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An efficient method for the isolation of intramuscular collagen
Mea? Science, Vol. 41, No. I. pp. 97-100, 1995 Copyright 0 1995 Elsevia Scicn@ Limited Printed in Great Britain. All rights lxacwal 0309-1740/95 s9.50+0.00
ELSEVIER
0309-1740(94)00067-O
Research Note An Efficient Method for the Isolation of Intramuscular Collagen
Nicholas C. Avery 6r Allen J. Bailey Muscle and Collagen Research Group, Department of Veterinary Medicine, University of Bristol, Langford, Bristol, UK (Received 18 March 1994; in revised form 29 September 1994; accepted 6 October 1994)
ABSTRACT Uitrasonic fragmentation of the myojibrils and subsequent solubiiisation in bufler has been used to isolate intramuscular collagen (IMC) in high yield and purity. The method is superior to previously reported techniques in providing both a high yield of collagen and intact fibres. The material obtained is suitable for both physical and biochemical analysis in attempts to demonstrate its role in determining the tenderness of meat.
INTRODUCTION Tenderness is the single most important parameter for the eating quality of meat. It is generally agreed that meat tenderness depends on a number of biological factors, primarily muscle type, age and sex of the animal. Over the past few years we have developed an explanation for the variation in texture based on the mechanical and thermal properties of the intramuscular collagen (Bailey & Light 1989; Sims & Bailey 1992~). The collagenous tracts in muscle are distinguishable as three separate hierarchies, each possessing a different morphology. The epimysium or outer muscle sheath, the”perimysium binding muscle fibre bundles together and the endomysium which surrounds each individual muscle fibre. In practical terms this means that the major tract affecting texture from the consumer’s viewpoint is the intramuscular perimysium. It is therefore important to be able to determine the properties of perimysial collagen, for example, its 97
98
N. C. Avery, A. J. Bailey
susceptibility to both heat denaturation during cooking and to proteolytic attack by the degradative enzymes involved in conditioning. The susceptibility to damage and consequently its influence on the texture of the meat is dependent upon the properties of the collagen fibres making up the perimysium. These in turn depend on the collagen types present and the nature and extent of intermolecular cross-linking. The amount of perimysial collagen varies across the whole range of bovine muscles from about 0.5 to 10% (Bendall, 1967). However, the perimysial collagen content of the majority of the muscles most commonly sold as fresh meat averages between 2 and 4%. This low collagen content precludes detailed analysis of the properties of the perimysium without isolation from the muscle. It is therefore important to be able to isolate the collagen both in high yield and as intact fibres in order to study the variation in properties of the perimysium between different muscles, and the changes in these properties with age. However, the efficient removal of such a large excess of muscle proteins has posed problems. A variety of methods for the isolation of the connective tissue tracts from myofibrillar proteins of muscle have been reported in the literature, each with varying degrees of success. For example, early studies (Carmichael & Lawrie, 1967; Jackson & Cleary, 1967) used powerful extracting solvents such as urea or sodium hydroxide to remove the myofibrillar proteins, but these are likely to denature or degrade the collagen and prevent further characterisation of the perimysium. For example, O-1 M sodium hydroxide solubilised 20% of the collagen. Fragmentation of the myofibrils was attempted by McCollester (1962), and by McClain (1969) the latter using a high speed blender on frozen meat in the presence of dry ice followed by sieving. Most of these and other reported techniques do not provide details of the collagen yield or purity of the product. Light and Champion (1984) by employing sodium dodecyl sulphate (SDS) achieved a high yield and a pure intramuscular collagen product however the SDS was difficult to remove prior to subsequent studies. Fujii and Murota (1982) used a blender followed by sieving and dissolution of the muscle proteins with buffered KI/Na&03 for three sequential 12 h periods to achieve high yields (90%) and a high collagen content (60%) for intramuscular collagen. Our main requirement was for a rapid method producing a high purity product and capable of simple modification in order to deal with larger amounts of tissue or a greater number of samples. In this brief note we report an efficient method for isolating pure intramuscular collagen with intact collagen fibres suitable for thermal stability analysis by differential scanning calorimetry (Judge & Aberle, 1982), susceptibility studies with degradative enzymes (Stanton & Light, 1987), solubility determinations and cross-link analysis (Sims & Bailey, 1992 a and 6).
