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Early post-mortem degradation of intramuscular collagen
Meat Science 26 (1989) 115-120
Early Post-mortem Degradation of Intramuscular Collagen* E. W. Mills,:]: S. H. Smith & M. D. Judge Department of Animal Science, Purdue University, West Lafayette, Indiana 47907, USA (Received 15 December 1988; revised version received 15 February 1989; accepted 16 February 1989)
ABSTRACT Post-mortem changes in physical and thermal stabilities of bovine intramuscular connective tissue were studied during the first 24h postmortem. Collagen thermal shrinkage temperature (T~) decreased (P < 0.01) and collagen solubility increased (P < 0.01) during the first 24 h following slaughter with greatest amount of change occurring in the first 8 h post mortem. The dynamic nature of intramuscular connective tissue during the very early post-mortem (VEP) period is compared to the VEP-tenderness relationships proposed by Marsh and others ( Lochner et al., 1980; Marsh et al., 1981, 1987, 1988).
INTRODUCTION The effects of post-mortem aging on intramuscular connective tissue are not well understood. Most studies of post-mortem aging processes have dealt only with the myofibrillar proteins. There is evidence, however, that connective tissue proteins are also affected during rigor onset and postmortem aging. Field et al. (1970) and Judge and Aberle (1982) reported that collagen thermal stability declined and physical characteristics changed * Journal Paper No. 11801. Purdue Agricultural Experimental Station. :~ Present address: Department of Dairy and Animal Science, The Pennsylvania State University, University Park, PA 16802, USA. 115 Meat Science 0309-1740/89/$03"50 © 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain
116
E. W. Mills, S. 11. Smith, M. D. Judge
during the first 24 h after slaughter while Kruggel and Field (1971), Pfeiffer et al. (1972) and Dutson (1974) found increased amounts of collagen subunits in isolated intramuscular connective tissue after 21 days of aging. Most studies ofpost-mortem aging effects on connective tissue have been designed to detect changes occurring over an extended period of time. Degradation of myofibrillar proteins, however, can be quite extensive in the first 24h after slaughter (Wilding et al., 1986; Koohmaraie et al., 1987). Connective tissue degradation may be occurring at this time as well. This study was designed to determine the nature of changes occurring in intramuscular connective tissue during the first 24 h after slaughter. Efforts were focused on the measurement of changes in the thermal and physical stabilities of intramuscular collagen.
EXPERIMENTAL Twelve crossbred beef steers weighing 454 to 618 kg were used to determine the effect of post-mortem aging on muscle collagen solubility and shrinkage temperature. The animals were slaughtered and within 45min post exsanguination the hot carcasses were placed in a cooler ( - 2°C, air velocity 0.6 to 1.0 m/s). Infraspinatus muscle samples (c. 150 g) were excised from the right side of each carcass after 0, 1, 2, 4, 6, 8, 12 and 24h in the cooler. Each excised muscle sample was immediately trimmed of all epimysial connective tissue and heavy fat seams. Samples were then frozen in liquid nitrogen, placed in a tightly sealed polyethylene freezer bag and stored at - 4 0 ° C for later connective tissue isolation. The isolation of intramuscular connective tissue was performed using a modification of a procedure described by McClain (1969). Frozen muscle samples were removed from the freezer and held at room temperature until they reached c. - 3 ° C . (In preliminary trials this temperature was found to give maximum yield of isolated connective tissue.) The muscle (c. 100 g) was placed in a Waring blender with c. 125 g of crushed dry ice and powdered at high speed for 45 s. Any connective tissue which had formed a cottonlike ball in the blender jar was saved. The remaining powdered muscle was passed through a No. 16 sieve (1.18 mm) to recover additional connective tissue. Isolated connective tissue saved from the blender jar and the sieve were combined and stored frozen (-29°C) in sealed polyethylene pouches. Collagen thermal shrinkage temperature (T~) was determined using the procedure described by Judge et al. (1981). Frozen connective tissue samples were allowed to thaw at room temperature for c. 15 rain. Approximately 10 mg of the material was moistened with distilled water, hermetically sealed in an aluminium sample pan and analyzed using a differential scanning
