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Observations on the Microstructure of Veal and on the Detection of Cooked Pork
Con. Ins!. Food Sci. Techno/.J. Vol. 18. No. I, pp. 89-93, 1985
RESEARCH
Observations on the Microstructure of Veal and on the Detection of Cooked Pork H.J. Swatland Oepartment of Animal and Poultry Science, University of Guelph, Guelph, Ontario NIG 2Wl
Abstract
Introduction
The microstructure of raw and cooked meat was examined in frozen sections of samples from 14 veal calves (carcass weight < 150 kg) and eight older bovines (carcass weight ~ 150' kg). None of the veal carcasses had a muscle fiber cross sectional area that exceeded 0.0025 mm 2 (raw semimembranosus, n = 13, and raw pectoralis profundus, n = 9). Histochemical fiber types were categorized on the basis of their myofibrillar adenosine triphosphatase (ATPase) and mitochondrial succinate dehydrogenase (SOH) activity into three fiber types: (I) high ATPase with low SOH, (2) high ATPase with high SOH, and (3) low ATPase with high SOH. Fiber typers were scattered throughout their fasciculi (pectoralis and diaphragm) and there was no exclusive grouping of high SOH fibers to a central location in their fasciculi (as in pork). The mitochondrial distribution indicated by SOH activity in raw veal was mirrored, after cooking, in the distribution of hematoxylin-stained particles within muscle fibers. In a survey of 35 samples of pale-colored meat sold as veal, several instances (n = 5) of the substitution of pork were detected by the central grouping within fasciculi of muscle fibers containing hematoxylin-stained particles derived from mitochondria. The overall mean fiber cross sectional area in these samples with fiber type grouping characteristic of pork samples was 0.00249 mm 2 with a range from 0.00155 ± 0.00054 to 0.00362 ± 0.00122 mm 2 . Thus, fiber diameters alone cannot be used to distinguish pork from veal.
Pork is generally less expensive than veal because of the higher costs associated with calf production. Before cooking, meat from different species may often be identified by its visual appearance, its antigenic properties (Ginsberg, 1948; Hay, 1962; Karpas et al., 1970) and its microstructure. However, it is possible to cook pork in such a way that it, superficially, resembles veal and identification of cooked meat proteins by immunochemistry is notoriously difficult. When cooked pork is falsely presented as veal, this pretence is not only illegal, but also constitutes a hazard to persons with either an allergic or a religious aversion to pork. In an incompletely cooked pork product, there might also be a risk of trichinosis. The cooking of meat produces some profound changes in the microstructure of myofibrillar proteins and connective tissues and, over the years, the topic has been extensively investigated by the histological analysis of paraffin-embedded tissues for light microscopy (Birkner and Auerbach, 1960). Electron microscopy has been used in more recent research (Stanley, 1983). The main focus of attention, however, has generally been on explaining the residual toughness of cooked meat, particularly beef. The present report describes observations that were made on frozen sections of raw and cooked veal. The overall objective was to facilitate the recognition of veal from its microstructure after cooking. The two main criteria chosen for investigation were muscle fiber size (cross sectional area) and the arrangement of histochemical fiber types.
Resume Cette etude se rapporte ala microsructure de viandes crues et cuites al'etat congele representatives de 14 veaux (poids de carcasse, 150 kg) et de 8 bovins plus ages (poids de carcasse ~ 150 kg). Aucune des carcasses de veau n'avait une surface de coupe transversale des fibres musculaires qui exceda 0.0025 mm 2 (semimembranosus cru, n = 13, et pectoralis profundus cru, n = 9). Les types de fibres histochimiques furent categorises sur la base de I'activite de la triphosphatase d'adenosine myofibrillaire (ATPase) et de celie de la dehydrogenase de succinate mitochondriale (SOH) en trois types de fibres: (I) ATPase elevee avec faible SOH. (2) ATPase elevee avec SOH eJevee, et (3) faible ATPase avec SOH elevee. Les types de fibre etaient distribues a travers leurs faisceaux (pectoralis et diaphragme) et il n'y avait aucun groupement exclusif de fibres riches en SDH a un point central dans leur faisceau (comme chez Ie porc). La distribution mitochondriale telle qu'indiquee par I'activite SOH de la viande de veau crue fut refletee, apres cuisson, dans la distribution des particules teintees al'hematoxyline dans les fibres musculaires. Oans une enquete comprenant 35 echantillons de viande pale vendue comme veau, plusieurs cas (n = 5) de substitution de viande de porc furent detectes par Ie gouvernement central a l'interieur des faisceaux de fibres musculaires contenant des particules teintees al'hematoxyline en provenance de mitochondries. La surface moyenne de la coupe transversale des fibres dans ces echantilIons avec Ie groupement fibrillaire caracteristique des echantillons de porc fut 0.00249 mm2 dans une marge de 0.00155 ± 0.00054 a 0.00362 ± 0.00122 mm 2 • En consequence, Ie diametre des fibres seul ne peut pas servir a la distinction entre Ie porc et Ie veau.
