ABSTRACT TENDERNESS OF FREEZE-DRIED CHICKEN WITH EMPHASIS ON ENZYME TREATMENTS By Gordon H. Wells, Jr. The effects of age on the tenderness of freeze-dried chicken breast muscle were determined, using chickens ll, 20 and 52 weeks of age. The old birds (toughest) were se- lected for proteolytic enzyme treatments and evaluation of muscle tissues. Tenderness was determined with a Warner-Bratzler shear by relating shear force to area of breast muscle sheared. A correlation coefficient of 0.59 was obtained between sensory panel scores and Warner-Bratzler shear values, using cooked and rehydrated freeze-dried muscle, and a correlation coefficient of 0.80 was calculated for non- freeze-dried muscle. Tenderness of muscles was inversely related to age of birds. Sensory evaluations indicated Juiciness to be directly related to tenderness. Percentage water uptake during rehydration was directly related to tenderness (as measured by the panel and Warner-Bratzler shear) and Juiciness (as measured by the panel). More sig- nificant differences were noted when breast muscles were measured by the sensory panel than when measured by the Warner-Bratzler shear. Gordon H. Wells, Jr. Papain, ficin, bromelin and Rhozyme P-ll were incor- porated directly into the rehydration solutions. All samples were rehydrated in the enzyme solutions for five minutes. A three-minute heating time at 100°C was used to inactivate the enzymes immediately after rehydration. In— activation was complete when no increases were found in non-protein nitrogen with time. A sensory panel and an Allo-Kramer shear press were used to determine optimum tenderness of breast meat treated with various enzyme concentrations by using several pH and temperatures. Enzyme concentrations of 0.02%, 0.0008%, 0.002% and 0.002% (calculated as weight of pure enzyme/ volume of buffer) were most suitable for Ehozyme P-ll, ficin, bromelin and papain, respectively. Rhozyme P-ll, ficin and bromelin were most active at pH 5.0, while papain had.maximum activity at pH 7.0. Optimum reaction tempera- tures were 50°, 50°, 600 and 70°C for Rhozyme P-ll, papain, bromelin and ficin, respectively. Control samples were significantly more tender when rehydrated at pH 7.0 than at higher or lower pH values. This may have been due to an increase in water uptake at pH 7.0 during rehydration. The percentage of water uptake of the control samples also increased with decreasing rehy- dration temperatures. After chicken breast samples were rehydrated in enzyme solutions under optimum conditions for tenderization, they were studied histologically. Masson's trichrome stain was Gordon H. Wells, Jr. modified for use on the cooked and rehydrated tissue. Ficin was most active on muscle fibers, while bromelin was least active. The effects of Rhozyme P-11 and papain were inter- mediate between those two extremes. Ficin produced the most activity on connective tissue, papain showed some activity, but bromelin and Rhozyme P-ll demonstrated little or no activity. Enzyme-induced tenderness seemed to be more re- lated to muscle fiber destruction than to dissolution of the connective tissue. Muscle fibers affected by enzymes showed a distinct swelling, dissolution of the sarcolemma, extensive granula- tion, disappearance of nuclei and loss of cross striations. TENDERNESS OF FREEZE-DRIED CHICKEN HITH EMPHASIS ON ENZYME TREATMENTS By Gordon H.“Wells, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1965 ACKNOWLEDGMENT The author is deeply indebted to Dr. Lawrence E. Daw— son for his assistance in conducting this study and for his counsel and understanding in the preparation of the manu- script. The author also wishes to express his appreciation for the assistance rendered him by Dr. Robert K. Ringer and his laboratory assistants, Mrs. Ellen Sanders and Mrs. San- dra Pangborn. The author's appreication is extended to Dr. Lawrence Dawson, Dr. James Price, Mr. John Steinhauer, Mr. Raleigh Wilkinson, Mr. Carter Crigler, Mr. Julius Miller and Mrs. Maurice Bitchey for their interest and participation in sensory evaluations. An expression of thanks is extended to Dr. W. D. Eaten for his aid and advice in analyzing the data and to Pro- fessor James Davidson for his assistance in the procurement of chickens. A sincere eXpression of appreciation is reserved for my wife, Nora, for her proof reading, helpful suggestions and typing assistance in the preparation of the manuscript and for her patience throughout this study. 11 TABLE OF CONTENTS Page ACKNOWLEDGMENT 11 LIST OF TABLES v LIST OF FIGURES vi LIST OF APPENDICES viii INTRODUCTION 1 LITERATURE REVIEW 5 Freeze-Drying of Meats and Its Relationship to Meat Quality 5 Factors Affecting the Tenderness of Poultry Meat 12 A. PhysiolOgy and Chemistry of Muscle 12 B. Slaughter 16 C. Scalding 17 D. Picking 18 E. Excising of Muscle 19 F. Aging 20 G. Freezing 23 H. Cooking 25 Objective and Subjective Evaluation of Tenderness 2 A. Objective Measurements (Mechanical and/or Chemico—Physcial Methods) 27 B. Subjective Measurements (Sensory Methods) 29 Structure of Muscle and Connective Tissue 31 A. Muscle Structure 31 B. Connective Tissue Structure 33 Tenderization by Commercial Proteolytic Enzymes 3b Histological Considerations 39 MATERIALS AND METHODS nu Selection of Chickens an Processing and Sample Preparation #4 Rehydration 46 Chemical Determinations MB A. Protein Nitrogen #8 B. Moisture 50 Physical Determinations 50 A. pH Measurements 50 B. Heat Penetration 50 Measurements of Tenderness 51 A. Warner—Bratzler Shear 51 B. Sensory Evaluation 52 C. Kramer Shear Press 5“ Tissue Preparation for Microsc0pic Examination 56 EYESULTS AND DISCUSSION 60 Part I. Effects of Age on Tenderness 60 Part II. Effects of Enzyme Treatments on Chicken Breast Muscle 7O SUI‘M‘IARY 9 9 ill Page LITERATURE CITED 102 APPENDIX 116 iv Table # 10 11 LIST OF TABLES Effects of freeze-drying on tenderness of chicken breast muscle as determined by shear values Effects of freeze-drying on tenderness of chicken breast muscle as determined by panel scores Analysis of variance and Duncan's Multiple Range Test for Warner-Bratzler shear values of freeze-dried breast meat from birds of three different ages Analysis of variance between initial and residual panel tenderness scores of freeze— dried breast meat from birds of three different ages Analysis of variance and Duncan's Multiple Range Test for panel tenderness scores of freeze-dried breast meat from birds of three different ages Analysis of variance and Duncan's Multiple Range Test for panel tenderness scores of non-freeze-dried breast meat from birds of three different ages Analysis of variance and Duncan's Multiple Range Test for panel juiciness scores of freeze—dried breast meat from birds of three different ages Analysis of variance and Duncan's Multiple Range Test for percentage water uptake by freeze-dried breast meat from birds of three different ages Correlation analyses for panel tenderness scores and Warner-Bratzler shear values Effects of proteolytic enzyme concentration on tenderness and acceptability of freeze- dried chicken breast meat as determined by a panel Change in pH of rehydration solutions during reconstitution 61 61 63 5-5 65 67 67 69 69 71 77 Figure a 10 11 12 13 LIST OF FIGURES Shear press values of freeze-dried chicken rehydrated in papain and ficin solutions at various pH values Shear press values of freeze-dried chicken rehydrated in bromelin and Rhozyme P-ll solutions at various pH values RelationShip between water uptake of freeze— dried chicken and shear force values Shear press values of freeze—dried chicken rehydrated in ficin and bromelin solutions at various temperatures Shear press values of freeze—dried chicken rehydrated in papain and Rhozyme P-ll solutions at various temperatures Percentage water uptake of freeze-dried chicken at various temperatures Enzyme inactivation determined by micro—Kjeldahl method for non-protein nitrogen Longitudinal section of cooked freeze-dried chicken breast muscle rehydrated in 0.0008% ficin solution at pH 5.0 and 70°C. 430X. Longitudinal section of cooked freeze—dried chicken breast muscle rehydrated in 0.002% bromelin solution at pH 5.0 and 60°C. u3ox. Longitudinal section of cooked freeze—dried chicken breast muscle rehydrated in 0.002% papain solution at pH 7.0 and 50°C. U3OX. Longitudinal section of cooked non-freeze— dried and non—enzymatically treated chicken breast muscle. h3OX. Cross section of cooked freeze—dried chicken breast muscle rehydrated in 0.0008% ficin solution at pH 5.0 and 70 C. #30X. Cross section of cooked freeze-dried chicken breast muscle rehydrated in 0.002% bromelin solution at pH 5.0 and 60 . lOOX. vi \3 \O 79 87 87 90 \L) \J.) F i gu r e la 15 Horizontal section of uncooked and once- frozen connective tissue. Q30X. Horiztonal section of cooked freeze—dried connective tissue rehydrated in 0.0008% ficin solution at pH 5.0 and 70°C. vii 430K. F (J D e 96 96 Appendix Table l 4:- IO 11 12 LIST OF APPENDICES Effects of age on the rehydration of freeze-dried chicken breast meat Effects of age on the tenderness of freeze-dried chicken breast muscle as measured by the Narner—Bratzler shear press Effects of age on the tenderness of freeze-dried chicken breast muscle as measured by a sensory panel Effects of age on the tenderness of breast muscle from control chickens as measured by the Warner-Bratzler shear press and sensory panel Effects of proteolytic enzyme con- centration on water uptake, tender— ness and acceptability of freeze— dried chicken breast meat Water uptake and tenderness of freeze- dried chicken breast muscle rehydrated in enzyme solutions at various pH Water uptake and tenderness of freeze- dried chicken breast muscle rehydrated in enzyme solutions at various temper— atures Moisture content of freeze—dried chicken breasts Protein content of proteolytic enzyme preparations Moisture content of proteolytic enzyme preparations pH of buffer solutions before and after rehydration Panel score card viii Page 117 123 126 128 131 135 138 139 140 1&1 114-2 INTRODUCTION Freeze—drying, drying by sublimation, or lyOphilization is the process of removing moisture from a substance while in the frozen state. The frozen product is placed in a vacuum chamber and a controlled amount of heat is applied. Heat is used to keep the temperature of the product higher than the temperature of the ice at the chamber condenser, but not high enough to melt the product. The condenser is used to collect the moisture vapor by condensing and freezing it and thus preventing moisture from being drawn into the vacuum pump. Dehydration depends on the difference in water vapor pressure between the dry immediate environ- ment of the product and the ice in the frozen interior of the product. When a prOper relationship exists, water vapor is continuously transported away from the substance, but the ice within never melts. The rapid sublimation of moisture cools the product sufficiently to prevent thawing. Thus, product shrinkage is minimized. The structure of the dried material permits a sometimes slow but practically complete reconstitution. Conventional freeze-drying, in which meat remains frozen throughout the drying cycle, permits a re- duction in moisture content to less than 2% without an .appreciable change in product appearance. Burke and Decareau (1964) revealed that at least 20 InaJor food processors market freeze-dried foods, while in 1960 there were only two. Despite this rapid growth, in 2 some respects the process is still in its infancy as a means of food preservation. The main commercial use for freeze-dried chicken is in the manufacture of dehydrated soups, although chicken in serving-size pieces is now marketed for consumption in other prepared foods. The primary advantage of using freeze-dried chicken in dehydrated foods is its highly acceptable flavor. It has excellent color and storage stability, but a dry texture often remains after rehydration. Even more serious is the toughness of rehydrated meat, and this factor has been an important limiting factor in successful commercial production of freeze-dried meats and poultry. Not only freeze-drying but all methods of drying are known to toughen meat and poultry. One method of increasing the tenderness of meat and poultry involves the use of proteolytic enzymes. Commercial tenderizers are not completely effective in tenderizing meat. Many problems exist, including those associated with penetration of the tenderizers, uniform action, and flavor changes. Although penetration of enzymes into meat is usually limited, penetration into freeze-dried meat is quite satisfactory when the meat is rehydrated in enzyme solutions. Most of the research on freeze—dried foods--particu- larly meats-~has been published within the past ten years. IPractically all of the meat research has been concerned ‘with freeze-dried beef, while research on freeze-dried 3 poultry meat has been neglected. The following study was undertaken in an effort to provide basic information on some of the factors affecting the tenderness of freeze-dried poultry. Attention has been directed to both preslaughter and post—rigor conditions with emphasis on the latter. Preslaughter conditions were concerned with the ultimate tenderness of reconstituted freeze-dried chicken as affected by age of birds. The Warner—Bratzler and Allo-Kramer shear presses were used to evaluate tenderness objectively, while a sensory panel was used for subjective evaluation of tenderness. The Warner—Bratzler shear press requires the use of a core of meat taken at right angles to the muscle fiber plane. Since many birds have a very shallow breast muscle, it is difficult to obtain a satisfactory core for evaluation. Thus, a method for adapting the Warner-Bratzler press to chicken breast muscle was a necessary objective. Post-rigor conditions were concerned with the effects of commercial proteolytic enzymes on tenderness. Optimum conditions for enzyme activity were desirable for this study. Thus pH, concentration of enzyme, and temperature were necessary factors to control. The rehydration times were planned to be of equal duration. Microscopic observation of histolOgical sections is an exnellent method for studying the physical structure of tnuscle and the structural alterations caused by various treatments. Since microscopic examination of structural it changes brought about by these enzymes has been utilized to determine the site(s) and mode of enzyme action, histolOgi- cal sections were planned for the present study. LITERATURE REVIEW Low-temperature evaporation of water under vacuum to produce freezing, followed by sublimation of the ice was known before 1813, when William Hyde Wallaston apologetically discussed the process befOre the Royal Society of London (Flosdorf, 1945). The freeze-drying method of studying tissue structure was introduced by Altmann (1890). The first clearly recorded use of sublimation for preserving biological substances was reported by Shackell (1909). How- ever, it was not until 1935 that foods were preserved by such a method (Flosdorf, 19U5). Freeze—dried foods have been commercially produced and marketed for consumption during the past several years. Nair (1963) estimated that one billion pounds of freeze- dried foods would be produced on four million square feet of freeze-dryer shelf area by 1970. Freeze-Drying of Meats and Its Relatignship_to Meat Quality Precooked, freeze-dried chicken with good functional and organoleptic prOperties was prepared by Yao gt 5;. (1956), Tappel 33 £1. (1957), Seltzer (1961), Howe (1961) and Wells 22 gl. (1962). Yao gt 5;. (1956) found that precooked chicken meat samples dried faster than uncooked samples, due to a lower initial moisture content. They also reported that the usual mode of heat transfer to chicken was predominately by radiation. When heat transfer by conduction was increased, 5 6 the drying rate was noticeably increased. Optimum drying cycles for diced and cooked chicken meat were later pre- pared (Anonymous, 1962a). Toumy gt El. (1962) reported that platen temperatures of 65.5°, 80.0° and 93.300 did not significantly affect tenderness, Juiciness or cutting ease of precooked, sliced beef. They also stated that overdrying by two hours was not critical. The advantages of freeze—dried chicken as reported by Bird (1963) were: (1) When the best known drying techniques were used, most flavor constituents remained in the food; (2) the physical structure remained the same, so that rehy- dration was easy and rapid; (3) the product was relatively 'stable at room temperature; and (h) the product could be shipped more economically than frozen or canned food. He stated that the general impression of freeze-dried chicken was that it was not as good as the processed standard. How- ever, the addition of other ingredients (as in creamed chicken, soups, or salads) greatly improved palatability. The USDA Marketing Economics Division (1963) substantiated these findings. Flosdorf (l9u5) found that the biOIOgical activity of labile components was generally unaffected by freeze—drying. Even vitamin C was reported to be completely stable. Rowe (1961) reported substantiating results. According to Hunt and Matheson (1958), long before muscle consumption, the actin and myosin combined to form actomyosin, which was present in greater quantity than any 7 other protein. Since actomyosin was rather labile, they suggested its survival of any particular treatment meant that many other proteins and enzymes also remained undamaged. HOpkins (1955) reported that the contractile system of living frOg muscle was not destroyed by freeze-drying al- though the rate of contraction was slower. Harper and Tappel (1957) stated that the texture of freeze-dried meat was drier than the frozen control and that this dry texture was one of the principal problems which re— mained to be solved in the field of lyophilization. This deficiency in texture has been attributed to the loss of water-holding capacity by the muscle proteins, and protein denaturation during drying has been suggested as the cause of this loss (Brooks, 1958). The effects of fast and slow freezing on freeze-dried beef were reported by Luyet (1960, 1961). Water penetrated more freely through solid parts than through cavities. Fast-frozen tissues did not shrink as much as slowly frozen tissues which swelled back to their original size when re- hydrated. Smithies (1961) related that rapid freezing in a dry ice-acetone mixture yielded a product which rehydrated more slowly than was typical of slowly-frozen meat. After cooking, the fast-frozen meat was tougher and drier than more slowly frozen meat. Meryman (1961) stated that mammalian tissues could not be frozen rapidly without mechanical injury from intracellular crystal