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Utility of immunohistochemical identification of muscle proteins in microstructural studies of comminuted meat products
Meat Science 27 (1990) 55-60
Utility of Immunohistochemical Identification of Muscle Proteins in Mierostructural Studies of Comminuted Meat Products Monique H. G. Zijderveld & Peter A. Koolmees Department of the Science of Food of Animal Origin, Faculty of Veterinary Medicine, The University of Utrecht, PO Box 80 175, 3508 TD Utrecht, The Netherlands (Received I February 1989; revised version received 3 May 1989: accepted 26 May 1989)
A BSTRA CT A series of experiments was conducted in an attempt to immunohistochemicalh' ident([~v specific musch" proteins #t raw bovine muscle, meat hatters and .fineO" conuninuted meat pro~htcts. Three different antibodies were investigated--monoclonal anti-actin ( IgG ), poO,clonal anti-desmh~ ( IgG ) and polyclonal anti-myoglobin ( IgM ). In addition, the fluorescent compound nitrobenzooxadiazole (NBD)-phallacidin was tested. The utility of the antibody anti-desmin proved to be poor. Anti-myoglobin and NBDphallacidin were useful in muscle tissues that had been technologicall.v treated to a limited extent. Anti-actin reacted with actin present in raw muscle tissue, in muscle samples commhtuted with and without additives and in muscle samples that had been comminuted with additives and subsequently heated to 80"~C and 115 ~C. However, its reaetivi O' was markedly more disthwt #r raw than ht processed samples. The utility oj current immunohistochenfical techniques to study the microstructure of processed meats seems to be limited due to the rapid denaturation of the specific muscle proteins.
INTRODUCTION Microscopy is increasingly applied to investigate the relationship between microstructure and functional properties of comminuted meat products (Lee, 1985; Schmidt et al., 1985; Cassens et al., 1987; Hermansson, 1987). One 55 Meat Science 0309-1740/90/$03"50 ,~ 1990 ElsevierScience Publishers Ltd, England. Printed in Great Britain
56
Monique H. G. Zijdert'eld, Peter A. Koolmees
major drawback in microstructural studies is the absence of conventional histochemical techniques to specifically identify certain muscle proteins. For the identification and localization of these proteins in meat batters and meat products one has to rely mainly on morphological characteristics. Immunohistochemical techniques include the use of antibodies as highly specific reagents for the detection of certain proteins. These reactions are visualized microscopically by the use of marked antibodies. In histopathological and biological research these mono- or polyclonal antibodies are commonly used (DeLellis, 1988). Different methods of antibody-labelling are available, such as conjugation with a fluorescent substance, an electron dense material or with enzymes. Antibody-labelling to an electrondense compound enables identification and localization at the electronmicroscopical level. The colloidal gold technique, in which antibodies are labelled with colloidal gold probes, represents such a high-resolution marking (Beesley, 1987). Conjugation with enzymes involves an enzymehistochemical technique in order to demonstrate the location of the antibody-enzyme complex. By applying these techniques to meat research microscopy, fundamental information about the role of muscle proteins in fat- and waterbinding may be provided. The recent review by Asghar and Bhatti (1987) on post-mortem changes in muscle proteins does not include any specific information about the immunological detection of muscle proteins under commercial storage conditions. In bovine post-mortem muscle, other authors observed changes of desmin and titin, by applying immunological techniques such as Elisa and immunofluorescence on isolated myofibrils. Weber (1984) reported a decrease ofdesmin after 3 and 6 days. Ringkob et al. (1988) described an alteration of the titin localization (the appearance of double bands) after 2 days. The present paper reports a series of experiments in which the utility was tested of some antibodies that bind specifically to the muscle proteins actin, desmin and myoglobin. Simultaneously, the applicability of nitrobenzooxadiazole (NBD)-phallacidin, which specifically binds to actin, was examined.