Copyright
©
Materials and Methods Muscle samples were obtained at approximately 1 h post mortem (pre-rigor) from 23 bovines ranging in live weight from 59 to 604 kg. Samples were obtained from either the semimembranosus (n = 13), both the pectoralis profundus and diaphragm (n = 9), or the longissimus dorsi (n = 1). The longissimus dorsi sample was used to examine the effect of cooking heat on mitochondrial distribution patterns and it was not included in the morphometric study on fiber diameters. Thirty-five samples of cooked meat were collected
1985 Canadian Institute of Food Science and Technology
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from restaurants. The vendors claimed that the meat was veal and the portions were described as either veal cutlets or by some indiscrimate name such as veal sandwich. Semimembranosus samples were stained with methylene blue (Swatland and Cassens, 1971) and frozen sections were cut at a thickness of about 150 Jlm so that whole muscle fibers could be examined in the depth of the section. Fiber diameters were measured on whole fibers and cross sectional areas were calculated on the assumption that fibers were approximately cylindrical in shape. Veal pectoralis and diaphragm samples, and a sample of pork biceps femoris (approximately 5 x 5 x 10 mm) were tied to plastic splints at their approximate length in the carcass and were frozen in liquid nitrogen. Transverse frozen sections were cut at a thickness of 14 Jlm at - 20°C in a cryostat. Serial sections were picked up on cover slips taken from room temperature and were reacted for myofibrillar adenosine triphosphatase (ATPase) by the method of Guth and Samaha (1970) and for succinate dehydrogenase (SDH) by the method of Nachlas et al. (1957). The stumps of four of the cut tissue blocks (pectoralis) were removed from the microtome chuck and were allowed to thaw in enclosed miniature beakers. Thaw shortening with the immediate production of exudate occurred in all four samples. The blocks were dropped into boiling distilled water for 2 min and then remounted on the chuck to be sectioned longitudinally. After this, they were remounted and sectioned transversely. Cooked samples of pectoralis were stained with hematoxylin as described below. A sample (approximately 250 g) of the longissimus dorsi was frozen (post-rigor) to approximately - 20°C (to simulate commercial storage) and was rapidly thawed in a microwave oven (to simulate maximum commercial tissue damage). The purpose of this part of the study was to determine if mitochondrial distribution patterns survived severe cooking. The replication (5 area blocks x 4 depths in each block) was planned to study within-sample variation rather than between-animal variation. The sample was wrapped in aluminum foil and cooked for 1 h in an electric oven at 175°C until it reached an internal temperature greater than 90°C (to simulate over-cooking). Five tissue blocks (approximately 1 x 1 x 3 cm) were cut from the sample so that the muscle fibers ran the length of each block. The tissue blocks were frozen in liquid nitrogen and transverse sections (20 Jlm) thick were cut with a cryostat at - 20°C. Frozen sections were picked up on microscope slides taken from room temperature. The slides had previously been lightly coated with egg albumin. Sections were dried with warm air (at approximately 65°C) in a slide drier for 5 min. Each block was sectioned at four different depths. Sections were stained with hematoxylin (Gill Formulation, Fisher Scientific, Fair Lawn, NJ) for 30 min. The staining solution contained 0.2070 W IV hematoxylin, 0.02% sodium iodate, 1.8% aluminum sulfate, 2% glacial acetic acid and 25% ethylene glycol. Sections were washed in distilled water, treated with dilute ammo90 / Swatlantl
nium hydroxide (1 drop of ammonium hydroxide in 50 mL water) for 5 min, and then dehydrated in an ascending series of ethanol solutions (70, 90, 100 and 100% for 5 min each). Sections were cleared in xylene for 5 min and mounted with Permount (Fisher Scientific). The same cooking and histological methods were also used for a 250 g sample of pork longissimus dorsi which was also examined at 20 different sites. Similar histological staining techniques were used for the 35 samples of cooked meat obtained from restaurants. Measurements of meat microstructure were made with a MOP-3 digitizer (Carl Zeiss, Oberkochen, W. Germany) connected directly to a light microscope with a camera lucida. A light-emitting diode on the tip of the digitizer pen was visible in the field of view of the microscope.