formation. Neither could they be frozen slowly without chemical injury 8 from the concentration of solutes. Auerbach (1960) found that a much better product resulted when meat was frozen slowly before drying, while meat that was frozen rapidly by evaporative cooling yielded a product that reconstituted poorly. Luyet and MacKenzie (1960) found that meat frozen rapidly had smaller channels and absorbed less fluid than material having larger channels. They found that meat re— hydrated in NaCl solutions gave higher absorption values than when rehydrated in water. A procedure was also described for rehydrating freeze—dried meat in a vacuum to speed up absorption. In general, they referred to fast freezing as intra-fiber freezing and slow freezing as extra- fiber freezing, when considering fiber-contained water. Worland and Urbin (1960) reported that some water in meat remained unfrozen even at very low temperatures. Luyet (1961) suggested several reasons why this water remained unfrozen: (1) because its temperature had not been lowered enough to cause the crystallization of all freezable water; (2) because it contained bound water, which does not freeze at any temperature; or (3) because it was cooled so rapidly that some of its water remained amorphous or in a state of incomplete crystallization. The binding energy for electro~ static bonds was not high according to Meryman (1961). He stated that this water was probably removed during the latter stages of freeze-drying. Also, since many organisms sur- vived the loss of at least one-half their bound water, the removal of this weakly-bound water was apparently not entirely irreversible. According to Hamm and Deatherage (1960a), the decreased ability of meat proteins to rehydrate was usually due to the formation of an excessive amount of electrostatic and hydro- gen bonds between actin and myosin in the myofibrillar fila— ments. According to the authors, formation of these bonds might be counteracted by relaxation of the muscle as well as by an increase of pH away from the isoelectric point. Thus, the least possible overlapping of the actin and myosin threads should occur. Connell (1957) suggested that increases in toughness and a loss of gel-forming ability in dehydrated fish might be due to increased cross-linking of protein chains. Re prOposed that this could also cause dryness, due to poor water—binding capacity. Connell (1962) stated that even small amounts of cross-linkages could produce large textural changes. According to Ziemba (1960), chicken meat tends to de- teriorate very rapidly after drying when exposed to oxygen and/or elevated temperatures. Therefore, an inert gas or a high vacuum must be present for the successful storage of freeze-dried chicken, to prevent oxidative deterioration of the protein. The shelf life for freeze-dried chicken products packaged in polyethylene lined, lever-locked fiber drums was reported to be approximately six months at room temperature (Anonymous, 1962). The average shelf life of freeze—dried products, gas-packed in hermetically sealed 10 containers, was extended to about one year. According to Flosdorf (l9b9), freeze-dried products were stable for five years when dried to a minimal moisture content and stored at about 5°C. Connell (1962) suggested that the carbonyl-amine browning or Maillard reaction was not entirely inhibited by freeze-drying to low moisture levels. Olcott (1961) re— ported that oxidative changes usually occurred more rapidly at low moisture content (1 - 2%) than when higher amounts of water were present. When water was absent, the most im- portant single deteriorative reaction in freeze-dried meat or fish was the browning reaction. The brown reaction products resulted from the reaction of reducing sugars with proteins and probably accounted for losses in solubility and rehydratability. Sidwell 33 a1. (1962) substantiated the results of Connell (1962) and in turn found that the oxygen content of freeze—dried chicken was lower when it was precooked. According to Connell (1957). many freeze-dried foods showed high ratios of rehydration, or more accurately, water uptake, but much of this water was easily expelled, presumably because it was held only by weak capillary forces. Auerbach 23 El. (1954) stated that freeze-dried meat generally rehydrated rather well. He reported that one-inch- thick samples of freeze-dried beef rehydrated from 80 to 90% of the original water content. Wang (195ha) stated that freeze-dried muscle tissues from beef rehydrated from 85 to ll 90% of their original moisture content. The muscle fibers also returned to 95% or more of their original diameter. Norman and Auerbach (1963) found that the level of re- hydration in 72°F water was in the 90 to 95 percentile range, while in 180°F water the rehydration level was in the 70 to 80 percentile range. According to Steinberg (1960a), the rehydration ratio for beef samples precooked to a center temperature of luo°s to 150°? was slightly higher than for samples precooked to a center temperature of l80°F to 1980F. Suden gt 31. (l96h) reported that the percentage rehydration of freeze-dried pork fillets ranged from ua.5 to 92.u% with a mean of 73.8%. Rehydration was not influenced signifi- cantly by either pH of the rehydrating solution or pH of the rehydrated meat. However, the fillets were rehydrated for DE hours at QOC. To detect small changes in texture, Smithies (1961) found it useful to rehydrate samples for only five minutes before cooking and before presentation to a panel. Ground poultry breast meat rehydrated in 30 seconds in 180°F to 200°F water, while dark poultry meats required from 1% to 2 minutes (Anonymous, 1965). Wang ££,§l- (1945b) used the re- appearance of distinct cross striations in the muscle fibers as a criterion for true reconstitution of muscle tissue. Wismer-Pedersen (1965a) reported that after injection with solutions of EDTA (ethylenediaminetetraacetic acid) and perphosphate, freeze-dried pork samples had improved rehydration capacities and texture. The main effect of EDTA 12 appeared to be better penetration of water into the dried meat structure. This effect was probably associated with removal of calcium and magnesium ions from the fibrillar proteins through chelation before drying. Pyrophosphate appeared to cause swelling of the wetted areas rather than improvement of water diffusion into the dried meat. Wismer-Pedersen (1965b) noted that when calcium and magnesium ions were added to pork myofibrils, the pH before drying influenced the water-holding capacity after rehydra— tion. At pH 7.0, the rehydrated myofibrils had the same water holding capacity as the corresponding fresh myofibrils. Factors Affecting the Tenderness of Poultry Meat Miyada and Tappel (1956a) and Parrish £3 El, (1962) stated that tenderness was the foremost factor considered in meat acceptability. A. Physiology and Chemistry of Muscle The data of Blakeslee and Miller (1948) demonstrated that beef short loins were less tender at the rib end than at the porter house steak end. Ramsbottom gt 2;. (1944) found a variation in tenderness of beef muscles in different muscles of the same commercial cut. According to White 33 21. (1964), tenderness differences in turkeys were smaller between inner breast muscles than between outer breast muscles. Thus when inner breast muscles were used, they accounted for greater difficulty in detecting tenderness differences. Koonz and Robinson (1946) reported that varia- tions existed among principal muscles of the poultry carcass. 13 Wise (1961) found significant variations in the tenderness of poultry skeletal muscle tissues. Breast meat was reported to be significantly more tender than meat from the thigh (Goodwin g; g1., 1962). Wise and Stadelman (1959) reported that resistance to shear force was related, at a highly significant level, to the depth at which samples from poultry were taken. Various components of muscle tissue have been found to contribute to tenderness. Deatherage and Harsham (1947) re- ported that both connective tissue and muscle plasma affected tenderness. Their results with beef indicated that initial post mortem changes involved the muscle plasma rather than connective tissue, and they postulated that changes in the plasma were more important during the initial aging period. They also prOposed that in later post mortem stages, muscle plasma was less important than connective tissues in contributing to toughness. Lowe (1948) stated that meat from young birds, when aged for the same period of time, was more tender than that from older birds. In general older birds, as shown by histological sections, were found to con- tain more connective tissue within a given muscle than younger birds. Ramsbottom gt g1. (1944) found a significant correlation between shear press readings and the amounts of collagen and elastin in beef muscle, and between panel re- sults and the amounts of collagen and elastin in the muscle. Koonz and Robinson (1946) found that elastic connective tissue was almost completely absent in poultry breast muscle. 14 A relationship between the amount of nitrogen extracted by buffer solution and tenderness of beef was reported by Wierbicki g; al. (1954). Paul 33 El. (1958) found that a correlation between tenderness scores and percent nitrogen extracted by buffer solution was statistically significant, but too low to indicate a decided usefulness for measuring tenderness in chickens. The study of meat tenderness covers the transition of muscle from the living state through the dead state, a period of time which includes the condition known as rigor mortis. Bendall (1963) related that the energy for muscle contraction came directly from the splitting of ATP (adeno- sine triphosphate) and that the opposite process, relaxation,. occurred when certain specific conditions inhibited this splitting. Bendall (1963) reported that when birds were slaughtered, their muscles became soft and pliable. Immediately after death, ATP was broken down and its concentration in muscle diminished. He reported that this splitting was the direct result of a sarcoplasmic ATPase which was probably associated with mitochondria. As a result of this breakdown, ADP (adenosine diphosphate) was produced and glycogen was de— pleted. Creatine phosphate, which served to phosphorylate ADP in muscle to ATP, continued to perform this function. At this state, blood circulation through the muscle limited its ability to maintain aerobic metabolism. Thus, the re- maining metabolism of post-mortem muscle was forced to depend 15 completely upon anaerobic glycolysis which led to an accumu- lation of lactic acid. This accumulation of lactic acid caused a decrease in the muscle pH from its initial pH (7.2) to a pH of approximately 5.6 to 5.8. The decrease in pH caused a decrease in anaerobic metabolism, since the enzymes involved were no longer at their cptimum pH. As the concen- tration of ATP fell, the muscle slowly hardened until it be- came quite stiff. This latter state of stiffness was called rigor mortis, although rigor mortis was really the result of the entire series of changes which started to occur at the moment of death. The rate of development of rigor mortis and related bio— chemical changes in chicken muscle were studied by DeFremery and Pool (1960) in relation to ultimate tenderness of the cooked muscle. They found that correlations between the loss of ATP and the onset of rigor mortis were the same for chicken as for other species. Muscles from 10- to 16-week- old chickens, held at room temperature, passed into rigor from 2 to 4% hours post—mortem and reached an ultimate pH of 5.8 to 5.9. They found that muscle toughness was induced with every treatment which caused a rapid loss of ATP, more rapid drop in pH, more rapid develOpment of rigor mortis and more rapid loss of glycogen. In other words, toughness in- creased as the rate in the onset of rigor increased. DeFremery and Pool (1959, 1960) postulated that the relative toughness of cooked muscle in uniform groups of chickens was directly related to the rate of development of l6 rigor mortis. In addition, the following pre-rigor treat- ments, which accelerated the onset of rigor mortis, also de- creased tenderness; freezing and thawing, exhaustive electri- cal stimulation, and electron irradiation. Weinberg and Rose (1960) suggested that upon the resolution of rigor, the re— sulting tenderization was not just a random autolysis but instead resulted from a specific cleavage of the actin asso- ciation responsible for the maintenance of the muscle matrix. Lethal doses of sodium monobromoacetate were injected into chickens by DeFremery (1959). These injections accel- erated the onset of rigor mortis and caused a marked increase in the rate of ATP depletion, but had little influence on pH or glchgen levels. However, the tenderness of the cooked meat was the same as the injected controls. He suggested that this ruled out the rapid loss of ATP as the determinant of increased toughness. The pH of these muscles (pH 6.5) was appreciably higher than normal. He reported that the iso- electric point of actomyosin was near pH 5.3, and a higher pH might lead to greater water-binding of actomyosin and, presumably, more tender meat. Gawronski gfi g1. (1964) obtained data which indicated that the oxidation of muscle sulfhydryl groups to disulfides contributed to the onset of rigor. They concluded that sulfhydryl/disulfide exchange had an important role in post- rigor-tenderization. B. Slaughter Goodwin 2E.§l- (1961) found that the method of slaughter 17 had no effect on the tenderness of breast muscles. However, humane slaughter treatments resulted in increased shear values for thigh muscles. Lineweaver (1959) stated that pre-mortem exercise, electric stunning, full feeding versus 24—hour fasting, and post-mortem delay before scalding had little or no effect on poultry tenderness. The struggling effect has been somewhat controversial but most researchers agreed that under normal processing con- ditions, struggling did not exert an effect on post-mortem tenderization (Dodge, 1959 and Dodge and Stadelman, 1960a). However, Stadelman (1959) did state that excitement before slaughter should be avoided, since it altered the normal rigor pattern and caused more birds to be tough, even though others were tender. Gainer g; g1. (1951) previously reported that the muscles of birds which struggled during slaughter were more tender than muscles from birds of the same lot that did not struggle. C. Scalding Koonz 23 El. (1954), Stadelman and McLaren (1954), Lineweaver (1955, 1959), Klose g; gi, (1956a, 1959), Shannon 22 al. (1957), Pool gt El. (1959) and Wise and Stadelman (1959) reported that chicken breast muscle was toughened by excessive scalding. Longer scald times and higher tempera— tures were found to significantly reduce the tenderness of poultry meat. Wise and Stadelman (1959) reported that the toughening effect of high-temperature, long—time scalds was related to the depth to which the scald heat penetrated the 18 muscle tissue. Variations in scalding temperature were found by Klose and Pool (1954) to have no effect on the tenderness of roasted muscles from Broad Breasted Bronze turkeys. However, in the case of roasted skin, increased scalding temperatures produced a marked increase in toughness and wrinkling. Wise (1961) concluded that the toughening effect of ex- cessive scalding was a direct function of the tissue tempera- ture during the early post—mortem period. Stadelman and McLaren (1954) concluded that the layer of fat surrounding the breast muscle on mature birds acts as an insulator to minimize any change in muscle tone or tenderness during scalding. They also related that the time in the scald water was more important than the scald water temperature. D. Picking Stadelman and McLaren (1954), Wise and Stadelman (1959) and Lineweaver (1955, 1959) agreed that ultimate toughness after aging increased with the extent of beating action in- curred by the carcass during feather removal. Beating was reported by Pool 23 El. (1959) to exert its greatest toughening effect when applied immediately after slaughter. Beating de- layed from one to three hours after slaughter had less effect. Klose gt al. (1956a) reported that toughness induced in chickens and turkeys by excessive beating could not be re— solved by prolonged aging. The authors found the effects of beating to be cumulative and stated that they could be re- duced by limiting the beating action to the minimum required 19 for sufficient feather removal. Gainer 23 El. (1951) found that muscles from hand picked birds were significantly more tender than those from machine picked birds. Klose §t_gl. (1959) and DeFremery and Pool (1959) found similar results. The latter authors also noted that machine picking markedly accelerated the rate of onset of rigor mortis. Goodwin and Stadelman (1962) reported that after two hours of muscle flexing and hand masaging of turkeys, significantly higher shear values were recorded than for controls. Massaging for shorter times affected toms and fryers more than hens. E. Excising of Muscle Lowe and Stewart (1948) noted that when breast muscles of chicken were out soon after slaughter, the shock of cutting induced a turgidity and roughness of the cut surface which persisted even after 24 hours of carcass aging and subsequent cooking. In general, the sooner after slaughter the muscle was cut, the greater the effect. However, when rigor de- veloped before the muscle was cut, turgidity did not develop. DeFremery and Pool (1960) substantiated these findings. Koonz 33 31. (1954) altered the tenderness pattern by cutting into muscles of dispatched birds. Under these condi- tions toughness, which was presumably associated with rigor, was maintained over a relatively long period of time. Pool 2§_gl. (1959) also reported that~cutting up the carcass in the early post-mortem period had a small toughening effect. 20 F. Aging Perhaps the most important single factor affecting poultry tenderness is aging. Chajuss and Spencer (1962) ob- tained results which indicated that certain oxidation reac- tions played an important role in chicken meat tenderization during post-mortem aging. Muscles treated with sodium sulfite (a redox agent) were more tender than controls. They indi- cated that the probable action of this compound on meat pro- tein was to reduce the disulfide bonds. The sulfhydryls thus formed were probably reoxidized so that the final products were the S-sulfonates. At the present time practically all ready-to-cook chickens are aged for a period of time in a slush ice—water mixture in order to maintain high quality during the resolution of rigor. Stadelman (1959) stated that aging at 55°F took approx- imately three times as long to resolve rigor as at 32°F. Most authorities agreed that aging for a period up to 24 hours provided maximum tenderness and that after this period no increase in tenderness was obtained (Lowe, 1948; Carlin g; g1., 1949; Koonz §§_§1.; 1954; Lineweaver, 1955; Stadelman, 1956; Klose 33 51., 1956b; and Dawson gt g1., 1958.) Dawson 23 a1. (1958) found that a holding time of between three to six hours for lO-week-old fryers was sufficient. Several investigators found that at chill temperatures, most tenderization took place within twelve hours post mortem and that very little occurred after this time (Anon., 1957; Pool 32 2.1. 