MATERIALS A N D METHODS Experiments were conducted with five different batches of beef neck muscle (M. trapezius). The first batch consisted of native muscle. Samples were taken 2 days post mortem. The meat used for the other batches was stored at -40°C. Batches two and three contained neck muscle tissue that had been comminuted with and without the addition of water (15%), salt (20) and polyphosphates (0-5%). Batches four and five consisted of comminuted
lmmunohistochemical identification of muscle proteins
57
muscles with added water, salt and polyphosphate, subsequently heated at 80 and 115°C, respectively. With the exception of the first batch, all batches were comminuted in a 30-1itre Alexander bowl chopper. Samples were taken at four time intervals; namely, after 2 min, 5 min, 7 min and 12 min. From two samples of each batch several paraffin and cryostat sections were sliced at 5/am thickness. Four different antibody dilutions were used to determine the optimal antibody concentration. In each of the tests one section served as a positive and one as a negative control. The frozen sections were fixed in acetone at - 2 0 ° C for 10 min after drying in air at room temperature for 5 min. In testing the anti-actin a second fixation method was used---drying in air for 2 h followed by fixation in acetone at room temperature for 10 min. For paraffin sectioning formalin-fixed samples were used. Additionally, samples were fixed in Carnoy's fluid in testing the anti-desmin. The monoclonal anti-actin antibody (IgG class) was developed at the Department of Molecular Cell Biology, Subfaculty of Biology, University of Utrecht. The polyclonal anti-desmin (IgG class) was obtained from Eurodiagnostics (Apeldoorn, The Netherlands). The polyclonal antibody anti-myoglobin (IgM class) was purchased from Australian Monoclonal Development (Artarmon, Australia). All antibodies used reacted with actin, desmin and myoglobin of different animaI species. Anti-actin and antimyoglobin were tested by means of an indirect immunoperoxidase technique. Anti-desmin was applied in an immunoperoxidase-antiperoxidase method (Bourne, 1983). NBD-phallacidin was obtained from molecular probes (Eugene, USA). This compound is a conjunction of phallacidin, which is the most abundant phallatoxin of the American variant of the Amanita phalloides mushroom, and the small fluorescent molecule nitrobenzooxadiazole, NBD-phallacidin, which can only be applied on cryosections, was used according to procedures provided by molecular probes. All sections were evaluated microscopically and photomicrographs of specific features were taken.
RESULTS AND DISCUSSION The results of the reactivity of the different antibodies and NBD-phallacidin with beef neck muscle are listed in Table 1. Anti-actin showed distinct positive reactions in paraffin sections from the unheated muscle samples, although a slight decrease of colour intensity was observed in the sections of samples that had been comminuted more intensively. In the sections from muscle samples comminuted and subsequently heated at 80 and 115°C,
Monique H. G. Zijderveld, Peter A. Koolmees
58
TABLE 1 Reactivity of Antibodies and NBD°phallacidin with Beef Neck Muscle Under Different Technological Conditions
Technological treatment No Comminution Comminution Comminution Comminution treatment + additives + additives + additives + heating + heating
(80°C)
Anti-actin Anti-desmin Anti-myoglobin NBD-phallacidin
P
F
P
+ . +
. + +
+ . -/+
F
P
.
. -/+ +
+ . .
F
P
-
-/+
. .
. .
-/+
F
P
F
-
-/+
-
. .
(11YC)
. .
. -
P = paraffin s e c t i o n s ; F = frozen s e c t i o n s ; + = d i s t i n c t p o s i t i v e r e a c t i o n ; + / p o s i t i v e r e a c t i o n ; - = n e g a t i v e reaction.
