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Ej'U'jm .;.-'<'_:_ gig-WK; eff-"=3 I31} ‘5: '- u.) “‘3' gut-L; u: ~ ‘ - flfl-r “w u-.., THE EFFECT OF MINERAL SUPPLEIfiINTATION ON THE COLOR AND MYOGLOBIN CONCENTRATION OF PORK MUSCLE By Wayne Edward Henry AN ABSTRACT Submitted to the College of Agriculture Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1959 /7 / "7” O [3; .1.‘//‘]«‘W I M Approved: '/ ' “’ ' I Wayne Edward Henry ABSTRACT 'l'wo trials were conducted using 36 purebred Hanpshire and 44 crosstred hogs to determine the effects of breed, and zinc, iron, and copper supplementation on color and moglobin content of pork muscle using sections of the Long; ssiaus 9253;. Surface color measurements were amde by use of Hunsell spinning disks and myoglobin values were determined spectrophotometrically. Analysis of variance indicated no significant effect of mineral supplementation upon myoglobin concentration of the Logissimus £12151. (Trial 1. F - 0.30; Trial II, F - 0.45). In addition, non-significant relationships were found upon comparing breed to the moglobin concen- tration of fresh sample (F ' 0.42) and mghbin concentration on fat- ‘free, moisture-free basis (F " 2.62). The latter comparison was found to be approaching significance. By use of the correlation coefficient, highly significant rela- tionship was found between index of fading and mglobin concentration (r ' -0.69) indicating the feasibility of using the Maxwell spinning disks as a method of predicting myoglobin content of pork auscle. Non- significant correlation coefficients were obtained upon comparing index of fading with ether extract (0.21) and total moisture (-0.16). Cor- relation coefficients obtained upon comparing mglobin concentration to ether extract and total misture were non-significant (-0.29 and -0.l3, respectively). The results indicated a very low relationship between fat and misture content of pork muscle and its color as measured spectrophotometrically or by disk calorimetry. THE E‘FECT OF MINERAL SUPPImENTATION ON THE COIDR AND HYOGLOBIN CONCENTRATION OF PORK MUSCLE By flayne Edward Henry ATHBIB Submitted to the College of Agriculture Michigan State University of Agriculture and .Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1959 ACKNOHLEDGMENT The author wishes to express his sincere thanks and appreciation to L. J. Bratzler, Professor of Animal Husbandry, for his continuous guidance, unfailing interest and encouragement during the course of this study. His inspiration and help will always be appreciated. Sincere thanks and acknowledgment are due to Dr. A. M. Pearson and Dr. R. J. Deans for their assistance in killing and cutting the animals in this experiment, to Mrs. Dora Spooner for her aid in the statistical analysis and to Mrs. Beatrice Eichelberger fer typing this manuscript. .Above all, the author wishes to express his gratitude to his wife, Bonnie, for her encouragement, sacrifices, understanding, and aid in making this manuscript possible. TABLE OF CONTENTS I mTRODUC TION . O O C O O O O O O O 0 II mm OF‘ HTMTURE . O Q O O O O O A. B. C. Color of Fresh Muscle Tissue . 0010!. Measurements e e e e e e Factors Affecting Color of Muscle Tissue III EXPERIMENTAL PROCEDURE . . . . . . . A. B. C. D. E. F. SourceofAnimals . . . . . . Sampling Procedure . . . . . . Color Measurements . . . . . . Myoglobin Determination . . . Fat and Moisture Determination Statistical Analysis . . . . . IV RESULTS AND DISCUSSION . . . . . . . A. B. C. D. V SUMMARY AND CONCLUSIONS Preliminary Studies . . . . . Relationship of Spinning Disks With Fat, andMyoglobinContent .............. Moisture, Effect of Zinc, Iron, and Copper Supplementation on Myoglobin Concentration. Trial I . . . . . . . Effect of Zinc and Copper Supplementation of Calcium Levels of 0.55 and 1.31 Percent. Trial II Effect of Breed on Myoglobin Concentration . . . . Page ~1th til: 14 14 17 17 19 24 32 Page VIBmummmoOOOOOOOOOOOOOOOO0....33 VII APPENDD O O O O O O O O O O O O 0 O O O I O O O O O 0 O 37 INTRODUCTION Desirable color of lean meat, although it does not affect the nutritive value, is recognised and demnded by the consumer. Wilson 3]:- L1. (1959) stated that the ideal color of fresh pork is grayish- pink. Lsrselere 3t. 21. (1956), in a consumer preference study, found that 61.8 percent of the dark colored pork chOps were selected over grayish-pink pork chops. hoglobin, the pigment primarily responsible for meat color, is affected by many factors. Such factors as age, pH, breed, species, exercise, and the ration fed have been found to influence the concen- tration of myoglobin in the muscle. Bray _e;t_ 5.1;. (1959) found that the supplementation of copper and iron in veal calf rations had a significant effect on the color and moglebin content present of the muscle. "ho-toned" hams and off-colored pork is a problem which has confronted the meat packer and retailer for years. Therefore, if a method could be found to