1959; and Klose gt _a_:_l_., 1959). Klose g a_l_. (1956b) 21 reported that most tenderization in poultry took place in the first six hours. Pool §£_g1. (1959) found that most tender- ization took place within four hours at chill temperatures and that the rate then decreased up to about twelve hours after which no appreciable tenderization occurred. They found that no appreciable tenderization took place in hard- frozen carcasses held at 25°F to 27°F for several days. Stadelman and Spencer (1955) indicated that turkeys packaged warm from the eviscerating line and cooled in the package for 24 hours prior to freezing resulted in a satis- factory frozen appearance. These turkeys were as tender as turkeys cooled in ice-water and then packaged and frozen. Dodge and Stadelman (1959) stated that the temperature of the aging medium appeared to affect the pattern of rigor and the level of tenderness at a given time post-mortem. Lowe (1948) reported that the onset of rigor in chickens usually began within one to two hours post—mortem and the greatest rigidity usually occurred between six and twelve hours after death. She found a direct relationship between temperature and the onset of rigor as well as its resolution. No signs of rigor were observed in the cooked carcasses of fowl aged for three hours or longer before cooking. None of the birds aged for periods up to one hour were in rigor when they went into the oven but all were in this state when they were removed. Effects of aging without freezing were compared with effects of aging, freezing, and thawing on the palatability 22 of roasters and fowl (Carlin gt a;., 1949). The unfrozen birds tenderized rapidly. Freezing resulted in a marked in- crease in tenderness of those halves aged less than six hours. When halves were aged for 2h hours, there was little difference in tenderness between frozen and unfrozen halves. Koonz gt El- (l95h) found that freezing interfered with the tenderness pattern and that complete tenderization was delayed until the tissues were defrosted. In another experiment these same authors immersed dispatched birds in hot water for various time periods. The muscles became significantly less tender as the time of immersion increased. A slight increase in potassium content in poultry tissue after an eight-hour aging period at 32°F was found by Stadel- man (1959). A slight increase in tenderness and Juiciness was also detected. Chicken stags (12-month-old cross-brads) aged in 2% K01 were as tender after two hours of aging as the controls were after eight hours. Dodge and Stadelman (1960b) showed that water uptake and rates of cooling during aging did not affect tenderness. Tenderization was closely associated with pH. Total moisture content of the tissue was not associated with water uptake, nor was it related to tenderness. Dodge (1959) showed similar results. Pippen and Klose (1955) reported that aging of poultry in ice water, although beneficial from the tenderness stand- point, caused a leaching-out of flavoring components when the holding period was prolonged. 23 pH adjustment of intact meat to pH 7.0 to 7.b with phosphate salts enabled meat fibers to take up and hold their normal water content (Swift and Ellis, 1956; Morse, 1955, and Kamstra and Saffle, 1959). An increase in tenderness was associated with this water—holding capacity. Carpenter gt 3;. (1961) found that tenderness in beef was improved by pre- rigor infusions of sodium hexametaphosphate. May gt_§;. (1962a) and Spencer and Smith (1962) reported that chilling chickens in polyphOSphate solutions resulted in significant increases in tenderness. In contrast, Klose gt 5;. (1963) found that shear force values of cooked fryer meat, after either three-hour or 22-hour chilling periods, did not show a significant effect of polyphosphates on tenderness. They stated that polyphosphates controlled cooking shrink and preserved quality. Mountney and Arganosa (1962) and Scher- merhorn and Stadelman (1962) reported that phosphates in the aging solutions increased water retention. Swift and Berman (1959) reported that increased pH values in beef were closely correlated with increased water retention. G. Freezing Koonz and Ramsbottom (1939) found that the rate of freezing affected the size, number and location of ice forma- tions. Nearly instantaneous freezing produced minute, evenly distributed ice columns within the fibers. When the rate of freezing was slower, the ice columns within the fibers were larger in diameter and fewer in number. The importance of ice crystal size was emphasized by Birdseye (l9h6). He 24 proposed that large ice crystals, as a result of slow freezing, resulted in physical damage to the cell (cell rupture) or in a physio-chemical change which he termed "salt dehydration." Structural changes in muscle tissue upon repeated freezing and thawing were observed by Nichols and McIntosh (1952). Repeated freezing and thawing caused an increased amount of drip loss. As the number of broken muscle fibers increased, more fluid was released. Both the intra- and inter-cellular ice formations contributed to the fragmentation of fibers. Dubois gt gl. (1942) stated that by normal observation, it was difficult to differentiate between rapidly and slowly frozen chickens. However, they noted that birds frozen by these two methods could be differentiated through the use of histological cross-sections of tissue viewed microscOpically. Early investigations showed that freezing allowed for the continuation of the aging process with a resultant increase in tenderization (Carlin, 19u9; Carlin gt 91..., l9u9; Hepburn, 1960; Monzini, 1953; and Swanson and Sloan, 1953). However, more recent research has shown that the tenderizing process was arrested and that complete tenderization was delayed until the tissues were defrosted (Koonz gt gl., 1959; Spencer gt_g;., 1956; and Klose 9.2 alo. 1956a. 1959). Klose gt _a_l_._.. (1959) reported that holding inadequately aged, frozen turkey fryers for as long as nine months at O°F had no effect on tenderness. Marion and Stadelman (1958) evaluated tenderness of poultry breast muscle by four different freezing methods. Method of freezing did not significantly affect tenderness. 25 Deatherage (1959) reported that the freezing rate affected the water-holding capacity of meat; tenderness of meat was related to the ability of meat proteins to hold water. Deatherage and Hamm (1960) substantiated these re— sults. They reported that quick freezing and thawing of beef resulted in no appreciable denaturation of muscle pro- tein. However, quick freezing caused a significant increase in the water—holding capacity of the meat, probably by a mechanical loosening of tissue structure due to the formation of tiny ice crystals within the cells. Slow freezing caused a significant decrease in the water-holding capacity, probably due to some destruction of protein structure by formation of large ice crystals between the cells. H. Cooking Hamm and Deatherage (1960b) detected a mild denatura- tion in muscle after the temperature reached 30°C to uo°c. This denaturation resulted in an unfolding of protein chains with the formation of new salt and/or hydrogen bonds. The denaturation and formation of new cross-linkages in muscle continued until about 65°C at which temperature the denatura— tion was almost complete. The step—wise change in the water- holding capacity of meat and in pH during heating was deter- mined by following a corresponding decrease in the acidic groups of muscle proteins. Heat denaturation did not cause a significant decrease in the amount of basic groups in muscle proteins. Kahn and van den Berg (1965) recently reported that the 26 sulfhydryl group content and tenderness of chicken muscle decreased simultaneously during cooking and frozen storage. They suggested that the sulfhydryl groups which survived heat denaturation in the muscle tissue contributed in some way to maintaining the eating quality of meat. They proposed that the loss of this sulfhydryl group content during storage might serve as an index of tenderness. Tenderness changes became apparent when the sulfhydryl group content of muscle tissue had decreased to about 50% of its value in the fresh cooked meat. Mickelberry and Stadelman (1962) reported that pre-cooked, frozen chicken meat was significantly less tender than chicken cooked after freezing. Goodwin gt gt. (1962) found that all turkey muscles became more tender when cooked. Koonz and Robinson (1946) found similar results with chickens. However, they also found that moderate cooking of beef caused many muscles to become tougher. Although Goodwin gt gt. (1962) found that the rate of cooking had no significant effect on shear values, there appeared to be a trend toward lower average shear values for the chicken breasts cooked at the lower temperatures. May gt gt. (1962b) observed that broilers and roasters cooked in an electronic range had slightly higher shear values than similar birds cooked by a moist heat method. Dawson gt g1. (1959) found that in general, dry heat methods yielded more tender beef than moist heat methods. Mickelberry and Stadelman (1962) found that birds fried in deep fat were less tender than birds cooked by other methods. 27 Shear values for pressure cooked breast meat were reported by Kahlenberg and Funk (1961) to be significantly lower than shear values for either boiled or simmered breast muscle. Tenderness of old fowl cooked in various salt solutions was similar to tenderness of birds cooked in plain water. How— ever, Goodwin gt gl. (1962) reported that the method of cooking had no statistical effect on the shear values of turkey meat. The methods used included cooking by microwave oven, deep fat frying, steam pressure, rotary reel oven and combinations of steam and deep fat frying and of microwave heating and deep fat frying. For a comprehensive review of the literature concerning cooking and tenderness of meat other than poultry, the reader is referred to a discussion by Paul (1963). Objective and Subjective Evaluation of Tenderness A. Objective Measurements (Mechanical and/or Chemico- Physical Methods) Pearson (1963) related that mechanical methods were more widely accepted for measuring meat tenderness objectively than were chemical and histological methods. Of the mechani- cal methods, the Warner—Bratzler shear was most widely used (Deatherage, 1951). However, the Warner-Bratzler machine necessitates the use of a cross-sectional core of meat for evaluation (Bratzler, 1932). In chicken breast, it is often difficult to obtain such a core due to the relative thinness of this muscle. Thus the Kramer shear press has proven to be the most satisfactory device for measuring tenderness of 28 chickens (Cameron and Ryan, 1955). The Warner-Bratzler shear has been fully described by Bratzler (1932) after his adaptation of the original instru- ment develOped by Warner in 1928. The Kramer shear press is an instrument which was originally deve10ped for fruits and vegetables by Kramer gt gt. (1951). Paul gt gt. (1958) reported a negative correlation of .0.71 between the average tenderness scores of a taste panel and the Warner-Bratzler shear values from chicken meat. Deatherage and Garnatz (1952) also compared sensory panel scores to Warner-Bratzler shear values. Although shear values measured a property of meat fairly satisfactorily, a poor re- lationship existed between shear press values and sensory panel scores when broiled steaks were evaluated. Although the Warner-Bratzler shear has several short- comings, results have revealed that correlation coefficients between shear values and sensory evaluations generally lie in the range of 0.60 to 0.85, with an average value of about 0.75 (Pearson, 1963). Shannon gt_gt. (1957) reported a correlation coefficient of 0.86 between Kramer shear press values and taste panel scores. Dodge and Stadelman (1960c) obtained a correlation coefficient of 0.97 when cooked meat was evaluated by the same two methods. Bailey'gt gt. (1962) measured 258 beef steaks and found a correlation coefficient of -0.89 between taste panel scores and Kramer shear press values for all steaks evaluated within grades and cuts. Disregarding grade 29 or cut, a -0.74 correlation coefficient was calculated. Dodge and Stadelman (1960c) found significant correla— tions between Kramer shear values on raw meat and panel evaluation of cooked samples from the same poultry carcass. However, the relationship was not as high as that found be- tween shear values and panel scores of cooked samples. Cameron and Ryan (1955) reported that sample size affected tenderness as measured by the Kramer shear press. Wells gt gt. (1962) found a poor relationship between Kramer shear values and taste panel scores when tenderness of freeze—dried poultry breast muscle was evaluated. Low correlations were obtained by Steinberg (1960a) between ob- jective and subjective texture measurements of freeze-dried beef. B. Subjective Measurements (Sensory Methods) Sensory methods for tenderness evaluation approximate the actual sensation realized by consumers. Two types of panels have been used in sensory evaluations (Pearson, 1963): (1) the large consumer or acceptance panel and (2) the smaller expert or difference panel. Consumer panels have been more expensive to conduct and have not always been applicable due to sample size and availability of personnel. The reactions of a large, un- trained panel of 355 people and of a small, trained panel of seven judges were reported by White gt gt. (l96h) while evaluating toughness differences in turkeys. The small panel used a triangle test method and distinguished differences in 30 tenderness of light meat which varied in shear resistance by 4 pounds in a 9 to 22 pound range. The small panel dis- tinguished differences more accurately than the consumer panel. Lowe (1949) stated that four judges were a minimum number for a trained panel. She proposed that a small, sensitive panel was preferable to a larger, less sensitive one for measuring textural differences. According to Pearson (1963), selection of a panel was best achieved by use of a triangle testing procedure, where- by each judge was given three samples of meat, two of which were alike. He stated that the chew count was the most ob- jective of the sensory procedures for studying meat tender- ness. The chew count method consisted of the number of chews required to completely masticate a sample before it was swallowed. Lowe (1949) preposed that the triangle test was an accurate and reliable method for tenderness evaluation. Peryam and Pilgrim (1957), in turn, preferred the hedonic scale method. A numerical rating was used and each panel member selected a description best fitting the sample in- volved. The hedonic scale method was designed for use with subjects having little experience in food tasting. These authors stated that the hedonic scale method was develOped on the assumption that direct responses, which were assumed to be based considerably on feelings, were more valid for predicting actual behavior toward food than were responses which depended more on reasoning. The authors stated that 31 long or short lines, vertical or horizontal orientation, or terminology such as "like" or "dislike", did not appear to be significant. Cover gt,gt. (1962) identified six separate components of tenderness and related them to shear force and fiber ex- tensibility. These six components included connective tissue, juiciness, mealiness, softness to tongue and cheek, softness to tooth pressure and ease of fragmentation and adhesion. Sartorius and Child (1938) and Deatherage (1951) re— ported significant positive correlation coefficients between tenderness and juiciness scores in meat. Structure of Muscle and Connective Tissue A. Muscle Structure Maximow and Bloom (1954) presented a comprehensive out- line of tissue structure. They classified muscle in verte- brates as smooth and striated muscle. In general, smooth muscles contracted independently of voluntary control, while striated muscles were of voluntary control. Cardiac muscle, although striated, was involuntary. Smooth muscle displayed a close relationship to ordinary connective tissue and was found primarily in the internal organs. The muscles attached to the mammalian skeleton consisted of striated muscular tissue. The authors stated that the individual muscle fiber was the functional unit of a muscle. In striated muscle Where these fibers were large, multinucleated cells, the 32 thickness of the individual fiber varied from 10 to 100 microns. This depended on the type and age of the animal and on the particular muscle. The fibers were relatively long, some of which extended the full length of the muscle. An average-sized skeletal muscle fiber contained several hundred nuclei. The striated fiber was covered with the sarcolemma, a thin, elastic, transparent and stuctureless membrane which completely envelOped the fiber. Muscles were formed of parallel muscle fibers cemented together by networks of connective tissue. The muscle fibers combined to form the so-called primary bundles, and several primary bundles com- bined to form secondary bundles. According to COpenhaver (1964), the sarcolemma encom- passed the nuclei and a cross-striated substance composed principally of the myofibrillae. Surrounding the fibrillae and accumulated near the nuclei was the sarcOplasm, the more fluid portion of the fiber. The myofibrillae imparted to the muscle fiber as a whole the appearance of longitudinal striation. Each of the myofibrils was composed of a number of thinner, thread-like elements known as myofilaments. The striations appeared as alternating light and dark bands. The dark band was labeled the A band or Q band. The light band was designated by the letter I or J. Each of these bands was bisected by a narrow line, which stained deeply in the I band and was designated Z; the line bisecting the A band was pale and was designated H. Within the H line or disc was a.narrow stripe designated by the letter M. In these 'various bands, actin and myosin filaments combined to form 33 actomyosin during contraction. B. Connective Tissue Structure Ham and Leeson (1961) classified the connective tissue into certain main types: loose, dense fibrous, adipose, cartilage, bone, dentin and hemOpoietic. Only the loose connective tissue was of concern in the present study. According to Ham and Leeson (1961), loose, irregularly- arranged connective tissue bound structures tOgether loosely and held them in position. It acted as a pathway for nerves and blood vessels and served as a padding. Loose connective tissue, like all other connective tissues, was composed of cells, intercellular fibers, and ground substance which was the material forming the background. It contained most of the cell types and all of the kinds of fibers found in the other varieties of connective tissue. Birkner and Auerbach (1960) stated that individual muscle fibers were separated by very thin networks of con— nective tissue called the endomysium. Primary muscle fiber bundles varied in the number of fibers per bundle, depending on the muscle, and were encompassed by larger sheets of connective tissue, the perimysium. The epimysium was the large outer layer of connective tissue which surrounded the entire muscle. Cepenhaver (1964) reported three types of fibers in adult connective tissue: white or collagenous fibers, reti- cular fibers and elastic fibers. The collagenous fibers were in bundles of indefinite length and variable thickness ranging 34 from 10 to 100 microns or more. Each collagenous fiber was composed of fibrillae. The fibrillae lay parallel to one another and imparted a longitudinally striated appearance to the fiber. They, themselves, did not branch but the fibers and bundles did. The course of the fibers was usually wavy. They consisted of collagen, a substance which stained easily with most acid dyes and yielded gelatin upon cooking. Reticular fibers were small fibers which branched to form a supporting framework or reticulum. Their magnitude was so small that they were masked by surrounding structures in ordinary stained preparations.' Reticular fibers were often found to be continuous with collagenous fibers and had a very close resemblance to the latter.