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Y a}; Michigan Sta {3 niversity gj This is to certify that the thesis entitled EFFECT OF SLAUGHTER METHOD, ELECTRICAL STIMULATION AND HOLDING TEMPERATURE ON THE TENDERNESS 0F BEEF STEAKS presented by Emanuel Uche Odume has been accepted towards fulfillment of the requirements for MSc degree in Food Science / '1‘ N. #744144. 0;) l \V‘ I i- ' ‘ 1 ,i" / Major professor DateDCIQbfiLmJEZLC , 0-7639 OVERDUE FINES ARE 25¢ PER DAY ‘ PER ITEM Return to book drop to remove this checkout from your record. EFFECT OF SLAUGHTER METHOD, ELECTRICAL STIMULATION AND HOLDING TEMPERATURE ON THE TENDERNESS OF BEEF STEAKS By Emmanuel Uche Odume A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1978 W" 7019/ ABSTRACT EFFECT OF SLAUGHTER METHOD, ELECTRICAL STIMULATION AND HOLDING TEMPERATURE ON THE TENDERNESS OF BEEF STEAKS By Emmanuel Uche Odume The effect of slaughter method of steers, electrical stimulation (100 volts for approx. 100 secs.) of the carcass halves and holding temperature of the carcasses on the tenderness of beef steaks were evaluated. The influences of these treatments on changes in pH, ATP, internal temperature and microbial load were also deter- mined. 12 grass-fed steers were involved in this study. 6 were slaughtered conventionally and the other 6 were confined, haltered and throat slashed with a sharp knife. Each carcass was split into sides and one side was electrically stimulated. 12 carcass sides were held in a 1°C chilling cooler and the other 12 in a 20°C storage atmosphere. Panel evaluation, Warner-Bratzler and Allo-Kramer shear values indicated that longissimus muscle samples from electrically stimulated sides of all steers were significantly (P < 0.01) more tender than Emmanuel Uche Odume samples from untreated sides. Differences in tender- ness due to slaughter method were not significant. Panel ratings favored the 20°C held over the 1°C held carcasses. ATP and pH decline were faster in electrically stimulated and 20°C-held carcasses. Higher microbial load was recorded for 20°C-held carcasses. To You, Obi ii ACKNOWLEDGMENTS The author wishes to express sincere appreciation and thanks to his major professor, Dr. J. F. Price, for his guidance and involvement in this study and for his assistance in preparing the Dissertation. Appreciation is expressed to Dr. A. M. Pearson, Dr. R. A. Merkel, Dr. M. Zabik and Dr. L. L. Bieber for serving on the guidance committee. Special thanks are expressed to the graduate students and staff of the meat laboratory for serving as panel members, and also to Mr. J. R. Anstead and his staff for slaughtering all cattle used in this study. The author is especially grateful to his parents for their continuous understanding and encouragement and lastly to the Federal Government of Nigeria for sponsoring his studies here in Michigan State University. 111 TABLE OF CONTENTS Chapter Page LIST OF TABLES. . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . viii INTRODUCTION. LITERATURE REVIEW . Pre—slaughter Handling. Slaughter Methods Post-Mortem Changes in Meat (DNQEJI'H Chemical Changes. Physical and Biophysical Changes . . . . 11 pH Changes. . . . . . . . . . . . . . . . . 1A Muscle Shortening . . . . . . . . . . . . . 16 Factors Affecting Muscle Tenderness . . . . . . 19 Genetic Factors . . . . . . . . . . . . . . 19 Sex Influence . . . . . . . . . . . . . . . 19 Age Influence . . . . . . . . . . . . . . . 2O Connective Tissue Effects . . . . . . . . . 21 Shortening Effect . . . . . . . . . . . . . 22 Aging Effect... . . . . . . . . . . . . . . 23 Marbling Effect . . . . . . . . . . . . . . 25 Meat Tenderization. . . . . . . . . . . . . . . 27 MATERIALS AND METHOD. . . . . . . . . . . . . . . . 33 Experimental Animals. . . . . . . . . . . . . . 33 Slaughter Procedure . . . . . . . . . . . . . . 34 Electrical Stimulation. . . . . . . . . . . . . 35 iv Chapter Page Measurement of pH and Temperature . . . . . . . . . . . . . . . . . 37 Microbial Count . . . . . . . . . . . . . . . 37 Holding and Sampling. . . . . . . . . . . . . 38 Cooking Method. . . . . . . . . . . . . . . . 39 Measurement of Tenderness . . . . . . . . . . 39 Panel Evaluation. . . . . . . . . . . . . 39 Warner-Bratzler Shear Force . . . . . . . A0 Allo-Kramer (Texture Recorder) Shear Force . . . . . . . . . . . . . . . A0 Adenosine Triphosphate (ATP) Determination . . . . . . . . . . . . . A0 Statistical Analysis. . . . . . . . . . . . . Al RESULTS AND DISCUSSION. . . . . . . . . . . . . . AU Postmortem Changes of pH. . . . . . . . . . . AA ATP Changes . . . . . . . . . . . . . . . . . 50 Postmortem Temperature Changes. . . . . . . . 55 Tenderness Evaluation . . . . . . . . . . . . 60 Panel Scores. . . . . . . . . . . . . . . 6O Shear Values. . . . . . . . . . . . . . . 62 Microbial Count . . . . . . . . . . . . . . . 66 SUMMARY 0 o o o o o o o o o o o o o o o o o o o o 7 2 LITERATURE CITED. . . . . . . . . . . . . . . . . 75 APPENDIX. . . . . . . . . . . . . . . . . . . . . 91 Table LIST OF TABLES Group distribution of experimental animals. Muscle pH changes as affected by electrical stimulation, slaughter method and holding temperatures. Simple correlation coefficients between various physical measurements, chemi- cal and microbial analysis of beef steaks Longissimus muscle ATP (pMoles/gm tissue) changes as affected by electrical stimulation, slaughter method and holding temperature Longissimus muscle temperature (°C) changes as affected by electrical stimulation, slaughter method and holding temperature. Comparison of means and standard deviations of panel tenderness ratings for beef longissimus muscle . . . . . . . . . . . Comparison of means and standard deviations of Warner-Brazler shear vi Page 34 A5 51 SA 56 61 Table force values (Kg/cm2) for beef longissimus muscles. Comparison of means and standard deviations of Allo-Kramer shear force values (Kg/gm) for beef longissimus muscles. Microbiological count (loglo) of loin surface as affected by electrical stimulation, slaughter and holding temperature. vii Page 63 6A 67 Figure LIST OF FIGURES Diagram of the electrical set—up used for stimulating the carcasses The effect of electrical stimulation on the rate of longissimus muscle pP and ATP decline. . . . . . The effect of slaughter method on the rate of longissimus muscle pH and ATP decline. The effect of holding temperature on the rate of longissimus muscle pH and ATP decline. Rate of temperature decline in longissimus muscles of stimulated and control carcasses. . . . . Rate of temperature decline in longissimus muscles of stunned and unstunned steers . . . . . . . . . Rate of temperature decline in longissimus muscles of carcasses held at 1°C and 20°C. Microbial growth rate on loin sur- faces of stimulated and control carcasses . viii Page 36 A6 A7 A8 55 56 57 68 Figure 10 Page Microbial growth rate on loin surfaces of stunned and unstunned steer carcasses. . . . . . . . . . . . . 69 Microbial growth rate on loin surfaces of carcasses held at 1°C and 20°C . 7O ix LIST OF APPENDIX TABLES Appendix II III IV VI VII Raw data of panel scores between stimu- lated and unstimulated beef longissimus muscles. Raw data of Warner—Bratzler shear force (Kg/cm2) between stimulated and unstimulated beef longissimus muscles. . . . . . . . . . Raw data of Allo—Kramer shear force (Kg/gm) between stimulated and un- stimulated beef longissimus muscles. Raw data of pH decline in beef longissimus muscles of stimulated and unstimulated carcasses Raw data of Adonosine triphosphate (ATP- Moles/gm tissue) in beef longissimus muscles of stimulated and unstimulated carcasses Raw data of temperature (°C) decline in beef longissimus muscles of stimulated and unstimulated carcasses. Raw data of microbial count (loglo) of loin surfaces of stimulated and un- stimulated carcasses Page 90 91 92 93 9A 95 96 INTRODUCTION In Nigeria, unpublished information indicates that steaks from the local Zebu cattle (Bos indicus) which had been shackled and slaughtered by direct throat slashing are generally tougher to bite when compared with steaks obtained from steers which had been immobilized pre- slaughter in packing houses. Work in the U.S. (Carlo gt al., 1970) shows that steers of Brahman (Bos indicus) breeding produce roasts and steaks which are less tender than most of the cattle of standard British breeds. The toughness difference has been attributed to "genetic" feactors and shown to be moderately heritable (Brackelsberg 32 11., 1971). It is also known that the fat deposition pattern in Egg indicus breeds (Zebu or Brahman) is different from that in Bos taurus (humpless) breeds. Cold induced muscle shortening is now known to result in a decrease in the tenderness of steaks (Marsh and Leet, 1966). Since the elucidation of this fact, little re- search has been directed toward discovery of how much of the breed related tenderness difference can be ex- plained by differences in chill rate or fat deposition pattern. Lee (1976) have shown that steaks from slowly chilled beef carcasses were more tender than those from fast chilled carcasses, and that steaks from fat carcasses tended to be more tender than those from thin carcasses. The relevance of chill induced muscle toughness in the cattle as they are produced and slaughtered in most slaughter houses in Nigeria, is open to question. While most of those cattle are not very fat, since they are principally grass fed and have to be trucked for very long distances in search of lush grass (Oyenuga, 1968) and may be old (chronologically) for their weight, the carcasses or parts may never be subjected to refrigera- tion prior to cooking. Some other stresses imposed upon the animals (Sorin— made gt al,, 1978) or the muscles during slaughter and handling could induce muscle shortening or rigor type of toughness problems. Recent studies involving electrical stimulation of carcasses or muscles immediately post- mortem rely upon the concepts that pre-rigor accelera— tion of glycolysis (ATP disappearance) will avoid or negate the effects of cold induced shortening (Carse, 1973; Chrystall and Hagyard, 1975; 1976) or increased activity of acid proteases (Savell e§_a1., 1978). It is thus conceivable that electrical stimulation might also affect the ultimate tenderness of beef muscle which is never chilled. The technique of immobilization or slaughter of the animal is also believed to influence, to a large extent, the tenderness of the steaks and roasts that result. Various techniques of immobilization and slaughter as defined by the Humane Slaughter Act of 1958, include electrical, chemical and mechanical methods, and their effects on meat quality are properly documented (Sybesma and Groen, 1970; Leest 33 al., 1970; Overstreet, 1975). This study is therefore designed to investigate the effects which a variation in slaughter technique, post- mortem holding temperature conditions and electrical stimulation of beef carcass halves, have on tenderness of beef steaks from "grass-fed" cattle. A comparison of the Zebu and British type cattle would be desirable but impracticable due to the lack of availability of cattle with Bos indicus breeding of the nature similar to those native to Nigeria. LITERATURE REVIEW Pre-slaughter Handling Several stress factors are brought to bear on beef steers during procedures required to convert the tissues into edible food. These are essentially environmental in origin (Forrest gt gt., 1975). Some studies on beef (Houston gt gt., 1962; and Henrickson gt gt., 1965) suggest that high energy content diets yield meat with increased flavor, tenderness and Juiciness, although