Results and Discussion Fiber size and microstructure The relationship between carcass weight and muscle fiber cross sectional area is shown in Figure 1. Under the present beef carcass grading system (Canada Gazette, 1983) the cut-off point between veal and beef is set at a hot carcass weight of 150 kg, although most of the carcasses aimed at the premium white veal market in Canada would weigh considerably less than 150 kg. Thus, raw veal might reasonably contain muscle fibers with a mean cross sectional area up to about 0.0025 mm2 • The pectoralis samples shown in Figure 1 were taken from premium white veal carcasses that were typical for southern Ontario (live weight 145.4 ± 14.9, hot carcass weight 103.1 ± 11.8). Studies on paraffin-embedded sections have shown that cooking mayor may not cause a decrease of muscle fiber volume (Birkner and Auerbach, 1960). In the four tissue blocks subjected to thaw shortening and cooking, fiber areas were increased in one sample (by approximately 50%, P < 0.005), were unchanged in another
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200
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Fig. I. Relationship between muscle fiber cross sectional area (mm 2) and carcass weight (kg) in 14 veal carcasses « 150 kg) and 8 beef carcasses (:3>' 150 kg). The two muscles shown are semimembranosus (.&) muscles in a range of carcasses from veal to beef, and pectoralis (e) muscles of white veal carcasses J. /nst. Can. Sci. Technol. Aliment. Vol. 18. No. I. 1985
sample, and were decreased in the remaining two samples (by approximately 25 and 49%, P < 0.005). Morphometric data (Figure 7-10 of Swatland, 1984a) indicate that a fiber diameter of about 0.05 mm and, hence, a fiber cross sectional area of about 0.002 mm 2 may be reached in a pork carcass by an early slaughter age of ISO days. Thus, because of biological variation, an overlap in fiber cross sectional area can be anticipated between heavy veal carcasses and light weight pork carcasses. Although a majority of veal carcasses might have fibers < 0.002 mm2 while a majority of pork carcasses might have fibers > 0.002 mm 2 , the criterion of fiber size by itself cannot be used to separate pork from veal. Older veal carcasses slaughtered as red veal might have fibers as large as those of a small pork carcass, and post mortem treatments such as cold shortening and cooking may act to change fiber areas in either direction. Raw veal muscles examined in thick longitudinal frozen sections conformed to the appearance of typical mammalian muscle, although some juvenile features were observed. Innervation zones in which intramuscular nerves passed perpendicularly across the muscle fibers were numerous and close together relative to adult beef muscle. Tapered intrafascicularly ter-
Fig. 2. Frozen sections of raw veal (diaphragm) reacted for SDH (A) and myofibrillar ATPase (B). Serial sections, both at same magnification. Can. InSf. Food Sci. Technol. J. Vol. 18. No.1. 1985
minating muscle fibers were abundant and conspicuously stained by methylene blue. Within the length of several millimeters these fibers decreased from their regular diameter down to a fine point so that their presence in large numbers would have imposed a negative bias on measurements of muscle fiber cross sectional area made from transverse sections. Tapered endings were anchored in the endomysium of fibers with a normal diameter. Intrafascicularly terminating muscle fibers are thought to be involved in the longitudinal growth of muscle fibers (Swatland, I984a), hence their abundance in the immature muscles of veal calves.
Histochemical fiber types A typical example of raw veal reacted for myofibrillar ATPase and SDH is shown in Figure 2. The three main fiber types of adult bovine muscle (Swatland, 1984a) were already well defined: (1) white fibers with strong ATPase and weak SDH, (2) intermediate fibers
Fig. 3. Frozen sections of pork, both at same magnification. Section A was from raw pork (biceps femoris) and was reacted for SDH. Section B, stained with hematoxylin, was from a sample of cooked meat admitted by the vendor to be pork (muscle unknown). Red and intermediate fibers with a peripheral concentration of mitochondria are grouped centrally within their fasciculi.
Swatland / 91
with strong ATPase and strong SOH, and (3) red fibers with weak ATPase and strong SOH. In both the pectoralis (a white muscle) and the diaphragm (a red muscle) the fiber types were scattered throughout the muscle fasciculi with no evidence of the grouping of red and intermediate fibers in the centers of fasciculi as occurs in pork muscles (Figure 3). Since SOH activity is restricted to mitochondria, the particulate nature of the diformazan reaction product in the SOH reaction showed the distribution of mitochondria. Large particles probably resulted from SOH activity in small clumps of mitochondria. Serial sections of raw muscle reacted for SOH and stained with hematoxylin showed that sarcoplasmic mitochondria were darkly stained with hematoxylin. Thus, although the histochemical reactivity of mitochondria was lost after cooking, the location of mitochondria after cooking could often be determined from the distribution of hematoxylin-stained mitochondria. When this was possible, veal and pork could be separated by the distribution patterns of their histochemical fiber types. The tight grouping of aerobic fibers in the fascicular axis is unique to pork (Swatland, 1984a). In the red and intermediate fibers of raw pork, it was confirmed that
Fig. 4. Frozen sections of cooked veal (pectoralis) stained with hematoxylin. The red and intermediate fibers are scattered within their fasciculi and there is little or no concentration of mitochondria in the periphery of each fiber.