MATERIALS
AND METHODS
Materials Samples of Longissimus dorsi muscle, obtained from cattle slaughtered under humane, controlled conditions in the University abattoir, were weighed and dissected free of normally associated fat and epimysium before all treatments.
An eficient methodfor
the isolation of intramuscular collagen
99
Reagents used to prepare the buffer solutions for myofibrillar protein extraction and for hydroxyproline analyses were all purchased from Merck Ltd (Leicestershire, UK). Methods Isolation
of Perimysial
Collagen
Fresh, bovine muscle was initially coarsely cornminuted using a Moulinette S food processor (Moulinex Ltd) to produce an homogeneous mince. Equal aliquots (5 g) of this material were treated: (i) fresh; (ii) after heating at 70°C for 20 min; (iii) after freezing in liquid nitrogen, freeze-drying and powdering in the food processor; (iv) after long-term frozen storage. Ultrasonic Homogenisation:
Each 5 g aliquot (or equivalent) was sonicated using an Ultrasonicator XL (Heat Systems Ltd) initially for 1 min at maximum setting in 50 ml of cold, HasselbachSchneider (1951) buffer containing dithiothreitol. (0.6 M potassium chloride, 0.1 M disodium hydrogen phosphate, 0.01 M sodium pyrophosphate, O+Ol M magnesium chloride and 0.005 M dithiothreitol: pH 7.9). The son&ted muscle was then filtered through a 380 micron copper sieve (Endecotts Ltd, London) and the material retained was added to a further 50 ml of cold Hasselbach-Schneider buffer and re-treated for 30 s. This sieving and re-sonication was repeated again to give a total of 2 m ultrasonication. Finally the material retained on the sieve was dialysed overnight against distilled water prior to freeze-drying, weighing and hydrolysis in constant boiling HCl for 24 h at 115°C. Both the filtrate and the product were analysed for collagen content, as a check for losses during processing, by hydroxyproline assay. This amino acid is present in fibrous collagens at approximately 140 residues per thousand. Consequently a factor of 7.14 times the hydroxyproline content (Etherington & Sims, 1981) is commonly used as an estimate of collagen content. Hydrolysates were assayed for hydroxyproline content using a ChemLab Autoanalyser (Essex, UK) based on the method of Bannister and Burns (1970). This involves the oxidation of all hydroxyproline present in the hydrolysate with chloramine T followed by reaction of the product with dimethylamino- benzaldehyde and heating at 70°C to give a final coloured product which absorbs at 550 nm.
RESULTS
AND DISCUSSION
A number of previously reported methods produced partially denatured or chemically contaminated collagen, whilst others were too slow for processing multiple samples. The homogenisation technique of McLain (1969), in our hands, produced reasonable yields of collagen but of low purity (less than 10%). The use of ultrasonics was attempted in order to fragment the myofibrils and render them more susceptible to dissolution in buffer. Using fresh muscle with an average collagen content of 2% w/w (Longissimus dorsi) IMC preparations of about 70% collagen, with an extraction efficiency of 95% or better were obtained. Similarly freeze-dried, powered, fresh muscle gave a
N. C. Avery, A. J. Bailey
100
TABLE 1 % Collagen and % Yield of Beef Intramuscular Sample
L. dorsi muscle Collagen prep (Fresh muscle) Collagen prep (Freeze-dried muscle) Frozen stored muscle Cooked muscle
Collagen After Ultrasonication
% Collagen of dry weight
% Yield of original collagen
2.1 63.6 73.7 40.0 45-l
924 97.9 80.0 40.6
(Figures represent the average of at least three analyses.)