Paul gt gt. (196A) found no such trend in lamb. Dif— ferent dietary schedules may, however, produce widely differing growth rates (Garringus gt gl., 1969; and Sherry fl git, 1978). Starvation is known to reduce the size of muscle fibers (Yeates, 196A) with consequent increase in percentage of connective tissue, and meat toughness. But a period of refeeding after starvation permitted recovery of meat quality (Yeates, 196A; Hill, 1967). Kirton gt g_1_., (1972) recorded a loss of edible meat and excess fat from the carcasses of starved cattle as well as weight loss from non-carcass components of live weight. Carr gt gt. (1971) observed that fasting reduced slaughter weight of bovine carcasses in the first 2“ hours, but no further effect on carcass yield up to 2 days. They also noted no detrimental effects on marbling score, maturity score or grade but fasting improved the color of lean. Lambs fed on white clover species were reported to have stronger flavor of fat and lean and intense odor than those fed on perennial rye grass (Shorland gt gl., 1970). Animal body muscles tend to be tough if subjected to very heavy exercise. The effects of various stress con- ditions on the palatability characteristics of the result- ing meat have been investigated. The basic cause for many of the effects observed appears to be treatment in- fluence on glycogen stores, with consequent alteration in the rate and extent of postmortem glycolysis and in the ultimate pH attained by the muscle. Exercise immediately ante-mortem reduced the glycogen level and influenced postmortem glycolysis (Lawrie, 1966; Sorinmade gt gt., 1978). Briskey gt gt (1959) showed that severe exercise of hogs immediately before slaughter depleted the muscle glycogen and produced high pH meat that was dark in color and dry in appearance. 0n the other hand, short- term excitement and exercise of swine immediately prior to slaughter produced rapid postmortem glycolysis and yielded muscle with inferior water binding capacity and low color and texture scores (Sayre gt gl., 1963). Electrical stimulation (Lewis gt_gl., 1963; Chrystall and Devine, 1978), abrupt change to a cold environment (Sayre gt gl., 1961) or elevated environmental tempera— ture (Sayre gt_gl., 1963) and the fatness of carcasses (Lee, 1976) are known to influence the depletion of muscle glycogen reserves, alter the ultimate pH of the muscle, and influence water binding, color and firmness of the resulting meat. Slaughter Methods In the U.S., the Humane Slaughter Act of 1958 resulted in the adOption of humane methods of pre-slaughter im- mobilization in most packinghouses requiring that live- stock must be insensible to pain at the time of exsang— uination. As a result, three categories of immobiliza- tion techniques were approved: (1) Electrical, (ii) Chemical, and (iii) Mechanical. Marple (1977) has in- vestigated which of these methods was most desirable for use since none of these methods of stunning meets the requirements of an ideal method. Althen gt gt. (1975) and Ono gt _t. (1976) suggested that the mechanical methods of stunning stimulate the release of epinephrine and norepinephrine and induce a corresponding increase in muscle cyclic-AMP levels eight times those of elec— trically stunned pigs and 28 times those of non—stunned pigs. They concluded that electrical stunning is less stressful to pigs than captive-bolt stunning. Sybesma and Groen (1970) have studied the comparative responses of animals to C02 immobilization using a com- mercial tunnel facility, 70 volt electrical stunning and 70 volt stunning after animals had passed through the tunnel with CO2 present. They observed that the stress of getting the animals into the CO2 tunnel accelerated the rate of postmortem glycolysis. C02 use for immobiliza- tion has also been linked to reduced bleed-out of car- casses (Leest _t gt., 1970). They also observed that animals immobilized with 002 and shackled prior to ex- sanguination had significantly lower blood and muscle pH at death, and lower muscle pH at 35 minutes postmortem. Muscle rigor value was also higher at 35 minutes and muscle ATP levels were lower at 35 minutes postmortem. Overstreet gt gt. (1975) noted that pigs slaughtered without restraint or stunning had the slowest rate of post— morten glycolysis over those subjected to CO2 immobiliza- tion. McLoughlin (1971) found that muscle ATP concentra- tions declined to their minimum value by two hours post- mortem in pigs slaughtered normally, although little depletion of ATP occurred until three hours postmortem in muscle from pigs anesthetized prior to exsanguination. Postmortem Changes in Meat Marked alterations in palatability characteristics are known to be caused by a number of factors. These result from the changes that occur in muscle postmortem as well as certain inherent differences. Chemical Changes. The chemistry of postmortem changes in muscle is essentially that of high energy phosphate compounds and the mechanisms involved in their synthesis and degradation (Pearson, 1970). This is anaerobic glycolysis and it involves the conversion of glycogen to lactic acid and is easily monitored by following pH decline (Bendall, 1960 and Cassens, 1966). Postmortem glycolysis in mammalian muscle frequently results in the pH decline to an ultimate level of 5.“ - 5.5, if glycogen content is adequate. However, values higher than this have been recorded (Lawrie gt gt., 1959; Bendall and Rhodes, 1976) or even less than 5.1 in pig muscle (Lawrie gt gt., 1958). Complete inhibition of glycolysis has been known to result at pH levels of 5.3 (Bate-Smith and Bendall, 19A9). This was attributed to the possibility that one or more enzymes involved in glycolysis was increasingly inhibited as the pH declined. In some muscles where glycolysis ceases at high pH rates, Briskey and Lawrie (1961) suggested that either the phosphorylase was in- activated more readily or the glycogen was less accessible to attack. Postmortem decline in pH of certain skeletal muscles varies considerably (McLoughlin, 1963; Elliot, 1965). Muscle temperature significantly influences the rate of pH and ATP change (Moeller gt gt., 1977; Dutson, 1977). The greatest decline in pH and loss of ATP occurs during the first 2 to 3 hours postmortem (Kastner gt gt., 1973). Cassens and Newbold (1967) found that in the range of 5°C to 37°C the pH of the ox sternomandibularis (neck) muscle fell more slowly at lower temperatures and there was a period of several hours during which the pH of muscle stored at 1°C fell faster than that of muscle at 5°C. The ultimate pH attained at 1°C or 5°C was significantly higher than that attained at 15°, 25° or 37°C. Moeller _t _t. (1977) reported similar results, and Bendall (1960) stated that the lower the temperature in the range of 0°C to 37°C, the more slowly the pH fall in the ggggg muscle of the rabbit. The levels of ATP (Bendall and Rhodes, 1976) and/or glycogen present in the muscle at time of slaughter may also influence the extent of pH decline. The depletion of glycogen can be affected through starvation, exhaus- tive exercises or even by struggling at the time of slaughter (Lawrie, 1966). Preslaughter injection of insulin (Howard and Lawrie, 1957) or adrenalin (Klose gt gt., 1970; Khan and Nakamura, 1970; Bouton gt_gt., 1971) can also reduce the level of muscle glycogen. Commercial handling practices after slaughter can influence the subsequent quality of meat but only within limits set by the physiological and biochemical characteristics of an animal before and at the time of slaughter. After death, the minimal rate of breakdown of ATP is probably that 10 required by the Ca++ pump (Bendall, 1969), for it is known that myofibrillar adenosine triphosphatase activity is strongly inhibited by falling pH in the range found during rigor onset (Wismer-Pedersen, 1966). Bate—Smith and Bendall (1956) suggest that to ensure a slow rate of decline in the pH of muscle after death, it is necessary to have high levels of ATP and creatine phosphate (CP) present in the muscle initially. To meet these criteria, therefore, it is essential, both before and after death, that the rate of breakdown of ATP in muscle must be adequate for current needs and to maintain an appreciable balance of CP. Needham (1960) concludes that any condition which increases the rate of breakdown of ATP, (e.g., the increase of myofibrillar adenosine triphosphatase activity when muscle is stimulated) or which reduces the rate of resynthesis, must bring about a reduction in pH. Struggling at death will accelerate the rate of postmortem glycolysis because under such condi- tions phosphocreatine is depleted and the resynthesis of ATP is primarily dependent on the anaerobic breakdown of glycogen (Bendall, 1960). Goll (1968), Lawrie (1968) and, Buege and Marsh (1975) have similarly reviewed postmortem chemical changes in muscles and have identified some major chemical events. In stunned animals, ATP hydrolysis is caused by mus- cular contractions that occur as a result of neural 11 stimuli during and immediately after stunning (Van der Wal, 1978). Eikelenboon and Sybesma (1969) had earlier observed that this decrease in ATP concentrations simul- taneously led to a drOp in pH. This hydrolysis of ATP may itself contribute up to 10% of this pH decrease through the protons which are thus set free (Honikel and Hamm, 197A). Bendall and Rhodes (1976) reported that at pH 6, 50% of the resting content of ATP had disappeared, and at pH 5.7 more than 90% and that rapid cooling of muscles to 2°C could be started without danger of cold shortening. But since rigor mortis manifests itself as soon as ATP concentration drops below a certain value — 2 to A uMoles/gm - (Bendall, 1960) it means that the on- set of rigor mortis is related to the initial ATP content of muscular tissue. Paul (1972) explains that the de- gradation of ATP leads initially to accumulation of ADP, followed by AMP, then IMP, and eventually inosine. When ATP decreases to 50—80% of its initial concentration, the muscle loses extensibility and becomes hard. Physical and Biophysical Changes. The thermodynamic equilibrium of living muscle is profoundly altered when the animal is slaughtered. At time of slaughter, muscle is plastic and highly extensible. Paul (1972) stated that as the oxygen supply is exhausted, the oxidation-reduction potential drops, and l2 aerobic energy production ceases. These changes are ac- companied by loss of ability to maintain, (1) body tem- perature, (ii) tg tttg osmotic equilibrium, (iii) membrane permeability and polarization, and (iv) normal ion con- centrations in various parts of the tissues. The tg tttg controls on muscle contraction are disrupted by the diminishing supply of ATP, so actin and myosin unite to form actomyosin, the irritability and extensibility of the muscle decrease, and the muscle becomes hard. This is rigor mortis, a condition of rigidity or contrac- ture which develops in a matter of hours after death (Wierbicki gt gt., 195A). The shortening that results from the sliding of the thick and thin filaments is the primary cause of carcass stiffening during the develop- ment of rigor (Okubanjo and Stouffer, 1975). A study by Buck and Black (1967) on bovine longissimus muscle strips in which two degrees of stretch-tension were applied during rigor indicated that the average muscle fiber diameter was significantly smaller in the stretched muscle strips. Rigor onset involves a period of increasing isometric tension development (Jungk gt gt., 1967; Goll gt gt., 1970; 1971). Huxley (1969) found that the active tension generated