92 / Swatland
mitochondria were more concentrated in the periphery of the fiber than in the axis (Swatland, 1984b; Figure 3A). In veal, this distribution pattern was far less conspicuous and most muscle fibers contained evenly distributed mitochondria (Figure 4A). The peripheral intracellular distribution of mitochondria in pork survived the cooking process (Figure 3B) and provided an additional criterion for the separation of veal from pork after cooking.
Survey of commercial samples All the commercial samples with the scattered fibertype distribution characteristic of bovine muscle (n = 20) also had relatively small fibers. The overall mean fiber cross sectional area in these samples was 0.000903 mm 2. The range in mean values was from 0.000397 ± 0.000188 to 0.001343 ± 0.000834 mm 2 • On microstructural grounds, the possibility of a substitution of pork for veal in these samples could safely be rejected. The microstructure of the remaining samples was more ambiguous. Some (n = 7) of the remaining samples were restructured meat products that contained randomly oriented fragments of muscle fasciculi in a matrix of unidentified material. In some cases the fascicular fragments were only found after long intervals of searching so that the product contained mostly matrix. In products with a high proportion of fascicular fragments, the fragments exhibited a typical bovine fiber type distribution. Although some of these fibers were small enough to be from veal carcasses, others were large enough to have originated from beef carcasses. In both natural and restructured meat, evidence was found for the inclusion or complete substitution of pork for veal. The muscle fibers were larger than those found in the veal carcasses examined in this study, and the distibution of fiber types fitted the pattern that is typical of pork muscle. In pork that was detected by the central grouping of fiber types within fasciculi (n = 5 samples), the overall mean fiber area was 0.00249 mm 2 with a range in mean values from 0.001558 ± 0.000537 to 0.003617 ± 0.00122 mm 2 • Microstructural changes caused by cooking The microstructure of meat in frozen sections was in agreement with earlier research undertaken on paraffin-embedded tissue (Birkner and Auerbach, 1960). The main features were: (I) an increased space between muscle fibers, (2) appearance of a gap between the endomysium and the boundary of the. muscle fiber, (3) expansion and fusion of perimysial collagen fibers to form sheets of homogeneously staining material, and (4) escape of triglyceride and the collapse of adipose cells. The increased space between fibers due to shrinkage often allowed intrafascicularly terminating muscle fibers to be seen clearly in transverse sections. The appearance of several meat components, such as muscle fiber nuclei, arteries, veins and nerves, was virtually unchanged in cooked samples. The preservation of hematoxylin-stained particles in locations occupied by mitochondria in uncooked pre-rigor muscle was dependent on the degree of cooking, as indicated by the amount of microstructural change. J. Ins!. Can. Sci. Technol. Aliment. Vol. 18, No. I, 1985
Although it was shown earlier that fiber types and patterns of mitochondrial distribution may survive even severe heat treatment, it does not automatically follow that these features are completely heat stable. The study of microstructural changes in cooked meat showed fiber types and mitochondrial distribution patterns are more likely to survive if the heat treatment is less severe, but then incompletely cooked pork samples sold as veal are precisely the ones that pose the greatest health hazards.
Acknowledgements Research supported by the Ontario Ministry of Agriculture and Food, and Consumer and Corporate Affairs Canada. The help and advice of Mr. Roy Mason and his colleagues is gratefully acknowledged. References Birkner, M.L. and Auerbach, E. 1960. Microscopic structure of animal tissues. In: The Science of Meat and Meat Products, American Meat Institute Foundation. W.H. Freeman and Company, San Francisco and London. Canada Gazette, 1983. Regulations respecting the grading of beef carcasses. Canada Gazette, Part I, January 8, pp. 288-298.
Can. [nsf. Food Sci. Technol. J. Vol. 18. No. I, 1985
Ginsberg, A. 1948. The differentiation of meats by the precipitation test. Vet. Rec. 60:683. Guth, L. and Samaha, F.J. 1970. Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Neurol. 28:365. Hay, D. 1962. Identification of the origin of animal protein by agargel diffusion. Nature 196:995. Karpas, A.B., Muers, W.L. and Segre, D. 1970. Serologic identification of species of origin of sausage meats. J. Food Sci. 35:150. Nachlas, M.M., Tsou, K-C., de Souza, E., Cheng, CoS. and Selligman, A.M. 1957. Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem. 5:420. Stanley, D. W. 1983. Relation of structure to physical properties of animal material. In: Physical Properties of Foods. M. Peleg and E.B. Bagley (Eds). AVI Publishing Company, Inc., Westport, CT. Swatland, H.J. 1984a. Structure and Development of Meat Animals. Prentice-Hall, Inc., Englewood Cliffs, NJ. Swatland, H.J. 1984b. The radial distribution of succinate dehydrogenase activity in porcine muscle fibres. Histochem. J. 16:321. Swatland, H.J. and Cassens, R.G. 1971. Innervation of porcine and bovine muscle. J. Anim. Sci. 33:750.
