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The Histological Effects of Proteolytic Enzyme Addition on Freeze-Dehydrated Chicken Meat1
ANTIGENICITY OF PHOSVITIN McCully, K. A., C-C. Mok and R. H. Common, 1962. Paper electrophoretic characterization of proteins and lipoproteins of hen's egg yolk. Can. J. Biochem. Physiol. 40: 937-952. Mecham, D. K., and H. S. Olcott, 1949. Phosvitin, the principle phosphoprotein of egg yolk. J. Am. Chem. Soc. 71: 3670-3679. Mok, C-C, 1963. Studies on the proteins of hen's egg yolk and serum. Ph.D. Thesis, McGill Uni-
553
versity. Morse, J. H., 1959. Rapid production and detection of insulin-binding antibodies in rabbits and guinea pigs. Proc. Soc. Exptl. Biol. Med. 101: 722-725. Taborsky, G., and C. C. Allende, 1962. A rearrangement in the structure of phosvitin. Biochem. 1: 406-411. Williams, J., 1962. Serum proteins and the livetins of hen's egg yolk. Biochem. J. 83: 346-355.
M. E. SOSEBEE, K. N. MAY AND S. C. SCHMITTLE2 University of Georgia, A thens, Georgia (Received for publication September 30, 1963)
I
N THE past few years, there has been increased interest in enzymatic tenderization of meats. Correlations between morphological and panel observations have been conducted with particular reference to freeze-dehydrated meats, especially beef and pork. Gottschall and Kies (1942) made an early attempt to study some of the modes of proteolytic enzyme action as possible tenderizers of beef. Wang et al. (1954, 1955, 1958) conducted many studies concerning enzyme application techniques, comparison of action of various proteolytic enzymes, and characteristics of tissues of freezedried beef. Weir et al. (1958) made taste panel and histological analysis of meat treated with liquid tenderizers containing papain. It was felt that similar studies should 1
Journal Paper No. 315, College Experiment Station, University of Georgia College of Agriculture Experiment Station, Athens, Georgia. 2 Departmental affiliation of the authors are: M. E. Sosebee, Food Technology (current address, Dept. of Food Technology and Nutrition, University of Florida, Gainesville); K. N. May, Poultry and Food Technology; and S. C. Schmittle, Poultry Disease Research.
be possible with chicken meat. Therefore, the following histological study was undertaken to determine: (a) the effects, if any, of freeze-drying on tissue structure; (b) the nature of enzymatic action on the various constituents of muscular and connective tissue structure; and (c) a comparison of the action of two proteolytic enzymes of widely different origin on various components of meat. EXPERIMENTAL The pectoralis major muscle from 24month-old White Leghorn hens was used in all treatments. With the exception of raw controls, all hens were cooked in boiling water until internal breast muscle temperatures reached 88°C. Muscle samples were then excised, diced into f inch cubes, and, in most cases, freeze-dried. All dried samples were capable of rehydrating to 100% or higher water content in five to ten minutes. Raw, diced, non-freezedried samples served as controls. The proteolytic enzymes used in the study were papain and Rhozyme P-l 1. No attempt was made to control the pH. Application of the enzymes was conducted
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
The Histological Effects of Proteolytic Enzyme Addition on Freeze-Dehydrated Chicken Meat1
554
M. E. SOSEBEE, K. N . M A Y AND S. C. SCHMITTLE
by immersing the diced, freeze-dried cubes in aqueous solutions of the enzyme concentrations. This solution was held at the optimum temperature for the particular enzyme for standard time periods. The temperature was then raised to boiling for ten minutes in order to inactivate the enzyme and completely rehydrate the tissue.
1. 2. 3. 4.
Raw, fresh muscle tissue. Cooked muscle tissue. Freeze-dried, cooked muscle tissue. Freeze-dried, cooked muscle tissue with enzyme treatment: A. Rhozyme P-l 1 - 0 . 0 2 % for 30 minutes at 55°C. B. Papain —0.003% for 5 minutes at 65°C.