by a living muscle decreased to zero when the muscle was stretched enough to eliminate any overlap of actin and myosin filaments. Postmortem contraction depends on the muscle being l3 stimulated to contract before the ATP supply is exhausted, since ATP furnishes the immediate energy source to drive the contraction process (Newbold and Harris, 1972). Goll gt gt. (1970) attribute this contraction to the decline in ATP concentration which lowers the ability of the sar- coplasmic reticulum to accumulate calcium ions against ++ a concentration gradient and the release of Ca ions is required to initiate tension development. Busch gt gt. (1967) and Jungk gt gt. (1967) reported separately that, after a given period of postmortem storage, both rabbit and bovine muscle strips gradually develop tension when suspended isometrically. The onset of isometric tension development, however, depends on environmental temperature of the strips and on ante-mortem condition of the animal (Jungk gt gt., 1967). For rabbit muscle Bendall and Davey (1957) reported that ATP levels dropped to 25 and 50% of initial level at 17° and 37°C respectively at the time of rapid rigor onset. Busch gt gt. (1972) found that in por- cine and rabbit longissimus muscle, tension development was minimal and similar at 2°, 16° and 25°C, but increased greatly if the strips were incubated at 37°C. Bovine semi- tendinosus muscle strips developed most isometric tension at 16° to 25°C. Like rabbit and porcine muscle, isometric tension development in bovine muscle reached a maximum sooner after death at 37°C than it did at 2°, 16° or 25°C. Some workers (Busch gt gt., 1967; 0011 _t__t., 1970; 1971) have reported that carcass softening or the dissipation 1A of rigidity results from the loss of ability to maintain this tension development. The period of isometric de— cline is defined as resolution of rigor mortis. RH Chagges. The rate and extent of pH changes vary with a number of factors. McLoughlin (1965) and Mc- Loughlin and Davidson (1966) studied the effect of stunning on pH of the longissimus dorsi of pig muscle. They noted that stunning either electrically or with C02 increased the tendency for low pH ( 6.0) over that found in animals slaughtered without prior stunning measured at 30—A5 mins postslaughter. Van der Wal (1978) reported that muscle contractions which occur as a consequence of stunning lead to significantly increased concentrations of lactate in the animal body. He had earlier suggested that the process of stunning may be responsible for the sudden increase in the cathecholamine level of blood plasma and stated that this contributes to an improvement of pork quality depending on the level present in the blood. The most-used criterion for rigor is the attainment of ultimate pH. Although pH fall is usually close to linear over most of the range, the rate declines in the last stages, and it is not easy to give a precise time for the end point (Locker, gt gt., 1975). Marsh (195A) showed that beef longissimus reached ultimate pH (5.80) 15 in 20 hours at 7°C and in 16 hours at 17°C (ZpH 5.6) and considered 36 hours in a chiller adequate for com— pletion of rigor. Moeller gt gt (1977) observed an increased rate of pH decline for the muscles incubated at 37°C over those of 2°C. Cassens and Newbold (1967) found the rate of pH fall in beef sternomandibularis during the first 8 hours to be the same at 1°C and 15°C, and slightly less at 5°C. They considered the fall complete in 72 hours at 1°C or 5°C, and in 30 hours at 15°C. Marsh and Thompson (1958) had found a constant rate of pH fall over most temperature ranges in lamb tgggr issimus muscle. They suggest 10 hours at 15°C to approach rigor and found only an 11% decrease in the rate of pH fall at temperatures between 17°C and 7°C. Scopes (1972) reported that the rate—temperature curve for lamb muscles has a shallow minimum at 12°C. The rate of pH fall at 0°C equalled that at 15°C. Wenham gt gt (1973) reported that the pH of ewe longissimus falls within 0.05 unit of ultimate (mean 5.77) in 13 hours at 15°C while biceps femoris takes 1A hours (S.D. ~3). Stimulation of carcass is known to accelerate the fall of pH (Defremery and Pool, 1960; Chrystall and Hag- yard, 1976). Hallund and Bendall (1965) and McLoughlin (1970) found that, in pigs with a naturally slow glycolytic rate, 30 seconds of stimulation approximately doubled the rate of pH fall. Chrystal and Devine (1978) observed 16 that when prestimulation pH was about 7.0, the change in pH ( pH) was high, but dropped to less than 0.2 pH units at a pre-stimulation pH of 6.5 (3—A hr at 35°C) and ap- proached zero at pH 6.3. They concluded that stimulation has little effect on muscles below this value, and that even with prolonged stimulation periods, muscle pH rarely dropped below 6.3. Muscle Shortening. Locker and Hagyard (1963) were among the first to observe the fact that excised, un— restrained beef muscle shortened more rapidly at 0°C (32°F) than at any other temperature and that minimum shortening occurred at 1A°C to 19°C. Earlier, Bendall (1951) working with rabbit muscle and Marsh (195A) with beef muscles, had reported that the shortening of excised muscle during onset of rigor increased with storage tem— perature from 17°C to 37°C. Cassens and Newbold (1967) reported that the delay phase of rigor mortis increased as the temperature decreased from 37°C to 15°C but was shorter with lower temperature in the range of 15° to 1°C. Marsh (1966) suggested that cold shortening may be due to inactivation of the relaxing factor by Ca++ and there is evidence (Buege and Marsh, 1975) to suggest that the calcium ions which trigger the shortening are released from the mitochondria responding to normal postmortem anoxia, and not from the sarcoplasmic reticulum reacting 17 to cold. The phenomenon of cold shortening occurred, not only in beef sternomandibularis muscle, but also in beef loggissimus muscle and to a lesser extent for beef psaos major muscle. Ovine (Marsh, 1968), porcine (Hen- derson gt gt., 1970; Hendricks t 1., 1971), rabbit red semitendinosus (Henderson gt gt., 1970) and avian (Smith gt gt., 1969) muscles, have also been shown to cold shorten. The chemical changes involved in cold shortening indicate that shortening occurred while about A0% of the ATP still remained (Newbold, 1966). Bendall (1975) suggested that cold shortening was minimized by delaying exposure to cold temperatures until muscle pH reached a value below 6.0 and approximately 50% of the adenosine triphosphate (ATP) had been depleted. These observations confirmed findings of Locker and Hagyard (1963), and Marsh and Leet (1966) that cold shortening decreases as the period between slaughter and exposure to cold or freez- ing conditions is extended. Davey (1970) observed that rapid chilling of beef carcass sides produced an erratic toughening, with shear force values ranging from 10 to 20 — that is from very tender to very tough, and a large proportion of the contracted meat would not age. Butcher (1972); and Lee (1976) confirmed this in young bulls, cows and calves where there was progressive toughening in the loin as chilling rate increased. 18 Marsh gt gt (1968) evaluated the effectiveness of condition— ing intact lamb carcasses at 18° to 2A°C for 0.5 to 2A hrs before freezing. They found that there was little effect on tenderness if carcasses were conditioned less than 6 hrs, a rapid increase in tenderness as holding time in- creased from 6 to 16 hrs, and a plateau in tenderness values between 16 and 2A hrs of conditioning. Although the amount of conditioning time for prevention of cold shortening was similar at 7°, 10° or 18°C (Locker t al., 1975), an additional conditioning time was needed to pre- vent thaw rigor. Davey and Gilbert (1976) reported that even when very low levels of ATP (5 to 20% of initial con- centration) remain in the muscle upon freezing, severe thaw contracture can be induced. There is therefore, a need to allow sufficient conditioning time to reduce muscle pH to near its ultimate value (Locker gt gt., 1975) or extended frozen storage in order to reduce ATP levels to near zero (Davey and Gilbert, 1976). This should prevent thaw contracture and the extreme toughness accompanying it. 19 Factors Affecting Muscle Tenderness One quality of meat palatability is its tenderness. Consumer studies have shown that tenderness is the most important factor in the acceptance of beef and probably of other meats, including poultry and game (Bratzler, 1971). Genetic Factors. Brackelsberg t al. (1971) have shown meat toughness to be moderately heritable and Carlo gt__t. (1970) reported differences in tenderness scores between roasts and steaks from the humped (Zebu/Brahman) cattle and humpless British breeds. Breed variations within a species have been found to influence such items as yield of cuts, lean—fat ratio, intramuscular fat distribution (marbling), firmness of fat, and color, tenderness, and juiciness of cooked meat (Paul, 1972). Bryce-Jones _t _t (1963) reported differences in tenderness, flavor, juici— ness and iodine number of the fat due to sire. Paul (1962) listed variations in tenderness of beef muscle fibers among average size animals of similar heredity and management. Sex influence. Variations in growth rate and muscula- ture resulting from sex differences have been reported by many researchers. Locker and Hagyard (1963) reported that muscles from steer carcasses sustained greater shortening than samples from cow carcasses when stored at 19°C and 31°C, although when refrigerated at 2°C, the samples from 2O cow carcasses shortened to a greater extent than the muscle samples from steer carcasses. No significant differences were found in Warner-Bratzler shear values of steaks from bulls less than 16 months of age and steers and heifers of comparable chronological age (Hedrick gt gt., 1969), although shear values of steaks from more mature bulls were greater than those from steers or heifers at the same age. Bull meat appeared to be as acceptable as steer meat, although the cuts from steers were usually more tender, and in some cases had more flavor and juice; the differ- ences becoming more marked with increasing age of animals (Bailey gt gt., 1966;Woodhanm.and Trower, 1965; and Field, t 1., 1966). Champagne gt _t. (1969) and Warwick gt gt. e (1970) reported non—significant differences in tenderness ratings between steaks from bull and steer carcasses. Age Influence. Meat from very young animals would give cooked products differing in flavor, tenderness, juiciness and yield from mature animals (Paul, 1972), since the ratio of lean, and of fat to bone increases as the animal increases in age. Meat from older animals would therefore seem to be less tender due to increasing amounts of connective tissue. Helander (1966) reported a small increase in connective tissue protein during old age, although analytical studies have shown that the lean tissue of very young animals contains the largest percent 21 of connective tissue of any age group, and that after the animal is mature there is little quantitative change in percent connective tissue. In their study, Smith gt _t. (1978) found that yearling Angora goats produced chops and roasts which were (P < .05) juicer and more tender than those from young goats. Six-month-old Spanish goats produced chops and roasts that were (P < .05) more tender than those from more youthful or more mature goats in a majority of such comparisons. With increasing age, meat from bovines tends to be darker and redder in color, lower in pH, and less tender (Walter gt gt., 1965; Henrickson