Accepted July 18, 1984
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RESEARCH
Observations on the Microstructure of Veal and on the Detection of Cooked Pork H.J. Swatland Oepartment of Animal and Poultry Science, University of Guelph, Guelph, Ontario NIG 2Wl
Abstract
Introduction
The microstructure of raw and cooked meat was examined in frozen sections of samples from 14 veal calves (carcass weight < 150 kg) and eight older bovines (carcass weight ~ 150' kg). None of the veal carcasses had a muscle fiber cross sectional area that exceeded 0.0025 mm 2 (raw semimembranosus, n = 13, and raw pectoralis profundus, n = 9). Histochemical fiber types were categorized on the basis of their myofibrillar adenosine triphosphatase (ATPase) and mitochondrial succinate dehydrogenase (SOH) activity into three fiber types: (I) high ATPase with low SOH, (2) high ATPase with high SOH, and (3) low ATPase with high SOH. Fiber typers were scattered throughout their fasciculi (pectoralis and diaphragm) and there was no exclusive grouping of high SOH fibers to a central location in their fasciculi (as in pork). The mitochondrial distribution indicated by SOH activity in raw veal was mirrored, after cooking, in the distribution of hematoxylin-stained particles within muscle fibers. In a survey of 35 samples of pale-colored meat sold as veal, several instances (n = 5) of the substitution of pork were detected by the central grouping within fasciculi of muscle fibers containing hematoxylin-stained particles derived from mitochondria. The overall mean fiber cross sectional area in these samples with fiber type grouping characteristic of pork samples was 0.00249 mm 2 with a range from 0.00155 ± 0.00054 to 0.00362 ± 0.00122 mm 2 . Thus, fiber diameters alone cannot be used to distinguish pork from veal.
Pork is generally less expensive than veal because of the higher costs associated with calf production. Before cooking, meat from different species may often be identified by its visual appearance, its antigenic properties (Ginsberg, 1948; Hay, 1962; Karpas et al., 1970) and its microstructure. However, it is possible to cook pork in such a way that it, superficially, resembles veal and identification of cooked meat proteins by immunochemistry is notoriously difficult. When cooked pork is falsely presented as veal, this pretence is not only illegal, but also constitutes a hazard to persons with either an allergic or a religious aversion to pork. In an incompletely cooked pork product, there might also be a risk of trichinosis. The cooking of meat produces some profound changes in the microstructure of myofibrillar proteins and connective tissues and, over the years, the topic has been extensively investigated by the histological analysis of paraffin-embedded tissues for light microscopy (Birkner and Auerbach, 1960). Electron microscopy has been used in more recent research (Stanley, 1983). The main focus of attention, however, has generally been on explaining the residual toughness of cooked meat, particularly beef. The present report describes observations that were made on frozen sections of raw and cooked veal. The overall objective was to facilitate the recognition of veal from its microstructure after cooking. The two main criteria chosen for investigation were muscle fiber size (cross sectional area) and the arrangement of histochemical fiber types.
Resume Cette etude se rapporte ala microsructure de viandes crues et cuites al'etat congele representatives de 14 veaux (poids de carcasse, 150 kg) et de 8 bovins plus ages (poids de carcasse ~ 150 kg). Aucune des carcasses de veau n'avait une surface de coupe transversale des fibres musculaires qui exceda 0.0025 mm 2 (semimembranosus cru, n = 13, et pectoralis profundus cru, n = 9). Les types de fibres histochimiques furent categorises sur la base de I'activite de la triphosphatase d'adenosine myofibrillaire (ATPase) et de celie de la dehydrogenase de succinate mitochondriale (SOH) en trois types de fibres: (I) ATPase elevee avec faible SOH. (2) ATPase elevee avec SOH eJevee, et (3) faible ATPase avec SOH elevee. Les types de fibre etaient distribues a travers leurs faisceaux (pectoralis et diaphragme) et il n'y avait aucun groupement exclusif de fibres riches en SDH a un point central dans leur faisceau (comme chez Ie porc). La distribution mitochondriale telle qu'indiquee par I'activite SOH de la viande de veau crue fut refletee, apres cuisson, dans la distribution des particules teintees al'hematoxyline dans les fibres musculaires. Oans une enquete comprenant 35 echantillons de viande pale vendue comme veau, plusieurs cas (n = 5) de substitution de viande de porc furent detectes par Ie gouvernement central a l'interieur des faisceaux de fibres musculaires contenant des particules teintees al'hematoxyline en provenance de mitochondries. La surface moyenne de la coupe transversale des fibres dans ces echantilIons avec Ie groupement fibrillaire caracteristique des echantillons de porc fut 0.00249 mm2 dans une marge de 0.00155 ± 0.00054 a 0.00362 ± 0.00122 mm 2 • En consequence, Ie diametre des fibres seul ne peut pas servir a la distinction entre Ie porc et Ie veau.