produce a desirable and uniform color of fresh lean meat, one of the problems of merchandising could be eli- minated. The objectives of this investigation were to determine the effect of mineral supplementation of swine rations upon the color and myoglobin content of pork muscle. Surface color studies were made and the concentration of muscle pigment was determined spectro- photometrically. Moisture and fat contents were determined in order to study their relationship to surface color and moglobin concentra- tion. -2- REVIEU OF LITERATURE A. Color of Fresh Muscle Tissue Hoagland (1915), Brooks (1933) and Levers (1948), stated that the red color of lean meat was due to oxyhemoglobin. Brooks (1937) reported that the color of lean meat was due primarily to the awoglo- bin present within the muscle fibers. In the same paper, Brooks reported the presence of hemoglobin in mscle fiber, but in quantities too small to have any significant effect on color. In 1932, Theorell (1947) isolated and crystallized the muscle pigment, myoglobin. Morgan (1936) confirmed Theorell's findings by crystallising lyoglo- bin from horse heart. Hill (1933) observed spectrophotometrically that there was a difference between myoglobin and hemoglobin. Hill also pointed out that myoglobin had a greater affinity for oxygen than'did hemoglobin. Someigert (1956) stated that nyoglobin is a conjugated protein that contains a heme moiety (iron-containing porphyrin compound) attached to a globin molecule. Its function in the live animal is to accept oxygen from the hemoglobin of the blood for use in oxida- tive, energy-yielding reactions within the cell. Hawk gt a}, (1954) reported that mglobin has a higher affinity for oxygen than has hemoglobin. Thus, the intracellular pigment (nyoglobin) may be fully oxygenated at lower oxygen tensions than the blood pigment (hemoglo- bin). The relative amounts of hemoglobin and oxyhemoglobin (oxygenated hemoglobin) present in blood will depend upon the concentration of oxygen present, which in turn is proportional to the oxygen tension (Henry's In). Henry's law states that the dissociation of oxyhemo- globin per unit lowering of oxygen tension is greater at a low tension than at a high tension. Pool (1949) stated that myoglobin exists in the muscle as a distinct pigment and because of its high affinity for oxygen, ny act as a store of oxygen for use in muscle contraction. Schweigert (1954) reported that the heme portion of the porphyrin compound gives rise to the color of mglobin. Buever, the color of home is modified considerably by the attachment of the protein globin and other colorless constituents. Lewis (1954), using electrophoretic separation, reported that muscle pigment contains two compounds. He suggested that twenty per- cent of the muscle pigment was a hemprotein. Busaini gt 3.1. (1950), extracting total pigment from beef mscle, found that ninety to ninety- five percent was moglobin. Craig at. g_l_. (1956) found thirty-five percent of total muscle pigment to be hemoglobin and sixty-five per- cent to be myoglobin. Broumand (1953) concluded that the bright red color of beef is due to the presence of 90 to 100 percent oxymyoglo- bin and 10 to 0 percent metwoglobin. Currently, the opinion of nay meat workers is that the negle- bin of fresh mlscle tissue, which is purplish in color, exists in the reduced state. Upon exposure to air, the myoglobin becosms oxy- genated, giving the desirable bright md color. However, upon extended exposure to air, the moglobin is oxidised to me‘lmyoglobin, which is .5- (1956), the effect of nutrition and age on color of beef muscle. The Hunter Color and Color Difference Meter is a tristimulus calorimeter that measures color on three scales, brightness, redness, and yellow- mess, which are read directly from.the instrument. Kennedy _e_t a_]._. (1926), in studying the identity of mglobin, used a,Bausch and Lamb spectrophotometer for color:measurements. Bull ‘gg‘gg. (1942) determined the color of beef muscle by use of a spectro- photometer. Hall 23‘31. (1944) used color paddles to study dark-cutting cattle. Disk calorimetry was developed in the laboratories of the United States Department of Agriculture for close tolerance measurements of color and far facilitating the observation of numerous samples (Nick- erson, 1946). The Munsell renotations of hue, value, and chroma‘were applied to the data. The Munsell color system.is commonly used with the spinning disks, as well as with other systems far naming differ- ent colors. Mackintosh (1932), in a study on.dark-cutting beef, used the spinning disks in measuring the color of muscle tissue. Be class- ified the color of’beef muscle in terms of the percentage of red and black of the spinning disks used. Mackintosh gt a__l. (1935), in another study of dark-cutting beef, explained the effect of finish on color. They found significant carrelation.coefficients of .610 and .665 be- tween finish and brilliance (value), and between finish and chrome, respectively. In this study, Mackintosh‘gt‘gl. converted the percen- tages of red, yellow, black and white obtained from the spinning disks to Munsell renotations of hue, value and chroma. They suggested -8- findings in that no difference in myoglabin canc entratians was found between exercised and unexercised beef steers. Craig 33 gl_. (1958) stated that the differences in color of lean were due to varying amounts of fat and moisture rather than the difference in pigment concentration. In contrast to these findings, Shank _e_t 51; (1934), in a study of pasture versus dry lot fed cattle, reported that in- creased swaglabin concentration cannot be due to nutritional causes but may be explained by pasture fed animals having more exercise and hence a higher swaglabin content. Ginger 33 51. (1954) stated that the variation in muscle color my be attributed to differences in amount of exercise, however, they pointed out that it did not explain the variations observed within a particular muscle. According to lawrie (1950), mglobin increases with age and exercise. He concluded that activity is the fundamental factor responsible for controlling the amount of pignent found in any muscle. Lawrie (1950) reported that park muscle can vary in color and still have the same maglobin content. He observed that muscles ap- pear much darker at the pH 7.0 than at pH 6.3. Briskey (1958b), stucb'ing exercised hogs versus unexercised hogs, found that exercise increased pH and produced a darker color. However, the difference in swaglobin concentration between the control and treated lots was not significant. This is in agreement with Rongey (1958) who found that exercise was associated vith a slight increase in pH which resulted darker colored ham muscle. However, Jacobson 93 31. (1956) stated -9- that color variations could not be explained on the basis of pH. Briskey 35 L1. (1958c), in a later study, observed the effect of exercise and high sucrose rations on the chemical characteristics of park has meals. The gluteus _me_d_i_u_s_ (outer ham muscle) was C‘Indied. Maustive exer- cise with normal rations were found to produce dark muscles, whereas high sucrose rations and no exercise produced muscles that were pale in color. The basal ration (no exercise) and high sucrose ration (ex- haustive exercise) produced muscles which were similar in color. Hus- cle color was determined by the Hunter Color and Color Difference Meter. The average L values were 37.85, 39.45, 39.75, and 40.18 for the basal ration plus exercise, high sucrose ration plus exercise, basal ration, and high sucrose ration respectively. The higher the 1. values the lighter the color. Myoglobin comentration was determined but no sig- nificant differences were found between treatments. lilsan gt 5;. (1959) in a study of "two-toned“ hams, postulated that the concentration of swaglobin and the degree-of “two-toning" us heritable characteristics. The degree of "two-toning" was observed to be more closely related to the amount of myoglabin‘in the dark ms- cles than the amount of maglabin in the light muscles. According to Niedermeier _e_t gl_. (1959), iron and copper supple- mentation greatly influence the color and waglobin concentration of veal muscle. It was also pointed out that an iron deficient ration would reduce the muscle myoglabin concentration. Bray gt :1. (1959) have shown that iron and capper supplementation has a definite effect -10- on the color and amount of myoglabin in veal muscle. Calves were fed a basal ration supplemented with 240 mg. of iron as ferric pyraphas- phate and ‘7 mg. of copper as copper sulfate per dw. It was found that the iron and copper-supplemented animals had a significantly larger quantity of Ivoglobin, or 0.54 mg. per gram of lean tissue. The authors tarpothesised that the increased iron intake accounted for a greater production of myoglabin. In the same study it was observed that the animls having the highest quantity of myoglabin produced a darker colored muscle as indicated by the Hunter Color and Color Dif- ference Meter. The dark and light samples had an average I. value of 29.47 and 27.3, respectively. A significant correlation coefficient of -0.91 was found between the myoglabin concentrations and 1. values. In view of the studies reviewed above, the present study was ini- tiated to determine what effect, if any, mineral supplementation of swine rations would have on the color of pork muscle. The observations were made by use of Hansell spinning disks and spectrophotometric es- timations af swaglobin. ~11- EXPERIHENTAL PROCEDURE A. Source of Animals Animals used in this study were obtained from.an experiment at Michigan State University designed to determine the relationship of sins, iron and copper supplementation to the incidence of parakerata- sis in swine. Previous workers (Bray'gt‘gl., 1959) have indicated a possible relationship existing between high level