and Moore,l965 and Webb gtgt., 1967). Connective Tissue Effects. A major factor of muscle tenderness is the character and content of connective tis- l. (1967) and Field gt gt (1970) re- sue. Herring gt ported the existence of more collagen in some muscles than in others and that the amount of collagen in the various muscles had a negative relationship to tenderness. McClain gt gt. (1965) and Herring gt gt. (1967) have demon- strated that with decreased tenderness due to increased age of animal there was essentially no change in total amount of collagen present in the muscle, and that although tenderness of muscle increased with postmortem aging, there was little change in the total amount of collagen 22 over the aging period. Some workers have investigated the possibility of in- creased cross-linking of collagen with increased age in a number of different species. Partington and Wood (1963) proposed possible structures for such cross-links, and Heikkinen _t _t, (196A) suggested the existence of varying strengths of cross—links. Goll (1962) reported that in- creased cross-linking of the collagenous fibers makes the tissue more resistant to the softening influence of heat, and may be the reason why meat from older animals is often less tender. There are changes in the molecular struc- ture of collagen due to postmortem aging (Kruggel and Field, 1971; Pfeiffer gt gt., 1972; and Stanley and Brown, 1973). They noticed an increase in the amount of smaller molecular weight subunits and a decrease in the amount of larger molecular weight subunits that could be extracted from muscles with increased postmortem aging. Shorteninngffect. There is a well defined relation- ship between toughness and the degree to which a muscle has shortened (Marsh and Leet, 1966; Davey and Gilbert, 1975). Earlier workers (Paul and Bratzler, 1955) had indi- cated that muscles which were cut or excised before the onset of rigor were tougher than comparable muscles still attached to the skeleton. Locker (1960) suggested that 23 the toughening which occurred might have been due to shortening of the muscle during the onset of rigor mortis. He noticed that the pre-rigor excision of psaos major muscle resulted in a 20-30% shortening and there was a corresponding decrease in sacomere length. The relaxed muscles were more tender than partly contracted muscles. Marsh and Carse (197A) attempted to explain the length/ tenderness relationship in terms of the overlap of thick and thin filaments within the sacomere, but later studies (Rowe, 197A) suggested that this may not be so. Drans- field and Rhodes (1976) are of the opinion that there may be an interaction between the contractile filaments and the connective tissue, but the confirmation by Locker and Leet (1975, 1976) and Locker gt gt. (1976) of the presence of so-called "gap" filaments in the muscle has complicated the comparatively simple picture of a few years ago. Locker gt gt. (1977) believe this new component to be a major contributor to the tensile properties of meat. Aging Effect. Muscle characteristics change sig- nificantly during the period of development and resolu- tion of rigor. Experience has shown that tenderness, juiciness, and flavor of resulting meat is usually im- proved by a period of storage or aging especially in bovine and ovine species (Paul, 1972). The length and tempera- ture of aging are known to influence tenderness of meat. 2A Doty and Pierce (1961) reported an improvement in both fat and lean flavor of broiled beef rib eye aged for two weeks. Taki (1965) found that beef longissimus dorsi steaks were more tender at 1 hour than at 2A or A8 hours, but less tender than at 192 hours post mortem. Usually aging is done between 0-15°C to minimize bac— terial growth. Stanley gt gt. (197A) identified some pos— sible separate mechanisms taking place during postmortem aging to include specific chemical changes at the Z-line and probably at actin-myosin interaction sites, catheptic activity, degradation of collagen cross-links and general microbial action. The importance of aging temperature as an essential factor has been demonstrated by a number of workers. Busch gt gt. (1967) and West (1978) reported increases in tenderness with high temperature condition- ing (HTC) of freshly slaughtered animal carcasses. Hender- son gt gt. (1970) found that the Z-line and M-line de- gradation occurred more rapidly upon storage at 25°C or 37°C as against 2°C or 16°C in 2A hours postmortem in porcine muscles. Ultrastructural changes in myofibrils such as degradation of the Z—line and changes in the actin- myosin interaction might play very crucial roles in post- mortem tenderization of meat (0011 gt gt., 1970; Hay gt gt. 1973). Hegarty gt gt. (1973) reported structural changes or breakdown in myofibrils from "normally" aged and rigor- stretched turkey and porcine muscle. Goll gt gt. (197A) 25 and Penny gt gt. (197A) have indicated that a role of proteinases in tenderization of meat during post-rigor aging, since these enzymes are compartmentalized in most tissues and partial disruption, such as by freezing and thawing, expose them to their substrates and hence affect the rate of tenderization. Johnson and Bowers (1976) suggested that fragmentation may result from weakening of bonds between the actin filaments and the Z-line material. Abbott gt gt. (1977) reported that the I-band was the area of the myofibril most susceptible to autolytic breakdown while the white fibers appeared to be slightly more labile to autolysis than red fibers. Contrary to most findings, however, Wolfe and Samejima (1976) demonstrated that postmortem aging of muscle has no effect on the dissociation of actin and myosin, and these findings are incompatible with the hypothesis that the actin-myosin interaction undergoes a "weakening" during post—mortem aging. Marbling Effect. It has been generally accepted that marbling is. an indicator of desirable eating quality, especially of meat. McBee and Wiles (1967) found sig— nificant differences in tenderness, juiciness and flavor among carcass grades of prime, choice, good and standard. Dryden and Marchello (1970) found a significant correlation 26 between muscle fat content and taste panel tenderness. Some researchers (Henrickson and Moore, 1965; Norris gt gt, 1971; and Parrishgt 1., 1973) have, however, reported that tenderness was not significantly affected by marbling scores. Campion and Crouse (1975) reported low positive correlation between ether extract, marbling and tender— ness. The role of fatness in altering the rate of carcass chilling has been investigated. Pike (197A) observed that certain thinly finished and light—weight goat carcasses which produced very tough cooked meat had muscles with very short sacomeres. This suggests that carcass weight and/or fatness can affect tenderness via the cold shortening phenomenon. Cross gt _t. (1972) and Reagan (197A) reported significant (P < 0.01) correlations between intramuscular fat content and sacomere length in lamb and beef longis- gtggg_muscles, respectively, thus suggesting that marbling might also be related to tenderness via its insulatory effect in reducing the severity of cold shortening induced by low temperature or changes in postmortem chilling rate (Lee, 1976; Smith gt gt., 1976). There is now evidence (Meyer gt gt., 1977) that the beneficial effects of marbling and fat cover on quality may be little more than a reflection of the slower cooling rate (and hence reduced cold-shortening) of the larger and fatter carcass (Marsh, 197A). 27 Meat Tenderization Newbold and Harris (1972) have shown that methods of handling carcasses immediately postmortem, until the onset of rigor mortis, affects the ultimate tenderness of meat. A number of procedures such as the suspension via the pelvic bone, mechanical restraint of muscles, cooler aging, high temperature conditioning (West, 1978), delayed chill- ing, blade or needle tenderization, use of tropical plant or fungal enzymes, have been developed for increasing meat tenderness (Smith gt__t., 1977). It is generally known and accepted that pre-rigor meat is inherently more tender_than its post—rigor counter—part (Paul _t gt., 1952; Pearson gt gt., 1973). Attempts have been made to maintain muscle relaxation during the contrac- tile process of rigor mortis by injection of chemical muscle relaxants such as phosphates and magnesium chloride (Huffman gt gt., 1969; and Streitel gt gt., 1977). The use of sodium citrate to maintain muscle relaxation and tenderize pre—rigor beef has been suggested (Lardy, 1966). It is believed to retard the rate of postmortem glycolysis through the inhibition of the enzyme phosphofructokinase (Conn and Stumpf, 1972). This will prevent a pH decline, raise the water holding capacity and effect tenderization. A wide variety of enzymes are contained in the muscle, some of which are hydrolytic (Randall and MacRae, 1967). These include cathepsins and leucine aminopeptidase. 28 Maximum activity of these enzymes at low pH and inactiva- tion by heating to 65°C (Parish and Bailey, 1966) or aging at 15° to 20°C for 12 to 2A hours before chilling (West, 1978) has been known to cause or contribute to, the soften- ing of muscle post-rigor. They are believed to act on the sarcoplasmic proteins (Bodwell and Pearson, 196A) and increase with storage (Suzuki and Fujimaki, 1968). The position in which the carcass is suspended during cooling may stretch certain muscles while permitting others to shorten. The method of suspending intact car- casses through the obturator foramen has been described and evaluated by Orts gt gt. (1972) and Bouton and Harris (1972). Various other methods of carcass suspension have also been designed to place positive stress during rigor in order to optimize sacomere length and thereby improve tenderness in as many muscles on the intact carcass as possible (Hostetler gt gt., 1972; Bouton SE.§l-: 197A). Okubanjo (1978) reported that pre-rigor leg-twisting of mutton carcasses lowers shear force values, yields longer sarcomeres and increases fragmentation of test muscles. Over the years, there has been increased use of blade or needle tenderizing machines to improve the palatability of retail cuts (Bowes, 1975). This method increased tenderness above that achieved by aging alone for strip beef loin steaks, but did not generally affect flavor, juiciness or overall palatability ratings. 