Early post-mortem degradation of intramuscular collagen
117
calorimeter (DuPont model 990). The onset temperature of collagen shrinkage was reported as T~. The collagen solubility was determined using a modification of the procedure described by Hill (1966). A sample of isolated connective tissue was heated for 70min at 77°C in 1/4-strength Ringer's solution. After cooling, the sample was centrifuged (3000 x g for 10min at 5°C) and the supernatant was decanted. The pellet was washed with 1/4-strength Ringer's solution and centrifuged again, with the two supernatants being combined. The supernatant and pellet were hydrolyzed in 6M HC1 for 6 h at 121°C. The hydroxyproline content was determined using the procedure of Bergman & Loxley (1963) and converted to collagen using a factor of 7.25 (Goll et al., 1963). Per cent soluble collagen was reported based on total collagen for isolated connective tissue. The total collagen content was a summation of the values garnered for both the heat soluble and insoluble fractions. Statistical analysis of data was performed using the Least Squares and Maximum Likelihood analysis of variance program of Harvey (1975). When analysis of variance indicated a significant effect, treatment means were compared using the Newman-Keuls test (Anderson & McLean, 1974).
RESULTS AND DISCUSSION The collagen shrinkage temperature (Ts) of isolated connective tissue declined (P < 0-01) during the 24h aging period (Fig. 1). This is consistent with findings of Field et al. (1970) for pork epimysial connective tissue and 70 A
o
o v C
69
• O~
68
eu
0
67
0
66
"~
65
m Ill E
s4
o Fo
63
~
s2
c "1~
61
4
8
12
16
20
24
Time (h) Postmortem
Fig. I.
Thermal shrinkage temperature o f collagen in isolated connective tissue. Error bar shows mean value plus and minus one standard error.
118
E. I4I. Mills, S. H. Smith, M. D. Judge
Judge & Aberle (1982) for bovine intramuscular connective tissue. These researchers reported lower collagen Ts values after 24 h post mortem but did not take samples between slaughter and 24 h post mortem. From Fig. 1, this study shows that the most rapid decline in Ts was observed during the first 6 h post mortem, remaining essentially unchanged thereafter through 24 h. Collagen solubility increased significantly (P < 0.01) during the first 6 h after slaughter then remained unchanged (P < 0.05) through 24 h (Fig. 2). This is in general agreement with the results ofWu et al. (1982) who reported a non significant increase in collagen solubility between 12 and 24h post mortem but did not measure collagen solubility immediately post mortem. In the present study (Fig. 2) there is a slight but non significant increase in the per cent soluble collagen from 12 to 24 h post mortem. Yet, as seen by these data, measuring collagen characteristics after 12 h post mortem tends to miss the majority of the changes (Figs 1 and 2). Judge & Aberle (1982) noted a significant decrease in collagen Ts (4.4°C) after the first 24 h post mortem for beef animals having a mean age of 16 months. This 4-4°C drop in Ts at 24 h is very similar to the decrease of 3.5°C observed at 6 h and the 4.4°C decrease at 24 h aging in the present study. The very early post-mortem (VEP) period appears to be a key time frame in which intramuscular connective tissue undergoes alteration. This observation is indirectly reinforced by the work of Lochner et al. (1980) and Marsh et al. (1981) who determined that the rate of temperature and pH decline in muscle during the first 2-4 h post mortem greatly influenced the potential enhancement of meat tenderness. These workers suggested that an 30
¸
28 26 o Ot I O
o _o
24 22 2O
J~ O
o~
18 16 14 12 10 0
r
,
,
8
12
16
2'0
24
Time (h) Postmortem
Fig. 2. Solubility of collagen in isolated connective tissue. Soluble collagen is expressed as per cent of total collagen for isolated connective tissue; error bar shows mean value plus and minus one standard error.