mineral supplemen- tation and the color and myoglabin content of muscle tissue. Therefore, this study was conducted to determine the effect of supplementing swine rations with zinc, iron and copper at various levels of calcium on the color and woglobin content of pork muscle. After the initial parakeratosis experiment was concluded, the animals were fed to ap- proximtely 200 lbs., slaughtered, and pl'wsical and chemical. deter- minations made. In.addition, since animals of known breeding were used, it was possible to study the possible affect of breed on color. Two separate trials involving a total of 80 pigs were conducted. Trial I consisted of 35 purebred Hampshire hogs and Trial II consisted of 43 crossbred hogs of known breeding. Two additional animals were used in the study of the relationship between mglobin and color. The basic ration was a typical fattening ration consisting of corn, soybean meal, meat and bone scraps, fish meal, alfalfa meal, limestone, dicalcium phosphate, iadised salt, B-vitamin mix, A and D concentrate, 312, and Aurofac 10. -14- 0. Color Measurements Hunsell spinning disks were used to determine the surface color of the lean meat, and hue, value, and chroma renotations were calcu- lated. Essentially the same tectmique was employed as described by Voegeli (1952) and Saffle (1958). A sample consisting of a 2 cm. slice of the loggissimus 92133, taken at the 10th rib, was placed in a Cryovac bag and then placed in a 36-38‘F. cooler. Color measure- ments were taken at the end of two hours. The average Munsell reno- tations for each of the lots were calculated as described by Saffle (1958). For evaluation of desirable pork muscle color, a pork loin sam- ple was selected by members of the Michigan State Animal Husbandry department, The sample had a renotation of 4.3m 4.9/3.6 which was considered as an "ideal“ pork color. This color was considered as the standard for calculating the index of fading by the formula of Nickerson (1946). D. Win Determination The quantitative determination of myoglabin as outlined by Ginger at 2.10 (1954) was used in this study with the exception of a few and- ifications. The method used in its entirety follows. Duplicate 25 gram aliquots of the ground sample were minced in a Waring Blender for two minutes with 100 ml. of cold distilled water. This was contrary to the procedure of Ginger gt, 9;. (1954) of over- night extraction in a refrigerator by mixing 10 grams of ground meat with 10 ml. of water. After blending, the solution was transferred to a 250 ml. centrifuge bottle and centrifuged for 20 minutes at 2500 revolutions per minute. All cen‘lrifugation was carried out at temper- atures of 34 to 38'F. The supernatant was then filtered through cotton which retained some of the fat that had risen to the top of the solution. After filtering, the red supernatant was adjusted to pH 7.0 with 1 N sodium ludromide. Foreign proteins were precipitated by the addition of 0.25 volumes of saturated basic lead acetate. Lead acetate was added at room temperature because at lower temperatures the protein precipita- tion is incomplete and at high temperatures (38'C.), the myoglobin will also precipitate (Schweigert, 1954). ~The lead acetate solution was allowed to stand for 20 minutes to allow complete precipitation of the foreign proteins, followed by 20 mintues of centrifugation (2500 rpms). The resulting supernatant was brought to pH 6.6 and the phosphate concentration to 3 M by the addition of mono and dibasic potassium phosphate in the solid form. The phosphates facilitate the precipitation of hemoglobin, leaving myoglabin in solution (Ginger gt .a_1., 1954). Following the third cemtrifugation of 20 minutes, the supernatant was filtered through Whatman numer 41H filter paper into a 50 ml. volumetric flash. Two milliliters of the solution were re- moved by a volumetric pipette and potassium ferricyanide and sodium cyanide were added to the filtrate to final concentrations of 0.6 ml! and 0.8 ml! per liter, respectively. The potassium ferricyanide oxi- dizes all of the myoglobin into metmyoglobin; and the sodium cyanide -17- E. Fat and Moisture Determination Approximately 5 gram duplicate samples were taken from the ground Logissimxs 9.9.523! placed in disposable aluminum dishes and dried at 100‘0. for 24 hours for moisture determination. Ether extract was determined from the same samples used in moisture analysis. The fat was extracted with snm'drous ether for 4 hours in a Goldfish Fat k- tractor. All samples were weighed to the nearest .0001 gram. Formu- lae for calculating the percent moisture and fat were as follows: wt. of dried sample . . wt. 0 resh samp e I 100 3; moisture Ii: 2? 322?. $1?" 