Copyright
©
Materials and Methods Muscle samples were obtained at approximately 1 h post mortem (pre-rigor) from 23 bovines ranging in live weight from 59 to 604 kg. Samples were obtained from either the semimembranosus (n = 13), both the pectoralis profundus and diaphragm (n = 9), or the longissimus dorsi (n = 1). The longissimus dorsi sample was used to examine the effect of cooking heat on mitochondrial distribution patterns and it was not included in the morphometric study on fiber diameters. Thirty-five samples of cooked meat were collected
1985 Canadian Institute of Food Science and Technology
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from restaurants. The vendors claimed that the meat was veal and the portions were described as either veal cutlets or by some indiscrimate name such as veal sandwich. Semimembranosus samples were stained with methylene blue (Swatland and Cassens, 1971) and frozen sections were cut at a thickness of about 150 Jlm so that whole muscle fibers could be examined in the depth of the section. Fiber diameters were measured on whole fibers and cross sectional areas were calculated on the assumption that fibers were approximately cylindrical in shape. Veal pectoralis and diaphragm samples, and a sample of pork biceps femoris (approximately 5 x 5 x 10 mm) were tied to plastic splints at their approximate length in the carcass and were frozen in liquid nitrogen. Transverse frozen sections were cut at a thickness of 14 Jlm at - 20°C in a cryostat. Serial sections were picked up on cover slips taken from room temperature and were reacted for myofibrillar adenosine triphosphatase (ATPase) by the method of Guth and Samaha (1970) and for succinate dehydrogenase (SDH) by the method of Nachlas et al. (1957). The stumps of four of the cut tissue blocks (pectoralis) were removed from the microtome chuck and were allowed to thaw in enclosed miniature beakers. Thaw shortening with the immediate production of exudate occurred in all four samples. The blocks were dropped into boiling distilled water for 2 min and then remounted on the chuck to be sectioned longitudinally. After this, they were remounted and sectioned transversely. Cooked samples of pectoralis were stained with hematoxylin as described below. A sample (approximately 250 g) of the longissimus dorsi was frozen (post-rigor) to approximately - 20°C (to simulate commercial storage) and was rapidly thawed in a microwave oven (to simulate maximum commercial tissue damage). The purpose of this part of the study was to determine if mitochondrial distribution patterns survived severe cooking. The replication (5 area blocks x 4 depths in each block) was planned to study within-sample variation rather than between-animal variation. The sample was wrapped in aluminum foil and cooked for 1 h in an electric oven at 175°C until it reached an internal temperature greater than 90°C (to simulate over-cooking). Five tissue blocks (approximately 1 x 1 x 3 cm) were cut from the sample so that the muscle fibers ran the length of each block. The tissue blocks were frozen in liquid nitrogen and transverse sections (20 Jlm) thick were cut with a cryostat at - 20°C. Frozen sections were picked up on microscope slides taken from room temperature. The slides had previously been lightly coated with egg albumin. Sections were dried with warm air (at approximately 65°C) in a slide drier for 5 min. Each block was sectioned at four different depths. Sections were stained with hematoxylin (Gill Formulation, Fisher Scientific, Fair Lawn, NJ) for 30 min. The staining solution contained 0.2070 W IV hematoxylin, 0.02% sodium iodate, 1.8% aluminum sulfate, 2% glacial acetic acid and 25% ethylene glycol. Sections were washed in distilled water, treated with dilute ammo90 / Swatlantl
nium hydroxide (1 drop of ammonium hydroxide in 50 mL water) for 5 min, and then dehydrated in an ascending series of ethanol solutions (70, 90, 100 and 100% for 5 min each). Sections were cleared in xylene for 5 min and mounted with Permount (Fisher Scientific). The same cooking and histological methods were also used for a 250 g sample of pork longissimus dorsi which was also examined at 20 different sites. Similar histological staining techniques were used for the 35 samples of cooked meat obtained from restaurants. Measurements of meat microstructure were made with a MOP-3 digitizer (Carl Zeiss, Oberkochen, W. Germany) connected directly to a light microscope with a camera lucida. A light-emitting diode on the tip of the digitizer pen was visible in the field of view of the microscope.
