH effect in the case of the sarcoplasmic fraction, however, the variation due to pH in the case of actomyosin was small. The amount of sarc0plasmic protein accumulating at the interface was twice as great at the low pH (5.7) than at the higher pH's. This was reflected in the greater stability observed in emulsions prepared with sarcoplasmic proteins at low pH. At the neutral and alkaline pH, the sarcoplasmic fraction showed a great deal of variation between duplicates which was not observed with any of the other proteins studied. The amount of actin recovered from broken emulsions was in the same range as the amount of sarcoplasmic protein recovered. This was also reflected in the poor stability qualities of emulsions prepared with actin. The important observation in table 17 is the small amounts of protein recovered in the aqueous phase of actomyosin and myosin stabilized emulsions in relation to the actin and sarcoplasmic fractions. It appears that the -94- amount of protein denatured at the oil-water interface is directly related to the stability of the resulting emulsion. Interfacial tension data have been utilized for many years to furnish an objective measurement of the interfacial activity of various emulsifying agents. Clayton (1928) reported that the method yielded acceptable results with soap stabilized emulsions, however, it was found in the current experi- ment that dr0p volume was not a valid method for the study of the interfacial activity of the muscle proteins (data from this eXperiment in table 18). This same conclusion was reached by Briggs and Schmidt as early as 1915 when they studied emulsifying prOperties of gelatin. They stated that the great elasticity and strength of the semi-solid protein film allowed the dr0p the expand as if it was contained in a rubber bag which was firmly attached to the delivery pipette. Such draps become very large because they are detached from the pipette with considerable difficulty. Table 18. Relative interfacial tension of some protein solution-oil systems determined by the drOp volume method. Relative interfacial Type of protein ApH Ionic strength tension Myosin 6.8 0.35 0.89 " 8.0 0.35 0.72 " 6 M urea 0.57 " 7.2 0.30 0.52 " 8.0 0.05 0.68 Sarcoplasmic 5.5 0.0 0.63 " 5.6 0.60 0.58 " 7.0 0.05 0.83 " 9.0 0.05 0.28 " 6 M urea 0.57 Actomyosin 6.7 0.35 0.82 " 8.0 0.35 0.72 SUMMARY AND CONCLUSIONS Part I A procedure was developed to fractionate the major nitrogen containing components of muscle. This procedure was utilized to determine the protein composition of the infraSpinatus muscle of a group of 12 beef carcasses com- posed of bulls, heifers and cows, and also to determine the protein composi- tion of the longissimus dorsi of 20 yearling bulls. Four of the group of 20 bulls were the same bulls from which the infraSpinatus was fractionated. Results showed that bulls had a higher percent of fibrillar protein and more total nitrogen per unit of muscle tissue than the other two groups stu- died. The longissimus dorsi contained more total nitrogen per unit of muscle tissue and almost twice as much sarc0plasmic protein as the infraspinatus. The longissimus dorsi contained less fibrillar protein and non-protein nitro- gen than the infraspinatus. Variation in protein solubility at three periods post mortem was also investigated in connection with the fractionation studies of the infraspinatus. A slight variation was noted in the extractability of the sarc0plasmic frac- tion with a general increase from slaughter to seven days. Fibrillar protein solubility was greatest at slaughter, and decreased significantly to a minimum at 24 hours. After seven days, fibrillar protein solubility had increased only slightly in the case of cows and heifers, while in the case of bulls it increased to almost the level observed immediately after slaughter. Fibrillar protein solubility was higher for bulls than cows and heifers at all times studied. 