A number of photomicrographs were made in order to illustrate the results. RESULTS AND DISCUSSION
The frozen technique of sectioning proved very satisfactory. Since no embedding was necessary, sectioning was quite rapid. Tissues were fragile, however, and tended to break easily. I t is believe t h a t if the tissues had been embedded, the samples would have been less fragile. Wang et al. (1954, 1955, 1958) and Weir
FIG. 1. Raw, untreated skeletal muscle. X400.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
All samples were histologically processed by use of the freezing technique of sectioning, making it unnecessary to fix or embed the tissues prior to cutting. Specimens were frozen to the block carrier by use of short, intermittent blasts of liquid carbon dioxide. An International Cryostat microtome designed specifically forcuttingspecimens in the frozen state was used. Samples were cut ten microns thick, affixed to the slides with egg albumin, and allowed to dry overnight at 52°C. Tissues were then stained with hematoxylin and Van Gieson's stain. Each sample was represented by a number of slides.
The number and type of experiments conducted were as follows:
HISTOLOGY OF CHICKEN MEAT
et al. (1958) used a celloidin embedding method with freeze-dried samples. However, this process was not chosen because processing is extremely slow and great skill is often required. The times required for staining were much shorter than those suggested in past literature. Samples were placed in hematoxylin for one minute, washed, and placed in Van Gieson's stain for 30 seconds. These times gave the desired red stain for collagen and yellow-orange for skeletal muscle. The necessary variation in staining times was probably influenced by the preliminary procedures, including cooking, freeze-drying, or freezing the tissues. A sample of normal, untreated skeletal muscle is illustrated in Figure 1. Freezing and cutting the specimen allowed the muscle fibers to remain intact showing prominence of cross-striations with only a few breaks. The connective tissue (Figure 2) absorbed the dye readily and evenly, giving a pinkish-red color which is typical
of collagen. The tissue consisted of longitudinally striated, unbranched units of fiber. Upon cooking the muscle fibers showed a slight pulling-away of the sarcolemma from the body of the fiber. As a result, a slight granular material appeared in the spaces between. This is typical of cooked muscle (Figure 3). Cooking definitely altered the structure of connective tissue. Merged or fused fibers appeared in many areas. In most of the tissue, there was an unequal distribution of the stain indicating hydrolysis of the collagen as a result of heating (Figure 4). The main difference noticed between freeze-dried and cooked muscle tissues was the brittleness and fragility of the freeze-dried samples during sectioning. Due to the similarity of these two, only one representative picture was made of the two (Figure 3). The tissues treated with papain showed extensive degradation of the collagen (Figure 5). In many cases, the entire body
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
FIG. 2. Raw, untreated connective tissue. X100.
556
M. E. SOSEBEE, K. N. MAY AND S. C. SCHMITTLE
FIG. 4. Cooked, untreated connective tissue showing fusion of fillers and unequal staining due to hydrolysis. X400.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
FIG. 3. Cooked, untreated skeletal muscle showing some granulation and breakage. This figure is also typical of cooked, untreated, freeze-dried muscle. X400.
HISTOLOGY OF C H I C K E N M E A T
557
of the tissue appeared destroyed with only an unorganized network remaining. In other areas, the tissue appeared extensively vacuolated, and, in all cases, samples lost a great deal of the staining capacity. Little change appeared in the muscle fibers with cross-striations remaining prominent. Granulation of the fibers was more extensive than in the cooked or freeze-dried samples (Figure 6). Rhozyme samples were also subject to enzyme attack. Action was prominent on muscle tissue, with a great deal more granulation than in papain treatments (Figure 7). Connective tissue was also somewhat vacuolated and unevenly stained (Figure 8). In this study, it was found that enzyme concentrations used for beef were much too high for chicken. When 10%, 5 % , 1%, and 0 . 1 % enzyme concentrations used by Wang el al. (1954, 1955, 1958) and Weir el al. (1958) were used on chicken samples, the meat was completely broken down
leaving only a stringy mush. A number of factors may have contributed to greater enzyme resistance with beef and pork. First, both beef and pork have greater amounts of connective tissue interspersed throughout the muscle than chicken. T h e connective tissue found in poultry is also thinner and finer than beef. Second, usually the beef was sectioned in larger cubes or steak sizes for drying as compared with the chicken cubes used in this study. With a larger surface area, the enzyme was given more opportunity to attack the meat components. Wang el al. (1958) stated that greater enzyme concentrations arc necessary to produce histological changes than are required to produce significance in relation to tenderness evaluations. The enzyme concentrations used in this study were the same as those used in a separate study to evaluate tenderness by taste panel and shear press measurements, thus, if larger amounts of the enzymes were used, even
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
FIG. 5. Cooked, freeze-dried, papain treated, connective tissue showing extensive vacuolation and loss of staining ability. X400.