29 Another method for increasing tenderness and/or pre— venting cold shortening may exist in the form of electrical stimulation of the carcasses shortly following slaughter. This idea was first suggested by Harsham and Deatherage (1951). Some workers (Carse, 1973; Davey gt gt., 1976; and Chrystall and Hagyard, 1976) have suggested that elec- trical stimulation could be used to prevent cold shorten- ing or increase the rate of conditioning of carcasses. Chrystall and Hagyard (1975), Grusby gt gt. (1976), Savell gt gt. (1977), Savell t gt (1978), Sorinmade gt gt. (1978) and Cross (1978) reported substantial increases in tenderness of lamb, beef or goat meat by use of elec- trical stimulation. Investigations on electrical stimula- tion have documented its effect on the acceleration of postmortem glycolysis (McLaughlin, 1970; Carse, 1973; Chrystall and Hagyard, 1976; Grusby gt_gt., 1976; Cross, 1978; Savell gt gt., 1978). Smith gt _1- (1977) found that electrical stimulation using low voltage (100 V, 5 amps) increased tenderness by 12 to 55 percent, decreased variability in tenderness in 5 of 6 test animal groups and did not negatively affect muscle color, condition or pH. They noted, however, that prevention of cold shortening, as determined by relative sacomere lengths, did not explain all of the tenderization effects achieved by electrical stimulation, suggesting that enhanced auto- lytic proteolysis may also be involved. Dutson gt gt. 30 (1978) and Sorinmade gt gt (1978) inferred that the en- hanced activity of the autolytic enzymes of muscles, in stimulated sides, may partly be responsible for some of the tenderization benefit. Chrystall and Hagyard (1975) suggested that electrical stimulation induced acceleration of post—mortem glycolysis, caused the muscle fibers to enter into rigor mortis before the effects of cold-shorten- ing can take place. Cassens and Newbold (1967), Moeller gt gt. (1976) and West (1978) reported a more rapid drop in pH with high temperature conditioning. With a lower pH at a high tem- perature, disruption of the lysosomal membrane and the con- current release of acid hydrolases into the muscle tissue occurs. Savell gt gt. (1978) are, however, of the View that physical disruption of muscle fibers resulting from the massive contractions during stimulation may be a mechanism for the tenderness improvement associated with electrical shock rather than high temperature conditioning ESE gg. Harsham and Deatherage (1951) had earlier investi— gated the influence of electrical stimulation on aging of beef and observed that application of voltages as high as 3000 volts also produced a fall in the pH of muscle to 6.1 in one hour. The meat was as tender, after 2 hours at 1°C, as that from unstimulated controls after 18 days at 1°C. Forrest and Briskey (1967), McLoughlin (1970) and Tarrant gt gt. (1972), reported that with approximately 31 30 seconds of electrical stimulation, the pH decline of naturally slow glycolytic rate pigs doubled. The glycolysis of freshly slaughtered lambs was accelerated by the applica- tion of high voltages (3600 V) of electrical stimulation (Carse, 1973; Chrystall and Hagyard, 1976), and the longis- gtggg muscle pH in stimulated carcasses fell to below 6 within one hour of slaughter, compared with lA hours re— quired by unstimulated muscle. Shear force values for muscles from leg and loin area cuts of stimulated car- casses roasted from the frozen state were about half of those from unstimulated carcasses and there were no deleterious effects due to stimulation. Davey gt gt. (1975) stimulated beef sides for l to 2 minutes period with high-voltage electrical stimulation immediately after carcass dressing and noted that the time for the development of rigor was reduced from 2A hours to about 5 hours. The stimulated carcasses, even though chilled rapidly, were still warm at rigor onset. Cold shortening or toughening would not develop under these conditions and the meat could be aged to a high degree of uniform tenderness. Although the electrical parameters chosen to hasten rigor in lamb carcasses (Carse, 1973; Chrystall and Hagyard, 1975, 1976) and beef sides (Davey t al., 1976) were empirically derived, they effectively reduced the time needed to achieve rigor to such a degree 32 that it was possible for all risk of toughening from cold and thaw shortening to be avoided and tenderness main- tained. Chrystall and Devine (1978) suggested a two-stage process involved in the accelerated onset of rigor in electrically stimulated muscles based on the pH changes. The first, occurring during stimulation, induces a remark— able 0.5 to 0.7 pH unit drop (ApH) in 120 seconds, repre- senting a 100- to 150-fold increase in the rate of under- lying biochemical reactions. In the second stage, oc- curring after cessation of stimulation, the rate is much slower, but is still almost twice as fast as in non- stimulated muscle over the same pH range. They observed that pH change (ApH) decreased with pre-stimulation pH, although Bendall gt gt. (1976) had claimed that rates of pH fall after stimulation do not differ from those in non-stimulated muscles if temperature corrections are applied. MATERIALS AND METHOD This experiment was a two-phased study for which 12 steers purchased on four different sale dates at a local auction (Howell, MI) and judged to be "grass-fed" by virtue of their condition and character and color of feces were selected. They were considered "Feeder" steers at the market place. The first part was a study to determine the influence which the technique of slaughter of the beef animals, electrical stimulation of the carcasses and temperature storage conditions had on the tenderness of steaks ob— tained from the short-loin region. The second part was to ascertain the relationships between these treatments and the changes in muscle pH, adenosine triphosphate (ATP) levels and total microbial load. Experimental Animals These were thin, humpless (Bos taurus) breed of cattle averaging between 31A.10 and AA8.70 Kg live weight, with a dressed weight of between 152.56 and 259.90 Kg. A total of 12 animals in 3 replications of A animals per replica— tion were involved. All the animals averaged between 18 and 2A months of age. Because of the distortion of the longissimus due to warm cutting of the loin, marbling 33 3A and other grade factors were not recorded. The quality grade range was estimated to be between mid point U.S. utility and end point U.S. standard. Table 1. Group Distribution of Experimental Animals. Dressed Weight (Kg) Animal Live Weight (Kg) Left Side Right Side 1 A2A.A0 121.30 117.50 2 AA8.70 129.A0 130.50 3 A2A.80 115.70 117.00 A A36.10 116.60 119.30 5 AO2.80 100.90 103.00 6 386.10 90.77 93.91 7 37A.A0 103.19 lOA.5A 8 363.60 96.89 98.69 9 335.30 86.72 87.17 10 391.50 108.59 110.57 11 357.30 96.17 96.17 12 31A.10 7A.8A 77.72 Slaughter Procedure The twelve animals were slaughtered in the abbatoir at Michigan State University Meat Laboratory. Each was fasted for at least 2A hours prior to slaughter but were provided with drinking water ggqttt, The throat slashed steers were held A days without feed. Two animals were slaughtered on each scheduled day. Six of the steers were stunned with a captive bolt pistol prior to bleeding. Each of the other 35 six was trapped in a steel bleeding chute and haltered. The animal's head was raised by the halter rope and then throat slashed with a sharp knife. They were exsanguinated and dressed within 50 minutes following death and split into right and left sides. Electrical Stimulation The right side of each carcass was subjected to elec- trical stimulation within 60 minutes postmortem. The source of electrical stimulation was a set-up consisting of a rheostat (a Powerstat variable autotransformer, Type 116B—50/60 Hz) ampmeter and a voltmeter to which were connected two cables which ended terminally in two metal probes (Figure 1). This modification was to facilitate the extension of these electrodes to the hind limb and the neck regions of the sides to be electrically stimulated. These probes were inserted into, and electricity was ad- ministered to: (1) Round and fore-arm (10 bursts at 5 secs/burst); (ii) Neck and Gluteus muscle (10 bursts at 5 secs/ burst). The current of the electrical impulse was single phase, 100 volts and 60 cycles similar to that used by Savell gt gt. (1977). However, the amperage of the current passing through the carcass was noted on the ammeter. 36 J 110_¥ LIN RHEOSTAT (100 V) M I'll E S 0 10 AMP FUSE > AMMETER *SINGLE POLE 0t SWITCH GROUND "CHARGED" PROBE HOT PROBE .. .. .. 4-— CARCASS SIDE Figure 1. Diagram of the electrical set-up used for stimu- lating the carcasses. 37 Individual carcass sections received a grand total of 100 seconds of electrical stimulation. The amperage noted ranged from 0.6 to 1.0 amp at each burst. Measurement ofng and Temperature Initial temperature and pH of each carcass side were noted before and immediately after electrical stimulation. The left and right sides of the carcasses of one group were wheeled into the cooler corridor whose temperature had earlier been regulated to 20°C, while the left and right sides of the carcasses of the other group were wheeled into the cooler set at —1°C. Internal temperature and pH of the longissimus dorsi muscles, at a point opposite the 6th lumbar vertebra of each side, were monitored every hour for the first 6 hours and later at 2A and 96 hours post-mortem. The thermocouples were attached to a Honey— Well brand "Brown Electronic" potentiometer. A type 7GR 23l/100L combination electrode, with a Type 36101 portable pH meter (Kerotest Manufacturing Corp., Pittsburg, PA) was used to measure pH. Microbial Count Standard plate counts were performed on all samples using the methods described by the American Public Health Association (1966) and Patterson (1971). Sterile cotton 38 swabs were used to obtain samples from each of three ran- 2 areas described by a sterile tem- domly selected 6.5 cm plate on the inner body cavity surfaces of each carcass side at 0, 5, 2A, 96 and 168 hours postmortem. 0 hour samplings were from the rib cage (ll-13th rib) and flank areas, while subsequent samplings were from the internal surfaces of the shortloin region. The tips of the cotton swabs were broken into tubes containing 10 ml citrate buffered distilled water (pH 7.0) and these were vigorously shaken. Serial dilutions of the samples were plated on Plate Count Agar and incubated at 32°C for A812 hours. Microbial counts were recorded as the mean of duplicate plates. Holding and Sampling For each treatment, the whole shortloin (13th thoracic through the 6th lumbar vertebra) was removed from each side of the carcasses at the end of 5 hours postmortem. In the first treatment, the shortloin was placed in a plastic container (approx. 32 gals. capacity) filled with cold (21°C) tap water, covered with a lid and left at room temperature (20°C) till the end of 2A hours postmortem before being placed inside the cooler (1°C) for the remain- ing period of the experiment. Shortloins from the rapidly chilled sides were left in cooler storage (1°C). Thermo- couples were inserted into each of the Shortloins and the 39 internal temperatures monitored by a poteniometer over a period of 2A hours. Two 3.8 cm thick steaks were cut out of each shortloin proceeding serially from the anterior end at the end of 5, 2A, 96 and 168 hours postmortem, for cooking. Cooking,Method The 3.8 cm thick steaks from the longissimus muscles were cooked in deep fat ("Crematex" - an all purpose shortening made from hydrogenated vegetable oils) with a deep fat fryer (Hotpoint Co., brand SER. B-58A51, CAT 201 HK3) set at lAA°C to 62°C internal temperature of the steaks. Cooked steaks were wrapped in aluminum foil and stored in the cooler at A°C for 2A hours. Measurement of Tenderness At the end of 2A hours of storage, the cooked steaks were laid out for panel evaluation and physical measure- ments of tenderness. Panel Evaluation. A 9-point hedonic scale was used by the twelve panel members which was made up of faculty, staff and graduate students of the Meat Laboratory at Michigan State University. They evaluated the tenderness of cubes taken from the steaks, obtained by cutting through sections. A0 Warner-Bratzler Shearforce Values. Six 1.25 cm cores were obtained parallel to the muscle fibers from each of the steaks. They were removed from the medial, central and lateral positions of the longissimus muscle. Each core was sheared 3 times by the Warner-Bratzler Shear instrument and the mean values calculated. 