‘ They were particu- larly sparse in the loose connective tissues except for regions around muscle fibers. According to Copenhaver (1964), the elastic fibers were usually thinner than white fibers. They branched freely and were a distinct yellow when seen in the fresh state. Chemically, these fibers consisted of elastin, which had a remarkable resistance to most agents. Elastin was not affected by boiling. The elastic fibers reacted very poorly with most stains, but were colored deeply with certain Specific dyes such as orcein and resorcin-fuchsin. Tenderization by Commercial Proteolyttc Enzymes In June, 1955, the Meat Inspection Division (MID) of the USDA officially permitted the use of enzymatic tender- izers in MID-inspected meats (Bavisotto, 1958). 35 A serious problem associated with the use of these pro- teolytic enzymes was that of penetration, especially in cooked meat (Auerbach, 1960). Enzyme-treated meat often showed overtenderization and a mushy appearance on the out- side but little or no effect on the inside. The problem in raw meat was partially solved by injecting enzyme tenderizers into the animal prior to slaughter. This problem was also minimized with freeze-dried meat, when the enzyme was incor- porated directly into the rehydration media. Hamm (1960) stated that freeze-dried.meat showed a better rehydration than meat dried by other methods. Penny (1960) reported that when meat was first dried by the accelerated freeze-drying method and then reconstituted with proteolytic enzymes, a resultant tenderized product was achieved. Sosebee gt,gt. (1963) reported similar results with freeze—dried chicken breast muscle. They also stated that much lower concentrations of enzyme were required to produce tenderness in chicken than those reported necessary for tenderizing beef. According to Schweigert (1960), much higher concentrations of enzyme preparations were needed to show histological changes than for differences to be detected by a taste panel. Weiner gt gt. (1957) reported that it was possible for tenderization to occur before the proteolytic effect was measurable. Thus, organoleptic testing should be used for the determination of tenderization. Hang gt gt. (1957) studied the relative potencies of twelve enzyme preparations on the tenderness and muscle 36 structure of beef. They reported that the amount of enzyme needed to produce meat of desirable tenderness varied with the initial tenderness of the meat. The same amount of en- zyme made a tender steak mushy, whereas it improved a very tough steak. They classified the enzymes into three cate- gories, depending on their origin: those of plant origin, such as papain, bromelin and ficin: those of bacterial or fungal origin, such as Rhozyme P-11; and those of animal origin, such as trypsin and Viokase which were not used in the present study. According to Bavisotto (1958), papain was the dried latex of the fruit of the Carica tataya which was cultivated extensively in Ceylon and in British East Africa. The fig tree of the genus Etgthwas the source of the fig latex from which ficin was isolated. It was grown in Central and South America. Bromelin was produced commercially from the stem of the pineapple, Ananas comosus, and was imported from Hawaii. Rhozyme P-ll was obtained by isolation from a se- lected species of fungus in the Astergillus ttgvus-oryzae group. Weir (1959) rehydrated freeze-dried beef steaks in the above—mentioned enzyme solutions for five minutes at 130°F. An extension of the holding time at 130°? after rehydration from 5 to 30 minutes did not affect the tenderness of, or amounts of residue from, the steaks. Greater enzyme concen- trations were needed to produce measurable changes in cooked beef than in raw beef. 37 The optimum temperature for an enzymatic reaction, according to Weiner 2E,§l- (1957), was closely related to the time which the reaction covered. In general, the shorter the digestion time, the higher was the cptimum temperature for that reaction. Teen and Tappel (1959) reported that the heat stability of papain generally permitted a maximum hydrolysis of pro— teins at 60°C. Tappel gt_gt. (1956a) found the cptimum temperature for papain digestion to be between 60°C and 80°C. Optimum temperature ranges for ficin, bromelin and papain were 30°C to 50°C, 30°C to 60°C, and 60°C to 85°C, respect- ively (Anonymous, 1963). At the same time, the cptimum range for Rhozyme P-ll was reported to be 43°C to 60°C. Labora— tory tests indicated that most of the tenderization took place during cooking. Maximum solubilization of all beef protein fractions occurred at pH 7.0 and 80°C with ficin and bromelin, according to El-Gharbawi and Whitaker (1963). These workers also stated that it was not practical to add buffer to influence the pH of raw, fresh meat, but that this could be done readily during the rehydration of freeze-dried meat. According to Kimmel and Smith (1957). the pH cptimum for the digestion of fibrin by papain was 7.0. Cohen (1958) de— termined the cptimum activity of ficin using 0.01 M cysteine as an activator. Hetobserved a broad cptimum pH from pH 6.5 to pH 9.5. Wang and Birkner (1957) stated that ficin was active on beef muscle over a pH range from 5.0 to 9.0 with an optimum at around pH 5.0 to 6.0. The optimum pH for 38 Rhozyme P—ll was reported to be between pH 5.5 and 6.0 (Anon- ymous, 1963). Yatco-Manzo and Whitaker (1962) found ficin- catalyzed hydrolysis of elastin to be optimum at a pH 5.0 to 5.5 and at a temperature of 55°C. Sosebee gt gt. (1963) obtained sufficient tenderization of poultry breast muscle with concentrations of papain and Rhozyme P-ll equal to 0.003% and 0.02%, respectively. A 30-minute rehydration time was used. Wang gt gt. (1958) used ficin, bromelin, papain, and Rhozyme P-ll on beef at concentrations of 0.0002%, for all enzymes except Rhozyme P-ll whose concentration was 100 times stronger. These in- vestigators stated that hemoglobin and gelatin assay methods of expressing enzyme activity might not reflect the meat tenderizing properties of the enzymes used. According to Thomas and Partridge (1960), the plant enzymes required a reducing agent such as cysteine for acti- vation. In the absence of cysteine, there was a marked de- crease in activity towards elastin and gelatin. Kimmel and Smith (1957) reported that all activators of papain were capable of reducing disulfide bonds; they included compounds such as H23, HCN, and other reducing agents. Free thiol groups were considered essential for papain activity. They stated that papain contained eight atoms of sulfur per mole of papain but that only six of these could be accounted for as half—cysteine. It was further acknowledged that removal of heavy metals was essential for maximal papain activity. Liener (1961a, 1961b) concluded that ficin contained at 39 least two sulfhydryl groups, only one of which was directly involved in the catalytic site of the enzyme. Ficin con- tained at least one disulfide bond which appeared to be un- essential for the maintenance of activity. Hammond and Gutfreund (1959) concluded that three reactive groups were necessary for the catalytic action of ficin; - SH, NH3+, and C02-. These investigators also proposed a reaction sequence between enzyme and substrate. The sequence involved the rapid formation of a loose enzyme-substrate compound, a sub- sequent acylation of the enzymic sulfhydryl group by the car- bonyl of the substrate, and finally the decomposition of the acyl enzyme. HistolOgical Congtderations For a brief review of muscle histology, the reader is referred to an article by Venable (1963). Histologically, freeze-dried muscle tissue was char— acterized by a system of interfibral spaces (Wang, 1954). These spaces were found to arise, in most cases, as a result of muscle fiber shrinkage without a corresponding alteration of the tissue volume. Wang gt gt. (1953) found similar re- sults with the additional finding that conventional drying resulted in a gradual loss of both longitudinal and trans- verse striations. Also, the nuclei were reported to have stained poorly, and there was a merging of individual muscle fibers. Similar findings were reported by Doty gt gt. (1953) when slices of raw beef were dehydrated at 70°C in an air oven. These investigators found that the histological 40 appearance of freeze-dried meat was almost indistinguishable from that of fresh raw meat. Sosebee gt gt. (1963) found that freeze-drying of chicken did not significantly affect the histological appearance. Ramsbottom and Strandine (1949) described the presence of granular protein material between the muscle fibers. During cooking, both longitudinal and transverse breaks occurred in the muscle fibers, and at these points greater breakdown of the muscle fibers resulted. Collagenous fibers, when cooked, underwent first a swelling and then a shrinkage and disintegration. Chemical changes in collagenous fibers were reported to have occurred during cooking as evidenced by changes in the affinity of the fibers for dyes. For years cooking was known to cause a decrease in muscle fiber diameter due to shrinkage (Sartorius and Child, 1938). Doty and Pierce (1961) referred to the granulation which occurred during cooking as the "erosion" of muscle fibers. They stated that this "erosion" or granulation started at the edges of muscle fibers and, when heating was continued, progressed to complete granulation of the fiber. The endomysial reticulum remained relatively intact. Al- though collagenous fibers were affected by heating, cooking did not appreciably alter the structure, staining affinities or physical properties of elastic fibers (Birkner and Auer- bach, 1960; Winegarden gt_gt., 1952; and Weir gt gt., 1958). Paul (1963) summarized the effects of cooking and the influ- ence of cooking methods on tenderness. Photographs of several 41 histological sections of cooked tissue were included. Bavisotto (1958) stated that proteolytic enzymes of microbiological origin exhibited potent activity on muscle fibers and in some cases slight activity on collagenous fibers. Wang and Maynard (1955a) reported that Rhozyme P-ll had no effect on collagenous or elastic fibers from freeze-dried pork muscle. Miyada and Tappel (1956b) found that papain and ficin hydrolyzed elastin and that bromelin, ficin, trypsin, papain and Rhozyme P-ll hydrolyzed collagen. Thomas and Partridge (1960) found that bromelin also had elastolytic activity. Wang and Maynard (1955b) found that papain and Rhozyme P-ll had very similar effects on muscle tissue com- ponents. Both attacked muscle fiber protein, the nuclei of muscle fibers and of cells located in the endomysia, but the enzymes were inactive on collagenous and elastic fibers at room temperature. Wang gt gt. (1957) conducted a comprehensive study on the influence of enzyme tenderizers on the microscopic structure of freeze-dried beef. Among the twelve enzymes used were ficin, papain, bromelin, and Rhozyme P-ll. Steaks were rehydrated in an enzyme solution of known concentration for 15 minutes. The earliest change in the muscle fibers was the dissolution of the sarcolemma followed by the dis- integration of the connective cell nuclei (mostly fibrocytés). Continued enzyme action resulted in the complete disappearance of cross-striations. Since the fibers had lost their sarco- lemma, they tended to merge. This merging was accentuated 42 by the swelling caused by the enzyme treatment. Enzymatic collagenase activity was manifested by a decrease in staining capacity with acid fuchsin and a decreasing discreteness in the fibrillar nature of collagen. The latter change was be- lieved to have resulted from the liquefaction of ground sub- stance which normally holds the collagenous fibers into defi— nite bundles. In the presence of elastase, the elastic fibers underwent a process of segmentation (linear breakage), which made the fibers appear beaded. When the enzyme activity continued, complete digestion of elastin was noted. This point was reached when the fibers were no longer stained. Sometimes a trail of "ghosts" was detected after complete elastin digestion, which indicated the presence of fibers prior to the treatment. Wang and Maynard (1955a, 1955b), Tappel gt gt. (1956b), Wang and Birkner (1957) and Sosebee gt gt. (1963) reported similar results. Tappel gt gt. (1956b) reported that papain hydrolyzed the sarcolemma and muscle cell nuclei before there was any appar- ent digestion of the muscle fibers themselves. They postu- lated that the heat labile muscle proteins denatured before the relatively heat-stable papain, and that papain then hy- drolyzed these denatured proteins with maximum effect. Tenderization by papain was not ascribed to one specific re- action but rather to a general hydrolysis of all the structural components of beef muscle. According to McIntosh and Carlin (1963), papain affected the mucoprotein and collagen more than the other skeletal muscle proteins. Collagen suspensions were converted to thick gels by the action of papain. 43 Heir gt gt. (1958) rehydrated freeze-dried beef in solu- tions of commercial tenderizers for 30 minutes. They found that the granulation invariably occurred in the interfibral spaces. However, this was the only manifestation of the treatment noted. In all other respects the tissue appeared indistinguishable from normal tissues. The granulated ma- terial was probably derived from endomysial collagen and the muscle fiber envelopes. Both structures made early contact with the liquid tenderizer and were disintegrated. A rela- tionship between sarcolemma destruction and an increase in tenderness was demonstrated. Studies by Wang and Maynard (1955a) showed the effects of papain and Rhozyme P—ll on freeze-dried pork. Rhozyme mani- fested a greater sensitivity on the muscle fiber nuclei than on those nuclei of the connective tissue cells, while the reverse was true of papain (in the form of Adolph's Meat Tenderizer). At present, the only published research found by this researcher concerning the effects of proteolytic enzymes on freeze—dried poultry was published by Sosebee gt gt. (1963). Solutions of papain and Rhozyme P-ll were used in rehydration. Rhozyme P-ll was used at a concentration of 0.02% and papain at 0.003%. Both enzymes altered the appearance of skeletal muscle and collagen. Papain caused extensive degradation and loss of staining ability of collagen and some granulation of muscle fibers. Rhozyme P-ll caused granulation of muscle fibers and some vacuolation and loss of staining ability of collagen. MATERIALS AND METHODS Selection of Chickens A total of 111 White Leghorn females of three different ages (11, 20 and 52 weeks) were used in Part I of the study (effects of age on tenderness). All birds were from the same brood and were raised under identical management practices. Two hundred twenty-six White Leghorn females (17 months of age) were processed for Part II of the study (effects of enzyme treatments on chicken breast muscle). The chickens used in this latter study were from three different broods. However, all of the birds used in each treatment were from the same brood. Eggcessigg and Sample Preparation A11 birds were slaughtered on a killing wheel by means of a semi-Kosher cut which severed the jugular vein and carotid artery on one side of the neck. They were bled and then scalded in a Rotomatic (basket-type) scalder at 59°C for 70 seconds. Feathers were removed by an Ashley auto- matic rubber-fingered picker. Seven birds were placed in the picker at a time for a period of 45 seconds. Following the picking operation, the birds were eviscerated and placed in tanks containing slush ice and water for a 24-hour aging period. The ready-to-cook birds, in lots of 15, were simmered in a steam-jacketed kettle. They were cooked to center breast temperaturesof 82°C. The temperatures were registered by a 44 45 Brown recording potentiOmeter. Thermocouples were inserted in six of the 15 birds in each lot. The thermocouples were connected by a series circuit. Thus, an average temperature from the six birds was obtained. After cooking, the birds were cooled in water for five minutes. The two pectoralis majgr muscles from each bird were removed, packaged in polyvinylidene-chloride bags, frozen .at -35°C for four hours, and transferred to -18°C for storage. After 24 hours of storage at -18°C, each breast in Part I was cut into a single uniform piece on a band saw. A 3 1/2- by l 3/16-inch wooden block was used as a guide to cut standard samples from the center of each breast. The block was placed on the breast, and a scalpel was used