558
M. E. SOSEBEE, K. N. MAY AND S. C. SCHMITTLE
FIG. 7. Cooked, freeze-dried, rhozyme treated skeletal muscle showing rather extensive granulation. X400.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
FIG. 6. Cooked, freeze-dried, papain treated skeletal muscle showing some granulation due to treatment. X400.
HISTOLOGY OF CHICKEN MEAT
559
more dramatic changes than those described would be expected.
REFERENCES Gottschall, G. Y., and M. W. Kies, 1942. Digestion of beef by papain. Food Research, 7: 373— 381.
SUMMARY Freeze-drying of cooked pecloralis major muscle did not significantly alter the histological appearance. Treatment of the freeze-dried meat with Rhozyme P-ll (0.02% for 30 minutes at 55°C.) altered the appearance of both skeletal muscle and connective tissue. Papain caused extensive degradation, vacuolation and loss of staining ability of connective tissue and some granulation of muscle fibers. Rhozyme caused granulation of muscle fibers and some vacuolation and loss of staining ability of connective tissue.
Wang, H., C. E. Weir, M. Birkner and B. Ginger, 1958. Studies on enzymatic tenderization of meat. III. Histological and panel analysis of enzyme preparations from three distinct sources. Food Research, 23:423-438.
ACKNOWLEDGMENTS This work was partly supported by the Thomas J. Lipton Foundation and the Georgia Egg Commission.
Weir, C. E., H. Wang, M. Birkner, J. Parsons and B. Ginger, 1958. Studies on enzymatic tenderization of Meat. II. Panel and histological analysis of meat treated with liquid tenderizers containing papain. Food Research, 23: 411-422.
Wang, H., V. Bates, E. Auerbach, D. Doty and H. Kraybill, 1954. Histological and histochemical study of beef dehydration. IV. Characteristics of muscle tissue dehydrated by freeze-drying techniques. Food Research, 19: 543-556. Wang, H., and N. Maynard, 1955. Histological observations on the action of proteolytic enzyme preparations on striated muscle. Anat. Rec. 121: 379.
SEPTEMBER 8-13. FIFTH INTERNATIONAL CONGRESS ON ANIMAL REPRODUCTION AND ARTIFICIAL INSEMINATION, TRENTO, ITALY.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
FIG. 8. Cooked, freeze-dried, rhozyme treated connective tissue showing some vacuolation and uneven staining. X400.
553
versity. Morse, J. H., 1959. Rapid production and detection of insulin-binding antibodies in rabbits and guinea pigs. Proc. Soc. Exptl. Biol. Med. 101: 722-725. Taborsky, G., and C. C. Allende, 1962. A rearrangement in the structure of phosvitin. Biochem. 1: 406-411. Williams, J., 1962. Serum proteins and the livetins of hen's egg yolk. Biochem. J. 83: 346-355.
M. E. SOSEBEE, K. N. MAY AND S. C. SCHMITTLE2 University of Georgia, A thens, Georgia (Received for publication September 30, 1963)
I
N THE past few years, there has been increased interest in enzymatic tenderization of meats. Correlations between morphological and panel observations have been conducted with particular reference to freeze-dehydrated meats, especially beef and pork. Gottschall and Kies (1942) made an early attempt to study some of the modes of proteolytic enzyme action as possible tenderizers of beef. Wang et al. (1954, 1955, 1958) conducted many studies concerning enzyme application techniques, comparison of action of various proteolytic enzymes, and characteristics of tissues of freezedried beef. Weir et al. (1958) made taste panel and histological analysis of meat treated with liquid tenderizers containing papain. It was felt that similar studies should 1
Journal Paper No. 315, College Experiment Station, University of Georgia College of Agriculture Experiment Station, Athens, Georgia. 2 Departmental affiliation of the authors are: M. E. Sosebee, Food Technology (current address, Dept. of Food Technology and Nutrition, University of Florida, Gainesville); K. N. May, Poultry and Food Technology; and S. C. Schmittle, Poultry Disease Research.