1‘ 10° ' 7‘ fat F. Statistical Anew}: Statistical analysis of data included means, analysis of vari- ance, standard error of estimate, simple correlation coefficients, and predicting formulae. The following formulae used in analyzing these data are in ac- cordance with Snedecor (1958): Aggis of variance (12 " (€192 - Total sum of squares x2+ x2+_ —(£ 2-(Xz-Btw trt‘ment Total sum of squares - between meme sum of equal-es - Error -18- Correlation co efficient ”r" - (xv- (1x Y Véxz-(jhgfl)x «rt-(3&2) Slaps of regression line B - (KY - (gxaqn Lei ”Y" intercept A - Y - 137 Standard error of estimte 6.‘ Vgurz-Au-nzxr N-Z Predicting formula ?- A + B (x) Standard deviation of D (slope of line) 6' e Wimp? 63. -19- REM/1‘5 AND DISCUSSIW 5- Prelim-11.319.11.22 Several preliminary studies were conducted in order to become familiar with mglobin determination and the use of the Munsell spinning disks. Color renotations and mglobin concentrations were determined on pork muscle using sections of the loggissimus 513323. The technique and apparatus usedwere as outlined by Voegeli (1952) and Saffle (1958). The preliminary studies indicated that a thor- ough understanding of the three-dimensional concept of color was necessary to make rapid and accurate measurements of fresh lean tissue. The three color qualities, hue, value, and chroma, are de- fined by Hunsell (1916) as follows: Hum - is the quality by which we distinguish one color from another, as a red from a yellow, a green, a blue, or a purple. Value - is the quality by which we distinguish a light color from a dark one. Chroma - is the quality by which we distinguish a strong color from a weak one. ' The three dimensional concept of color required the operator using the Hunsell spinning disk apparatus to make three distinct comparisons when matching a sample. -21- the disk mixture from darker than the sample to lighter than the sam- ple. In order to facilitate the use of whole units of disk area in calculating renotation values, it was decided to use the lighter readings for purposes of standardisation. A method of expressing color differences is useful in man cases. Several workers have derived formulae for expressing small color differences, however, Nickerson's (1946) index of fading, which is based upon the Hunsell scale of hue, value, and chroma is applicable for expressing color tolerance, color fading, etc. The formula is as follows: I (index of fading) -§. (zen) + sov + sac C ' chroma of sample A H - difference in hue between sample and standard A Y - difference in value between sample and standard A C ' difference in chroma between sample and standard Butler (1953b) indicated that there is a slight error in the application of this formula. The formula assumes that at a chrome of 5, a one unit change in value has the sue effect on the overall color as do two units change in chroma or three units change in hue. moglobin determination is a time consuming and tedious proce- dure. Therefore, if disk calorimetry could be applied as a suitable method for estimating muscle pigment concentration, studies involv- ing woglobin content of muscle tissue would be markedly acceler- '1“. Upon comparing myoglabin, fat, and moisture content and index of fading of all samples studied, the following relationships were found: index of fading versus myoglabin content produced the highly significant correlation coefficient of -0.69 (Figure I) which sup- ports the concept of myoglabin being highly associated with muscle tissue color. Although over one-half of the variability (l-rz) be- tween the tro methods was unaccounted for, the predicting formula of I II 1.01 - .023 (X) appears to be a useful estimtor for predict- ing mglobin content from disk calorimetry readings of fresh pork loin muscle. Comparing the fat content with index of fading and woglobin concentration, the low and insignificant correlation coefficients of 0.21 and -0.29, respectively, were obtained, indicating that little relationships exist between fat content and muscle color. Such ap- pears to be true whether color is measured as a surface phenomenon by disk calorimetry or as total myoglabin content determined spec- trophotomeh‘ically. The effect of moisture content upon index of fading and moglo- bin was found to be low and in a negative direction as indicated by the non-significant correlation coefficients of -0.16 and -0.l3, respectively. The slight effect upon color due to variations in fat and moisture content appears to be somewhat of a lightening effect (increase in value) with an increase in fat, and a slight darkening of muscle color (decrease in value) with an increase in moisture (Appendix Tables A and B). The usual high relationship between fat -24- and moisture content was found as indicated by the highly significant correlation coefficient of -0.88. These findings are not in accordance with Craig .e_t_ 3.1. (1958), who indicated that differences in color of lean meat were due to varying amounts of fat and moisture rather than differences in pig- ment concentration. However, the results of the present study would indicate that color differences cannot be explained on the basis of fat and moisture concentration. C. Effect