to trace the outline of the block on the breast. The samples were cut with the band saw along the tracing. The samples were freeze-dried for 18 to 20 hours in a Stokes freeze—dryer, Laboratory Model 2003 F—2, using a vacuum of 100 to 150 microns of mercury and a plate temperature of 30°C. The freeze-dried meat was packaged under partial vacuum in polyvinylidene—chloride bags and stored at -18°C until used. The chicken breasts used in Part II were handled in the same manner, with one exception. Just prior to freeze-drying, the frozen breasts were removed from the polyvinylidene— chloride bags and diced into 3/8-inch cubes by a Toledo one- horsepower meat saw. Non-freeze-dried control birds were prepared by the same methods. To maintain constant conditions, these breast 46 samples were subjected to the same rehydration procedure as the freeze-dried breasts. Rehydration The muscles used in Part I were rehydrated in 100°C distilled water at a water—to-sample ratio of 6:1. The sam- ples were rehydrated for 15 minutes. After rehydration, the samples were emptied into a 20-mesh sieve and drained for five minutes before Warner-Bratzler shear press and sensory evaluations were conducted. In Part II the diced, freeze-dried samples were rehy— drated in various buffer solutions to provide the desired pH. The various buffers used in this study with their re- spective pH values were as follows: (1) pH U.0 __ 0.2 M acetic acid and (2) pH 5.0 0.2 M sodium acetate (3) pH 6.0 __ 0.2 M monobasic sodium phosphate (a) pH 7.0 ' and 0.2 M dibasic sodium phosphate (5) pH 8.0 _g 0.2 M tris (hydroxymethyl) amino (6) pH 9.0 methane and 0.2 M HCl The above solutions were made up according to the speci- fications of Gomori (1955). A total of 200 m1 of each buffer was placed in a 500 m1 wide-mouthed Erlenmeyer flask. A 40- inch air condenser was inserted into the drilled hole in a #10 rubber stOpper and fitted into the neck of the reaction flask. The condenser was used to prevent water evaporation from the reaction flask. Thus, it prevented changes in pH due to evaporation, or changes in the water-to-sample ratio which would affect rehydration. #7 The entire apparatus containing the buffer was then placed in a Magni Whirl constant-temperature water bath and held at the desired temperature until the buffer solution and water bath temperatures equilibrated. The temperatures used in this study were 00°, 50°, 60°, 70°, and 80°C. Just prior to the addition of the enzyme and substrate, five ml of a stock solution of 0.5M l-cysteine hydrochloride was added to the 200 ml of buffer solution. This gave the buffer solution an actual concentration of 0.0125 M cysteine. The cysteine was added to activate the enzyme. The enzymes used in this study were papain, ficin, bromelin and Rhozyme P-ll.1 The first three enzymes were obtained from the Nutritional Biochemical Corporation and the latter one from the Rohm and Haas Company. All enzymes were stored at 2°C and low relative humidity. Immediately after the addition of cysteine to the buffer, a predetermined amount of enzyme was added with a pre—weighed freeze-dried meat sample. The enzymes were weighed accurately to the third decimal place on a Mettler analytical balance. The freeze-dried chicken samples were weighed accurately to the first decimal place on a Torsion balance. The reaction mixture was allowed to incubate at the de- sired temperature for five minutes. After incubation, the reaction flask was removed from the bath and heated to boiling over a hot flame to step the reaction. The reaction 1Hemoglobin assay units of these enzymes as received from the suppliers were: Rhozyme P-ll 3,200; papain 10,100; bromelin 15,000 and ficin 27,200. #8 mixture was allowed to boil gently for 3 l/H minutes. Immediately after removal from the water bath, a water-cooled condenser was inserted into the neck of the reaction flask to prevent evaporation, which would be greatly accelerated at the higher temperatures. The meat sample was weighed prior to and following re- hydration to determine the amount of water absorbed. Imme— diately following enzyme inactivation, the reaction mixture was emptied into a 20-mesh sieve and the buffer collected for pH determination. The meat sample was weighed and a constant weight was then subjected to Kramer shear press analysis. Chemical Determinations A. Protein Nitrogen The commercial proteolytic enzymes used were sold as a crude mixture which was diluted with a filler to a specific activity. Previous workers, when dealing with meat tenderi- zation through the use of proteolytic enzymes, expressed the enzymes used in terms of percentage concentration (weight/ volume). As a result it was necessary to conduct protein- nitrogen determinations on the commercial preparations to specify the concentrations of enzyme used. For each enzyme, six protein-nitrogen determinations were made. All deter- minations were carried out by using the micro-Kjeldahl pro- cedure (Ogg, 1960). Boric acid was used as the receiving agent. Non-protein nitrogen determinations were also used as ”9 a method of determining enzyme inactivation. Sosebee 2£.§l' (1963), after reconstituting freeze—dried chicken in proteo— lytic enzyme solutions, used an enzyme inactivation time of ten minutes at 100°C. It was believed that the added effects of heat on tenderness could be reduced by reducing the in- activation time. Thus, it was reasoned that if after the heating period the enzyme was not destroyed, then there should be continued proteolysis with a resultant increase in non-protein nitrOgen. This non-protein nitrOgen could then be determined by the micro-Kjeldahl method. A procedure was developed which involved heating the reaction mixture at 100°C for three minutes. The reaction mixture was transferred from the reaction flask to a Waring blender and blended for 60 seconds. Two lO-ml aliquots were pipetted from the blended mixture and deposited in two large test tubes. Twenty m1 of 20% trichloroacetic acid was added to each of the test tubes, which were then shaken vigorously. This resulted in the precipitation of all of the protein. The samples were held for five minutes and filtered twice through Whatman #3 filter papaer. Ten m1 of the fil- trate was pipetted directly into a micro-Kjeldahl flask for nitrogen determination. During the five-minute time interval mentioned, the re- maining blended mixture was transferred to a clean 500 ml wide-mouthed Erlenmeyer flask and returned to the thermo- statically controlled water bath. Additional, duplicate aliquots of this mixture were taken at intervals of 30 and 60 minutes. 50 B. Moisture Moisture determinations were made immediately after freeze-drying on samples selected at random. Such determina- tions were also conducted on the crude enzyme preparations to calculate their initial moisture content. Moisture contents were determined according to the pro- cedure outlined by the A.O.A.C. (1960). This method involved drying a ground sample for 16 to 18 hours in a dry—air oven at 100°C. Physical Determinatigns A. pH Measurements All pH determinations were made with a Beckman Zero— matic pH meter. pH of rehydration solutions were measured 60 minutes after reconstitution in enzyme solutions. The pH of each freeze-dried chicken breast was measured after first. rehydrating for five minutes in distilled water at a water- to-sample ratio of 6:1 and then blending in a Waring blendor for 60 seconds. A 25-ml aliquot of the blended mixture was used to determine pH. B. Heat Penetration Several control samples from Part II were rehydrated as usual, except without enzymes. Before rehydration, six thermocouples from a Brown recording potentiometer were connected in series and one thermocouple inserted into each of six pieces of freeze-dried chicken breast samples. One thermocouple measured the temperature of the reaction mixture. 51 The meat containing the thermocouples was immersed in the buffer-cysteine rehydration solution; the condenser was attached; the reaction flask was placed in the controlled water bath; and the meat was rehydrated for five minutes. A multipoint potentiometer, recording every 30 seconds, pro- vided data for the time-course curve which consisted of the temperatures required to bring the reaction mixture to water- bath temperature. Similar data were obtained for all five temperatures used in the study. Samples were taken directly from the water bath and placed on a hot flame without removal of the thermocouples and heated to 100°C. The air condenser was replaced by the water condenser during the "come—up" time. The time-course curve obtained showed the time involved at the various re— action temperatures to bring the reaction mixture up to 100°C. The data obtained showed that the individual meat cubes reached 100°C 15 seconds after the buffer-cysteine solution. Thus, 3% minutes was the actual time used for enzyme inactivation rather than 3 minutes--the excess 15 seconds allowed for the delay in heat transfer. Measurements of Tenderness A. Warner—Bratzler Shear Tenderness of rehydrated freeze-dried chicken breast muscle from birds in Part I was measured with a Warner— Bratzler shear. This shear press consists of a steel blade 1/32 of an inch thick, with a triangular-shaped aperture 52 slightly larger than the sample of meat. Each sample was placed in the aperture, and the blade pulled through a rigidly supported Opening formed by two steel plates. The steel plates were Just wide enough to allow free passage of the blade. The gear system was powered by a constant-speed, l/lZ-horsepower motor. The shear readings were taken from a spring scale calibrated in pounds. Each sample was sheared four times at intervals of approximately ll/16 inches. The first shear was obtained at the anterior end of the muscle. Since a cross-sectional core of breast muscle was very difficult to obtain from chicken, a different method was used to eXpress the shear force from the chicken meat. After each shear, the muscle section was pressed lightly against an ink blotter (a piece of coarse Whatman filter paper would be satisfactory). The moist outline remaining was traced with an ink pen, and the area was measured by taking dupli- cate readings with a Keuffel and Esser compensating polar planimeter. The force was thus ultimately expressed in pounds of force to shear each square inch of cross-sectional muscle area. An average of the four shear values was used as the tenderness value for each individual sample. B. Sensory Evaluation The "triangle test", as recommended by Pearson (1963). was used to select the panel. Five panel members, as recommended by Ohlson (1955), were used. Panel members were selected from the staff of the Food 53 Science Department, who had previous experience on other taste panels. The project, objective, score sheet, and tasting procedure were explained to panel members during training. Trial practice sessions were held using dehydrated freeze-dried chicken several times before the actual project began. A seven-point hedonic scale was used in this study. Although space was provided on the score card for the evalua— tion of four factors--initia1 tenderness, residual tender- ness, juiciness and flavor--only the first three factors were considered. (Initial tenderness was defined as the sensation realized after completing the first chew through the sample, while residual tenderness referred to the sensa- tion realized after the complete mastication of the sample.) Even though more than one factor was evaluated, the panelists were asked to evaluate each sample independently from other samples. Descriptions for initial and residual tenderness ranged from "extremely tender" to "very tough" (Appendix Table 12). The word descriptions for juiciness ranged from "very juicy" to "very dry". When the tenderness of breasts from birds of different ages were evaluated by this panel and calculated statistically, the initial and residual tenderness factors were not sig- nificantly different. Therefore, only the residual tender- ness was used for the evaluation of enzyme-treated chicken. For statistical purposes each description was assigned a point value; i.e., the toughest category was assigned 5h seven points and the most tender category, one point. Since the Warner-Bratzler shear was used to shear each sample of chicken breast four times, this provided five in- dividual sections of breast muscle for immediate panel evaluation. The first shear was obtained from the anterior end of the sample and the remaining three shears in succes- sion. Each panel member obtained approximately the same section of muscle every time. Five samples were rated by each panelist at each sitting. Panel members were provided coded plates with five randomly selected numbers marked on each plate. At each sitting, each panel member was provided a plate with a different code. Samples were rotated so that at no two successive sittings were samples of the same age on the same number. The panel members were asked to chew each sample across the grain of the meat. All samples were evaluated at room temperature. The performance of panel members was checked periodically by providing identical samples for evaluation. C. Kramer Shear Press An Allo-Kramer shear press with a Model SP—12 recording attachment was used to measure the tenderness of rehydrated chicken breast muscles in Part II. The Kramer shear press measured the maximum pressure required to force the shearing ram through the material. The instrument contains an electronically sensitive pressure plate, which registers through a proving ring. The pressure— 55 sensitive plate was connected through an amplifier to a recording chart. As the force was applied to the sample in the shearing cell, the proving ring compressed (deflected) by an amount preportional to the force. This compression was detected by an electrical transducer, whose output was an electrical signal of amplitude proportional to the de— flection. The output signal of the proving ring was ampli- fied to the recording mechanism. It was possible to record continuous pressure as the press ram completed its downward stroke. The shearing head was located on the end of the press and contained several thin, rectangular-shaped blades which passed through the product. A pressure-time curve was obtained from the recorder, and this curve was used to calculate the work required to penetrate the product. Wells 32 3;. (1962) found that the peak of the pressure-time curve was as accurate as the area under the curve for measuring tenderness of rehydrated freeze-dried chicken. All of the samples were placed in a single layer in the shearing cell with the grain of the meat perpendicular to the shearing blades. Measurements were based on the pounds of force required to shear the sample. In order to simplify comparisons, all of the results were expressed in pounds of force necessary to shear each gram of sample. A proving ring setting of 1,000 pounds was used, which required that each shear value be multiplied by a factor of 10. Since the recorder chart paper was divided into single-unit increments from one to 100, this multiplication factor allowed the 1,000 56 pounds to be equally dispersed over the entire scale. A 3,000-pound proving ring was used and the shear press Speed was standardized so that each downward stroke of the ram was completed in 15 seconds. Tissue Preparation for MicroscOpic Examination Samples of cooked, rehydrated, freeze-dried chicken breast muscle were placed in tissue buttons and subjected to the following treatment in an Autotechnicon as suggested by the Armed Forces Institute of Pathology (1960): Step Procedure Time 1 Dehydrated in 70% ethyl alcohol Holding point 2 " " 80% ethyl alcohol 1 hour 3 " " 95% ethyl alcohol 1 hour a " " 95% ethyl alcohol 1 hour 5 " " 100% ethyl alcohol 1 hour 6 " " 100% ethyl alcohol 2 hours 7 Cleared in 100% ethyl alcohol + xylene (50:50) 1 hour 8 Cleared in Methyl benzoate 1 hour 9 Cleared in Xylene o 1 hour 10 Infiltrated in Paraffin (50-52 C) 1% hours 11 Infiltrated in Paraffin (56-58°C) 1% hours Since the above samples were from cooked muscle, the samples were not fixed in 10% Formalin. Cooking, by itself, is a fixing procedure. However, raw samples were placed in 10% Formalin for six hours preceding the above procedure. After the above cycle was completed, the container of paraffin and tissue samples was removed from the Autotechni— con and placed in a vacuum oven at 60°C for 20 minutes. The tissue samples were then immersed in new paraffin and returned to the vacuum oven for 20 minutes. They were then imbedded in paraffin (56-5800), which was allowed to solidify. Six 57 individual tissue samples were placed in each embedding mold. After solidifying the mold in running tap water, tissue blocks were cut, trimmed and labeled. A 12-hour period of soaking in distilled water was required prior to sectioning, because of the extremely brittle nature of the cooked tissues. Tissues were sectioned at six microns on a Spencer #820 microtome. Slides were coated with Mayer's egg albumin so tissues would adhere. This solution was composed of blended egg white and glycerine in a 1:1 ratio. The ribbons of sections were floated on water at UBOC. The coated slides were then immersed in the bath and the sections floated onto the slides. The slides were placed in staining racks and air dried for at least 12 hours. The racks containing the slides were placed in a 5600 oven for one hour. This melted the paraffin so that it would not interfere with staining. All slides prepared in this experiment were stained with a modification of Masson's trichrome stain (Masson, 1929) according to the following procedure: Step 1. Deparaffinized in Xylene for 5 minutes 2. Deparaffinized in Xylene for 5 minutes 3. Deparaffinized in 100% ethyl alcohol for 3 minutes. A. Hehydrated in 95% ethyl alcohol for 3 minutes 5. Rehydrated in distilled water for 3 minutes 6. Stained in Weigert's iron hematoxylin solution for 10 minutes 7. 8. 9. 10. ll. 12. 15. 16. 17. 18. 19. 58 Rinsed in running tap water for 10 minutes Rinsed in distilled water Stained in Biebrich scarlet-acid fuchsin solution for 10 minutes when raw tissues were used or for 5 minutes in the case of cooked tissues. The solution was saved. Hinsed in distilled water Differentiated in phosphomolybdic acid- phosphotungstic acid solution for 5 minutes. The solution was discarded. Stained in aniline blue solution for U0 seconds in the case of raw tissue or for 10-15 seconds if cooked tissue was used. The solution was saved. Hinsed in distilled water The excess aniline blue stain was removed by placing.in 1% acetic acid solution for h minutes. The solution was discarded. Dehydrated in 95% ethyl alcohol for 3 minutes Dehydrated in 100% ethyl alcohol for 3 minutes Dehydrated in 100% ethyl alcohol for 3 minutes Clearing in Xylene for 5 minutes Clearing in Xylene for 5 minutes The tissue sections were mounted in Permount, dried for wo hours and labeled. According to the procedure of Masson (1929), the prepara— tion was stained with Biebrich scarlet-acid fuchsin solution. 