1.25 cm cores were chosen so as to facilitate the removal of 6 cores from each steak. Paul and Bratzler (1955) found that there was close agreement between shears of 1.25 and 2.5A cm in diameter and suggested that either size may be used to measure shear force. Allo-Kramer (Texture Recorder) Shear Force. Sections measuring approximately 2.50 x 1.50 cm were obtained from each steak and weighed. Each of these was sheared with the flexible blunt multiple blades of the standard shear com- pressor cell using a 135 Kg (300 lb) force ring and a range of 20. The mean of six shears per steak was used for com— putation. Adenosine Tri-phosphate (ATP) Determination ATP determination was based on the Luciferase enzyme technique described by Strehler and Totter (1952). Sam- ples were excised from portions of the fresh short-loins at 0 hr 10 mins, 5 hr and 2A hr poststimulation and frozen in liquid nitrogen. They were placed in Whirlpack Al polyethylene bags and stored in a freezer at -36°C. The frozen muscle pieces were powdered by grinding with dry-ice in a Waring brand commercial blender in a ~23 to ~29°C atmosphere. 500 mg of each of the powdered samples were put in test—tubes each containing 15 ml of boiling dis- tilled water and allowed to boil for 5 minutes. The test- tubes were immediately transferred into ice-cold water. 0.1 m1 volumes of the meat ATP—extracts were mixed with 0.25 ml of the luciferase enzyme (a Commercial fire-fly extract — an Aldrich Chemical Co., Inc. product), which had been standardized with known concentrations of the ATP standard (from Equine Muscle - a Sigma Chemical Co. product) inside the dark compartment of the Aminco-Bowman Spectro- photofluorometer (An American Instrument Company, Inc. product). The emission wavelength was set at 550 and the degree of light emission from the mixture was recorded as percent of needle deflection in the light detecting portion of the Farrand photofluorometer. The values so obtained were expressed in terms of uMoles ATP per gm of meat tissue sample. Statistical Analysis The statistical analysis was done by a CDC 6500 com- puter at the Michigan State University (MSU) Computer Center using the MSU STAT Systems Group (1977). Simple correlation coefficients were determined as described A2 under the BASTAT program (STAT Systems Group, 1977) following Snedecor and Cochran (1973). Data were analyzed by a split plot analysis of variance using the STAT Systems program. A3 RESULTS AND DISCUSSION Postmortem Changgs ofng The rates of pH decline for all treatments are present- ed in Table 2 and illustrated in Figures 2-A. These results show the electrically stimulated beef sides to have the most rapid rate of decline (P < 0.01) while the unstimulated (control) group had the slower decline rate. This is in line with the findings by Chrystall and Hagyard (1976), Chrys- tall and Devine (1978) and Sorinmade gt gt. (1978). Both stimulated and unstimulated groups had an average 0 hr (post-stimulation) pH value of 6.85. This falls within the range of 6.A8-7.0A reported in literature (Moeller gt gt., 1976; McCollum and Henrickson, 1977; Tarrant and Mothersill, 1977; and Sorinmade _t _t., 1978). In both the stimulated and control treatments, the slope of the pH curves (Figure 2) decreased gradually after 5 hours post-stimulation storage until they attain their ultimate 96 hour values. The pat- tern of pH drop, however, shows an average of 0.5A pH units fall within the first 60 minutes of storage for the stimu- lated sides as against 0.26 units for the control, rep- resenting about 2 times the rate of pH decline of longis- gtggg muscles from stimulated carcass sides over the control. This agrees with the findings of Bendall (1976) who reported a rate of fall 2-3 times greater than normal for electrically stimulated rabbit and lamb carcasses, A5 .mo. v m at emnpo some Eopm unvoMMHc szconMchHm no: one poppoH oEmm one an pmSOHHom .pcoEpmoep co>Hw m :H .cssHoo beam on» :H moSHm>m oom.m on.m om>.m own.m omw.m ooo.m OON.m om=.m omm.m NH oooN oou.m omm.m on.m omo.m OMH.m on.m ow:.m 0H~.m omm.m NH ooH nom.m nmm.m nm>.m nm>.m mm.m nmm.m 93H.m nnz.m m~.m NH UoCCSmeD eme.m hme.m hae.m hmm.m em.m ema.e emm.e emm.e mm.e ma eaeeaam m>.m :>.m Hm.m mm.m mm.m mH.w o:.m om.m mmm.m NH pouMHSEprCD wm.m mm.m mo.m m>.m mm.m mm.m No.0 om.m mzw.m NH UmpmHserm 8 am e m a m m H e e Regatta Awenv oEHB wcHw< COHpmHSEHmepmom .opdpmmoQEop wsHUHoc paw ponpoe empswsmHm .QOprHBEHpm HmOproon mp popommmm mm wowcmno *mQ oHomsz .N mHnt A6 “’91 7‘0 0 pH STIMULATED 0 pH UNSTIM‘ULATED VATP ST IMULATED VATP UNSTIMULATED I 20- '73 ATP (uMoles/gm TISSUE) q: 9’ 6'0! -4-O- l 1 4 ll ' I O 2 4 6 fit '34 I 96 TIME POST-STIMULATION (hr) Figure 2. The effect of electrical stimulation on the rate of longissimus muscle pH and ATP decline. A7 160- 70» 0 pH UNSTUNNED . pH STUNNED V ATP UNSTUNNED V ATP STUNNED ATP (uMoles/gm TISSUE) 100- mi 3°0~ (rim “8- l l l l 60 6'0:- 1 *\ 4‘04 58;- l\0 |\ \ \ 2-0- 56» \ \v o. 5.4. 5 3 4:1 H2; £t———¢6 ls)!- TIME POST-STIMULATION (hr) Figure 3. The effect of slaughter method on the rate of longissimus muscle pH and ATP decline, 160! "H 120- 100- 13 ATP (uMoles/gm TISSUE) 5 Q‘ P 9 20‘ cl 5% Figure A. 48 1 1 l 1 l 4 e 1% 24 ”—16 TIME POST-STIMULATION (hr) Md- The effect of holding temperature on the rate of longissimus muscle pH and ATP decline. A9 and those of Chrystall and Devine (1978) who reported that the rate of fall in stimulated bovine sternomandibularis muscles is more than one and one—half times that in non- stimulated muscles over the same pH range. The value for the stimulated sides dropped to 5.68 within 6 hours while the control fell to 5.81 thus representing a highly sig- nificant (P < 0.01) difference due to electrical stimula- tion. Gilbert and Davey (1976) obtained a pH of 5.A in 5 hours (stimulated) using a higher voltage (3600 volts) and Sorinmade gt gt. (1978) obtained a value of 5.A5 within 5 hours post—stimulation. At 96 hours post-stimu- lation the pH of the unstimulated carcass sides (5.78) was significantly higher (P < 0.05) than that for the stimu- lated sides (5.57). The unstunned steer longissimus muscles exhibited a significantly (P < 0.05) lower 0 hour (post-stimulation) pH as compared to the stunned steers (Table 2). This conflicts with the observations of McLoughlin (1965), and McLoughlin and Davidson (1966) working on porcine loggissimus dorsi and Van der Wal (1978), but may be attributed to the fact that the unstunned steers, at time of slaughter, struggled furiously thus inducing a faster breakdown of ATP and/or glycogen present in the muscles and influencing a faster decline in pH levels (Bendall and Rhodes, 1976) due to lactate accumulation from accelerated glycolysis (Lister, 1970). 50 pH decline was slower in muscles held at 1°C when com- pared with the rate of decline for muscles held at 20°C (Figure A). Ultimate pH of 5.70 was reached in 96 hours at 1°C while the value of 5.60 was attained in 2A hours at 20°C. These results are of identical pH change pattern as those reported by Marsh (195A), Moeller gt gt, (1977) and Jeacocke (1977) who observed that the rates of pH decline and ATP depletion occur faster as temperature in- creases above 10°C. This suggests that the higher the holding temperature, the more rapid the glycolytic rate, thus influencing the time of attainment of ultimate pH levels, although Cassens and Newbold (1967) reported that the rate of pH decline at 1°C was not slower than that at 15°C until several hours postmortem. ATP Changes The 0 hour post-stimulation (50 minutes postmortem) ATP levels and the extent of glycolysis are well reflected in the postmortem pH profile (Figures 2-A). They also illustrate the relationship between ATP degradation and pH decline during period of aging for all treatments. Positive correlations exist between pH decline rate and ATP disappearance (Table 3). This is particularly evident at A hour pH and 5 hour ATP (r=.65, P<.01). As with most postmortem changes, ATP depletion was precipitous (Figures 2—A) and although some residual ATP (~1.1A uMoles/gm 51 R. u 8. w. a S. - R. u S. v me smile. H OO.H we. ow. m5. «H.I mm.n mm.s No. «m.u 0H.| mm.l mH.u Nm.| xv.| vn.n mm. (m. A; «N .HomnoLOWE oo.H «S. «b. mo.n HH.| mH.I 0N.n mN.I mH.n NH.| Om.n Om.n mv.n mv.| hm. mm. L: m .Hownocemfi oo.H mm. OH.u mm.n Om.n 5H.u bv.n mm.n em.u mN.u mm.c Hc.n cm.u nv. 0v. p: cm .LEte Oo.H HH. HN.: bN.I «H.| hm.u 0N.n 0H.u On.| mv.| Cv.u Hm.n Nm. Nm. L: m .eEwg oo.H or. no. ow. Hm. Om. HN. on. em. Hv. HH. mm.u av.u A: vm :e cc.H Ho. be. ov. 90. On. On. no. Go. hm. cc.n cu.u L: m :; oo.H Nv. Nv. no. mv. cc. cc. Cm. Cm. ©@.n cv.: p; v :; OO.H mm. OH. co. mu. or. am. Ho. Hm.u 00.: p: c I; oc.H mm. hm. CH. On. 65. Cb. cc.| nv.| c: «N Le< cc.H mm. mm. mm. cc. xv. $0.: mm.a a: m L+< cc.H 5H.I «v. Nq.. NH. vm.n «v.n L: c de< cc.H He. mo. m. co.u bv.: A: co Lumen xn< co.H Cb. mm. mc.c ox.u L: 0 Lemma xu< :c.H ch. cc.u vo.u e: co ccozm mu; CC.H a¢.i cc.n L; m Lawns mi; oc.H Ho. L: co mwmceovzme Accra CC.H L: m mmeccmcceu Hmccd mm gm ma gm mm gm 7m om mm gm om %m gs mm gm we we a u.c .w u.m u. m. u. .d "H.d "H.d “A "Ann . flu .Anu s u s 3 J J J u. J.d u. J J H. J J u. J u m s a :.e O O J 8 a J J J CO C... J C. C. I wt 0. o. J J s "w "H "A "4 ,0 ..Lo .L. B 8 a a a a 97w (.7— B B .4 a... E E E B a a T. T. n n J J J J u.u 1.1 J J J D. w c. a a a “d J J . . n.m.uxrogc down do uwczHccc Hcmncee_e new HmoHEmzo .cacoEopsamoE Hcomczza uscwgw> comapon uezcmemeeoco :omSmHoLLcc oHLEmm .m.canH 52 tissue) remained after 2“ hours in the longissimus of the unstimulated carcass sides, practically all the ATP in the electrically stimulated carcasses had disappeared (0.M5 Moles/gm tissue) and had dropped to about l/3 of the 0 hour value at 5 hours post-stimulation. This relationship is in agreement with the results reports by Davey 23.31- (1976) and Sorinmade _t _l. (1978). At pH 5.88 (5 hours post-stimulation) about 60% of the ATP level in the muscles of unstimulated carcasses had disappeared reaching a residual low level of about 10% of 0 hour content at pH 5.78. Bendall and Rhodes (1976) reported that at pH 6, 50% of the resting content of ATP had disappeared, and at pH 5.7 more than 90%. Samples from stimulated sides showed an 86% ATP depletion at pH 5.73 post—stimulation and fell to a low residual level of 5% of the original at pH 5.57. These values were reached at 5 hour and 2M hour, respectively. The muscle samples from stimulated sides contain uuz less ATP than those from control samples at 10 minutes post-stimulation showing that electrical stimu- lation of carcasses immediately after slaughter is a very effective means of rapidly lowering ATP level and the pH of longissimus muscles of beef animals. 