Earl), post-mortem degradation of intramuscular collagen
119
intermediate rate of chilling (or temperature decline) as well as an intermediate rate of glycolysis, enhanced the eating-quality (e.g. tenderness) of lean beef. They further suggested that this tenderness improvement was a result of an 'as-yet unidentified mechanism' rather than via the prevention of cold shortening. More recently Marsh et aL (1987, 1988) found a quadratic relationship between glycolytic rate and tenderness. Their data suggested that the greatest degree of tenderness was obtained when a 3 h pH of approximately 6-1 (corresponding to an intermediate rate of glycolysis) was reached, and that tenderness was substantially reduced at points on either side of this pH value. Again, these studies indicate that an intermediate rate of VEP glycolysis produces a desirably tender end-product with more consistency. In fact, they proposed the possible use ofa 3 h pH as an industry indicator of eating-quality in the future. No direct comparisons can be made between the experiments of Marsh et al. (1987, 1988) and the present study, as muscle pH and temperature were not monitored in the latter. Yet, the results of the present study shed some light on the tenderness enhancement occurring in the VEP period. The dynamic nature of intramuscular connective tissue during the VEP aging period may offer a partial explanation for the effect of VEP chilling and glycolysis rates on beef tenderness. It would be, therefore, beneficial to explore the effects of varying cooler environments (thus affecting the rates of muscle temperature and/or pH decline) on the thermal and physical characteristics of intramuscular connective tissue during this crucial VEP aging period. REFERENCES Anderson, V. L. & McLean, R. A. (1974). Design of Experiments: A Realistic Approach. Marcel Dekker Inc., New York. Bergman, I. & Loxley, R. (1963). Anal Chem., 35, 1961. Dutson, T. R. (1974). Proc. Meat Industry Res. Conf., p. 99. Field, R. A., Pearson, A. M., Koch, D. E. & Merkel, R. A. (1970). J. FoodSci., 35, 113. Goll, D. E., Bray, R. W. & Hoekstra, W. G. (1963). J. Food Sci., 28, 503. Harvey, W. R. (1975). USDA Agricultural Research Service, originally published as ARS 20-8, 1960. Reprinted with corrections of minor errors as ARS H-4, 1975, 157 pp. Hill, F. (1966). J. Food Sci., 31, 161. Judge, M. D. & Aberle, E. D. (1982). J. Anim. Sci., 54, 68. Judge, M. D., Reeves, E. S. & Aberle, E. D. (1981). J. Anim. Sci., 52, 530. Koohmaraie, M., Seideman, S. C., Schollmeyer, J. E., Dutson, T. R. & Crouse, J. D. (1987). Meat Sci., 19, 187. Kruggel, W. G. & Field, R. A. (1971). J. FoodSci., 36, 1114. Lochner, J. V., Kauffman, R. G. & Marsh, B. B. (1980). Meat Sci., 4, 227.
120
E. W. Mills, S. 14. Smith, M. D. Judge
Marsh, B. B., Lochner, J. V., Takahashi, G. & Kragness, D. D. (1981). Meat Sci., 5, 479. Marsh, B. B., Ringkob, T. P., Russell, R. L., Swartz, D. R. & Pagei, L. A. (1987). Meat Sci., 21, 241. Marsh, B. B., Ringkob, T. P., Russell, R. L., Swartz, D. R. & Pagel, L. A. (1988). Proc. Recip. Meat Conf., 41, 113. McClain, P. E. (1969). Nature, 221, 181. Pfeiffer, N. E., Field, R. A., Varnell, T. R., Kruggel, W. G. & Kaiser, I. I. (1972). J. Food Sei., 37, 897. Wilding, P., Hedges, N. & Lillford, P. J. (1986). Meat Sei., 18, 55. Wu, J. J., Dutson, T. R. & Carpenter, Z. L. (1982). Meat Sci., 7, 161.