59 Differentiation was accomplished with the phosphomolybdic- phosphotungstic acid solution, which discolored the collagen and fixed the stain to the cytoplasm. Collagen was toned with aniline blue. In the modified procedure used in this study, the nuclei stained black; the cytoplasm, muscle fibers and intercellular fibers, red; and the collagen, blue. ESULTS AND DISCUSSION Part I. Effects of Age on Tenderness The initial phase of this study was conducted to evalu- ate the relationship between age of birds and the tenderness of freeze-dried and reconstituted breast muscle. Cooked pectoralis mgjgg muscles from White Leghorn hens ll, 20 and 52 weeks of age were freeze-dried in pieces measuring 3 1/2 inches long, 1 3/16 inches wide and normal muscle thickness. Each muscle sample was sheared four times with a Warner— Bratzler shear. Shear values were calculated in pounds of force to shear each square inch of sample. Water uptake during rehydration of freeze-dried samples was obtained and calculated as a percentage increase of the dried weight. Shear values for freeze-dried and control samples of breast muscle from different aged birds are reported in Table 1. Shear values from freeze-dried samples were higher than from control samples. Differences in tenderness due to freeze-drying were accentuated in samples from 11_week-old birds, and shear force was directly related to age of birds. The volume of water absorbed during rehydration was inversely related to shear force and age of birds. Shown in Table 2 are results from panel evaluations of tenderness and juiciness of freeze-dried chicken breast muscle from different aged birds. Tenderness and juiciness decreased with increasing numerical scores. Control samples were more tender than freeze-dried samples, and breast meat from young birds was more tender than from 20- and 52-week— 60 61 TABLE 1. Effects of freeze-drying on tenderness of chicken breast muscle as determined by shear values Ego of No. of Treatment Water Shear Fords Bird Samples U take Weeks % lbs/sq in lbs7gm 11 lb control 17.3 60 freeze-dried 212.5 M8.6 0.68 diff. 31.3 20 lb control 33.3 60 freeze—dried 173.5 A .8 0.67 diff. 10. 52 1h control M7.6 60 freeze—dried 1U2.7 56.8 0.9a diff. g9.2 TABLE 2. Effects of freeze-drying on tenderness of chicken breast muscle as determined by panel scores Age of No. of Treatment Tenderness Juiciness _Bird Samples Weeks ini- resid- (“f resid- tial ual ual ll 1U control 2.5 2.2 3.3 60 freeze-dried %:j. 3;_, 2;g diff.+ .0 +1.1 +0.1 20 14 control 2.3 3.0 3.3 60 freeze-dried ._a5 ._;2 3:2 diff.+l.2 +1.3 +0. 52 1h control 5.0 3.9 3 8 60 freeze-dried 5;: .h .%a2 __ diff.+0.7 +1. + .1 1Larger numbers represent less tender or less juicy meat. 62 old birds. Juiciness scores of freeze-dried and control breast samples from ll-week-old birds were similar. However, juiciness scores of breast samples from 20- and 52-week-old birds decreased with increasing age of birds. The data were evaluated by analyses of variance to de- termine significance between treatments. These analyses were conducted according to Snedecor (1956) and are presented in Tables 3-8. The mean panel scores and mean shear press values were compared by Duncan's Multiple Range Test (Duncan, 1955). Significance was determined by calculating the variance ratio (F). In each of the analyses significance was indi- cated by * (5%) and ** (1%) levels of probability. Two correlation coefficients were computed and are presented along with their parameters in Table 9. Tenderness was measured objectively by the Warner- Bratzler shear and an analysis of variance is reported in Table 3. Differences in tenderness due to age of birds were significant at the 1% probability level. Results obtained by using Duncan's Multiple Range Test showed that 52-week- old birds were toughest. Unlike the panel scores, shear loress values were not significantly different between breasts from birds of the two younger age groups. In the above analysis, no significant difference in tenderness was noted between right and left pectoralis major Imlscles, whether tenderness was evaluated by the panel or Niear, or using freeze-dried or control breast meat samples. 63 TABLE 3. Analysis of variance and Duncan’s Multiple Range Test for Warner-Bratzler shear values of freeze-dried breast meat from birds of three different ages Sourcemof” Degrees 6?“ Sum of Mean Variation Freedom Squares Square F Total 179 62381.48 Ages 2 5179.34 2589.6? 7-75** Breasts 1 101.55 101.55 0.30 Age x Breasts 2 66h.37 332.19 0.99 Replications 29 7985.65 275.37 0.82 Error 145 48%50.57 .334.lh Duncan's Multiple Range Test1 Age of birds“ " 26Veeks“' 11 Weeks 52 Weeks Mean shear value M3.8 #8.6 56.8 1Any two means not underscored by the same line are signifi- cantly different. Any two means underscored by the same line are not signifi- cantly different. 6U As mentioned previously, two tenderness scores were recorded by each panel member for each sample. One score was based on the sensation realized after the first chew, whereas the other score was based on evaluation after masti- cation of the sample. Differences in tenderness due to age of birds were highly significant (1% level) as shown in Table U. The younger birds were more tender. However, no significant difference was found between initial and residual tenderness scores (indicated as treatments) nor between the interaction of these tenderness scores and the differences due to age. Therefore, residual tenderness scores were used in the final analysis of tenderness. The statistical analysis of residual tenderness scores of the panel is presented in Table 5. Significant differ— ences (1% level) were found among birds of different ages, among panel members and among replications. A significant F value for replications was anticipated. Individual birds vary considerably in tenderness, and repli- cuations represent individual breasts. Although tenderness suzores varied significantly among panel members, individual tenderness scores were fairly consistent. Since there were significant differences in tenderness 'betnveen age groups, the Multiple Range Test was used to eVTiluate significant differences between means. It was found trmit ll-week-old birds were significantly more tender than tFRB 20-week—old birds, and the latter were significantly more tenlder than the 52-week-old hens. The lower numerical ratings TABLE h. Analysis of variance between initial and residual panel tenderness scores of freeze-dried breast meat from birds of three different ages Source of Degrees of Sum of Mean variation -~-*~rggggggy1 Squares Square _, F ‘q Ages 2 134.68 67.3h 60.67** Treatments 1 2.22 2.22 2.00 Age x Treatments 2 0.81 0.41 0.37 Replications 2 26.hu 0.91 0.81 Error .~_‘ .. 1&5, J 160.61 1.11 TABLE 5. Analysis of variance and Duncan's Multiple Range Test for panel tenderness scores of freeze-dried breast meat from birds of three different ages Source of Degrees of Sum of Mean ‘Variation Freedom Squares Square _E Total 899 2090.38 Breasts 1 1.77 1.77 1.17 Age 1 Breasts 2 0.52 0.26 0.17 Panel 1+ 129-79 32.0 21.49“ Age X Panel 8 12.29 1.5 1.02 Breasts x Panel it 0.22 0.06 0.04 :ge x Breasts x Panel 8 8.39 1001 0-67 Replications 29 87.78 3003 20°F Error 8&1 1261.89 lgjl Duncan's Multiple Range Test ITS—e of birds Mean Tenderness 11 Weeks 20 WéEks 52 Weeks Score 3.4 h.2 5.” 66 represent more tender samples. Similar results were obtained by an analysis of variance of panel tenderness scores from non-freeze-dried control samples (Table 6). Although no significant difference was found among replications, a significant F value was noted among mean scores of panel members and age of birds. Duncan's Multiple Range Test indicated that panel tenderness scores decreased with increasing age of birds. Panel members were asked to evaluate Juiciness of breast meat samples. The analysis of variance of Juiciness scores is presented in Table 7. Differences in juiciness scores due to age of birds were highly significant, as were differences among scores of panel members and replications (individual 'breasts). Results from Duncan's Multiple Range Test showed that Juiciness scores of samples from all three age groups 'were significantly different. Juiciness and tenderness of ‘breast meat decreased with an increase in age of birds. :Steinberg (1960b) found no significant correlation between (ibjective tests and sensory tenderness and juiciness scores (If freeze—dried, cooked beef. The results of this study did that confirm his findings but instead supported those of Deatherage (1951) who reported a positive correlation between tenderness and Juiciness scores. In the analysis of variance of the percentage of water updaake (Table 8), breast meat samples from birds of different aEKBS absorbed significantly different amounts of water. Per- CeTrtage of water uptake was inversely related to age of birds. 67 TABLE 6. Analysis of variance and Duncan's Multiple Range Test for panel tenderness scores of non-freeze-dried breast meat from birds of three different ages Source of Degreesfiof Sum of ‘Mean :Zariation _“~-_ Freedom Squares Square F Total 209 336.20 Ages 2 90.20 [+5.10 4303769” Breasts l 0.04 0.04 0.04 Age X BreaStS 2 061 2081 2070 Panel L" 3 .55 8061+ 8031*“ Age x Panel 8 13052 1069 1063 Breasts x Panel 11 3.70 0.33 0.89 .Age x Breasts x Panel 8 3.38 0. 2 0.40 Replications 6 3.43 0.57 0.55 Errgr 174 181.77 1.04 _____ Duncan's Multiple Range Test Age of Birds 11 Weeks 20 Weeks 52 weeks Mean Tenderness Score 2:3 340, 3L2 —--.--.~.- '— TABLE 7. Analysis of variance and Duncan's Multiple Range Test for panel Juiciness scores of freeze—dried breast meat from birds of three different ages Source of Degrees of Sum of Mean Eariation_ ,1, Freedom Squares_ Square _. F Total 899 1674.60 Ages 2 467.90 233.95 184.21** Breasts l 1.69 1.69 1.33 Age x Breasts 2 0.78 0.39 0.31 Panel 4 55.59 13.90 10.94** Ase x Panel 8 16.39 2.05 1.61 Breasts x Panel 4 0.71 0.18 0.14 A88 x Breasts x Panel 8 3.64 0.46 0.36 Replications 29 62.90 2.17 1.70* EEEQE, -1 A 841 1065.00 1.27 Duncan's Multiple Range Test Ase of Birds 11 weeks 20 weeks 52 Weeks Mean Juiciness Score 2:2 3.2 4.9 ~._ — 68 Since tenderness also decreased with increasing age (Table 2), a direct relationship was found between percentage rehydra- tion and tenderness. Two correlation coefficients were obtained from the data summarized in the analysis of variance tables and are in- cluded in Table 9. The panel tenderness scores for chicken breast meat from birds of each age group studied were corre- lated with the Warner-Bratzler shear values obtained from the same meat samples. A correlation coefficient r = 0.59 was calculated between panel tenderness scores and Warner— Bratzler shear values using freeze-dried chicken, as compared to a correlation coefficient r = 0.80 for similar control samples. Freeze-dried breast meat was noticeably tougher than control samples. Unlike the results of Wells 23 a1. (1962), shear values for freeze-dried chicken breast in this study agreed with panel scores. However, more significant differences were inoted when breast muscles were measured by the sensory panel than when measured by the Warner—Bratzler shear. With the shear, only the older birds were significantly different in tenderness, whereas the panel found all three age groups to differ significantly. Seltzer (1961) found that older, more mature birds pro- duced the most tender freeze-dried chicken meat. The results 0f tfldis study do not support his findings but instead agree With. the sensory results of Wells gt a1. (1962). Tenderness varied greatly between individual birds; most 69 TABLE 8. Analysis of variance and Duncan's Multiple Range Test for percentage water uptake by freeze-dried breast meat from birds of three different ages Source of Degrees of Sum of Mean Variation Freedom Squares, Square F Total 179 280294.1 Ages 2 146930.9 73465.4 100.4** BreaStS 1 760.9 760.9 100 Age x Breasts 2 260.3 130.2 0.2 Replications 29 26262.8 905.6 1.2 Error _”___-__ 145 106079.2 731.6 _w» _ Duncan's Multiple Range Test Age of Birds 52‘Weeks 20 Weeks 11 Weeks Mean 5: Water Uptake 142.7 173.5 212.5 TABLE 9. Correlation analyses for panel tenderness scores and Warner-Bratzler shear values Sample i Sx y;_ 8y lb» Sy°rZ r Freeze-Dried Chicken 4.28 1.27 49.24 18.39 8.49 150.77 0.59 NoneFreeze- jgried Chickenq3L06M_g,89‘ 32.71 18.25 16.46 123.56 0.80 70 of this variation was accounted for in the replications of the analyses. The remaining variation was present as part of the error term. In many instances, tenderness scores of individual panel members differed significantly. This significant variation was the result of lower-than-average scores from one panel member and higher-than—average scores from another. However, this should not imply that any one panel member was incon- sistent in evaluating samples; actually, a very high degree of consistency was noted. Part II. Effects of Enzyme Treatments on Chicken Breast Muscle Proteolytic enzymes were incorporated in rehydration solutions to cause proteolytic breakdown and increased tender— ness in the meat. Wang gt 21. (1958) found that Rhozyme P-11 and papain tsere similar in their ability to hydrolyze gelatin, but 150 times as much Rhozyme P-ll as papain was needed to affect significantly the initial tenderness of meat. Therefore, in the present study, it was decided to base the concentration of enzyme on percentage rather than on activity. Preliminary trials led to the establishment of an effective concentration Zange for each enzyme for optimum tenderization. Different enzyme concentrations were placed in the rehydration solutions, and zafter the samples were rehydrated, they were evaluated fru"tenderness by the sensory panel. Table 10 shows the re- sults; of these evaluations. The sample was considered 71 TABLE 10. Effects of proteolytic enzyme concentration on tenderness and acceptability of freeze-dried chicken breast meat as determined by a panel Concentration Average Tender- Accept— Emzyme _jfleightZVolume) ness Score ‘__ able ? Cfi) Iflhozyme P—ll 0.010 3.2 yes " 0.015 2.9 yes " 0.020 2.6 yes " 0.025 2.2 ? " 0.030 1.7 no £?1cin 0.0001 4.2 yes " 0.0005 3.3 yes ” 0.0010 2.” no ” 0.0020 ---3 no Bromelin 0.0005 #.1 yes " 0.0010 3.2 yes ” 0.0020 2.? yes " 0.0030 1.8 no Papain 0.001 3.3 yes " 0.002 2.6 yes " 0.003 2.3 no " 0.005 1.3 no aToo "mushy” to give to the panel and was given an automatic unacceptable rating. 72 acceptable when four of the five members agreed that it was not too soft or "mushy". The sample was rated as question- able when two panel members thought the sample was unaccept— ably tender. When more than two members found that a sample was too soft or "mushy", it was given an unacceptable rating. Thus, it was possible to find an enZyme concentration of optimum strength for producing the most tender yet acceptable product. An enzyme concentration of 0.02% was found cptimum for Rhozyme P-ll, whereas considerably smaller concentrations were most desirable for the other three enzymes. Ficin was most desirable at a concentration of between 0.0005% and 0.001%. A value of 0.0008% was used. Both bromelin and papain exerted maximum acceptable effectiveness at a con- centration of 0.002%. In most cases, average panel scores of 2.0 or below were not acceptable. Enzyme concentrations were inversely related to tenderness values. The most desirable concentration for each enzyme was used to find an cptimum pH. Also considered was the phenome— non of water uptake. These two factors could not really be separated, since an increase in the amount of solution ab- sorbed by the meat would result in more enzyme being absorbed into the structure of the meat, and it was conceivable that rehydration might be affected by pH. Effects of pH on tenderness of breast meat are presented in Figures 1 and 2. Each point plotted, except at pH 4.0, represents average tenderness values of five replicate samples 73 as determined by the Kramer shear press. The most desirable The same data were plotted for controls in each figure. enzyme concentrations (from Table 8) were selected for use. Temperatures of rehydration solutions were 50°C for Rhozyme P-11 and 70°C for the other three proteases. Two important observations are: (l) Ficin, bromelin and Rhozyme P-ll all produced the most tender samples at pH 5.0 while pH 7.0 was optimum for the most tender papain-treated samples. (2) The non-enzyme-treated control samples were :noticeably more tender at pH 7.0 than at the other pH values. The enzyme treated samples also showed a marked increase in tenderness at pH 7.0. Since pH 5.0 was the lowest pH originally selected for study and preliminary results indicated that maximum tender- fness occurred at this pH, it was considered desirable to test the results of the tenderizing action at pH 0.0. As eXpected, 'the shear values for bromelin and Rhozyme P-ll were higher sat pH h.0 than at pH 5.0. At pH n.0, these enzymes were in- sactivated, since the shear force required to penetrate the naeat samples was of the same magnitude as that required for the controls. Samples were not rehydrated in ficin solutions art pH u.o because of the lack of birds with the same back— is:round. All enzymes appeared to possess some activity over the 1311 range 5.0 to 9.0. However, ficin activity was greatly I"educed at pH 9.0. It is not entirely clear why the controls were more 7a léi Control in - 12 1 E \\ g a lO-m o a. . r U) f. 8 1" Ficin 6 L _1 4 l A: 510 610 Vic 320 9.0 Figure l. Shear press values of freeze—dried chicken rehydrated in papain and ficin solutions at various pH values. 164- Control luv-H- 12.- E) \\ g Bromelin g; 10-- a. a; :3 8+- Rhozyme '-ll 6 i 44 1 i i % h.0 510 610 7.0 8.0 9.0 Figure 0 Shear press values of freeze-dried chicken rehydrated in bromelin and Rhozyme P-ll solutions at various pH values. 