'Water-holding capacity followed the fibrillar protein solubility pattern very closely for each class of animals. The results obtained in this -95- -96- study are possibly somewhat different from the changes that would occur in an uncut‘muscle. A portion of the data obtained from the fractionation of the longissimus Eggs; of the previously mentioned group of 20 bulls was correlated with ten- derness measurements of an adjacent portion of the longissimus dorsi. The following factors were correlated with tenderness as measured by shear and panel: sarc0plasmic protein nitrogen/total fibrillar protein nitrogen; solu- ble fibrillar protein nitrogen/total fibrillar protein nitrogen; and percent total water released. Fibrillar protein solubility was highly correlated with tenderness (r - -0.69 for shear and r - 0.59 for panel). An r value of 0.49, significant at the 5 percent level, was found between water-holding capacity and tenderness as measured by the shear. Part II Emulsifying capacity curves for actin, myosin, actomyosin and the sarco- plasmic fraction of beef intracellular muscle proteins have been determined. Without exception, the emulsifying capacity increased as protein concentration decreased within the concentration range studied. The effect of pH on emul- sifying capacity was negligible in the case of the sarcoplasmic proteins at an ionic strength of 0.05. 'The pH may have a slight depressing effect on the emulsifying capacity of actomyosin at low pH. The presence of salt greatly depressed the emulsifying capacity of actin. Variation in salt concentration between 0.3 and 0.6 M did not affect the emulsifying capacity of actomyosin. Non-protein nitrogen compounds and the globulin X fraction of the sarc0plasmic proteins were shown to have no influence on emulsifying capacity. In regard to emulsifying capacity, the proteins studied ranked from greatest to least -97- as follows: actin (ionic strength = O), myosin, actomyosin, sarc0plasmic and actin (ionic strength - 0.3). Stability of emulsions prepared with these same fractions and with a crude extract was also studied under various conditions of ionic strength and pH. At neutral pH and at an ionic strength of 0.35, myosin and actomyosin produced similar emulsions with excellent stability. The sarcoplasmic fraction produced emulsions which varied greatly in stability. The most desirable emul- sions, from a stability standpoint, prepared with this fraction were at low pH (5.5 - 5.7) and 0.35 ionic strength, and the least desirable were those emulsions prepared with protein solutions at high pH and high ionic strength. Under no conditiOns did the emulsions prepared with the sarcoplasmic fractions compare favorably with myosin and actomyosin fractions at neutral pH. How- ever, at low pH (5.5 - 5.7), which is approximately the pH of meat, the sarco- plasmic fraction produced the most stable emulsions. Emulsions prepared with actin were very unstable and under all conditions studied broke within 24 hours. An analysis of the protein nitrogen remaining soluble in the aqueous phase after the maximum amount of oil was emulsified affords some interesting data. The amount of myosin and actomyosin accumulating at the oil-water interface was much greater than the amount of either actin or the sarcoplasmic protein. Such data are generally in agreement with the superior stabilizing qualities of actomyosin and myosin. The fibrous structure of myosin and actomyosin is probably an important factor in emulsion stabilization. Such molecules have a great number of avail- able polar and non-polar side groups essential for interfacial activity opposed -93- to the sarc0plasmic proteins