high yield of purified IMC. The yields of IMC from cooked meat however were poor, and highly variable averaging only approximately 40% yield with a collagen content of only 45% (Table 1). Prolonged frozen storage of the muscle prior to ultrasonication was also found to reduce the purity and yield of the IMC. The ultrasonication procedure when applied to muscle that had been frozen and stored for at least 6 months before processing was capable of producing IMC from 10 samples of 15 g per working day, with a collagen yield of approximately 84% of the collagen originally present and with an average purity of 50% collagen. These latter values although not as high as those obtained for fresh muscle are adequate for crosslink analysis and can be improved by a further ultrasonic treatment. The use of ultrasonics to fragment the myofibrils is by far the most effective method we have found for obtaining intramuscular collagen with a high collagen content, and, equally important, as a high yield of intact fibres.
REFERENCES Bailey, A. J. & Light, N. D. (1989) Connective Tissue in Meat and Meat Products. Elsevier Applied Science, London. Bannister, D. W. & Bums, A. B. (1970). Analyst, 95, 596. Bendall, J. R. (1967). J. Food Sci. Agric., 18, 553. Carmichael, D. J. & Lawrie, R. A. (1967). J. Food Technol., 2, 299. Etherington, D. J. & Sims T. J. (1981). J. Sci. Food Agric., 32, p. 539. Fujii, K. & Murota, K. (1982). Analytical Biochem., 127, 449. Hasselbach, W. & Schneider G. (1951). Biochem. Z., 321,462. Jackson, D. S. & Cleary, E. G. (1967). Methods of Biochemical Analysis, 15, 25. Judge. M. D. & Aberle. E. D. (1982). J. Animal Sci.. 54.68. List,’ N. D. 8c Champion, A. E. (1984). Biochem. Jl, 2i9, 1017. McClain, P. E. (1969). Nature, 221, 181. McCollester, P. L. (1962). Biochim. Biophys. Acta., 57, 427. Sims, T. J. & Bailey, A. J. (199242). Structural Aspects of Meat in Chemistry of Muscle Based Foodr (Eds. D. A. Ledward, D. E. Johnson & M. K. Knight). Royal Sot. Chem., London, pp. 106127. Sims, T. J. & Bailey, A. J. (19923). J. Chromatog., 582, 49. Stanton, C. & Light, N. D. (1987). Meat Sci., 21, 249.
ELSEVIER
0309-1740(94)00067-O
Research Note An Efficient Method for the Isolation of Intramuscular Collagen
Nicholas C. Avery 6r Allen J. Bailey Muscle and Collagen Research Group, Department of Veterinary Medicine, University of Bristol, Langford, Bristol, UK (Received 18 March 1994; in revised form 29 September 1994; accepted 6 October 1994)
ABSTRACT Uitrasonic fragmentation of the myojibrils and subsequent solubiiisation in bufler has been used to isolate intramuscular collagen (IMC) in high yield and purity. The method is superior to previously reported techniques in providing both a high yield of collagen and intact fibres. The material obtained is suitable for both physical and biochemical analysis in attempts to demonstrate its role in determining the tenderness of meat.