0 hour (post—stimulation) ATP levels for control carcasses were slightly higher (9.31 pMoles/gm tissue) than previous observations on bovine longissimus by Scopes and Newbold (1968) and Sorinmade 33 al, (1978). The 53 differences in ATP may be due to the different procedures used for the estimation of ATP or to the rapidity with which the muscles were removed from the animal and pre- pared for analysis. There is a high correlation coef- ficient between the 5 hour ATP level and 5 hour pH (r= 0.69, P<0.01) indicating that the simple measurement of pH would be sufficient to estimate the extent of glycolysis. There exists a significant difference (P < 0.05) in initial ATP levels (0 hour post-stimulation) due to tech- nique of slaughter of steers. The stunned steer carcass longissimus muscles contained a higher 0 hour (post—stimula- tion) ATP than the longissimus muscle from unstunned steers (Table A). This is most likely due to the effect of struggling at slaughter by the unstunned steers which is also reflected in the difference in 0 hour pH (Figure 3) resulting from accelerated rate of post—mortem glycolysis. ATP levels drop significantly with time (P < 0.01) with the unstunned steer longissimus muscles attaining a lower residual ATP (0.63 uMoles/gm tissue) than the stunned steer longissimus muscle (0.96 uMoles/gm tissue) at 2A hour post-stimulation time. Figure A illustrates the effect of a variation in hold- ing temperature on the decline inlflfl’levels in longissimus muscles of steer carcasses held at 20°C and 1°C over a 2M hour period. The muscles held at an elevated temperature (20°C) generally had lower ATP levels than those of muscles 5U .mo.o v N pm pcmpchHo mecmOHNHcmHm so: mpm poppmH meow map mp poonHom .pcoEpmmLp cw>Hw m CH .CESHoo meow map CH mmsHm> * 0mm.owmm.o omN.HHmN.N 0mm.HHHm.m oom.HHmm.w NH QOON on.owom.o omN.HHmN.N omm.HHmm.o om:.HHmw.m NH 00H nHm.owmo.o nzm.meo.N nmN.HHmw.m mm.Hme.w NH cmccsumc: pHo.OHmm.o nN:.HHmm.N pmm.HHmH.o mH.HHNH.oH NH cmcczpm N®.OH:H.H m:.HHON.m No.HHmN.N mom.HHHm.m NH empmHserwc: H:.owmz.o mm.oHNm.H oo.HHNN.H mmm.HHNN.m NH empmHzerm As: :NV OJHH Ac: mo com OH o c semapmmpe AcHzV meHe mch< cOHpmHsermnsmom .oLSprwQEop wchHoc paw ponuoE Lopcwszm .COHpmHSEHpm HmoHLpoon >9 ompommmw mm mowcmco AosmmHu &\moHozquv *me< oHomse mSEHmmHmdoq .: pomB 55 held at 1°C although the differences were not statistically significant even at the end of 2“ hours of holding time (P : 0.U8l). ATP level dropped to as low as 0.69 uMoles/gm tissue in the 20°C held muscles while those kept in 1°C environment averaged levels of 0.90 pMoles/gm tissue at the end of 2M hours of aging time. This observation may be due to the accelerated glycolytic process suggested by Moeller et al. (1977) and West (1978) which is enhanced at elevated temperatures in the range used in this ex- periment. Post—mortem Temperature Changes Changes in internal temperatures of the longissimus muscles were monitored during the first 2“ hours of aging and are illustrated in Figures 5-7 for all treatments. These results indicate that no significant differences exist in internal temperatures of longissimus muscles due to electrical stimulation, method of slaughter or holding temperature within the first 3 hours of aging time. At the end of 3 hours of holding time,a significant difference (P < 0.05) was noted between muscles held at 1°C and 20°C, and becomes highly significant (P < 0.01) at the end of 2N hours where internal temperatures averaged 12.08 and 22.79 °C, respectively. The average initial temperature of car- casses for all treatments was 37.79°C (0 hour post- 56 omOo v N pm ucmpmNNHo uoc ohm poppoH mEmm on» an UmonHom «pcmEpmmpp cm>Hm m CH CESHoo mEmm zHucmonchHm one CH mosHm> * mN.NN om.mN N:.om mH.Hm mN.Nm ooo.mm oo:.mm omm.~m NH coON wo.NH HN.ON NH.NN om.:N om.NN oom.om oHN.zm omm.wm NH 00H nmw.mH nm>.mN nm>.mN Qoo.mN no:.om QNH.Nm nmN.mm QwH.Nm NH poc25meD an.wH nom.:N omw.mN 2mm.NN cmm.mN nmw.Hm on.:m nwm.mm NH meQSpm mmw.mH moo.mN mNH.mN www.5N mmN.om mmN.Nm mmN.mm mmm.mm NH woumHSEHumCD mmm.oN mmN.mN mN:.mN www.wN mmm.om mNH.Nm mNm.:m www.5m NH UopmHSEHpm :N m m z m N H o : pCmEpmoLB Hugo meHe NCHNH COHpmHzermupmom .ogzpmpooEou wcHoHoc Ucm ponpoE pmpnmsmHm .QOHpmHserm HNOHLpooHo mp oopoommm mm mmmcmco Avov *mLSQNLoQEop oHomze mssHmmemoq .m oHnt 57 ' 4 O STIMULATED TEMPERATURE (°C) Figure 5. .‘UNSTIMULATED M Li! 1 "I' 20 U 15 lOr 00 j j g fi'\"'_"5'4 TIME POST-STIMULATION (hr) Rate of temperature decline in beef longissimus muscle of stimulated and control carcasses. 58 4° 0 STUNNED C UNSTUNNED N O TEMPERATURE (°C) 5' f 10' 0., 5 s a 4—74 TIME POST-STIMULATION (hr) Figure 6. Rate of temperature decline in beef longissimus muscle of stunned and unstunned steers. 59 30' N U! \ ’T \ c: 0 E20) E" 15 I11 9?! m 5" 15. 10‘ 5t- TIME PdgT—STIMULAEION (T) 00 *3 Figure 7. Rate of temperature decline in beef lon issimus muscle of carcasses held at 1°C and 20°C. 60 stimulation). The normal cattle body temperature is approximately 38.3°C to 39.l°C (Hafez, 1968). The internal temperature of carcasses used for this study falls short of this range, although this value (37.7900) does not vary much considering that the reading was obtained at 50 minutes postmortem. The 1°C stored carcasses showed a more rapid declinetnmdj_the internal temperature of the longissimus at 2“ hours post-stimulation reached l2.08°C. Muscle tem- peratures of carcasses that were held at 20°C dropped very gradually reaching an ultimate 2“ hour average internal temperature of 22.7900. Thus, the environmental temperature in which 13MB beef carcass is held has a significant in— fluence on the rate at which internal temperature of the muscle changes. There does not appear to be any in- fluence on changes in internal temperatures due to either electrical stimulation or method of slaughter. Tenderness Evaluation Panel Scores. Panel data as related to muscle tender- ness are presented in Table 6. These scores tend to indi- cate that the panelists were able to distinguish the steak samples that had been tenderized, as a result of the treat- ments, from the controls. Samples from electrically stimulated carcasses had significantly (P < 0.01) higher panel scores than the controls. This agrees with the findings of Savell gt 1. (1977) who reported significant .mo. v N pm pcmsmNCHo sHchOHCchHm pOC ohm LoppoH meow on an pmonHon .pCmEpmon Cm>Hw m CH aCECHoo 68mm me CH woCHm>N .Coocop mHmEoprm u m mnm50p hHoEonxo u H onom wCHpmp pCHOQIm w Co comma mCmoz 61 H mo.HHm©.© mo.HHmw.m om:.HHHo.m o:w.owzH.m NH OOON :H.HHmm.m um.HHN>.: omm.HHmo.: 0N0.OHNw.N NH 00H an.OHm:.o Hm.OHmw.m NO.HHH:.m m>.owmz.m NH UmCCSHmCD Dm:.HHmm.m H:.HHmm.: Hm.Hwa.m mm.OHom.N NH UmCCSpm mo.HHo:.m ON.HHm:.: :m.HHmw.m mN.OHom.N NH UmpMHSEHumCD mw.ow>m.m No.0Hmo.© om.HHmN.m m©.owoo.m NH UmHMHSEHpm mmH mm :N m C uCoEpmoLB AwCCV mEHB wCHm< COHpmHCEHmepmom .moHomCE Hmpoo mCEHmmeCOH moon Com N.HmeHme mmmCsooCoB Honm mo mCoHpmH>op ULNUCmpm UCw mCmoE mo ComHLwQEoo .w oHQMB 62 (P < 0.15 to P < 0.01) improvement in light-weight beef steak tenderness as a result of stimulation with 25, 50 or 75 electrical impulses, and Sorinmade £2 a1. (1978). Comparisons of panel tenderness for steaks aged for 168 hours indicate that tenderness difference (P < 0.01) could be detected as early as 5 hours post-stimulation. As expected, these panel scores show that improvement in tenderness progressed positively as aging time was pro- longed. Panel scores also rate the steaks from carcasses of unstunned steers significantly (P < 0.01) more tender than those from stunned steer carcasses up to 96 hours post-mortem. At 168 hours of post-stimulation time any differences due to method of slaughter had significantly (P < .15) disappeared. The differences of scores between steaks obtained from 1°C and 20°C held carcasses showed a tendency for steaks from carcasses held at elevated temperature (20°C) to be more tender than those from carcasses held at 1°C especially at 96 hours aging time (P < .05). This relationship is in line with the results reported by Locker gt 1. (1975) and also by West (1978). Shear Values. Mean values for shear force (Warner- Bratzler and Allo-Kramer) are presented in Tables 7 and 8. These values confirm the panel scores on the tenderness advantage of steaks from electrically stimulated carcass halves over those from unstimulated sides. They had 63 v m pm pCohommHo meCNOHmHCme HOC mam LmuumH oEmm on» ma UoBOHHom .quEpmep Cm>Hm m CH aCECHoo mean on» CH mosz> * owm.ome.m omm.ome.H 0H:.Hflmz.m omm.NHmH.N NH QOON OHN.HHow.: om:.HHm>.m om©.Hme.w OOH.MHmw.w NH 00H DMH.HHNO.: DNN.HHQ©.: an.HHNm.m DON.HHHN.m NH UmCCSmeD QNN.HHmN.: nqm.HHNN.m pqm.HHmm.m nmN.MHH:.m NH cmCCSHm ww.OHbo.m m:.HHNm.m ®@.HHNw.® ww.NHmw.m NH UmpmHSEHmeD NO.HHmN.m NH.HHom.: mo.HH::.m mm.NHHm.N NH UmpwHSEHpm me mm :N m C pCoEpmmCB AmLCV mEHB wCHm< COHpmHCEHumIpmom .moHomCE wCEHmmeCoH moon Log ANEo\wxv mmCHm> *oopow CmoCm LmHNPMLmILmCCC3 no mCOHpmH>mp ULMUCmpm UCm mCmoE No COmHLmQEoo .n oHCmB 61$ .mo.o v N pm pcmsmNNHc NHpcm0HNHcmHm poC ohm LoppmH oEmm on No UoBOHHom .pCoEpwoCu Co>Hw m CH .CECHoo beam on» CH mmCHm> * onm.HHON.N OHN.NHom.m omN.mwom.NH omz.:Hm©.HN NH OOON ONm.mem.OH omN.mHNH.NH ONN.mH:m.mH omN.mHHw.mN NH 00H Dom.NHmm.w 9mm.MHN:.OH Hw.Nme.MH 92H.NH>o.mH NH UmCCSmeD DHo.MH©w.m QHm.MH>H.HH w©.:H©m.zH Dmm.:HHo.mN NH Umcczpm ow.MHo>.OH mm.mwwN.NH mw.me:.mH mw.mez.MN NH UmpmHSEHmeD NN.NHHH.m :>.NHNm.m 0H.MHmm.NH OH.:HO®.HN NH UmpmHSEHpm me mm :N m C pCoEpmoCB AmCCV oEHB NCHw< CoHpmHCEHpmlpmoN .moHomCE msEHmmHmCOH moon Cog AEw\wxv moCHm> *mosom CmoCm CoEmCxIOHH< mo mCOHpmH>oU opwocmum UCm maoE mo ComHCmCEoo .w oHCmB 65 significantly (P < 0.01) lower shear force values than the controls, and as with panel scores, the tenderness dif- ferences were detectable as early as 5 hours post-stimula- tion (5 hours) of differences in tenderness tends to give further credence to the theory by Savell 33 al. (1978) that physical disruption of muscle fibers is a major contributor to muscle tenderness by electrical stimulation rather than the lysosomal enzyme theory proposed by Dutson t 1. (1978). dence to the theory by Savell _t al, (1978) that physical disruption of muscle fibers is a major contributor to muscle tenderness by electrical stimulation rather than the lysonsomal enzyme theory proposed by Dutson 33 a1. (1978). Also in Tables 7 and 8 are shear mean values for com- parisons between steaks obtained from carcasses held at 1°C and 20°C. Detectable differences (P < 0.065) were observed for Warner-Bratzler shear at the end of 2“ hours post- stimulation, but Allo-Kramer shears found rm) significant (P 1 0.215) differences for same samples. Shear values show that steaks from 2“ hour samples from stimulated sides were almost as tender as those obtained at 96 or 168 hours post—stimulation aging time from the control sides. Simi- lar observations were reported by Parrishgq §_l_. (1973) who demonstrated that longissimus samples from choice carcasses conditioned 2“ hours at 16° before subsequent chilling were 66 as tender as samples from control carcasses chilled and aged 7 days at 2°C, and also by Smith et a1. (1971) using 12 to 18 month old steer carcasses. One reason for the tenderness advantage due to high temperature conditioning may be due to the fact that the ‘technique accelerates the rate of postmortem glycolysis and hastens the onset of rigOr mortis, thus preventing toughening due to cold shortening. At the resultant low pH and elevated tempera- ture conditions there is increased autolytic activity of the endogeneous proteolytic enzymes on the myofibrillar structures (Henderson gt al., 1970 and Olson et_al., 1976). Shear values did not identify any significant differences in tenderness of steaks due to method of slaughter. In this experiment, the simple correlations between panel tenderness and shear force values gave highly negative relationships as evidenced at 5 hours for Allo-Kramer (r=-.89, P<0.0l) or Warner-Bratzler (r=-.62, P<.01) and at 96 hours for Allo-Kramer (r=-.60, P<.01) or Warner-Bratzler (r=-.66, P<.01). These results therefore, tend to confirm the suggestion that either of these three methods could be used to estimate the tenderness of cooked steak samples. Microbial Count Results of microbial count (loglo) per cm2 of the sur- face of the short loin and standard deviations, for all treatments, are presented in Table 9 and illustrated in 67 HOC opm CoppoH mEmm mCu mp UmZOHHom .mo.o v m pm HCmHmMMHU .pCoEpmoHp Cm>Hw m CH .CECHoo oemm NHHCCOHHHCme on» CH mmsHm> * No.HH::.m mm.oumm.w wo.HHNo.o mN.OHmm.m omo.HHNm.N NH OoON N>.oHom.m mm.OHom.m mu.OH:m.m >2.0HmH.N 02>.OHHH.N NH 00H nom.HHoN.m 9mm.HH:N.: QN©.HHmm.: CNo.HHmm.N now.HHHm.N NH ooCCsmeD Dam.HH:H.m Qmw.HHmm.z me.HHmw.: mm.OHmw.N nam.OHNm.N NH UmCCSpm mm.HH:o.m me.HHNN.z mmm.HHNN.H mHo.HHNN.N mmoHHmmN NH smpmHssHumca w:.HHmN.m mom.HHNw.: mm>.HHmm.: mNm.OHNw.N mHm.OHom.N NH UmpmHSEHpm mmH mm :N m o C pCmEumoLE Asnv NEHH NCHNN coHpmHsermnmeN .mopsumHoCEop NCHUHOC UCm COCpoE HopCmsmHm .CoHpmHCEHpm HNOHLpomHm an Umpommmm mm mommpsm oHomsE mCEHmmeCOH mo AQHNOHV pCCoo *HNOHNOHoHnopoHE .m mHan 68 Figures 10—12. Statistical analysis show no significant difference in microbial load as a result of either elec- trical stimulation (P : 0.9U0) or method of slaughter (P i 0.760). Differences due to aging time were similarly insignificant for both treatments. Analysis of variance for counts obtained at 0 hour poststimulation also showed insignificant differences due to slaughter method (P 1 0.975), electrical stimulation (P 1 0.935) or holding temperature (P 3 o.u31). Counts (loglo) per cm2 obtained at 5, 2M, 96 and 168 hours (post-stimulation from samples held at l0 and 20°C showed highly significant differences (P < 0.01) in microbial load of samples held between these two tem— peratures. Maximum count (loglo) at 168 hours was 3.90 for samples held at 1°C, but this value is far below the recommended bacteriological limits that have been sug— gested by the USDA (1968) and Kotula (1970). That for samples held at 20°C (6.