which are of the globular type and are relatively spherical in shape and present a minimum of such side groups. As little is known about the structure of actin, it would be difficult to eXplain' its be- havior on this basis. Results of interfacial tension studies carried out by the drOp volume method indicated that this procedure was unsatisfactory for the study of emul- sions prepared from the muscle proteins. BIBLIOGRAPHY American Instrument Co. 1961. The determination of Nitrogen by theKjel- dahl Procedure including digestion, distillation and titration. Reprint No. 104. American Meat Institute Foundation. 1960. The Science'gijeat and Meat Products. W. H. Freeman and Co., San Francisco and London. Ardenne, V. M., and H. H. Weber. 1946. Blektronemnikroskapisnhe Untersuch- ung des Muskeleiweisskorpers "Myosin". 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Effects of added sodium chloride, potassium chloride, calcium chloride, magne- sium chloride and citric acid on meat shrinkage at 70°C and of added sodium chloride on drip losses after freezing and thawing. Food Tech. 11, 74. Wierbicki, E., L. E. Kunkle and F. E. Deatherage. 1957b. Changes in the water-holding capacity and cationic shifts during heating and freez- ing and thawing of meat as revealed by a simple centrifugal method for measuring shrinkage. Food Tech. 1;, 69. APPENDIX Appendix A. Composition of solutions used for the fractionation and iso- (deionized water used in all cases) lation of proteins. -107- List of solutions: 1. weber Edsall solution (for protein fractionation, figure 2) u - 0.67, pH 9. 2. Phosphate buffer (for protein fractionation, figure l,and isolation of sarc0plasmic fraction) u - 0.05, pH 7.6. 3. K01 phOSphate buffer (for isolation of myosin) u - 0.57, pH 6.5. 4. Sodium bicarbonate (for isolation of actin) u - 0.05, pH 8.2. 5. Weber Edsall solution (for isolation of actomyosin) u = 0.67, pH 9. Composition: Solution Salt Mblarity g./1iter 1. NaCl 0.60 35.04 Na2003 0.01 1.06 2. K2HPO4 0.156 2.71 KHzPO4 0. 0035 0.475 3. KCl 0.30 22.35 KH2P04 0.09 12.24 KzHPO4 0.06 10.44 5. KCl 0.60 44.70 Na2003 0.01 1.06 -108- Appendix B. Complete calculated data from infraspinatus muscle of 12 beef animals. Soluble Sarc0plasmic fibrillar protein protein nitrogen nitrogen Animal (mg./g. tissue) (mg/g. tissue) No. Slaughter 24ihgurs 7 days Slaughter 24 hours 7 days Cows: l 4.7 5.5 6.0 10.6 5.0 5.9 2 4.3 5.3 4.9 9.7 4.9 5.1 3 5.2 | 5.6 5.6 10.9 3.2 3.4 Heifers: l 6.4 7.8 7.7 10.8 5.0 3.9 2 5.0 7.1 5.0 15.0 3.8 7.8 3 5.6 6.5 5.6 10.7 2.7 7.2 4 6.6 5.0 7.1 9.9 4.1 4.8 5 5.1 4.4 7.7 10.5 4.4 4.7 Bulls: 1 4.6 5.9 8.0 12.8 5.9 9.0 2 5.7 7.0 - 14.8 7.8 - 3 4.5 7.0 7.8 12.6 4.7 11.7 4 5.0 8.4 7.3 13.0 6.0 14.1 -109- Appendix B. Complete calculated data from infraspinatus muscle of 12 beef animals. (continued) Percent of total water released pH Animal No. Slaughter 24 hours 7 days Slaughter 24 hours 7 days Cows: 1 42.7 48.3 44.9 6.6 6.1 5.9 2 48.6 50.2 48.9 6.7 5.7 5.9 3 45.7 50.3 47.8 6.4 5.8 5.7 Heifers: 1 43.9 47.5 45.5 6.45 5.8 6.0 2 44.5 47.1 47.9 6.35 5.6 5.8 3 46.8 45.8 47.7 6.70 5.85 5.8 4 48.8 52.5 47.7 6.50 5.70 5.8 5 44.8 51.6 49.3 6.50 5.70 5.7 Bulls: 1 44.7 48.5 47.5 6.7 5.55 5.65 2 47.0 44.2 -- 6.4 -- -- 3 47.9 48.5 47.1 6.5 5.5 5.4 4 47.1 47.1 44.6 6.65 5.55 5.5 -110- Appendix B. Complete calculated data from infraspinatus muscle of 12 beef animals . (continued) Total Stroma fibrillar protein Total Non-protein protein nitrogen nitrogen nitrogen Animal nitrogen (mg. /g. (mg. /g . (mg. /g. Fat No. (mg.