Early Post-mortem Degradation of Intramuscular Collagen* E. W. Mills,:]: S. H. Smith & M. D. Judge Department of Animal Science, Purdue University, West Lafayette, Indiana 47907, USA (Received 15 December 1988; revised version received 15 February 1989; accepted 16 February 1989)
ABSTRACT Post-mortem changes in physical and thermal stabilities of bovine intramuscular connective tissue were studied during the first 24h postmortem. Collagen thermal shrinkage temperature (T~) decreased (P < 0.01) and collagen solubility increased (P < 0.01) during the first 24 h following slaughter with greatest amount of change occurring in the first 8 h post mortem. The dynamic nature of intramuscular connective tissue during the very early post-mortem (VEP) period is compared to the VEP-tenderness relationships proposed by Marsh and others ( Lochner et al., 1980; Marsh et al., 1981, 1987, 1988).
INTRODUCTION The effects of post-mortem aging on intramuscular connective tissue are not well understood. Most studies of post-mortem aging processes have dealt only with the myofibrillar proteins. There is evidence, however, that connective tissue proteins are also affected during rigor onset and postmortem aging. Field et al. (1970) and Judge and Aberle (1982) reported that collagen thermal stability declined and physical characteristics changed * Journal Paper No. 11801. Purdue Agricultural Experimental Station. :~ Present address: Department of Dairy and Animal Science, The Pennsylvania State University, University Park, PA 16802, USA. 115 Meat Science 0309-1740/89/$03"50 © 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain
116
E. W. Mills, S. 11. Smith, M. D. Judge
during the first 24 h after slaughter while Kruggel and Field (1971), Pfeiffer et al. (1972) and Dutson (1974) found increased amounts of collagen subunits in isolated intramuscular connective tissue after 21 days of aging. Most studies ofpost-mortem aging effects on connective tissue have been designed to detect changes occurring over an extended period of time. Degradation of myofibrillar proteins, however, can be quite extensive in the first 24h after slaughter (Wilding et al., 1986; Koohmaraie et al., 1987). Connective tissue degradation may be occurring at this time as well. This study was designed to determine the nature of changes occurring in intramuscular connective tissue during the first 24 h after slaughter. Efforts were focused on the measurement of changes in the thermal and physical stabilities of intramuscular collagen.
EXPERIMENTAL Twelve crossbred beef steers weighing 454 to 618 kg were used to determine the effect of post-mortem aging on muscle collagen solubility and shrinkage temperature. The animals were slaughtered and within 45min post exsanguination the hot carcasses were placed in a cooler ( - 2°C, air velocity 0.6 to 1.0 m/s). Infraspinatus muscle samples (c. 150 g) were excised from the right side of each carcass after 0, 1, 2, 4, 6, 8, 12 and 24h in the cooler. Each excised muscle sample was immediately trimmed of all epimysial connective tissue and heavy fat seams. Samples were then frozen in liquid nitrogen, placed in a tightly sealed polyethylene freezer bag and stored at - 4 0 ° C for later connective tissue isolation. The isolation of intramuscular connective tissue was performed using a modification of a procedure described by McClain (1969). Frozen muscle samples were removed from the freezer and held at room temperature until they reached c. - 3 ° C . (In preliminary trials this temperature was found to give maximum yield of isolated connective tissue.) The muscle (c. 100 g) was placed in a Waring blender with c. 125 g of crushed dry ice and powdered at high speed for 45 s. Any connective tissue which had formed a cottonlike ball in the blender jar was saved. The remaining powdered muscle was passed through a No. 16 sieve (1.18 mm) to recover additional connective tissue. Isolated connective tissue saved from the blender jar and the sieve were combined and stored frozen (-29°C) in sealed polyethylene pouches. Collagen thermal shrinkage temperature (T~) was determined using the procedure described by Judge et al. (1981). Frozen connective tissue samples were allowed to thaw at room temperature for c. 15 rain. Approximately 10 mg of the material was moistened with distilled water, hermetically sealed in an aluminium sample pan and analyzed using a differential scanning