of Zinc, Iron, and Copper Supplementation on Moglobin Concentration. Trial I. Myoglobin values and levels of mineral supplementation are pre- sented in Table III. It will be noted that greater variations exists between animls than between treatments. The loin eyes of animals receiving iron or iron plus zinc (treatments II 8: III) contained slightly more myoglabin (3.55 mg./g.) than did those of animls re- ceiving zinc, copper, or copper plus zinc (3.52, 3.44, and 3.27 mg./g., respectively). However, analysis of variance indicated no signifi- cant differences between treatments (Table IV). The results of this experiment are not in agreement with Bray at 11‘. (1959) who indi- cated that supplementation of iron and copper to veal calf rations increases moglobin concentration. In their work, 240 mg. iron and 7 mg. of capper were fed daily per animal. In the present study, minerals were mixed into the ration prior to feeding, and estimation of mineral intake per animal was based on daily feed consumption. maturity than veal calves that are dressed at about the same weight. Myoglobin concentration increases with age and maturity, therefore, at the degree of maturity of the hogs studied the treatment differ- ence in myoglobin content may have been minimized. D. Effect of Zinc and Copper Supplementation of Calcium Levels of 0.55 and 1.31 Percent. Trial II. In Trial 11 the average daily mineral intake per animal was: 114 mg. of zinc, 295 mg. of copper, 104 mg. of zinc and 218 mg. of copper for treatments I, II, III, and IV, respectively. As indi- cated in Table V, there is little variation between treatments. However, the loin eyes from the control animls generally contained less moglobin per gram than did those of treated animals. These data (Trial II) are very similar to Trial I in that copper or zinc had little effect on total myoglabin concentration. In addition, greater variation was observed between animals within treatments than between treatments. Analysis of variance (Table VI) indicates no significant differences between treatments. -28- TABLE V CONCDITIATION 0F MYOGLOBIN 0F PORK LONGISSIHUS DORSI MUSCLE. TRIAL II Number Myoglobin Number uygglobin Tins-ls.) (ago/g.) OControl lei-Treatment II sex-334 3.46 X-33-3 4.24 X-61-8 3.74 1-59-3 5.03 L60-9 3.71 X-62-5 2.88 1.58-10 3.34 1-33-9 4.64 L59-6 3.65 L61-7 3.02 L37-ll 4.13 1-65-1 3.19 1-66-1 3.09 1-35-4 3.99 I—Sl-ll 2.31 1-34-12 3.84 X-Tl-l 3.93 1-61-5 2.73 X-36-6 3.82 1-60-7 4.07 Av. 3.52 3.76 Treatment I Treatment III 1-61-1 4.09 X-33-lO 3.69 1-59-9 4.76 X-61-4 2.47 1-33-1 3.46 1-59-4 3.60 1-58-9 3.65 1-59-8 3.99 1-33-2 3.02 X-39-3 4.03 1-58-1 3.54 1561-1 3.52 1-77-2 4.16 1-58-4 3.44 X-64-l 3.71 1-34-7 4.31 1.33.1. 4.54 X-66-10 2.90 1-62-3 3.43 3.56 Av. 3.84 Treatment IV 1-59-2 4.02 X-33-5 2.96 X-61-2 4.66 x-34-11 3.12 Av. 3.69 Wong-SI received cm .557. calcium Mix ' crossbred hogs MTreatment I - Zinc 75 ppm + .5573 calcium Treatment II - Copper 125 ppm + .5575 calcium Treatment III - Zinc 75 ppm 4* 1.31% calcium Treatment IV - Copper 125 ppm * 1.31% calcium TABLE VI ANALYSIS OF VARIANCE OF MYOGIDBIN VALUES. TRIAL II """""""""""'lEi31E5f1EF""15Ef7EF""lfi;:f"""""""' Source of Variance Freedom Squares Square F Total 42 15.78 Between Treatments 4 .71 .178 i545 N.S. Within Treatments 38 15.07 .397 - iescat non-1sg variance , . . 0.05 level. B. Effect of Breed on Myofiglgbin Concentration. Wilson 23; a}. (1959) postulated that the concentration of swo- globin found within a particular muscle may be a heritable character- istic. Studying the light and dark colored muscles of “two-toned" hams from three breeds of hogs (Poland China, Duroc, and Chester ' White), a distinct difference in myoglabin concentration of the dark muscles was noted between breeds. The Poland China had the highest moglobin content with 2.35 mg./g., followed by the Duroc with 1.80 lg./g. and the Chester White with 1.58 mg./g. of fresh muscle tissue. However, the differences between breedsin mglobin concentration of the lighter muscles were found to be non-significant. Table VII sunmnarizes the myoglobin content of the Longissimus doggi- muscles from the breeds of hogs represented in this study. By examination of Table VII, it can be seen that when woglobin is expressed in Ig./g. of fresh muscle tissue, there is less differ- ence between breed means than when myoglabin is expressed on a moisture- -31- cance at the (p - .05) level. However, the F - ratio of 2.62 ap- proaches significance. TABLE 1! ANALYSIS OF MYCXHDBIN WNCEN'mATION BETWEEV BREEDS (Fat-free, Hois- ture-free Basis) variance Freedom. Squares Square F Total 77 26.94 Breeds 1 .89 .89 *2.62 N.S. Error 76 26.05 .34 e.. - non-s 3. ' ant at p ' .05 love . .Although no significant difference in.myoglobin concentration. was found between breeds, the data suggest that there may be a rela» ticnship between breeds and the amount of muscle pigment present within the muscle. AnsF - ratio of 3.96 is required for