[g. tissue) tissue) tissue) tissue) (%) Cows: 1 22.7 -0.9 31.3 4.0 5.0 2 19.4 2.2 30.2 3.3 8.6 3 20.8 5.0 33.5 2.4 4.9 Heifers: 1 22.0 -l.4 31.1 3.4 5.0 2 20.5 4.3 34.3 3.4 5.7 3 20.6 3.2 33.5 3.6 6.3 4 22.6 1.7 32.6 2.5 3.3 5 18.0 6.2 31.5 2.5 3.2 Bulls: 1 21.2 4.3 33.9 3.1 1.8 2 24.4 -0.4 33.2 2.9 2.8 3 23.2 0.6 32.6 3.1 2.3 4 22.6 1.3 33.7 3.1 2.3 -111- Appendix C. Complete calculated data from longissimus dorsi muscle of 20 bulls. Total Sarc0plasmic fibrillar protein Soluble fibrillar Animal protein nitrogen nitrogen protein nitrogen No. (mg./g. tissue) (mg./g. tissue) (mg./g. tissue) 247 10.1 22.2 8.2 23 10.4 21.6 10.6 3 10.2 20.4 13.4 37 10.1 20.9 15.0 32 10.7 21.3 8.2 15 11.3 20.7 14.6 25 11.1 20.7 8.0 50 11.9 21.3 17.4 20 10.2 20.6 11.2 700 10.8 19.6 11.6 361 10.1 20.9 12.4 22 10.1 20.5 14.2 21 10.7 21.5 15.0 672 10.7 23.3 9.6 47 11.5 21.3 18.2 6 10.2 21.4 12.2 720 11.2 22.2 10.2 29 11.8 21.0 14.0 42 10.4 21.6 8.2 12 10.4 21.4 11.4 -112- Appendix C. Complete calculated data from longissimus dorsi muscle of 20 bulls. (continued) Non-protein Stroma protein Animal nitrogen nitrogen Total nitrogen No. (mg./g. tissue) (mg./g. tissue) (mg./g. tissue) 247 2.2 0.0 34.5 23 2.2 0.1 34.3 3 2.2 1.3 34.1 37 2.4 0.1 33.5 32 2.2 1.1 35.3 15 2.2 0.6 34.8 25 2.4 0.4 34.4 50 2.2 -0.1 35.3 20 2.2 2.4 35.4 700 2.2 1.9 34.5 361 2.2 -0.9 32.3 22 2.2 1.3 34.1 21 2.2 -O.3 34.1 672 2.2 -2.9 33.3 47 2.4 -0.4 34.8 6 2.0 0.3 33.9 720 2.2 -0.4 35.2 29 2.2 0.0 35.0 42 2.2 -O.8 33.4 12 2.2 -0.2 33.8 -113- Appendix C. Complete calculated data from longissimus dorsi muscle of 20 bulls. .(continued) Animal % of total water Tenderness No. released pH Shear Panel 247 47. 6 5. 60 10. 60 5.4 23 46.5 5.55 12.30 4.1 3 45.3 5.60 9.05 7.3 37 45.4 5.45 9.27 7.4 32 46.3 5.70 12.50 4.7 15 46.6 5.60 9.68 6.0 25 43.9 5.55 9.82 6.8 50 42.1 5.45 7.33 6.9 20 43.6 5.60 10.64 5.4 700 46.3 5.50 7.41 7.7 361 44.3 5.60 8.29 7.6 22 44.1 5.60 8.37 6.3 21 43.3 5.50 7.78 6.8 672 45.6 5.50 9.40 5.8 47 45.0 5.50 7.30 7.3 6 46.2 5.60 9.58 5.8 720 46.7 5.60 9.39 6.5 29 44.7 5.50 8.83 6.5 42 47-1 5.60 10.91 3.5 12 47.1 5.50 8.57 7.0 -114- Appendix D. Protein concentration of aqueous phase and average amount (g.) of oil emulsified for various trials involving deter- mination of emulsifying capacity. Actomyosin at threeng's, u - 0.65. Protein nitrogen concentration 4g. of oil emulsified pH 7.2 10.7 5.8 0.77 263 222 237 0.60 266 213 191 0.45 265 220 162 0.40 260 212 172 0.30 254 236 150 0.20 227 237 150 0.10 156 208 81 Actomyosin at two ionic strengthsyng 7.2 Protein nitrogen concentration g. of oil emulsified salt concentration (KCl) 0.6 M 0.3 M 0.5 278 270 0.3 237 240 0.2 200 204 0.1 168 144 Buffer extracted sarcoplasmic fraction before and after dialysis. u - 0.05, EH 5.8. Protein nitrogen concentration g. of oil emulsified Before dialysis After dialysis 0.8 341 370 0.5 316 350 0.3 278 272 0.1 185 153 -115- Appendix D. Protein concentration of aqueous phase and average amount (g.) of oil emulsified for various trials involving deter- mination of emulsifying capacity. (continued) Sarcoplasmic fraction at two ionic strengths, pH 5.4. Protein nitrogen concentration g. of oil emulsified salt concentration;(KCl) 0 0.6 M 1.2 - 240 0.9 - 214 0.7 348 212 0.5 262 p 212 0.3 233 216 0.2 205 - 0.1 153 183 Actin at two ionic strengths, pH 7.2. Protein nitrogen concentration g. of oil emulsified salt concentration (K01) 0 0.3 M 0.5 344 252 0.3 302 194 0.2 268 190 0.1 203 147 Myosin in 0.3 M KCl, pH 6.8. Protein nitrogen concentration g. of oil emulsified 0.5 299 0.3 255 0.2 235 0.1 192 -116- Appendix D. Protein concentration of aqueous phase and average amount (g.) of oil emulsified for various trials involving deter- mination of emulsifying capacity. (continued) Sarcoplasmicyproteins at threeng's using two kinds of oil. u,= 0.05. g. of oil emulsified Protein nitrogen ,pH 5.7 ng 7.0 _pH 9.0 concentration Cotton Soy Cotton Soy Cotton Soy 1.0 300 0.8 373 320 356 318 264 0.7 366 334 0.6 303 323 230 300 228 0.5 270 0.4 283 213 293 180 200 0.3 298 0.2 231 143 225 146 180 0.1 195 Room uss 0m