Early post-mortem degradation of intramuscular collagen
117
calorimeter (DuPont model 990). The onset temperature of collagen shrinkage was reported as T~. The collagen solubility was determined using a modification of the procedure described by Hill (1966). A sample of isolated connective tissue was heated for 70min at 77°C in 1/4-strength Ringer's solution. After cooling, the sample was centrifuged (3000 x g for 10min at 5°C) and the supernatant was decanted. The pellet was washed with 1/4-strength Ringer's solution and centrifuged again, with the two supernatants being combined. The supernatant and pellet were hydrolyzed in 6M HC1 for 6 h at 121°C. The hydroxyproline content was determined using the procedure of Bergman & Loxley (1963) and converted to collagen using a factor of 7.25 (Goll et al., 1963). Per cent soluble collagen was reported based on total collagen for isolated connective tissue. The total collagen content was a summation of the values garnered for both the heat soluble and insoluble fractions. Statistical analysis of data was performed using the Least Squares and Maximum Likelihood analysis of variance program of Harvey (1975). When analysis of variance indicated a significant effect, treatment means were compared using the Newman-Keuls test (Anderson & McLean, 1974).
RESULTS AND DISCUSSION The collagen shrinkage temperature (Ts) of isolated connective tissue declined (P < 0-01) during the 24h aging period (Fig. 1). This is consistent with findings of Field et al. (1970) for pork epimysial connective tissue and 70 A
o
o v C
69
• O~
68
eu
0
67
0
66
"~
65
m Ill E
s4
o Fo
63
~
s2
c "1~
61
4
8
12
16
20
24
Time (h) Postmortem
Fig. I.
Thermal shrinkage temperature o f collagen in isolated connective tissue. Error bar shows mean value plus and minus one standard error.
118
E. I4I. Mills, S. H. Smith, M. D. Judge
Judge & Aberle (1982) for bovine intramuscular connective tissue. These researchers reported lower collagen Ts values after 24 h post mortem but did not take samples between slaughter and 24 h post mortem. From Fig. 1, this study shows that the most rapid decline in Ts was observed during the first 6 h post mortem, remaining essentially unchanged thereafter through 24 h. Collagen solubility increased significantly (P < 0.01) during the first 6 h after slaughter then remained unchanged (P < 0.05) through 24 h (Fig. 2). This is in general agreement with the results ofWu et al. (1982) who reported a non significant increase in collagen solubility between 12 and 24h post mortem but did not measure collagen solubility immediately post mortem. In the present study (Fig. 2) there is a slight but non significant increase in the per cent soluble collagen from 12 to 24 h post mortem. Yet, as seen by these data, measuring collagen characteristics after 12 h post mortem tends to miss the majority of the changes (Figs 1 and 2). Judge & Aberle (1982) noted a significant decrease in collagen Ts (4.4°C) after the first 24 h post mortem for beef animals having a mean age of 16 months. This 4-4°C drop in Ts at 24 h is very similar to the decrease of 3.5°C observed at 6 h and the 4.4°C decrease at 24 h aging in the present study. The very early post-mortem (VEP) period appears to be a key time frame in which intramuscular connective tissue undergoes alteration. This observation is indirectly reinforced by the work of Lochner et al. (1980) and Marsh et al. (1981) who determined that the rate of temperature and pH decline in muscle during the first 2-4 h post mortem greatly influenced the potential enhancement of meat tenderness. These workers suggested that an 30
¸
28 26 o Ot I O
o _o
24 22 2O
J~ O
o~
18 16 14 12 10 0
r
,
,
8
12
16
2'0
24
Time (h) Postmortem
Fig. 2. Solubility of collagen in isolated connective tissue. Soluble collagen is expressed as per cent of total collagen for isolated connective tissue; error bar shows mean value plus and minus one standard error.