significance. It is recognised, however, that variations in myoglobin content are found within breeds as'well as between breeds. Therefore, in.order to draw any valid conclusions, additional studies would be required. SUMMARY AND CONCLUSIONS The levels of zinc, iron and copper used to supplement swine rations in this study had no significant effect upon myoglabin conp centration of the Loggissilms 32531 muscle. In addition, the effect of breed upon nyoglobin.concentration ef pork muscle on both fresh tissue basis and fat-free, moisture-free basis, was found to be non- significant. However, on the fat-free, moisture-free basis, the relationship between nyoglobin content and breed approached signifi- cance, indicating that a sufficiently strong relationship exists be- tween breed and myoglobin content to warrant further investigation. A highly significant relationship was found between the index of fading and myoglobin concentration. From this, it may be concluded that disk calorimetry might be used as a.nethod for estimating awo- globin content of pork muscle. The effect of fat and total moisture content upon.color renota- tions and nyoglobin concentrations was found to be nonpsignificant. This indicates that little relationship exists between the fat and moisture content of pork.muscle and its color, whether it be measured as a surface phenomenon by disk calorimetry, or, as a pigment concen- tration by spectrophotometry. B ELIOGRAPHY Bray, R. W., Rupnow, E. H., Manning, F. M., Allen, N. N. and Nieder- meier, R. P. 1959. Effect of Feeding Methods on Veal Production and Carcass Quality. 11. Carcass Grades, Liver, Hide, Specific Gravity, Yield and Chemical Analysis of the Muscle. J. of An. 861. 188732-737e Briskey, E. J., Bray, R. IL, Hoekstra, H. G.,Q'ummer, R. H. and Phillips, P. H. 1959a. The Chemical and Physical Characteristics of Various Pork Ham Muscle Classes. J. of An. Sci. 18:146-152. Briskey, E. J., Bray, R. 11., Hoekstra, ll. G.,G:ummer, R. H. and Phillips, P. H. 1959b. The Effect of Various Lovels of hercise in Altering the Chemical and Physical Characteristics of Certain Pork Ham Muscles. J. of An. Sci. 18:153-157. Briskey, E. J., Bray, R. 3., Hookstra, W. Gnammer, R. H. and Phillips, P. H. 1959c. The Effect of Exhaustive Exercise and High Sucrose Regimen on Certain Chemical and Physical Pork Ham Muscle Characteristics. J. of An. Sci. 18:173-177. Brooks, John. 1933. The Effect of Carbon Dioxide on the Colour Changes or Bloom of Lean Meat. Journal of the Society of Chemi- cal Industry. 52:17-19. Brooks, John. 1937. Color of Meat. Food Research. 3:75-77. Broumand, H. 1953. The Development of a Spectrophotometric Method for Estimation of the Pigmented Compounds of Meat. Ph.D. Thesis. Rutgers Univ. Bull, Sleeter and Rusk, H. P. 1942. Effect of Exercise on Quality of Beef. Illinois Agr. Expt. Sta. Bull. 4883107-120. Butler, 0. D., Bratzler, L. J. and Mallmann, 1". L. 1953a. The erct of Bacteria on the Color of Prepackaged Retail Beef Cuts. Food Tech. 7:397-400. Butler, 0. D. 1953b. Some Causes and Measurements of Color Changes in Fresh Retail Meat Cuts. Ph. D. Thesis. Michigan State Univ. Craig, H. B., Blumer, T. N. and Derrick, E. R. 1959. Effedt of Several Combinations of Grass and Grain in the Ration of Beef Steers on the Color Characteristics of Loan and Fat. J. of An. SOie 188241-248e Mackintosh, D. L. and Hall, J. L. 1935. Some Factors Related to Color of Meat. Am. Soc. An. Prod. Proc. 281-286. Millikan, G. A. 1939. Muscle Hemoglobin. Physiol. Reviews. 19: 503-523. Mitchell, H. H. and Hamilton, T. S. 1933. Effect of Long-Continued Exercise Upon the Chemical Composition of the Muscle and Other Tissues of Cattle. J. Agr. Res. 46:917-944. Morgan, V. E. 1936. Studies on Myoglobin I. The Solubility of Myoglobin in Concentrated Ammonium Sulfate Solutions. J. Biol. Chem. 112:557-563. Munsell, A. H. 1916. A Color NOtation. 5th Ed. Baltimore, Md. Nickerson, Dorothy. 1946. Color Measurement and Its Application to the Grading of Agricultural Products. U.S.D.A. Publication.580. Niederneier, R. P., Allen, N. H., Lance, R. D., Rupnow, E. H. and Bray, R. H. 1959. Effect of Feeding Methods on Veal Production and Carcass Quality. I. Rate of Gain, Stomach Capacity, Vitamin A, Iron and Hemoglobin Values. J. of An. Sci. 18:726-731. Poel, W. E. 1949. Effect of Anoxic Anoxia on Myoglobin Concentra- tions in Striated Muscle. Am. J. Physiol. 156:44-51. Rongey, E. H. 1958. Color Stability and Uniformity of Cured Hams. M.S. Thesis. Univ. of Missouri. Saffle, R. L. and Bratzler, L. J. 1959. The Effect of Fatness on Some Processing and Palatability Characteristics of Pork Car- casses. Food Tech. 13:236-239. Schweigert, B. S. 1954. Quantitative Chemical Measurements of Myo- globin. Proc. Seventh An. Reciprocal Meat Conf. 77-80. Schweigert, B. S. 1956. Chemistry of Meat Pigments. Proc. of the Eighth Res. Conf. of A.M.I.F. 61-65. Shenk, J. H., Hall, J. L. and King, H. H. 1934. Spectrophotometric Characteristics of Hemoglobin. I. Beef Blood and Muscle Hemo- globin. J. Bio. Chem. 105:741-752. Snedecor, G. H. 1957. Statistical Methods. 