INTRODUCTION Tenderness is the single most important parameter for the eating quality of meat. It is generally agreed that meat tenderness depends on a number of biological factors, primarily muscle type, age and sex of the animal. Over the past few years we have developed an explanation for the variation in texture based on the mechanical and thermal properties of the intramuscular collagen (Bailey & Light 1989; Sims & Bailey 1992~). The collagenous tracts in muscle are distinguishable as three separate hierarchies, each possessing a different morphology. The epimysium or outer muscle sheath, the”perimysium binding muscle fibre bundles together and the endomysium which surrounds each individual muscle fibre. In practical terms this means that the major tract affecting texture from the consumer’s viewpoint is the intramuscular perimysium. It is therefore important to be able to determine the properties of perimysial collagen, for example, its 97
98
N. C. Avery, A. J. Bailey
susceptibility to both heat denaturation during cooking and to proteolytic attack by the degradative enzymes involved in conditioning. The susceptibility to damage and consequently its influence on the texture of the meat is dependent upon the properties of the collagen fibres making up the perimysium. These in turn depend on the collagen types present and the nature and extent of intermolecular cross-linking. The amount of perimysial collagen varies across the whole range of bovine muscles from about 0.5 to 10% (Bendall, 1967). However, the perimysial collagen content of the majority of the muscles most commonly sold as fresh meat averages between 2 and 4%. This low collagen content precludes detailed analysis of the properties of the perimysium without isolation from the muscle. It is therefore important to be able to isolate the collagen both in high yield and as intact fibres in order to study the variation in properties of the perimysium between different muscles, and the changes in these properties with age. However, the efficient removal of such a large excess of muscle proteins has posed problems. A variety of methods for the isolation of the connective tissue tracts from myofibrillar proteins of muscle have been reported in the literature, each with varying degrees of success. For example, early studies (Carmichael & Lawrie, 1967; Jackson & Cleary, 1967) used powerful extracting solvents such as urea or sodium hydroxide to remove the myofibrillar proteins, but these are likely to denature or degrade the collagen and prevent further characterisation of the perimysium. For example, O-1 M sodium hydroxide solubilised 20% of the collagen. Fragmentation of the myofibrils was attempted by McCollester (1962), and by McClain (1969) the latter using a high speed blender on frozen meat in the presence of dry ice followed by sieving. Most of these and other reported techniques do not provide details of the collagen yield or purity of the product. Light and Champion (1984) by employing sodium dodecyl sulphate (SDS) achieved a high yield and a pure intramuscular collagen product however the SDS was difficult to remove prior to subsequent studies. Fujii and Murota (1982) used a blender followed by sieving and dissolution of the muscle proteins with buffered KI/Na&03 for three sequential 12 h periods to achieve high yields (90%) and a high collagen content (60%) for intramuscular collagen. Our main requirement was for a rapid method producing a high purity product and capable of simple modification in order to deal with larger amounts of tissue or a greater number of samples. In this brief note we report an efficient method for isolating pure intramuscular collagen with intact collagen fibres suitable for thermal stability analysis by differential scanning calorimetry (Judge & Aberle, 1982), susceptibility studies with degradative enzymes (Stanton & Light, 1987), solubility determinations and cross-link analysis (Sims & Bailey, 1992 a and 6).
MATERIALS
AND METHODS
Materials Samples of Longissimus dorsi muscle, obtained from cattle slaughtered under humane, controlled conditions in the University abattoir, were weighed and dissected free of normally associated fat and epimysium before all treatments.
An eficient methodfor
the isolation of intramuscular collagen
99
Reagents used to prepare the buffer solutions for myofibrillar protein extraction and for hydroxyproline analyses were all purchased from Merck Ltd (Leicestershire, UK). Methods Isolation
of Perimysial
Collagen
Fresh, bovine muscle was initially coarsely cornminuted using a Moulinette S food processor (Moulinex Ltd) to produce an homogeneous mince. Equal aliquots (5 g) of this material were treated: (i) fresh; (ii) after heating at 70°C for 20 min; (iii) after freezing in liquid nitrogen, freeze-drying and powdering in the food processor; (iv) after long-term frozen storage. Ultrasonic Homogenisation:
Each 5 g aliquot (or equivalent) was sonicated using an Ultrasonicator XL (Heat Systems Ltd) initially for 1 min at maximum setting in 50 ml of cold, HasselbachSchneider (1951) buffer containing dithiothreitol. (0.6 M potassium chloride, 0.1 M disodium hydrogen phosphate, 0.01 M sodium pyrophosphate, O+Ol M magnesium chloride and 0.005 M dithiothreitol: pH 7.9). The son&ted muscle was then filtered through a 380 micron copper sieve (Endecotts Ltd, London) and the material retained was added to a further 50 ml of cold Hasselbach-Schneider buffer and re-treated for 30 s. This sieving and re-sonication was repeated again to give a total of 2 m ultrasonication. Finally the material retained on the sieve was dialysed overnight against distilled water prior to freeze-drying, weighing and hydrolysis in constant boiling HCl for 24 h at 115°C. Both the filtrate and the product were analysed for collagen content, as a check for losses during processing, by hydroxyproline assay. This amino acid is present in fibrous collagens at approximately 140 residues per thousand. Consequently a factor of 7.14 times the hydroxyproline content (Etherington & Sims, 1981) is commonly used as an estimate of collagen content. Hydrolysates were assayed for hydroxyproline content using a ChemLab Autoanalyser (Essex, UK) based on the method of Bannister and Burns (1970). This involves the oxidation of all hydroxyproline present in the hydrolysate with chloramine T followed by reaction of the product with dimethylamino- benzaldehyde and heating at 70°C to give a final coloured product which absorbs at 550 nm.