Results and Discussion Fiber size and microstructure The relationship between carcass weight and muscle fiber cross sectional area is shown in Figure 1. Under the present beef carcass grading system (Canada Gazette, 1983) the cut-off point between veal and beef is set at a hot carcass weight of 150 kg, although most of the carcasses aimed at the premium white veal market in Canada would weigh considerably less than 150 kg. Thus, raw veal might reasonably contain muscle fibers with a mean cross sectional area up to about 0.0025 mm2 • The pectoralis samples shown in Figure 1 were taken from premium white veal carcasses that were typical for southern Ontario (live weight 145.4 ± 14.9, hot carcass weight 103.1 ± 11.8). Studies on paraffin-embedded sections have shown that cooking mayor may not cause a decrease of muscle fiber volume (Birkner and Auerbach, 1960). In the four tissue blocks subjected to thaw shortening and cooking, fiber areas were increased in one sample (by approximately 50%, P < 0.005), were unchanged in another
/
4
.. 3 '0 ~
••••
)(
E2
N
E
•
/
. .:... . 100
kg
200
300
Fig. I. Relationship between muscle fiber cross sectional area (mm 2) and carcass weight (kg) in 14 veal carcasses « 150 kg) and 8 beef carcasses (:3>' 150 kg). The two muscles shown are semimembranosus (.&) muscles in a range of carcasses from veal to beef, and pectoralis (e) muscles of white veal carcasses J. /nst. Can. Sci. Technol. Aliment. Vol. 18. No. I. 1985
sample, and were decreased in the remaining two samples (by approximately 25 and 49%, P < 0.005). Morphometric data (Figure 7-10 of Swatland, 1984a) indicate that a fiber diameter of about 0.05 mm and, hence, a fiber cross sectional area of about 0.002 mm 2 may be reached in a pork carcass by an early slaughter age of ISO days. Thus, because of biological variation, an overlap in fiber cross sectional area can be anticipated between heavy veal carcasses and light weight pork carcasses. Although a majority of veal carcasses might have fibers < 0.002 mm2 while a majority of pork carcasses might have fibers > 0.002 mm 2 , the criterion of fiber size by itself cannot be used to separate pork from veal. Older veal carcasses slaughtered as red veal might have fibers as large as those of a small pork carcass, and post mortem treatments such as cold shortening and cooking may act to change fiber areas in either direction. Raw veal muscles examined in thick longitudinal frozen sections conformed to the appearance of typical mammalian muscle, although some juvenile features were observed. Innervation zones in which intramuscular nerves passed perpendicularly across the muscle fibers were numerous and close together relative to adult beef muscle. Tapered intrafascicularly ter-
Fig. 2. Frozen sections of raw veal (diaphragm) reacted for SDH (A) and myofibrillar ATPase (B). Serial sections, both at same magnification. Can. InSf. Food Sci. Technol. J. Vol. 18. No.1. 1985
minating muscle fibers were abundant and conspicuously stained by methylene blue. Within the length of several millimeters these fibers decreased from their regular diameter down to a fine point so that their presence in large numbers would have imposed a negative bias on measurements of muscle fiber cross sectional area made from transverse sections. Tapered endings were anchored in the endomysium of fibers with a normal diameter. Intrafascicularly terminating muscle fibers are thought to be involved in the longitudinal growth of muscle fibers (Swatland, I984a), hence their abundance in the immature muscles of veal calves.
Histochemical fiber types A typical example of raw veal reacted for myofibrillar ATPase and SDH is shown in Figure 2. The three main fiber types of adult bovine muscle (Swatland, 1984a) were already well defined: (1) white fibers with strong ATPase and weak SDH, (2) intermediate fibers
Fig. 3. Frozen sections of pork, both at same magnification. Section A was from raw pork (biceps femoris) and was reacted for SDH. Section B, stained with hematoxylin, was from a sample of cooked meat admitted by the vendor to be pork (muscle unknown). Red and intermediate fibers with a peripheral concentration of mitochondria are grouped centrally within their fasciculi.
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with strong ATPase and strong SOH, and (3) red fibers with weak ATPase and strong SOH. In both the pectoralis (a white muscle) and the diaphragm (a red muscle) the fiber types were scattered throughout the muscle fasciculi with no evidence of the grouping of red and intermediate fibers in the centers of fasciculi as occurs in pork muscles (Figure 3). Since SOH activity is restricted to mitochondria, the particulate nature of the diformazan reaction product in the SOH reaction showed the distribution of mitochondria. Large particles probably resulted from SOH activity in small clumps of mitochondria. Serial sections of raw muscle reacted for SOH and stained with hematoxylin showed that sarcoplasmic mitochondria were darkly stained with hematoxylin. Thus, although the histochemical reactivity of mitochondria was lost after cooking, the location of mitochondria after cooking could often be determined from the distribution of hematoxylin-stained mitochondria. When this was possible, veal and pork could be separated by the distribution patterns of their histochemical fiber types. The tight grouping of aerobic fibers in the fascicular axis is unique to pork (Swatland, 1984a). In the red and intermediate fibers of raw pork, it was confirmed that
Fig. 4. Frozen sections of cooked veal (pectoralis) stained with hematoxylin. The red and intermediate fibers are scattered within their fasciculi and there is little or no concentration of mitochondria in the periphery of each fiber.