“0) also falls below the recom- mended limit, indicating that the holding conditions employed in this experiment were within reasonable sani— tary levels. Highly positive correlation coefficients were observed between microbial load and holding temperature at 5 hours (r=.7U, P<.01) and 2“ hours (r=.80, P<.01). 69 o 7-Or STIMULATED O UNSTIMULATED 6.0L 5 O ‘47. \ LOG MICROBIAL NUMBER 59 c: C) P0- ‘0 T 3 4' 53 i 36 " "138 TIME , hrs Figure 8. Microbial growth rate on loin surfaces of stimulated and control beef carcasses. LOG MICROBIAL NUMBER 70 7.0 . o STUNNED O UNSTUNNED 60* U! 0 \ L k. c: 5’ c: PO JL 4 1' I H ' 1' 24 41 9b 168 TIME, hrs Figure 9. Microbial growth rate on loin surfaces of stunned and unstunned steer carcasses. LOG MICROBIAL NUMBER 7iw (roy- .50F é- c> I %’ c: 71 ..H-ll fir '9 PO Figure 10. Mb ’,,r-4F--O 3 rt—fl—‘it—fio—‘ib—fie TIME, hrs Microbial growth rate on loin surfaces of car- casses held at 1°C and 20°C. SUMMARY Twelve grass-fed steers were used to conduct experi— ments to investigate the influence of slaughter technique, electrical stimulation and holding temperature on the tenderness of beef steaks from the longissimus muscles, and the influence of the treatments on changes in muscle pH, adenosine triphosphate (ATP), internal temperature and microbial load. Six steers were slaughtered conventionally while the other 6 were confined in a steel frame cattle chute, haltered and throat slashed with a sharp knife. All the steer carcasses were Split into right and left sides, and the right sides were subjected to 100 seconds of electrical stimulation (100 volts) each. Twelve steer halves (6 steer carcasses) were placed in a 1°C chilling cooler and the others in a 21°C storage atmosphere. In- ternal temperatures and pH of the longissimus muscle were monitored. ATP levels and total microbial count were also determined. At 5, 2H, 96 and 168 hours (post-stimulation), samples were obtained from the longissimus muscle in the lumbar region for tenderness determinations. Tenderness was determined using Warner-Bratzler and Allo—Kramer shear devices and by taste panels. ATP determination was based on the luciferase enzyme technique read spectrophotometric- ally as percent deflection and converted to uMoles ATP per gram tissue. 72 73 Panel evaluation and Warner-Bratzler and Allo-Kramer shear values indicate that longissimus muscle samples from electrically stimulated sides of all steers were sig- nificantly (P < 0.01) more tender than samples from the untreated sides at each sampling time. All the 2b, 96 and 168 hour samples had higher panel tenderness scores and lower shear values than did the 5 hour steaks. There ap— pears to be a tenderness advantage of steaks from the un- conventionally slaughtered steers over the conventional method as Judged by the panelists although this was not significantly detected by the shear measurements. Ele- vated holding temperature (20°C) did improve the tender- ness of steaks after 96 hours of post-stimulation aging as reflected in the panel scores, although this was not significantly so with the shear values. Changes in internal temperatures of the longissimus muscles were dependent on the holding temperatures. There were no significant differences in initial post—stimulation temperatures due to treatments within the first 3 hours. Highly significant difference (P < .01) after 5 hours of holding time were observed. Temperature decline in the 1°C held carcasses was faster than in those held at 20°C. Rates of temperature drop due to slaughter method or elec— trical stimulation showed no significant difference through- out 168 hours. Electrically stimulated sides had a faster pH decline than 74 did the controls, and 20°C holding temperatures caused pH to drop much faster in carcasses than did 1°C. There were no significant differences in 0 hour pH levels, although a slight difference (P < .02) exists as a result of method of slaughter with the conventional technique having a higher value. The ultimate pH reached in the electrically stimu— lated muscles were highly significantly (P < 0.01) different from the controls but showed no significant difference for either holding temperature (P 2 0.833) or slaughter method (P 1 0.531). ATP content and pH levels of muscles had identical change pattern through 2D hours post-stimulation for all treatments. 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Post-Stimulation Aging Time (Hrs) Stimulated Sides Unstimulated Sides 5 2“ 96 168 5 2“ 96 168 2.75 6.25 6.36 7.u5 2.50 “.27 5.36 5.27 1.75 u.u5 5.A5 6.60 1.10 2.18 3.00 6.27 3.36 6.82 7.00 7.25 3.55 5.27 5.A5 6.25 “.27 “.73 5.33 6.17 3.36 3.6“ “.00 5.83 3.15 5.13 6.u0 7.57 2.60 3.01 3.56 3.80 2.78 “.90 6.20 7.00 2.96 2.99 3.10 3.50 3.65 7.01 6.50 7.62 3.20 6.1“ 6.25 6.60 8.10 6.29 6.31 6.26 3.00 5.59 5.75 5.95 3.u1 5.79 7.21 7.8“ 2.85 “.19 5.10 5.“9 3.25 5.2“ 6.2“ 6.91 2.62 “.20 5.0“ 5.21 2.57 3.31 5.60 7.81 2.11 2.92 “.20 6.31 2.01 2.8“ 3.70 5.23 -1.29 1.85 2.63 “.30 * (Scale - l, extremely tough - 9, extremely tender). 92 Appendix II. Raw data of Warner-Bratzler shear force (Kg/cm2) between stimulated and unstimulated beef langissimus muscles. Post-Stimulation Aging Time (Hrs) |_.l O Stimulated Sides Unstimulated Sides 5 2“ 96 168 5 2“ 96 168 5.““ “.26 3.“5 2.60 6.97 5.52 “.92 “.51 5.80 “.93 3.37 3.07 8.63 6.93 6.51 5.“6 “.“3 3.51 2.59 2.03 “.93 3.69 2.77 3.59 5.29 “.57 3.3“ 2.57 8.30 6.98 “.85 “.07 8.05 5.“6 “.73 “.07 8.31 6.78 6.60 5.22 8.25 6.6“ 5.25 “.25 8.69 8.0“ 6.97 5.“9 6.67 5.16 5.11 “.05 7.00 5.82 5.63 5.27 7.16 6.06 5.85 “.59 7.22 6.8“ 6.7“ 5.“9 6.96 5.08 “.01 3.53 7.82 5.76 “.98 “.30 7.61 6.05 5.09 “.72 7.1“ 6.76 5.30 5.08 .2“ 6.08 5.01 “.21 13.06 8.2“ 6.9“ 5.32 .2“ 7.“6 6.20 5.76 15.52 10.“7 8.2“ 7.03 H J: Appendix III. 93 Raw data of Allo-Kramer shear force (Kg/gm) between stimulated and unstimulated beef longissimus muscles. Post-Stimulation Aging Time (Hrs) Stimulated Sides Unstimulated Sides 5 2“ 96 168 5 2“ 96 168 22.26 11.21 9.57 9.21 2“.53 16.86 13.71 13.7“ 23.63 15.66 11.55 10.92 30.86 22.09 18.50 l7.“1 17.65 10.69 9.18 7.38 15.62 1“.31 15.12 11.“6 16.60 1“.61 13.32 11.13 17.“5 16.50 15.18 1“.6“ 21.20 7.28 5.61 5.58 22.32 9.10 7.“7 6.““ 22.91 8.82 7.50 6.16 23.13 12.13 8.““ 7.51 20.21 9.96 5.51 5.11 20.59 11.1“ 7.“9 6.30 20.29 11.2“ 7.22 5.90 21.03 12.13 7.86 6.76 17.02 12.“6 8.2“ 7.3“ 19.2“ 16.27 10.19 9.0“ 20.“6 16.62 11.3“ 10.21 22.63 17.02 1“.“0 12.15 25.62 1“.51 9.0“ 7.37 28.9“ 17.36 12.“8 9.“8 31.37 17.2“ 13.80 11.03 35.“6 20.06 16.36 13.“2 9“ m.m m.m m.m o.m H.@ :.m N.@ m.m o.» m.m m.m N.m m.m o.m m.m m.m N.@ H.» m.m m.m m.m m.m o.m m.m m.m w.m H.N :.m :.m N.m N.m N.m m.m H.m m.w o.N m.m m.m m.m o.m H.m m.m m.m N.m m.m N.m w.m m.m N.m m.m m.m H.m m.o m.m w.m w.m m.m m.m N.m H.m N.m m.m N.m m.m m.m w.m m.m N.m m.m m.m 2.0 N.@ w.m N.m w.m m.m m.m H.o N.@ :.m m.m m.m m.m N.m m.m m.m m.m 0.0 H.@ m.m N.m N.m m.m w.m m.m o.m m.m m.m N.m m.m o.m m.m N.m N.m m.m m.m o.m m.m N.m N.m N.m m.m N.m o.m N.@ :.m m.m m.m m.m m.m N.m m.m N.m 0.6 0.0 N.m m.m m.m w.m m.m o.m H.w m.m m.o o.» m.m m.m N.m N.m m.m o.m H.@ N.@ m.m m.m m.m o.m m.m m.m H.m m.m m.m m.m m.m m.m m.m N.m w.m H.m o.m m.m N.m m.m N.m m.m m.m m.m o.m :.m w.m m.m m.m m.m m.m m.m N.m w.m H.m m.m N.m N.m N.m N.m o.m N.m N.m m.m N.m o.N m.m N.m m.m N.m o.m H.m N.@ 3.6 o.N N.m N.m w.m N.m H.m N.m m.m N.m m.m m.m m.m w.m w.m m.m 0.6 H.@ 3.8 m.m mm HN m m z m N H 0 mm 3N m m z m N H o mmeHm cmpmHssHpmcs mmeHm 66pmHzerm Ampmv mEHB wCHpmom .mmmmwopmo cmpmHSEHpm ICC UCm umpmHserm mo moHomCE mCEHmmeCOH moon CH mCHHomc ma mo dump 30m .>H xHocmNN< 95 Appendix V. Raw data of adenosine triphosphate (ATP- uMoles/gm tissue) in beef longissimus muscles of stimulated and unstimulated carcasses. Sampling Time (Mins)-Post-stimulation Stimulated Unstimulated 300 1““0 300 1““0 10 (5 hr) (2“ hr) 10 (5 hr) (2“ hr) 10.85 5.77 2.29 0.21 10.88 6.85 3.53 0.53 11.16 6.17 1.55 0.50 10.88 7.75 “.09 0.53 7.19 3.81 0.90 0.09 7.16 6.08 1.83 0.09 7.56 “.22 1.18 0.06 7.97 6.57 2.08 0.93 9.70 3.39 0.67 0.25 10.0“ 7.38 3.26 1.18 11.22 “.62 1.58 0.78 11.5“ 7.8“ 2.98 1.52 10.01 5.89 0.56 0.56 9.67 8.7“ 6.11 2.02 10.11 6.23 0.90 0.31 10.57 8.62 5.52 1.30 7.29 3.“7 0.3“ 0.09 7.0“ 5.“6 1.83 0.81 8.06 “.09 0.“3 0.22 7.66 6.13 3.29 1.05 7.91 “.22 1.76 0.81 8.25 6.29 “.25 1.61 9.58 5.38 3.69 1.“6 10.01 8.09 5.67 2.1“ Appendix VI. Raw data of temperature (°C) decline in 96 beef longissimus muscles of stimulated and unstimulated carcasses. Reading Time (Hrs) Treatment 0 2 3 “ 5 2“ Stimulated Sides 39.0 36.0 35.0 32.0 32.0 31.0 31.0 27.0 “0.0 37.0 3“.0 28.0 28.0 2“.0 23.0 16.0 36.0 35.0 33.0 32.0 30.0 30.0 28.0 28.0 35.0 32.0 29.0 25.0 23.0 22.0 21.0 28.0 “0.0 38.0 37.0 37.0 35.0 32.0 30.0 27.0 39.0 35.0 31.0 30.0 25.0 23.0 21.0 12.0 36.0 35.0 33.0 33.0 31.0 30.0 30.0 27.0 “0.0 37.0 35.0 3“.O 26.0 25.0 23.0 10.0 38.0 36.0 3“.0 33.0 31.0 31.0 30.0 28.0 38.0 35.0 31.0 29.0 25.0 2“.0 23.0 13.0 36.0 31.0 29.0 28.0 26.0 27.0 26.0 20.0 35.0 32.0 25.0 23.0 20.0 18.0 17.0 8.0 Unstimulated Sides “0.0 36.5 35.0 33.0 31.5 31.0 30.0 26.5 “0.5 36.5 33.5 28.0 26.0 2“.0 21.0 15.0 35.0 3“.0 33.0 31.5 31.0 30.0 30.5 28.0 35.0 33.0 29.0 25.0 23.0 20.0 20.5 15.0 “1.0 “0.0 39.0 38.0 36.0 3“.0 32.5 29.0 “0.0 35.0 31.0 30.0 26.0 22.0 20.0 11.0 38.0 37.0 35.0 3“.0 32.0 31.0 30.0 25.0 “0.0 36.0 3“.0 31.0 28.0 23.0 21.0 10.0 37.0 37.0 35.0 3“.0 32.0 31.0 30.0 26.0 39.0 36.0 30.0 28.0 2“.0 2“.0 22.0 1“.0 35.0 30.0 28.0 28.0 26.0 27.0 26.0 19.0 35.0 32.0 25.0 23.0 20.0 17.0 16.5 7.0 Table VII. Raw data of microbial count (loglo) of the 97 surface of loin muscle of stimulated and un- stimulated carcasses. Stimulated Sides Sampling Time (Hrs) Unstimulated Sides 0 5 2“ 96 168 0 5 2“ 96 168 2.“8 3.62 6.20 6.93 6.79 3.7“ “.“3 6.59 6.96 6.91 2.92 2.72 3.36 “.53 “.“1 l.“8 1.51 3.85 “.75 “.18 3.61 “.36 7.93 7.8“ 7.28 “.15 “.83 7.15 7.“0 6.80 3.18 2.53 “.“9 3.32 “.36 3.“8 2.08 “.3“ 2.15 3.56 3.36 “.20 5.52 5.79 “.82 2.11 3.30 6.“5 5.80 5.“0 2.38 2.72 3.87 2.“3 2.71 1.52 2.66 3.53 2.15 2.38 1.52 3.5“ “.“8 5.61 5.53 1.“9 3.“8 “.26 “.51 5.23 1.“9 2.32 2.56 3.“1 “.63 2.08 2.18 3.38 3.“9 3.“3 1.“8 2.23 5.63 6.“0 6.78 l.“8 3.60 5.0“ 6.“6 6.08 2.23 1.“8 2.“9 2.15 “.32 1.“8 2.51 3.36 “.15 “.3“ 1.“9 2.63 6.30 6.23 7.53 3.30 3.26 6.66 6.77 8.11 l.“8 1.60 2.15 3.11 “.3“ 1.60 2.00 2.66 3.96 “.08 1111917117711 9 6 4| 3 nu 3 9 2 1 l H " Illllll F.“ H " ||I|