5th Ed. The Iowa State College Press, Ames, Iowa. Theorell, H. 1932. Kristellinisches Myoglobin. Biochem. 272:1-7. Theorell, H. and DeDuve, E. 1947. The Crystalline Human Myoglobin in Heart Muscle and Urine. Arch. Biochem., 12:113-124. Townsend, H. E. 1958a. Effect of Temperature, Storage Conditions and Light on the Color of Pro-packaged Frozen.Meat. Ph.D. Thesis. Michigan State Univ. Townsend, H. E. and Bratzler, L. J. 1958b. Effect of Storage Condi- tions on the Color of Frozen Packaged Retail Beef Cuts. Food Tech. 12:663-666. Voegeli, M. M. 1952. The Measurement of Fresh Beef Muscle Color Changes by Disk Colorimetry. Ph. D. Thesis. Michigan State Univ. Wilson, G. D., Ginger, I. D., Schweigert, B. S. and Aunan, H. J. 1959. A Study of the Variations of Myoglobin Concentration in "Two-Tened" Hams. (In Press) Hinkler, C. A. 1939. Colour of Meat. I. Apparatus for its Measure- ment and Relation Between pH and Colour. Can. J. Research. 17 31-7. -33- APPENDIX A DATA COLLECTED FROM HILL I (Purebred Hampshire Hogs) “MM—4.... NW*rd—.-—oi - - l.. -. . . f'" Myoglobin moglobin' Index - Number ‘Moisture Fat Content Content Renotation Fadigg___ . ($5 1%) (i6./6of’ ~Elsie.) lot 1 H-83-3 71.28 5.86 .816 3.57 7.9 IR 5.5/3.0 9.7 3.31.2 75.29 1.63 .919 3.98 5.6 13.5.5/3.o 7.0 H-87-2 74.85 1.03 .728 3.02 8.6 TR.6.0/2.5 14.2 H-86-8 74.28 2.13 .867 3.68 5.4 13 5.2/2.7 5.7 H-82-4 73.07 ‘ 4.90 .844 8.83 7.5 TR 5.4/2.8 9.0 H-80-7 74.35 2.17 .793 3.38 5.8 IR 5.3/3.0 6.6 H-87-4 72.26 4.70 737 3.20 6.9 13.5.5 2.9 8.7 Mean "73'.“62 """"3.2o ‘T.8 5 "'"""'3.52 6.8 n 5.5 . ""'""'8.7 Lot 2 H-87-6 75.45 1.89 .718 3.17 7.2 YR 5.6/2.8 9.8 H-86-2 75.73 1.09 .760 3.28 5.9 23 5.8/2.5 10.1 H-86-5 75.50 1.82 1.041 4.59 5.0 12.5.8/2.5 9.4 3380.9 72.82 6.18 .611 2.91 6.8 YR.5.6/2.6 9.8 H-8l-4 74.96 2.62 849 3.79 7.0 IR 5.3/3.3 7.5 Mean 74.35 2.75 (7% 3.35 e 5e 2e 503 Lot 3 ‘ H-83-5 75.04 1.47 . .924 3.93 6.7 YR.5.8/2.5 11.1 H-84-3 73.26 4.21 .840 3.73 6.3 YR 5.0/3.0 4.3 H-81-6 72.32 3.78 .788 3.30 7.6'TR 5.9/2.9 11.9 H-85-2 73.64 4.54 .751 3.44 8.5 IR 5.8/2.7 12.6 H-80-6 74.59 1.63 .779 3.28 5.1 YR 5.2/2.7 5.4 H-87-1 .75.10 .93 .872 3.64 8.2 YR.5.7/2.7 11.7 H-86-8 , 74.60 2.21 .826 3.56 8.6 YR 5,453.1 9.3 _ Mean 72.08 2.35 .826 3.55 7.4 .7 e e Lot 4 H-86-4 75.34 .76 .802 3.36 8.0 YR 5.7/2.5 11.8 H-87-9 75.17 .34 1.230 5.02 3.9 73.5.1/3.1 -3.4 H-80-3 71.05 6.08 .658 2.88 6.4 YR.6.0/3.4 10.3 H-80-5 74.85 1.45 .840 3.54 8.4 YR.5.6/2.8 11.2 H-Bl-l 74.84 1.47 .793 3.35 8.4 TR 6.3/2.9 11.8 H-84-5 72.55 4.67 .732 3.21 6.2 12.5.3/3.4 5.6 H-83-4 73.78 3.07 .723 3.12 8.4 YR.6.3/2.9 15.8 [1-82-1 74.45 1.16 .746 3.06 4.7 YR. 5. 3.0 4.1 Mean 73:66 2737 '7616 3733 ‘676‘23'52§5326’ . -39.. APPENDIX A (Contimed) Trial I - Continued —_ 4:111:31 Myoglobina'L ' Myoglobinf Munsell 154 Si ‘oF' Number Moisture Fat Content Content Renotation Fadigg (7'7 (7.) (mg-I3.) (Ia/33 Lot 5 H-83-9 73.83 3.09 .770 3.34 9.7 m 6.0/ .8 14.0 H-83-6 74.44 2.34 .672 2.89 5.8 n 5.3/3.0 6.0 H-85-l 73.48 3.96 .732 3.24 6.3 m 5.2/2.9 6.2 [1-84.6 75.16 1.94 1.130 4.93 5.4 n 5.1/3.3. 4.1 H-80-8 75.26 .44 .597 2.46 8.2 m 5.1/2.7 11.7 H-86-3 73.68 3.24 .676 2.95 7.6 n 5.6/3.0 10.0 H-80-2 74.30 1.60 .849 3.52 7.0 m 5.4/3.0 8.0 H-87-8 73.20 3.63 .658 2.84 6.4 m 5.6/2.3 10.0 Mean 7Z.I§ 2.55 e 160 3.25 7e e e e H-87-3d 74.02 2.57 .672 2.87 7.9 m 5.9/2.5 12.9 0g obin express n mg. g. o resh musc 6 issue globin expressed in mg./g. on a moisture-free, fat-free basis cStandard ' Hue 4.3 Yellow-Red, value 4.9, Chroma 3.6 dUnassigned animal used in computing correlation coefficients -41- APPENDIX B (Continued) Trial II Continued AnimaI Myoglobifia Myoglobinl5 MunseIl Ifidex ofu Number Moisture Fat Content Content Renotation Fading__ (. /0 (me-f3.) Tag-[6.) Lot 4 10.33.10 72.81 2.44 .914 3.69 4.3 YR 5.5/ .6 7.6 1-61-4 71.96 7.26 .513 2.47 9.4 YR 6.5/2.7 - 17.8 1559.4 73.73 2.92 .840 3.60 6.4 YR 6.0/3.2 12.1 X-59-8 73.59 1.85 .980 3.99 3.7 YR 4.9/3.7 1.2 1.39-3 72.47 6.94 .830 4.03 5.7 YR 6.0/3.0 10.1 1561-12 73.52 2.38 .849 3.52 6.9 YR 5.4/3.1 7.7 X-58-4 71.32 6.50 .746 3.44 7.6 YR 5.6/3.0 10.0 x-34-7 71.89 6.68 .924 4.31 5.2 YR 5.5/3.1 6.2 X-66-10 73.77 3.72 .653 2.90 8.8 YR 6.0 3.2 13.6 mean 72.84 4.52 .805I 3.56 6.8 YR 5.7 3.1 . - Lot 5 x-59-2 73.89 2.90 .933 4.02 6.9 YR 5.6/2.5 7.7 x-33-5 74.30 1.95 .704 2.96 8.5 YR 5.7/2.7 12.0 X-61-2 74.01 4.11 1.020 4.66 6.5 YR 5.5/2.8 8.5 x-34-11 71.65 7.43 .653 3.12 6.9 YR 5.6 3.2 8.7 Mean 7"”53.4 “4.10 “""".823 ”3.69 7.2 YR 5.7 2. . 1-58-3d 74.20 .849 3.85 7.4 YR 5.5/3.1 9.2 Euyoglobin expressed in mg.7g. of fresh muscle tissue _bMyoglobin expressed in mg./g. on a moisture-free, fat-free basis 3.75 0Standard - Hue 4.3 Yellow-Red, Value 4.9, Chroma 3.6 dUnassigned animal used in computing correlation coefficients H": Lei. GRLY MICHIGAN STATE UNIVERSITY LI 0 3 1293 3085 164 II 0 ARIES ._4_ "<1 .I amilj-L