75 tender at pH 7.0, but it is probably due somewhat to the ex- tent of the rehydration. Auerbach 33 al. (1954) found that the highest level of rehydration of freeze—dried beef occurred in solutions in which the pH was near 7.0, regardless of the osmotic pressure. Similar results were obtained in the present study with the controls and are presented in Figure 3. (The scale for % water uptake was reversed for ease of plotting.) The greatest amount of water uptake occurred at pH 7.0 as did the lowest shear values. The lower tenderness scores of enzyme—treated meat at or near pH 7.0 may be attributed in part to the relationship of water uptake to shear force. Although water uptake may have accounted for the increased tenderness produced by papain at pH 7.0, this ij was cptimum since no other depression in that curve was lobtained. In the present study, the determination of cptimum pH :for enzymatic action on freeze—dried meat actually includes 'the consideration of both pH and rehydratability. This cpti— nnum pH for activity is not necessarily the same as that found bYgelatin or hemoglobin assay (Wang 33 al., 1958). Although Sosebee 33 a1. (1963) used papain and Rhozyme Il—ll during rehydration of freeze-dried chicken, no attempt tvsas made to control the pH of the solutions. Also, the re- Eicztion temperatures selected were not necessarily cptimum for the enzymes used for tenderization. The pH values of rehydration solutions were determined before and after reconstitution (Table 11). When pH was lbs. Force/gm 76 178 q-179 16v- ip180 13" ..181 \ .,182 la... 4-183 .3 s +181» 43* 131- 7 D H d) 4.) #185 g we 12*- , “r186 ~~187 114k 10 L i 5 ‘ t 189 5.0 600 700 81.0 9.0 pH Figure 3. Relationship between water uptake of freeze- dried chicken and shear force values. 77 TABLE 11. Change in pH of rehydration solutions during reconstitution Average pH after Initial‘pH Reconstitution Difference “.0 3.9 -0.1 5.0 5.0 0.0 6.0 5.6 -0.u 7.0 6.7 -0.3 8.0 7.5 -0.5 9.0 8.4 -0.6 78 measured after the normal five minutes of rehydration time but without the addition of meat, here was no change in pH. Therefore, when there were changes in pH of the solutions, they were attributed solely to the meat samples. All changes in pH were to the acid side of the initial pH. This was be- lieved due to the lower pH of the freeze—dried meat (pH 5.8) and to the increase in free acidic groups resulting from proteolysis. Figures u and 5 show the pounds of force to shear each gram of sample, when samples of freeze—dried breast muscle were rehydrated at temperatures from #00 to 80°C. Optimum rehydration temperatures of 50°, 50°, 600 and 70°C were se- lected for papain, Rhozyme P-ll, bromelin and ficin, re- Spectively. Each point recorded in these figures represents the average force obtained from five replicate samples. The same control data were used in each figure. Unlike the pH-shear force curves, temperature-shear ffiarce curves showed no sharp peaks. Variations in the tempera- tnxre of the rehydrating solution did not appear to be as czzsitical as variations in pH in the evaluation of tenderness. Ifimcin and bromelin were quite active at all the temperatures used. Papain and Rhozyme P-1l possessed little or no activity at: 80°C. In general, papain was less active than the other el'lzymes. The tenderness of control samples was not affected by rehydration temperature. The percentage of water uptake by the control samples W353 :pdotted against temperature (Figure 6). The percentage 79 Ian-M 16“ Control lum- I 12‘ Bromelin Ficin Lbs force/gm i—l <3 ab 5% 60 o 70 80 Temperature ( C) Figure 4. Shear press values of freeze-dried chicken rehydrated in ficin and bromelin solutions at various temperatures. Control I" 01‘ r Papain Rhozyme P-ll Lbs force/gm L I I 4 l J 40 50 30 o 70 80 Temperature ( C) Figure 5. Shear press values of freeze—dried chicken rehydrated in papain and Rhozyme P-ll solutions at various temperatures. Percentage of Water Uptake 200 190q 180* 17Q. 80 b I l 160 46 5b 8 do do 80 Temperature (°C) Figure 6. Percentage water uptake of freeze-dried chicken at various temperatures. 81 of absorbed water (based on the dry weight) decreased as the temperaturecfi'the rehydration solution increased. Steinberg (1960a, 19600) and Norman and Auerbach (1963) reported simi- lar results with freeze-dried beef. Although water uptake by control samples was higher at the lower temperatures used, tenderness remained fairly con- stant (Figures 4 and 5). Thus, shear force (tenderness) is probably not directly related to water uptake. However, Deatherage (1959) reported that tenderness of meat was re— lated to the ability of meat proteins to hold water. He also found that water-binding capacity decreased with increasing temperatures of the rehydrating solution. Kimmel and Smith (1957) reported that the pH cptimum for digestion of fibrin was pH 7.0, and results of the present work agree. Tappel gt al. (1956a) and Weiner gt at. (1957) reported that the cptimum temperature for papain digestion of beef was 60° to 80°C. An cptimum temperature of 50°C was reported for papain in the present study. Tappel gt al. (1956a) and Weiner gt at. (1957) also stated that the Optimum temperature for any enzymatic reaction was closely related to the length of time which that reaction covered. In general, ‘the shorter the digestion time, the higher the Optimal tempera- tnlre for that reaction. Weiner gt at. (1957) used a three— miJTute digestion period. Since a five-minute digestion period W343 used in the present study, it was expected that the cpti- mum: temperature would be comparatively lower. Wang (1957) stated that ficin showed a wide range of pH 82 activity (pH 5.0 to 0.0) on beef connective tissue with an optimum at around pH 5.0 to 6.0. Results from the present study with chicken muscle agree. Yatco-Manzo and Whitaker (1962) found that ficin-catalyzed hydrolysis of elastin was cptimum at a pH 5.0 to 5.5 and at a temperature of 55°C. In the present study, the optimum pH was 5.0 while the optimum temperature was 7000. However, ficin activity was not greatly affected by the temperature differences investi- gated. The optimum pH for Rhozyme P—ll was reported to be be- tween pH 5.5 and 6.0 with an optimum temperature range of 43° to 60°C (Anonymous, 1963). The results of the present study agree, since a 50°C optimum was obtained, although a slightly lower pH cptimum of 5.0 was determined. The cptimum temperature range for bromelin was reported to be between 30° and 60°C (Anonymous, 1963). An cptimum temperature of between 500 and 60°C was found in the present study. With cptimum concentrations, pH, and temperatures eg- ‘hiblished, chicken samples were rehydrated under these con— ditions and the resulting proteolysis was examined histolOgi- cally. It was necessary to inactivate the enzymes after rehy~ dration. If the enzymes were not completely inactivated, contiqnued proteolysis, after rehydration, would invalidate conclusions based on histological observations. iAs stated in the Procedure, preliminary results showed 83 that a three-minute heating period at 100°C was sufficient for enzyme inactivation. An increase in non-protein nitrogen would occur in the rehydrating solution when proteolysis continued after the three—minute destruction time. Any in- crease due to continued proteolytic breakdown could be measured by calculation of this non-protein nitrogen. Non-protein nitrOgen was measured in the present study by the micro—Kjeldahl method. The results are reported in Figure 7. Two replicate samples and a control were used for each of the four enzymes. The control samples were not heat treated, and proteolysis was allowed to continue to provide a basis for comparing the heat-treated replicates. In all heat—treated samples, there were no increases in non-protein nitrogen with time after heat treatment. Slight differences which did exist were attributed to experimental error, since the differences were always less than 0.5 ml of HCl titrated. A direct linear increase in non-protein nitrOgen occurred in control samples held up to 60 minutes of incuba— tion for ficin, bromelin, papain and Rhozyme P-ll, respect- ively. This was probably due to the reaction rates of the individual enzymes used. When the five-minute rehydration period was extended to 30 and 60 minutes, the reaction rate 0f1ficin was greater than the reaction rate of Rhozyme P—ll. Hist010gical sections were prepared from the freeze— driead.chicken samples treated with enzymes under cptimum cond itions for tenderness . an spews >H @9095 b» spews w wHoBoHps >H El weoeofis 5:: _. ~ . >N ween—apps ~::::E:::n m mmWWPS wmpmpa Pm mmpmps w wrouwso mIHH >H wzowwso wuHH bm wrouwso wnHH w WPWAHHJQ .Vo ~ 5 \NNHEE‘EPE:F:-E:D x b p I )- b I 0 5.5503 Sea 5:58 Dmo 35:33 a a menace ea Hoooo wow u zpssaom w n ooawwow m.o awe ewe . X 205nmwoaops ZPeHomo: “.0 mmo q.o manaao psmodpdmupou aodoHBPSoa dw spououwgowamsw season mow souneeouops swnwomos 85 Many preliminary trials were necessary to perfect a procedure for staining the cooked and freeze-dried sections. A staining technique was needed which, in one Operation, would differentiate nuclei, connective tissue and muscle fibers. A differential stain for elastic fibers and collagen (Margolena, 1951) was first attempted. Microscopic Observa- tions showed a minute amount of elastic tissue in breast muscle. Thus, a stain to discern elastic tissue was Of little value. Therefore, a general stain for connective tissue was used with the knowledge that it was mostly collagen. Pre- liminary results indicated that Masson's trichrome stain (Masson, 1929) could be used after considerable modification. After many preliminary trials, it was fOund necessary to re— duce the recommended staining times and concentrations. This was probably a result of staining cooked muscle tissue. Staining results indicated that cooked muscle tissue was very receptive to both acid and basic dyes and that the differential stainbng ability Of cooked tissue was very poor. Tissues were stained unevenly and excessive staining with the counter-stain(s) occurred readily. Thus, extreme care was necessary at this step in the procedure. Many of the histological sections showed similar effects of enzymatic breakdown of the tissue. There were considerable variations in this degree of breakdown, however. Four longi- tudinal sections of muscle, two horizontal sections of muscle and.two horizontal sections of connective tissue are presented 86 in Figures 8 through 15, respectively. Figure 8 shows a longitudinal section Of muscle that was exposed to the action Of ficin. This enzyme exerted the greatest effect on the muscle fibers of any of the four used. Disintegration of the sarcolemma was apparent, and complete dissolution is shown in a portion of the bottom two fibers. The extensive granulation was caused by the dissolution of the sarcolemma. A complete absence of nuclei and a gradual disappearance in cross striations was Observed. Overall, there was a slight swelling of muscle fibers as compared to controls which were rehydrated in buffer without the addition of enzyme. The effects of bromelin on chickenmuscle are shown in Figure 9. Bromelin was the enzyme least reactive on muscle fibers. Although bromelin definitely affected connective tissue (Wang gt g;., 1957). it is controversial whether or not bromelin activity can be detected on muscle fibers. Figure 9 shows that there was some action on the muscle fiber. The large, swelled fiber in the center of the photOgraph shows proteolysis of the sarcolemma with the disappearance Of nuclei and cross striations. Nuclei, although poorly stained, are evident in the intact fibers. The fiber was hydrolyzed at specific sites. These results indicate that bromelin did not hydrolyze the fiber in a progressive step— by-step manner, since it attacked various eXposed sites along the fiber simultaneously. Although fibers, such as the one shown, were relatively uncommon in chicken muscle treated 87 Figure 8: Longitudinal section of cooked freeze—dried chicken breast muscle rehydrated in 0.00083 ficin solution at pH 5.0 and 70 . 430K Figure 9: Longitudinal section Of cooked freeze-dried chicken breast muscle rehydrated in 0.002% bromelin solution at pH 5.0 and 60°C. 430x 89 with bromelin, several did exist. However, most of the fibers and the remaining structures in the figure were left rela— tively intact. The effects of bromelin activity discussed here support the data of Wang gt gt. (1957) who also found that bromelin had a trace of activity on muscle fibers. Figure 10 shows the action of papain on muscle fibers. The actions of Rhozyme P-11 and papain were similar--both possessed more activity than bromelin but less than ficin. Granular cytoplasmic material may be found throughout the photograph. As in the previous figures, some of this was due to the cooking process, although further granulation occurred with enzymatic activity. A sarOOplasmic breakdown was evidenced by the broken fiber in the center of the figure. There was a gradual loss of cross striations in other fibers. NO nuclei are evident since their disappearance was usually the next step in proteolysis after the loss of the sarcolemma. A micro-photograph of a section Of cooked, non-freeze— dried and non—enzymatically treated tissue is presented in Figure 11. Cooked, freeze—dried tissue not treated with enzymes had similar structural characteristics and therefore this photograph represents both types Of tissue. The cooked tissue had a certain amount of fiber shrinkage (not as evident here as in cross sections) when compared to raw tissue. The granular material, evident between the muscle fibers, was derived only from the periphery of the protOplasm of the muscle fiber and is distinct from that 90 Figure 10: Longitudinal section of cooked freeze-dried chicken breast muscle rehydratedoin 0.002% papain solution at pH 7.0 and 50 C. 430K. Figure 11: Longitudinal section of cooked non—freeze—dried Enzymatically treated chicken breast muscle. 30X. 92 caused by proteolysis. Wang (1957) reported that the sarco— lemma itself was not hydrolyzed during the cooking process. The muscle fibers appeared quite intact with the presence of very distinct cross striations. Although the nuclei were visible as small, dark, elongated areas in the fibers, they were not clear. In the center Of Figure 11 is a small amount of greyish connective tissue. It is mostly collagen which was coagulated into a gel—like mass by the cooking process. Enzymatic action on the muscle fibers was best Observed in longitudinal sections. The four enzymes had marked simi- larity in their activity when muscle was Observed in cross section. (Figure 12 is representative of activity by all four enzymes.) Again, ficin was most active, Rhozyme P-11 and papain were intermediate in activity, and bromelin was least active. The cooking process resulted in a shrinkage of the main body of the fiber away from the endomysium. The intact endo- mysium surrounding some of the individual fibers is distinct in the lower central portion of Figure 12. However, the en- zymes destroyed the endomysium and extensive fragmentation is visible around the remainder of the exposed fibers. The nuclei are present in some of the fibers, and granular ma- terial is evident throughout the compact tissue area and along the endomysium and its fragments, where the granules tend to collect. As evidenced by the more compact cross- sectional area, the enzymes do not completely separate the individual fibers. If they did, there would be no forces to Fi gar e 13: 93 Cross section of cooked I eeze —f;ied chicken 3“, *7 x» . breast muscle r€hydrate d in 0.000;; llOll solution at p? 5.0 and 70°C . 530x Cross section of cooked freeze-dried chicken breast muscle rehydrated in 0.002% bromelin solution at pH 5.0 and 60°C. lOOX 95 hold the tissue together, and the muscle would become "mushy" in appearance. A micro—photOgraph of a cross section Observed under 100X power is shown in Figure 13. The nuclei are evident as is the intact and fragmented endomysium. Of primary interest is the vast granular material, which "eroded" away from the periferal protOplasm and collected in the connective tissue of the perimysial spaces, completely camouflaging the peri- mysium. A distinct fiber shrinkage is also evident, resulting in rather large endomysial spaces. A micro-photograph of a section of raw, once—frozen connective tissue is presented in Figure 14. The connective -tissue (collagen) is evident as dark fibers with a wave-like appearance. The fibers were stained with aniline blue, which is an acid aniline dye. When this raw collagen or connective tissue was heated during the cooking process, it formed a compact gelaceous substance (Figure 15). The connective tissue on the left side of the figure is representative of this heat—coagulated material. No longer do the individual fibers appear wavy. Although elastic tissue was not considered in this study, Weir gt gt. (1958) stated that cooking did not visibly affect elastic fibers. During cooking, chemical changes occurred in the col- lagenous fibers as evidenced by the changes in their affinity for the aniline blue dye. Raw collagenous fibers did stain blue with this dye, but the cooked fibers solidified into a 96 Figure 14: Horizontal section of uncooked and once—frozen connective tissue. 