= slightly
some reactivity was visible, but the binding of antibodies to actin was less distinct. Actin appeared to be a relatively heat-stable muscle protein in comparison with other muscle proteins. This observation agrees with the findings of Den Hartog et al. (1988). In all frozen sections this antibody failed to bind to actin. The frozen sections were fixed in acetone, a commonly used fixative in immunohistochemistry. Two different ways of acetone fixation were tested, but failed to be successful in improving the antibody binding. In the experiments with anti-desmin, this antibody did not react with desmin in paraffin or with desmin in frozen sections. Fixation in Carnoy's fluid did not improve the antibody-antigen binding. The exclusive appearance of desmin in muscle tissue makes this protein particularly suitable for microstructural studies ofcomminuted meat systems. However, another property of desmin may have induced the negative results in our experiment. Robson and Huiatt (1983) reported that due to its susceptibility to degradation, nearly all desmin in post-mortem meat had disappeared after 1 week ofstorage. Freezing, thawing, comminution and other processes which are commonly applied in the meat industry may easily have accelerated the denaturation of desmin. Thus, even very specific antibodies against bovine desmin would fail to recognize the desmin molecule. The polyclonal antibody anti-myoglobin reacted very specifically with myoglobin in the sections of beef muscle that had been comminuted to a limited extent. No reaction was observed with myoglobin in sections of beef muscle that had been comminuted intensively or comminuted in
lmmunohistochernical identification of muscle proteins
59
combination with additives. Few intact muscle fibre fragments showed a positive reaction. Myoglobin is evenly distributed within aerobic fibres of striated muscle. Despite its solubility, the in-vivo distribution of myoglobin is preserved in post-mortem meat (Swatland, 1984). However, when muscle tissue is comminuted myoglobin molecules are partly released and are probably dissolved easily in the water phase outside the muscle particles. The observed reactivity of the antibodies tested indicates that immunohistochemical examination of fresh and undamaged muscle tissue yielded better results than that of treated muscle samples. This was also observed by Manz (1985) and Den Hartog el al. (1988) in demonstrating specific muscle proteins in heated meats by serological and chemical methods, respectively. Because of the complexity of the immunohistochemical reactions it is not easy to explain the precise cause of failure of antigen recognition by the antibody. Possibly, the antigenic sites of the protein molecules may be destroyed by the fixation method and/or by the technological processes. It is likely that the same problem will occur with antibody labelling to electrondense compounds that are used to identify and localize meat proteins by electron microscopy. The utility of immunohistochemical techniques to study the microstructure of treated muscle samples and processed meat products seems to be limited due to the rapid denaturation of the specific muscle proteins. Proper specimen fixation and appropriate antibody labelling, conditions mentioned by Cassens et al. (1984), are not the only prerequisites to immunohistochemically localize specific muscle proteins in processed meat products. Undesirable cross reactions and false positive reactions are likely to occur in complex mixtures of several denatured animal proteins. So far only the immunohistochemical identification of soya protein in meat products seems to be successful (Groves, 1988). With respect to proteins of animal origin in these products, the development of highly specific antibodies against processed proteins, like, for instance heated actin or myosin, may provide new possibilities. Fluorescent staining with NBD-phallacidin resulted in positive reactions in frozen sections of those muscle samples that had been comminuted, as well as in sections of samples comminuted in the presence of water, salt and polyphosphate. In the latter sections, however, the intensity of the staining was less distinct. Specific staining of actin in the heated samples was unsuccessful, with the exception of the actin in the tunica media of bloodvessels. Another disadvantage of this technique was the rapid loss of fluorescence intensity during microscopy and photography. The NBDphallacidin technique does not appear to be a useful alternative for the innunohistochemical identification and localization of muscle proteins in comminuted meat products.
60
Monique H. G. Z(iderveld, Peter A. Koolmees
A C K N O W L E D G E M ENTS The authors thank Dr W. J. C. Amesz and Miss A. Hey of the D e p a r t m e n t of Molecular Cell Biology, Subfaculty of Biology, University of Utrecht, for providing the anti-actin and for skilful help. This study was supported by the C o m m o d i t y Board for Livestock and Meat at Rijswijk, The Netherlands.