Earl), post-mortem degradation of intramuscular collagen
119
intermediate rate of chilling (or temperature decline) as well as an intermediate rate of glycolysis, enhanced the eating-quality (e.g. tenderness) of lean beef. They further suggested that this tenderness improvement was a result of an 'as-yet unidentified mechanism' rather than via the prevention of cold shortening. More recently Marsh et aL (1987, 1988) found a quadratic relationship between glycolytic rate and tenderness. Their data suggested that the greatest degree of tenderness was obtained when a 3 h pH of approximately 6-1 (corresponding to an intermediate rate of glycolysis) was reached, and that tenderness was substantially reduced at points on either side of this pH value. Again, these studies indicate that an intermediate rate of VEP glycolysis produces a desirably tender end-product with more consistency. In fact, they proposed the possible use ofa 3 h pH as an industry indicator of eating-quality in the future. No direct comparisons can be made between the experiments of Marsh et al. (1987, 1988) and the present study, as muscle pH and temperature were not monitored in the latter. Yet, the results of the present study shed some light on the tenderness enhancement occurring in the VEP period. The dynamic nature of intramuscular connective tissue during the VEP aging period may offer a partial explanation for the effect of VEP chilling and glycolysis rates on beef tenderness. It would be, therefore, beneficial to explore the effects of varying cooler environments (thus affecting the rates of muscle temperature and/or pH decline) on the thermal and physical characteristics of intramuscular connective tissue during this crucial VEP aging period. REFERENCES Anderson, V. L. & McLean, R. A. (1974). Design of Experiments: A Realistic Approach. Marcel Dekker Inc., New York. Bergman, I. & Loxley, R. (1963). Anal Chem., 35, 1961. Dutson, T. R. (1974). Proc. Meat Industry Res. Conf., p. 99. Field, R. A., Pearson, A. M., Koch, D. E. & Merkel, R. A. (1970). J. FoodSci., 35, 113. Goll, D. E., Bray, R. W. & Hoekstra, W. G. (1963). J. Food Sci., 28, 503. Harvey, W. R. (1975). USDA Agricultural Research Service, originally published as ARS 20-8, 1960. Reprinted with corrections of minor errors as ARS H-4, 1975, 157 pp. Hill, F. (1966). J. Food Sci., 31, 161. Judge, M. D. & Aberle, E. D. (1982). J. Anim. Sci., 54, 68. Judge, M. D., Reeves, E. S. & Aberle, E. D. (1981). J. Anim. Sci., 52, 530. Koohmaraie, M., Seideman, S. C., Schollmeyer, J. E., Dutson, T. R. & Crouse, J. D. (1987). Meat Sci., 19, 187. Kruggel, W. G. & Field, R. A. (1971). J. FoodSci., 36, 1114. Lochner, J. V., Kauffman, R. G. & Marsh, B. B. (1980). Meat Sci., 4, 227.
120
E. W. Mills, S. 14. Smith, M. D. Judge
Marsh, B. B., Lochner, J. V., Takahashi, G. & Kragness, D. D. (1981). Meat Sci., 5, 479. Marsh, B. B., Ringkob, T. P., Russell, R. L., Swartz, D. R. & Pagei, L. A. (1987). Meat Sci., 21, 241. Marsh, B. B., Ringkob, T. P., Russell, R. L., Swartz, D. R. & Pagel, L. A. (1988). Proc. Recip. Meat Conf., 41, 113. McClain, P. E. (1969). Nature, 221, 181. Pfeiffer, N. E., Field, R. A., Varnell, T. R., Kruggel, W. G. & Kaiser, I. I. (1972). J. Food Sei., 37, 897. Wilding, P., Hedges, N. & Lillford, P. J. (1986). Meat Sei., 18, 55. Wu, J. J., Dutson, T. R. & Carpenter, Z. L. (1982). Meat Sci., 7, 161.