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mitochondria were more concentrated in the periphery of the fiber than in the axis (Swatland, 1984b; Figure 3A). In veal, this distribution pattern was far less conspicuous and most muscle fibers contained evenly distributed mitochondria (Figure 4A). The peripheral intracellular distribution of mitochondria in pork survived the cooking process (Figure 3B) and provided an additional criterion for the separation of veal from pork after cooking.
Survey of commercial samples All the commercial samples with the scattered fibertype distribution characteristic of bovine muscle (n = 20) also had relatively small fibers. The overall mean fiber cross sectional area in these samples was 0.000903 mm 2. The range in mean values was from 0.000397 ± 0.000188 to 0.001343 ± 0.000834 mm 2 • On microstructural grounds, the possibility of a substitution of pork for veal in these samples could safely be rejected. The microstructure of the remaining samples was more ambiguous. Some (n = 7) of the remaining samples were restructured meat products that contained randomly oriented fragments of muscle fasciculi in a matrix of unidentified material. In some cases the fascicular fragments were only found after long intervals of searching so that the product contained mostly matrix. In products with a high proportion of fascicular fragments, the fragments exhibited a typical bovine fiber type distribution. Although some of these fibers were small enough to be from veal carcasses, others were large enough to have originated from beef carcasses. In both natural and restructured meat, evidence was found for the inclusion or complete substitution of pork for veal. The muscle fibers were larger than those found in the veal carcasses examined in this study, and the distibution of fiber types fitted the pattern that is typical of pork muscle. In pork that was detected by the central grouping of fiber types within fasciculi (n = 5 samples), the overall mean fiber area was 0.00249 mm 2 with a range in mean values from 0.001558 ± 0.000537 to 0.003617 ± 0.00122 mm 2 • Microstructural changes caused by cooking The microstructure of meat in frozen sections was in agreement with earlier research undertaken on paraffin-embedded tissue (Birkner and Auerbach, 1960). The main features were: (I) an increased space between muscle fibers, (2) appearance of a gap between the endomysium and the boundary of the. muscle fiber, (3) expansion and fusion of perimysial collagen fibers to form sheets of homogeneously staining material, and (4) escape of triglyceride and the collapse of adipose cells. The increased space between fibers due to shrinkage often allowed intrafascicularly terminating muscle fibers to be seen clearly in transverse sections. The appearance of several meat components, such as muscle fiber nuclei, arteries, veins and nerves, was virtually unchanged in cooked samples. The preservation of hematoxylin-stained particles in locations occupied by mitochondria in uncooked pre-rigor muscle was dependent on the degree of cooking, as indicated by the amount of microstructural change. J. Ins!. Can. Sci. Technol. Aliment. Vol. 18, No. I, 1985
Although it was shown earlier that fiber types and patterns of mitochondrial distribution may survive even severe heat treatment, it does not automatically follow that these features are completely heat stable. The study of microstructural changes in cooked meat showed fiber types and mitochondrial distribution patterns are more likely to survive if the heat treatment is less severe, but then incompletely cooked pork samples sold as veal are precisely the ones that pose the greatest health hazards.
Acknowledgements Research supported by the Ontario Ministry of Agriculture and Food, and Consumer and Corporate Affairs Canada. The help and advice of Mr. Roy Mason and his colleagues is gratefully acknowledged. References Birkner, M.L. and Auerbach, E. 1960. Microscopic structure of animal tissues. In: The Science of Meat and Meat Products, American Meat Institute Foundation. W.H. Freeman and Company, San Francisco and London. Canada Gazette, 1983. Regulations respecting the grading of beef carcasses. Canada Gazette, Part I, January 8, pp. 288-298.
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Ginsberg, A. 1948. The differentiation of meats by the precipitation test. Vet. Rec. 60:683. Guth, L. and Samaha, F.J. 1970. Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Neurol. 28:365. Hay, D. 1962. Identification of the origin of animal protein by agargel diffusion. Nature 196:995. Karpas, A.B., Muers, W.L. and Segre, D. 1970. Serologic identification of species of origin of sausage meats. J. Food Sci. 35:150. Nachlas, M.M., Tsou, K-C., de Souza, E., Cheng, CoS. and Selligman, A.M. 1957. Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem. 5:420. Stanley, D. W. 1983. Relation of structure to physical properties of animal material. In: Physical Properties of Foods. M. Peleg and E.B. Bagley (Eds). AVI Publishing Company, Inc., Westport, CT. Swatland, H.J. 1984a. Structure and Development of Meat Animals. Prentice-Hall, Inc., Englewood Cliffs, NJ. Swatland, H.J. 1984b. The radial distribution of succinate dehydrogenase activity in porcine muscle fibres. Histochem. J. 16:321. Swatland, H.J. and Cassens, R.G. 1971. Innervation of porcine and bovine muscle. J. Anim. Sci. 33:750.
Accepted July 18, 1984
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