be possible with chicken meat. Therefore, the following histological study was undertaken to determine: (a) the effects, if any, of freeze-drying on tissue structure; (b) the nature of enzymatic action on the various constituents of muscular and connective tissue structure; and (c) a comparison of the action of two proteolytic enzymes of widely different origin on various components of meat. EXPERIMENTAL The pectoralis major muscle from 24month-old White Leghorn hens was used in all treatments. With the exception of raw controls, all hens were cooked in boiling water until internal breast muscle temperatures reached 88°C. Muscle samples were then excised, diced into f inch cubes, and, in most cases, freeze-dried. All dried samples were capable of rehydrating to 100% or higher water content in five to ten minutes. Raw, diced, non-freezedried samples served as controls. The proteolytic enzymes used in the study were papain and Rhozyme P-l 1. No attempt was made to control the pH. Application of the enzymes was conducted
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
The Histological Effects of Proteolytic Enzyme Addition on Freeze-Dehydrated Chicken Meat1
554
M. E. SOSEBEE, K. N . M A Y AND S. C. SCHMITTLE
by immersing the diced, freeze-dried cubes in aqueous solutions of the enzyme concentrations. This solution was held at the optimum temperature for the particular enzyme for standard time periods. The temperature was then raised to boiling for ten minutes in order to inactivate the enzyme and completely rehydrate the tissue.
1. 2. 3. 4.
Raw, fresh muscle tissue. Cooked muscle tissue. Freeze-dried, cooked muscle tissue. Freeze-dried, cooked muscle tissue with enzyme treatment: A. Rhozyme P-l 1 - 0 . 0 2 % for 30 minutes at 55°C. B. Papain —0.003% for 5 minutes at 65°C.
A number of photomicrographs were made in order to illustrate the results. RESULTS AND DISCUSSION
The frozen technique of sectioning proved very satisfactory. Since no embedding was necessary, sectioning was quite rapid. Tissues were fragile, however, and tended to break easily. I t is believe t h a t if the tissues had been embedded, the samples would have been less fragile. Wang et al. (1954, 1955, 1958) and Weir
FIG. 1. Raw, untreated skeletal muscle. X400.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
All samples were histologically processed by use of the freezing technique of sectioning, making it unnecessary to fix or embed the tissues prior to cutting. Specimens were frozen to the block carrier by use of short, intermittent blasts of liquid carbon dioxide. An International Cryostat microtome designed specifically forcuttingspecimens in the frozen state was used. Samples were cut ten microns thick, affixed to the slides with egg albumin, and allowed to dry overnight at 52°C. Tissues were then stained with hematoxylin and Van Gieson's stain. Each sample was represented by a number of slides.
The number and type of experiments conducted were as follows:
HISTOLOGY OF CHICKEN MEAT
et al. (1958) used a celloidin embedding method with freeze-dried samples. However, this process was not chosen because processing is extremely slow and great skill is often required. The times required for staining were much shorter than those suggested in past literature. Samples were placed in hematoxylin for one minute, washed, and placed in Van Gieson's stain for 30 seconds. These times gave the desired red stain for collagen and yellow-orange for skeletal muscle. The necessary variation in staining times was probably influenced by the preliminary procedures, including cooking, freeze-drying, or freezing the tissues. A sample of normal, untreated skeletal muscle is illustrated in Figure 1. Freezing and cutting the specimen allowed the muscle fibers to remain intact showing prominence of cross-striations with only a few breaks. The connective tissue (Figure 2) absorbed the dye readily and evenly, giving a pinkish-red color which is typical
of collagen. The tissue consisted of longitudinally striated, unbranched units of fiber. Upon cooking the muscle fibers showed a slight pulling-away of the sarcolemma from the body of the fiber. As a result, a slight granular material appeared in the spaces between. This is typical of cooked muscle (Figure 3). Cooking definitely altered the structure of connective tissue. Merged or fused fibers appeared in many areas. In most of the tissue, there was an unequal distribution of the stain indicating hydrolysis of the collagen as a result of heating (Figure 4). The main difference noticed between freeze-dried and cooked muscle tissues was the brittleness and fragility of the freeze-dried samples during sectioning. Due to the similarity of these two, only one representative picture was made of the two (Figure 3). The tissues treated with papain showed extensive degradation of the collagen (Figure 5). In many cases, the entire body
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
FIG. 2. Raw, untreated connective tissue. X100.
556
M. E. SOSEBEE, K. N. MAY AND S. C. SCHMITTLE
FIG. 4. Cooked, untreated connective tissue showing fusion of fillers and unequal staining due to hydrolysis. X400.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
FIG. 3. Cooked, untreated skeletal muscle showing some granulation and breakage. This figure is also typical of cooked, untreated, freeze-dried muscle. X400.