430K Figure 15: Horizontal section of cooked freeze-dried con- nective tissue rehydrated in 0.0008% ficin solution at pH 5.0 and 70°C. 430x 98 substance which appeared grey. The enzymes did not possess very much collagenase activity at the concentrations used in this study. Ficin produced the greatest breakdown of connective tissue. Bromelin showed some activity, but Rhozyme P-11 and papain demonstrated little or no activity. Enzymatic action on cooked connective tissue is demon- strated in Figure 15. There is a gradual dissolution of the connective tissue on the right from the compact mass on the left. This would account for some of the increased tenderness due to enzymatic proteolysis. However, at the concentrations used in this study, the increased tenderness in chicken was probably due to dissolution of the muscle fibers. Since there is much more connective tissue in beef, proteolytic action on this tissue may be of greater importance. SUMMARY This study was designed to investigate tenderness of freeze—dried chicken breast muscle, as affected by age of bird and applications of commercial proteolytic enzymes. Proteolysis was observed in histological sections from enzyme-treated samples. Chickens ll, 20 and 52 weeks of age were selected for tenderness evaluations. A procedure was develOped to deter- mine tenderness with the Warner-Bratzler shear press by relating shear force to cross-sectional area sheared. Tender- ness data obtained from freeze-dried meat resulted in a cor- relation coefficient of 0.59 between mean panel scores and Warner-Bratzler shear press values. A correlation coefficient of 0.80 was obtained between similar data from non-freeze- dried muscle. Freeze-dried meat was less tender and more variable in texture than control samples. Tenderness of muscles was inversely related to age of birds. As age increased, tenderness decreased. Although results were similar, panel scores were better indices of tenderness than were shear press values. Panel scores indicated that Juiciness was directly re- lated to tenderness. The percentage of water uptake was calculated for each sample, and it was directly related to both tenderness and Juiciness of the rehydrated samples. Papain, ficin, bromelin and Rhozyme P-ll were incorpor- ated directly into the rehydration solutions. All freeze- dried samples were rehydrated in the enzyme solutions for 99 100 five minutes. A three—minute heating time at 100°C was used to inactivate the enzymes. Inactivation was determined by a micro-Kaeldahl method; non-protein nitrogen did not in- crease after three minutes of heating. Panel scores and Allo-Kramer shear press values were obtained to detect conditions of enzyme concentration, pH and temperature, which would produce the most tender yet acceptable chicken. Panel results indicated that enzyme con- centrations (weight/volume) of 0.02%, 0.0008%, 0.002% and 0.002% were suitable for Rhozyme P-ll, ficin, bromelin, and papain, respectively. Various buffers were used to control the pH of rehydration solutions. Shear press values showed that Rhozyme P-ll, ficin and bromelin were most active at pH 5.0. Papain was most active at pH 7.0. Optimum reaction temperatures were 50°, 50°, 60° and 70°C for Rhozyme P-ll, papain, bromelin and ficin, respectively. The cptimum conditions for tenderization by the enzymes used were found to be affected by a combination of water uptake and pH or temperature. Control samples were signifi- cantly more tender when rehydrated at pH 7.0 than at pH values higher or lower than pH 7.0. This may have been due to a simultaneous increase in water uptake at pH 7.0 during rehy- dration. In control samples, significant increases in water uptake were found with decreasing rehydration temperatures. Histological sections were obtained from chicken breast muscles after they were rehydrated in enzyme solutions under the most cptimum conditions for tenderization. Masson's 101 trichrome stain was modified for use on the cooked and rehy— drated tissues. Ficin was most active on muscle fibers, while bromelin was least active. Rhozyme P—11 and papain both produced effects which were intermediate between the above two ex- tremes. Ficin produced the most activity on connective tissue, papain showed some activity, but bromelin and Rhozyme P-11 showed little or no activity. Enzyme—induced tenderness seemed to be more related to muscle fiber destruction than to dissolution of the connective tissue in chicken breast muscle. Muscle fibers which were attacked by enzymes showed a distinct swelling, dissolution of the sarcolemma, extensive granulation, the disappearance of nuclei and the loss of cross striations. Some of the granulation present was due to "erosion" of the periferal protOplasm caused by cooking, rather than to enzymatic action. LITERATURE CITED Altmann, R., 1890. Die Elementarorganismen und ihre Bezie- hungen zur den Zellen. Veit, Leipsig. Anonymous, 1957. Research on tenderizing frozen poultry. Ind. Ref. 133:26. Anonymous, 1962. Stable, flavorful, dehydrated chicken. Food Process. 23:107. Anonymous, 1963. Enzyme Topics. Special Products Depart- ment of Rohm and Haas Company, Philadelphia. Anonymous 1965. Take pulse of freeze—drying. Food Eng. 37M: 45. A.O.A.C., 1960. Official Methods of Analysis, 9th ed. Asso— ciation of Official Agricultural Chemists. Washington, D.C. Armed Forces Institute of Path010gy, 1960. Manual of Histo- logic and Special Staining Techniques, 2nd ed. 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Yatco-Manzo, E. and J. R. Whitaker, 1962. Ficin-catalyzed hydrolysis of elastin. Arch. Biochem. and Biophys. 97:122. Ziemba, J. V., 1960. Freeze-drying. Food Eng. 32:57. all. izi iii]... 3 1 \III‘ III" III ill. :1 I APPENDIX 116 117 APPENDIX TABLE 1 Effects of age on the rehydration of freeze-dried chicken breast meat Weight Weight %_' Weight Weight ,% Sam- Before After Water Sam- Before After Water p16 Rehyd. Rehyd. Uptake __p1e Rehyd. Rehyd. Uptake (smST Tsmsl (ems) (emf ll-Week-Old Birds 1 7.4 22.1 198.6 31 6.0 20.0 23 .3 2 5.6 16.6 196.4 32 7.5 22.8 20 .0 3 8.3 25.5 207.2 33 6.9 20.0 189.8 4 7.5 22.9 205.3 34 7.5 24.6 228.0 5 5.5 17.8 223.6 35 7.1 20.6 190.1 6 5.6 15.9 183.9 36 7.7 24.2 214.3 7 8.4 25.8 207.1 37 8.1 24.6 203.7 8 7.8 25.8 230.8 38 6.9 21.6 213.0 9 7.3 22.7 210.9 39 6.6 21.0 218.1 10 6.4 29.9 367.2 40 7.4 21. 5 190.5 11 7.4 22.5 204.1 41 7.9 25.7 225.3 12 6.2 19.9 221.0 42 7.3 27 211.0 13 7.5 22.8 204.0 43 7.01.6 208.6 14 6.8 20.1 19 .6 44 .10.0 30.0 200.0 15 7.0 22.7 22 .3 45 9.0 26.6 195.6 16 7.9 24.2 206.3 46 9.0 28.4 215.6 17 8.2 24.4 197.6 47 7.4 24.8 235.1 18 7.1 22.2 212.7 48 7.7 23.7 207.8 19 7.4 21.6 191.9 49 7.7 23.0 198.7 20 6.1 19.1 213.1 50 7.2 22.9 218.1 21 6.5 19.2 195.4 51 6.9 23.0 233.3 22 7.8 24.2 210.3 52 7.6 22.7 198.7 23 7.5 23.0 206.7 53 8.7 27.2 212.6 24 7.7 24.7 220.8 54 7.3 23.4 220.5 25 6.7 22.8 240.3 55 6.6 20.2 206.1 26 6.7 21.4 219.4 56 6.7 20.6 207.5 27 7.2 23.0 219.4 57 6.5 21.4 229.2 28 6.4 19.1 198.4 58 5.9 20.0 239.0 29 6.0 19.0 216.7 59 6.6 21.6 227.3 30 9.9 26. 3 165.7 60 7.9 22.3 182.3 Average: 21225 20-Week-01d Birds 1 11.3 27.9 146. 31 9.5 28.1 195.8 2 10.4 30.1 189. 32 10.1 29.5 195.0 3 11.0 33.1 200.9 33 12.0 33.2176.7 4 9. 9 26. 171.7 34 10.8 32. 4 200.0 5 10. 8 32. 200.0 35 10.8 32.8 203.7 6 9.2 27.4 197.8 36 12.5 36. 2 189.6 '7 10.2 31. 3 206.9 37 10.0 29.4 194.0 118 Weight Weight %3 Weight Weight % Sam- Before After Water Sam- Before After Water pie Rehyd. Rehyd. ,gptake _ple Rehyd._flRehyd. Uptake Tansy (amsY TsmsT (ems)w 8 16.0 28.0 75.0 38 9.7 27.7 185.6 9 9.7 25.8 165.9 39 10.9 30.8 182.6 10 80"“ 2502 200.0 "4’0 1000 28.5 185.0 11 10.0 29.8 198.0 41 9.3 27.2 192.5 12 9.3 27.4 194.6 42 9.7 27.2 180.4 13 8.9 24.7 177.5 43 10.0 30.1 201.0 14 12.1 35.8 195.9 44 12.1 32.8 171.1 15 10.5 30.9 194.3 45 9.6 22.6 135.4 16 10.9 29.5 170.6 46 8.7 24.2 178.2 17 10.5 30.0 185.7 47 10.5 29.8 183.8 18 10.0 27.2 172.0 48 10.2 29.7 191.2 19 12.1 36.2 199.2 49 11.3 28.9 155.8 20 11.1 30.1 171.2 50 8.7 24.8 185.1 21 10.5 24.3 131.4 51 11.2 25.1 124.1 22 9.8 28.6 191.8 52 10.3 27.6 168.0 23 13.1 29.2 122.9 5 12.4 27.5 121.8 24 10.5 25.7 144.8 5 9.7 29.5 204.1 25 11.3 26.0 130.1 55 8.6 24.2 181.4 26 11.3 33.2 193.8 56 10.5 29.6 181.9 27 10.2 27.8 172.5 57 12.3 33.8 174.8 28 10.7 29.3 173.8 58 12.6 33.7 167.5 29 10.2 2 .0 125.3 59 13.0 31.5 142.3 30 13. 3 .0 146. 60 12.0 26.1 117.5 Average 173.5 52-Week-Old Birds 1 10.0 20.8 108.0 31 12.2 28.5 120.9 2 9.2 24.4 165.2 32 13. 30.0 123.9 3 12.2 25.6 109.8 33 10. 24.7 133.0 4 13.4 27.6 106.0 34 11.1 22.8 105.4 5 1 .1 30.0 98.7 35 14.0 33.7 140.7 6 1 .3 30.5 113.3 36 11.1 27.2 145.0 7 10.8 21.8 101.9 37 10.5 29.8 183.8 9 10.4 23.9 129.8 39 13.0 31.0 138.5 10 11.6 28.5 145.7 40 11.2 27.4 144.6 11 14.9 31.5 125.0 41 14.6 32.3 233.0 12 11.1 26.3 136.9 42 7.1 19.0 167.6 13 12.3 26.8 117.9 4 10.3 21.5 108.7 14 11.8 28.1 138.1 4 10.8 25.2 133.3 15 15.0 36.0 140.0 45 9.2 26.6 189.1 16 -11.5 23.6 105.2 46 6.9 20.0 190.0 317 12.5 28.5 128.0 47 10.0 28.4 184.0 118 9.0 26.5 194.4 48 11.4 27.1 137.7 119 9.7 25.1 158.8 49 8.8 25.9 194.3 220 9.1 19. 114.3 50 11.1 25.7 131.5 131 14.5 31.0 113.8 51 14.1 33.7 139.0 232 8.9 17.4 95.0 52 9.7 29.8 207.2 119 Weight Weight %' Weight Weight 3 Sam- Before After Water Sam- Before After Water 219 Rehyd. Rehyd. Uptake ,ple Rehyg. Rehyd. Uptake (811181 (ems) (211187 T8138) 23 11.2 27.8 148.2 53 10.1 30.0 197.0 24 10.9 32.2 195.4 54 8.1 20.1 148.1 25 9.7 27.2 180.4 55 9.0 29.0 222.2 26 9.7 26.4 172.2 56 12.0 26.0 116.7 27 12.3 29.0 135.8 57 8.5 16.4 92.9 28 10.2 23.1 126.5 58 9.1 20.0 119.8 29 10.7 24.1 125.2 59 12.1 26.4 118.2 30 11.7 24.2 106.8 60 12.0 24.7 106.0 Average: 142.7 Shear Force/ Force/ Gram (Ib/1n37(lbéi) Shear Area_A Sample Area Sheared (ind? Sam- ‘ple 120 APPENDIX TABLE 2 Gram Shear ll—Week-Old Birds ‘) (lb/8m) A_ Force/ Force/ Area Shear- “rib/in Area Sheared in fects of age on the tenderness of freeze-dried chicken Sample breast muscle as measured by the Warner—Bratzler shear E?“ .- Sam- 1e 5317318717523347381319309407528 4881799 4676577795664569596476896775556 8994574 00000 OOOOOOOOIOOOOOOOOOCOOO 00.0.00 0000000000000000000000000000000 0000010 1054924386174137 254644431033976 7393192 000.000.0000.... 0.0.0.0.... 000.... 4562926828311615420288510776798 7127911 3444355473443345384444565543334 5572343 1138514456147319813033196685702 6795394 3333333323333332323333323233333 4444444 00.00....OOOOOOOOOOOOOOOOOOOOOO 00.0.00 0000000000000000000000000000000 0000000 8 .m .0 123456789012345678901234567890e 1 1234567 333333333444444444455555555556fl B 3333333 r d e 1 A: 4262783276 0054166921820483091 k 6501679 5557458649 6875584678489798878 6 0646367 OOOOOOIOOOOOOOOOOOOOOO000.0... e 0.0.000 0000000000 0000000000000000000 M 1000000 0 2 650757330672468289276559008428 9288872 0.000....OOOOOOOOOOOOOOOOOOOOO .000... 915754474779116591701 53080894 3637357 344434623754753343556 56565555 7423246 887479016377664772828362457662 5544655 223333342222233333232222333323 3354433 0000... OOOOOOOOOOOOOOOOOOOOOO 0...... 000000000000000000000000000000 0000000 123456789012345678901234567890 1234567 111111111122222222223 121 Ow- Sample Shear Shear Sample Shear Shear Sam- Area Force/ Force/ Sam- Area Force/ Force/ pl§u_h§heared Area Gram t_#ple Sheared Arean Gram Iinz) 11b71n2) lib/gm) (1nd) (lb71n‘) Inwfim) 8 0.53 21.9 0.35 38 0.54 43.4 0.69 9 0.40 45.5 0.70 39 0.44 56.0 0.86 10 0.45 42.4 0.63 40 0.42 70.8 0.98 13 0.43 46.6 0.68 43 0.50 44.3 0.75 14 0.37 41.1 0.62 44 0.40 40.3 0.58 15 0.47 39.6 0.64 45 0.40 46.0 0.66 16 0.40 41.5 0.64 46 0.34 24.8 0.37 17 0.47 33.7 0.51 47 0. 5 33.0 0.54 18 0.37 34.4 0.47 48 0.61 45.5 0.93 19 0.39 42.0 0.60 49 0.45 25.6 0.41 20 0.51 40.5 0.68 50 0.40 39.3 0.63 21 0.48 24.0 0.35 51 0.44 79.1 1.27 22 0.34 67.8 0.91 52 0.40 76.7 1.19 23 0.44 30.8 0.48 53 0.48 33.9 0.53 24 0.47 34.3 0.53 54 0.46 87.1 1.36 25 0.41 37.4 0.52 55 0.59 20.9 0.34 26 0.39 45.2 0.72 56 0.51 40.4 0.68 27 0.47 25.9 0.38 57 0.41 38.1 0.58 28 0.38 60. 0.87 58 0.44 40.2 0.54 29 0.46 35. 0.55 59 0.40 96.8 1. 4 30 0.40 43.4 0.62 60 0.42 29.2 0.44 Average: 30.45, 43.8 0.67 52-Week-01d Birds 1 0.38 86.0 1.44 31 0.51 87.0 1.48 2 0.48 40.8 0.72 32 0.34 45.3 0.62 3 0.42 30.5 0.45 33 0.35 46.1 0.61 4 0.45 63.7 1.01 34 0.46 32.0 0.50 5 0.43 42.5 0.67 35 0.40 45.1 0.80 6 0.51 25.6 0.41 36 0.34 55.3 0.74 7 0.34 66.5 0.86 37 0.40 34.2 0.51 8 0.39 44.5 0.61 38 0.51 45.5 0.72 9' 0.31 49.6 0.77 39 0.43 37.2 0.53 AIC) 0.41 103.6 1.79 40 0.25 61.8 0.79 112 0.49 35.2 0.48 42 0.59 46.6 1.15 1:3 0.55 50.6 0.98 43 0.38 44.9 0.82 11+ 0.37 66.7 0.92 44 0.37 38.9 0.62 15 0.33 87.3 1.31 45 0.35 85.0 1.40 165 0.39 57.9 0.88 46 0.57 48.4 0.89 17 0.56 37.9 0.66 47 0.55 53.1 1.07 153 0.51 31.7 1.01 48 0.42 63.2 0.94 159 0.40 52.7 0.86 49 0.43 82.8 1.24 2C) 0.47 42.3 0.7 50 0.40 40.0 0.61 31 0.39 86.0 1.5 51 0.37 79.3 1.36 23 0.50 46.1 0.82 52 0.52 25.0 0.48 122 -*--— - Sample Shear Shear Sample Shear Shear Sam- Area Force/ Force Sam- Area Force/ Force/ ple Sheared__ AreaA Gram .iPl§_11§h§9refi_m.§T32 _ Gram- (in‘) ( 1b71r175) le/ng Tin?) (lb71n27 7111/5417 23 0.37 142.3 2.41 53 0.43 70.8 1.15 24 0.29 38.0 0.59 54 0.56 88.2 1.47 25 0.51 82.9 1.37 55 0.42 71.1 1.08 26 0.53 65.7 1.40 56 0.50 34.0 0.56 27 0.42 101.7 1.66 57 0.48 36.0 0.67 28 0.40 41.1 0.57 58 0.34 69.4 1.43 29 0.38 53.5 0.84 59 0.39 63.6 1.02 30 0.50 28.4 0.50 60 0.40 88.0 1.50 Average: 0.43 ‘56.8 0.94 123 APPENDIX TABLE 3 breast muscle as measured by a sensory panel Effects of age on the tenderness of freeze—dried chicken 55131: Tenderness Initial Residual Sam- ,ple Juici- Tenderness Initial Residual Sam- ple O.» 1.....- ness ness ll-Week-Old Birds 14.002208048222204020222068684042 3314 3332 332 BBQ/333333333332 2 2 34 2 3 144614646 2488240008224846806/04444 1432332232324 044423432332423442J .4064608866866AU0214082480620684625 1432 33323232314441.4324 32333423414214 12314567890 2345/07 3333333334 144414144 4800884 04624 2600141414 06406620204 V 00000. oooooooooooooooooooooooA 32 332 3333333333333332 332 2 33333 4406264842822022461686820606814 141J42333232234143423233423423333 14814014nW88000000140/O/O4884822/004O6 3314 3314 3214 2331414034232 3314 23140433143 123.4 5/0 718 90123-456 7.801012314567890 11111111112 :2 2:2 22223 20-Week-Old Birds 2x01482284 h4515233159?4 64482820 666914 3.4 5 014880244. n/o/O/021414I4 5 12 B456 78 33 33333 86/fl-nU02022 hm 511%331441U 8141442608 5653144142 06246602 666441443 123145678 Juici- ness Tenderness Initial Residual Sam; 124 Juici— ness gple Tenderness Initial Residual Sam- ple 4642466226624L2M8268009 O OJ44 3333/06 5333333332 2 333. l nUonUo48000/0448282000480003 0342344000 24345634234414 0208028048802u402080285 63523437662444563523434 0 L1 IL #7 0/ 123450 89012024507890 3 444444 44 55555555556 erage: 2428482006224440608602v 0000000 000000 coo-0A 4 3332 3355534 34 34 333233 8044488824426084844488 34. 4422356534414 333144223 0026/00/00 0824/0/00 022 nU02/0/0/0/0 44 .44223666444433444223 52-Week-Old Birds 840442204800420022400824 2 34 5.4 55554 554/0 54 3354/0 5/0 5 0280888806 00848060608042 0 O O O C 0 O O 0 O O O O O O 0 O O 0 O O O O 34 3/04 555/0 306 5/0 534 35/0 56/00 006260820440060204424004 344/050 56/060 7/0/0/0 3530/0/06 7/0 8444 06028604004866264220 2 34 354 555400600 2 33354 555 £02624824840244482282684 O C O O O O O 0 O O O O O O O O O 0 O O O O O 34 334456646 7060 34 34 5/0 550 804646080060688204446048 34 334 56/0 756 7060 3534 56/0/00 123456789 012345/0 78 90123.4 111-1111*}. 122222 125 Sam- Tenderness Juici- Sam- Tenderness Juici- h ple Initial Residual ness ple Initggl Regiflual ness 25 u.6 u.2 3.6 55 6.n 6.0 h.8 26 6.6 6. 6.2 56 6.u 6.0 5.2 27 7.0 7. 6.2 57 7.0 6.6 6.0 28 6.6 6.2 6.2 58 5.8 5.u h.8 29 6.4 6.2 5.0 59 6.8 6.4 6.2 30 6.2 5.4 5.8 60 6.2 5.0 a.u [LELQSEB‘ 5.7 ij ‘13,.9 126 APPENDIX TABLE 4 Effects of age on the tenderness of breast muscle from control chickens as measured by the Warner-Bratzler shear and sensory panel ghear Press Values Panel Scores Sample Shear Area Force/ 8a551e Sheared Area A "715?) (lb/ind) ll—Week-Old Birds Initial Residual Tenderness Tenderness Juiciness 1 0.29 19.3 2.2 2.2 3.4 2 0.47 22.7 2.4 2.4 2.4 3 0.51 11.5 1.8 1.8 2.8 4 0.30 22.0 2.2 1.6 2.4 5 0.58 16.1 2.4 2.0 3.8 6 0.63 12.6 2.4 2.0 3.2 7 0.42 1 .5 2.2 2.2 3.8 8 0.30 29.6 3.6 3.4 2.8 9 0. 9 16.9 2.6 2.0 3.0 10 0.59 14.6 2.6 3.0 4.0 11 0.47 15.3 3.2 2.6 4.4 12 0.61 11.6 2.8 2.0 3.0 13 0.57 16.6 2.8 2.6 3.8 14 0.44 16.2 2.2 2.4 3.6 Average: 0.49 17.3 2.5 2.3 #3.3_ 20-Week-01d Birds 1 0.56 15.2 2.2 2.2 2.4 2 0.40 18.6 2.6 2.6 3.2 3 0.42 25.6 3.2 2.6 3.0 4 0.40 28.9 3.4 2.8 3.0 5 00,41" 2808 304 300 300 6 0.24 23.5 2.4 2.4 4.8 7 0036 12009 [4’02 308 300 8 0.51 23.3 4.0 3.6 3.4 9 0.36 46.8 4.8 3.8 3.2 10 0.30 52.1 2.6 2.6 2.6 11 0.31 46.8 2.6 2.4 2.2 12 0.35 76.5 5.4 4.6 4.0 13 0.39 17.8 2.4 2.6 3.8 14 0.26 21.2 2.6 2.6 4.4 New 91:19:18-- -----_3_.3;3-.. 3.3 2.0 2.2 52-Week-01d Birds 1 0038 3308 [4’06 “'06 3.2 2 0.29 30.6 3.0 3.0 4.0 3 0.30 46.2 4.4 4.8 4.0 um“.— 127 Shear Press‘Valuesw jfygfl.80ores .. Sample Shear Area Force/ Initial Residual Sample Sheared Area ‘5 Tenderness Tenderness Juiciness (ind) (lb/ind) 4 0.44 31.4 4.8 3.8 3.0 5 0.40 77.3 4.8 5.2 3.6 6 0.23 68.0 3.2 3.4 3.0 7 0.37 33.2 3.6 2.8 3.8 8 0.33 42.1 3.6 3.4 4.0 9 0.27 70.6 4.4 4.6 3.4 10 0.27 53.9 4.0 3.6 3.6 11 0.39 33.2 3.2 3.2 3.8 12 0.27 56.1 5.0 5.0 4.8 13 0.32 39.3 3.6 3.4 4.4 14 0.28 50.8 4.4 4.2 4.6 Average: 0.32 47.6 _5.0 _3.9 3‘8 128 APPENDIX TABLE 5 Effects of proteolytic enzyme concentration on water uptake, tenderness and acceptability of freeze-dried chicken breast meat ' -..-—-.. —._.._ ‘-.-- -- — ~ - -. -fi‘ -. o—- - -“-- Shear Ave. Accept- Enzyme Water Shear Force/ Panel able to §ggple Conc.1 Uptake Weight Gram Score Panel 7 (%3 ($7 ‘Tsm8) (lb/2m) Rhozyme P-11 1 0.010 202.1 17.7 7.1 3.2 yes 2 0.010 173.7 20.0 4.0 2.8 yes 3 0.010 166.4 17.8 5.3 3.0 yes L" 00015 17907 26.2 1.5 300 yes 5 0.015 210.0 22.0 7.0 2.4 yes 6 0.015 213.3 18.2 6.6 2.8 yes 7 0.015 201.9 20.0 4.5 2.6 yes 8 0.020 180.3 17.4 5.2 2.8 yes 9 0.020 211.7 18.4 5.4 2.0 yes 10 0.020 180.7 16.3 4.6 2.0 .nO 11 0.020 194.3 23.2 8.0 4.2 yes 12 0.020 205.3 23.1 3.5 2.2 yes 13 0.025 185.2 14.0 8.6 2.0 ? 14 0.025 214.9 19.5 3.1 2.2 ? 15 0.025 202.8 17.8 3.4 2.2 ? 16 0.025 204.5 21.4 4.2 3.2 yes 17 0.030 194.1 19.3 2.3 3.0 no 18 0.030 242.0 20.0 1.8 1.2 no 19 0.030 178.3 17.8 1.7 2.0 no 20 0.030 222.8 25.5 2.5 1.0 no. 21 0.030 207.4 24.1 1.7 1.4 no Ficin 1 0.0001 161.0 24.7 16.1 3.8 yes 2 0.0001 166.5 21.0 1 .5 4.6 yes 3 0.0001 167.0 20.3 1 .5 3.8 yes 4 000003 17205 29.6 1006 3.4 yes 5 0.0005 163.3 23.8 14.5 3.4 yes 6 0.0005 176.0 21.7 15.2 3.2 yes 7 000005 203.“ 21.3 703 2.8 yes 8 0.0005 189.0 21.5 16.7 4.0 yes 9 0.0007 142.9 20.7 14.8 3.0 yes 10 0.0010 162.3 26.9 4.1 1.2 no 11 0.0010 205.1 20.0 3.5 1.4 no 12 0.0010 162.0 20.5 11.7 4.4 yes 13 0.0010 254.1 22.8 10.1 2.8 ? 14 0.0010 207.3 21.8 3.9 2.2 ? 15 0.0010 205.0 20.4 3.5 --- no 16 0.0020 190.1 25.0 3.3 --— no 129 *- —— M.-—._.-—. V. Shear Ave. Acceptla Enz‘1E Water Shear Force/ Panel able to Sample Conc. Uptake weLflhpnu §?a@.1,1§£9£§M_HR§EEl_Z_ (%) (%’ {ems} (lb/2m? 17 0.0030 200.0 20.0 3.2 . --- no 18 0.0050 ..... ---- --- --- no Bromelin 1 0.0005 166.5 ”24.0 22.1 5.0 yes 2 000005 16305 1505 907 3.2 yes 3 0.0005 18 .1 15.8 6.3 4.2 yes 4 0.0005 18 .0 17.2 20.9 3.6 yes 5 0.0005 161.6 23.7 14.3 4.6 yes 6 0.0010 194.0 25.2 8.7 2.8 yes 7 0.0010 190.3 18.3 11.7 3.6 yes 8 000010 18007 16.“ 703 3014’ yes 9 0.0010 206.7 15.8 12.7 3.6 yes 10 0.0010 193.5 25.6 6.8 2.6 yes 11 0.0020 212.1 25.2 16.1 3.8 yes 12 0.0020 234.0 18.2 6.0 1.6 yes 13 0.0020 202.5 18.1 4.1 3.0 yes 14 0.0020 25 .0 24.1 14.9 3.0 yes 15 0.0020 237.5 23.0 5.0 2.2 yes 16 0.0030 279.0 26707 , “'05 2.0 no 17 0.0030 243.0 26.5 12.1 2.4 yes 18 0.0030 225.5 18.7 7.8 1.6 no 19 0.0030 246.7 20.0 4.3 1.6 no 2 0.0030 260.5 24.0 2.5 1.6 no again 1 0.001 159.8 36.2 11.0 3.2 yes 2 0.001 240.3 27.7 11.1 2.6 yes 3 0.301 271.3 27.4 9.8 3.4 yes [’4’ 00001 J. 807 27.3 1905 4.0 yes 5 00001 1 9.0 3705 9.3 3014’ yes 6 0.002 182.5 35.5 8.2 3.0 yes 7 0.002 244.1 39.8 5.7 1.8 no 8 0.002 201.0 37.2 8.7 3.6 yes 9 0.002 238.4 35.3 12.6 1.4 yes 10 0.002 224.8 31.5 5.0 2.6 yes 1]. 0.002 21207 3500 6.9 3.2 yes 12 0.003 208.7 35.8 3.9 2.0 no 13 0.003 260.6 30.0 3.5 --- no 14 0.003 231.5 37.9 10.8 3.4 yes 15 0.003 228.3 36.7 3.8 1.6 no 16 0.005 247.6 21.8 3.5 1.4 no 17 0.005 221.6 36.0 5.3 1.2 no 130 Sample W \OC1)\JO\U\{:\-QNH thozyme P-ll was used at pH 7.0 and 50°C. and papain were employed at pH 7.0 and 70°C. -0- ---- ' ' — “““““‘§55§5“‘ Enzyme Water Shear Force/ Panel Conc. __;QE%QK§__WK9¥88§1_1Q£QQ. ‘MSgore C6) l ‘ ': lendernesd 1.1deget lent, y tron other sanples. Residual .. :3 r-n . i ‘ ‘ v, ) ‘|.) sane Way initially.. across tte grain of e Juiciness toe n ut. 3) Score in fable 2 the Flavor numoer from laole l best representing the sample . Comments: 31 lgwmufiyujuum 3 1293 o m Hm I“ I!" H M. MI m