REFERENCES Asghar, A. & Bhatti, A. R. (1987). Adv. Food Res., 31,343. Beesley, J. E. (1987). Int. Analyst, !, 20. Bourne, J. A. (1983). Handbook of Immunoperoxidase Staining Methods. Dako Corp., Santa Barbara, p. 11. Cassens, R. G., Carpenter, C. E. & Eddinger, T. J. (1984). Food Microstructure, 3, 1. Cassens, R. G., Sebranek, J. G. & Loveday, H. D. (1987). Rec. Meat Conf. Proc., 40, 29. DeLellis, R. A., Ed. (1988). Advances in lmmunochemistry. Raven Press, New York. Groves, K. (1988). Immunocytochemistry for food research. Paper presented at the Food Microstructure Conference, University of Reading, UK, 20--22 Sept. Hartog, J. M. P. den, Jonker, M. A., Roon, P. S. van, Haasnoot, W. (1988). Meat Sci., 22, 293. Hermansson, A.-M. (1987). Proe. 33rd International Congress Meat Science & Technology. Helsinki, Finland, p. 290. Lee, C. M. (1985). Food Microstructure, 4, 63. Manz, J. (1985). Fleischwirts., 65, 497. Ringkob, T. P., Marsh, B. B. & Greaser, M. L. (1988). J. Food Sci., 53, 276. Robson, R. M., Huiatt, T. W. (1983). Rec. Meat Conf. Proc., 36, 116. Schmidt, G. R., Acton, J. C. & Ray, F. K. (1985). Rec. Meat Conf. Proc., 38, 44. Swatland, H. J. (1984). Food Microstructure, 3, 9. Weber, A. (1984). Proc. 30th European Meeting Meat Research Workers, Bristol, UK, p. 135.
Utility of Immunohistochemical Identification of Muscle Proteins in Mierostructural Studies of Comminuted Meat Products Monique H. G. Zijderveld & Peter A. Koolmees Department of the Science of Food of Animal Origin, Faculty of Veterinary Medicine, The University of Utrecht, PO Box 80 175, 3508 TD Utrecht, The Netherlands (Received I February 1989; revised version received 3 May 1989: accepted 26 May 1989)
A BSTRA CT A series of experiments was conducted in an attempt to immunohistochemicalh' ident([~v specific musch" proteins #t raw bovine muscle, meat hatters and .fineO" conuninuted meat pro~htcts. Three different antibodies were investigated--monoclonal anti-actin ( IgG ), poO,clonal anti-desmh~ ( IgG ) and polyclonal anti-myoglobin ( IgM ). In addition, the fluorescent compound nitrobenzooxadiazole (NBD)-phallacidin was tested. The utility of the antibody anti-desmin proved to be poor. Anti-myoglobin and NBDphallacidin were useful in muscle tissues that had been technologicall.v treated to a limited extent. Anti-actin reacted with actin present in raw muscle tissue, in muscle samples commhtuted with and without additives and in muscle samples that had been comminuted with additives and subsequently heated to 80"~C and 115 ~C. However, its reaetivi O' was markedly more disthwt #r raw than ht processed samples. The utility oj current immunohistochenfical techniques to study the microstructure of processed meats seems to be limited due to the rapid denaturation of the specific muscle proteins.
INTRODUCTION Microscopy is increasingly applied to investigate the relationship between microstructure and functional properties of comminuted meat products (Lee, 1985; Schmidt et al., 1985; Cassens et al., 1987; Hermansson, 1987). One 55 Meat Science 0309-1740/90/$03"50 ,~ 1990 ElsevierScience Publishers Ltd, England. Printed in Great Britain
56
Monique H. G. Zijdert'eld, Peter A. Koolmees
major drawback in microstructural studies is the absence of conventional histochemical techniques to specifically identify certain muscle proteins. For the identification and localization of these proteins in meat batters and meat products one has to rely mainly on morphological characteristics. Immunohistochemical techniques include the use of antibodies as highly specific reagents for the detection of certain proteins. These reactions are visualized microscopically by the use of marked antibodies. In histopathological and biological research these mono- or polyclonal antibodies are commonly used (DeLellis, 1988). Different methods of antibody-labelling are available, such as conjugation with a fluorescent substance, an electron dense material or with enzymes. Antibody-labelling to an electrondense compound enables identification and localization at the electronmicroscopical level. The colloidal gold technique, in which antibodies are labelled with colloidal gold probes, represents such a high-resolution marking (Beesley, 1987). Conjugation with enzymes involves an enzymehistochemical technique in order to demonstrate the location of the antibody-enzyme complex. By applying these techniques to meat research microscopy, fundamental information about the role of muscle proteins in fat- and waterbinding may be provided. The recent review by Asghar and Bhatti (1987) on post-mortem changes in muscle proteins does not include any specific information about the immunological detection of muscle proteins under commercial storage conditions. In bovine post-mortem muscle, other authors observed changes of desmin and titin, by applying immunological techniques such as Elisa and immunofluorescence on isolated myofibrils. Weber (1984) reported a decrease ofdesmin after 3 and 6 days. Ringkob et al. (1988) described an alteration of the titin localization (the appearance of double bands) after 2 days. The present paper reports a series of experiments in which the utility was tested of some antibodies that bind specifically to the muscle proteins actin, desmin and myoglobin. Simultaneously, the applicability of nitrobenzooxadiazole (NBD)-phallacidin, which specifically binds to actin, was examined.