RESULTS
AND DISCUSSION
A number of previously reported methods produced partially denatured or chemically contaminated collagen, whilst others were too slow for processing multiple samples. The homogenisation technique of McLain (1969), in our hands, produced reasonable yields of collagen but of low purity (less than 10%). The use of ultrasonics was attempted in order to fragment the myofibrils and render them more susceptible to dissolution in buffer. Using fresh muscle with an average collagen content of 2% w/w (Longissimus dorsi) IMC preparations of about 70% collagen, with an extraction efficiency of 95% or better were obtained. Similarly freeze-dried, powered, fresh muscle gave a
N. C. Avery, A. J. Bailey
100
TABLE 1 % Collagen and % Yield of Beef Intramuscular Sample
L. dorsi muscle Collagen prep (Fresh muscle) Collagen prep (Freeze-dried muscle) Frozen stored muscle Cooked muscle
Collagen After Ultrasonication
% Collagen of dry weight
% Yield of original collagen
2.1 63.6 73.7 40.0 45-l
924 97.9 80.0 40.6
(Figures represent the average of at least three analyses.)
high yield of purified IMC. The yields of IMC from cooked meat however were poor, and highly variable averaging only approximately 40% yield with a collagen content of only 45% (Table 1). Prolonged frozen storage of the muscle prior to ultrasonication was also found to reduce the purity and yield of the IMC. The ultrasonication procedure when applied to muscle that had been frozen and stored for at least 6 months before processing was capable of producing IMC from 10 samples of 15 g per working day, with a collagen yield of approximately 84% of the collagen originally present and with an average purity of 50% collagen. These latter values although not as high as those obtained for fresh muscle are adequate for crosslink analysis and can be improved by a further ultrasonic treatment. The use of ultrasonics to fragment the myofibrils is by far the most effective method we have found for obtaining intramuscular collagen with a high collagen content, and, equally important, as a high yield of intact fibres.
REFERENCES Bailey, A. J. & Light, N. D. (1989) Connective Tissue in Meat and Meat Products. Elsevier Applied Science, London. Bannister, D. W. & Bums, A. B. (1970). Analyst, 95, 596. Bendall, J. R. (1967). J. Food Sci. Agric., 18, 553. Carmichael, D. J. & Lawrie, R. A. (1967). J. Food Technol., 2, 299. Etherington, D. J. & Sims T. J. (1981). J. Sci. Food Agric., 32, p. 539. Fujii, K. & Murota, K. (1982). Analytical Biochem., 127, 449. Hasselbach, W. & Schneider G. (1951). Biochem. Z., 321,462. Jackson, D. S. & Cleary, E. G. (1967). Methods of Biochemical Analysis, 15, 25. Judge. M. D. & Aberle. E. D. (1982). J. Animal Sci.. 54.68. List,’ N. D. 8c Champion, A. E. (1984). Biochem. Jl, 2i9, 1017. McClain, P. E. (1969). Nature, 221, 181. McCollester, P. L. (1962). Biochim. Biophys. Acta., 57, 427. Sims, T. J. & Bailey, A. J. (199242). Structural Aspects of Meat in Chemistry of Muscle Based Foodr (Eds. D. A. Ledward, D. E. Johnson & M. K. Knight). Royal Sot. Chem., London, pp. 106127. Sims, T. J. & Bailey, A. J. (19923). J. Chromatog., 582, 49. Stanton, C. & Light, N. D. (1987). Meat Sci., 21, 249.