HISTOLOGY OF C H I C K E N M E A T
557
of the tissue appeared destroyed with only an unorganized network remaining. In other areas, the tissue appeared extensively vacuolated, and, in all cases, samples lost a great deal of the staining capacity. Little change appeared in the muscle fibers with cross-striations remaining prominent. Granulation of the fibers was more extensive than in the cooked or freeze-dried samples (Figure 6). Rhozyme samples were also subject to enzyme attack. Action was prominent on muscle tissue, with a great deal more granulation than in papain treatments (Figure 7). Connective tissue was also somewhat vacuolated and unevenly stained (Figure 8). In this study, it was found that enzyme concentrations used for beef were much too high for chicken. When 10%, 5 % , 1%, and 0 . 1 % enzyme concentrations used by Wang el al. (1954, 1955, 1958) and Weir el al. (1958) were used on chicken samples, the meat was completely broken down
leaving only a stringy mush. A number of factors may have contributed to greater enzyme resistance with beef and pork. First, both beef and pork have greater amounts of connective tissue interspersed throughout the muscle than chicken. T h e connective tissue found in poultry is also thinner and finer than beef. Second, usually the beef was sectioned in larger cubes or steak sizes for drying as compared with the chicken cubes used in this study. With a larger surface area, the enzyme was given more opportunity to attack the meat components. Wang el al. (1958) stated that greater enzyme concentrations arc necessary to produce histological changes than are required to produce significance in relation to tenderness evaluations. The enzyme concentrations used in this study were the same as those used in a separate study to evaluate tenderness by taste panel and shear press measurements, thus, if larger amounts of the enzymes were used, even
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
FIG. 5. Cooked, freeze-dried, papain treated, connective tissue showing extensive vacuolation and loss of staining ability. X400.
558
M. E. SOSEBEE, K. N. MAY AND S. C. SCHMITTLE
FIG. 7. Cooked, freeze-dried, rhozyme treated skeletal muscle showing rather extensive granulation. X400.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
FIG. 6. Cooked, freeze-dried, papain treated skeletal muscle showing some granulation due to treatment. X400.
HISTOLOGY OF CHICKEN MEAT
559
more dramatic changes than those described would be expected.
REFERENCES Gottschall, G. Y., and M. W. Kies, 1942. Digestion of beef by papain. Food Research, 7: 373— 381.
SUMMARY Freeze-drying of cooked pecloralis major muscle did not significantly alter the histological appearance. Treatment of the freeze-dried meat with Rhozyme P-ll (0.02% for 30 minutes at 55°C.) altered the appearance of both skeletal muscle and connective tissue. Papain caused extensive degradation, vacuolation and loss of staining ability of connective tissue and some granulation of muscle fibers. Rhozyme caused granulation of muscle fibers and some vacuolation and loss of staining ability of connective tissue.
Wang, H., C. E. Weir, M. Birkner and B. Ginger, 1958. Studies on enzymatic tenderization of meat. III. Histological and panel analysis of enzyme preparations from three distinct sources. Food Research, 23:423-438.
ACKNOWLEDGMENTS This work was partly supported by the Thomas J. Lipton Foundation and the Georgia Egg Commission.
Weir, C. E., H. Wang, M. Birkner, J. Parsons and B. Ginger, 1958. Studies on enzymatic tenderization of Meat. II. Panel and histological analysis of meat treated with liquid tenderizers containing papain. Food Research, 23: 411-422.
Wang, H., V. Bates, E. Auerbach, D. Doty and H. Kraybill, 1954. Histological and histochemical study of beef dehydration. IV. Characteristics of muscle tissue dehydrated by freeze-drying techniques. Food Research, 19: 543-556. Wang, H., and N. Maynard, 1955. Histological observations on the action of proteolytic enzyme preparations on striated muscle. Anat. Rec. 121: 379.
SEPTEMBER 8-13. FIFTH INTERNATIONAL CONGRESS ON ANIMAL REPRODUCTION AND ARTIFICIAL INSEMINATION, TRENTO, ITALY.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 26, 2015
FIG. 8. Cooked, freeze-dried, rhozyme treated connective tissue showing some vacuolation and uneven staining. X400.