MATERIALS A N D METHODS Experiments were conducted with five different batches of beef neck muscle (M. trapezius). The first batch consisted of native muscle. Samples were taken 2 days post mortem. The meat used for the other batches was stored at -40°C. Batches two and three contained neck muscle tissue that had been comminuted with and without the addition of water (15%), salt (20) and polyphosphates (0-5%). Batches four and five consisted of comminuted
lmmunohistochemical identification of muscle proteins
57
muscles with added water, salt and polyphosphate, subsequently heated at 80 and 115°C, respectively. With the exception of the first batch, all batches were comminuted in a 30-1itre Alexander bowl chopper. Samples were taken at four time intervals; namely, after 2 min, 5 min, 7 min and 12 min. From two samples of each batch several paraffin and cryostat sections were sliced at 5/am thickness. Four different antibody dilutions were used to determine the optimal antibody concentration. In each of the tests one section served as a positive and one as a negative control. The frozen sections were fixed in acetone at - 2 0 ° C for 10 min after drying in air at room temperature for 5 min. In testing the anti-actin a second fixation method was used---drying in air for 2 h followed by fixation in acetone at room temperature for 10 min. For paraffin sectioning formalin-fixed samples were used. Additionally, samples were fixed in Carnoy's fluid in testing the anti-desmin. The monoclonal anti-actin antibody (IgG class) was developed at the Department of Molecular Cell Biology, Subfaculty of Biology, University of Utrecht. The polyclonal anti-desmin (IgG class) was obtained from Eurodiagnostics (Apeldoorn, The Netherlands). The polyclonal antibody anti-myoglobin (IgM class) was purchased from Australian Monoclonal Development (Artarmon, Australia). All antibodies used reacted with actin, desmin and myoglobin of different animaI species. Anti-actin and antimyoglobin were tested by means of an indirect immunoperoxidase technique. Anti-desmin was applied in an immunoperoxidase-antiperoxidase method (Bourne, 1983). NBD-phallacidin was obtained from molecular probes (Eugene, USA). This compound is a conjunction of phallacidin, which is the most abundant phallatoxin of the American variant of the Amanita phalloides mushroom, and the small fluorescent molecule nitrobenzooxadiazole, NBD-phallacidin, which can only be applied on cryosections, was used according to procedures provided by molecular probes. All sections were evaluated microscopically and photomicrographs of specific features were taken.
RESULTS AND DISCUSSION The results of the reactivity of the different antibodies and NBD-phallacidin with beef neck muscle are listed in Table 1. Anti-actin showed distinct positive reactions in paraffin sections from the unheated muscle samples, although a slight decrease of colour intensity was observed in the sections of samples that had been comminuted more intensively. In the sections from muscle samples comminuted and subsequently heated at 80 and 115°C,
Monique H. G. Zijderveld, Peter A. Koolmees
58
TABLE 1 Reactivity of Antibodies and NBD°phallacidin with Beef Neck Muscle Under Different Technological Conditions
Technological treatment No Comminution Comminution Comminution Comminution treatment + additives + additives + additives + heating + heating
(80°C)
Anti-actin Anti-desmin Anti-myoglobin NBD-phallacidin
P
F
P
+ . +
. + +
+ . -/+
F
P
.
. -/+ +
+ . .
F
P
-
-/+
. .
. .
-/+
F
P
F
-
-/+
-
. .
(11YC)
. .
. -
P = paraffin s e c t i o n s ; F = frozen s e c t i o n s ; + = d i s t i n c t p o s i t i v e r e a c t i o n ; + / p o s i t i v e r e a c t i o n ; - = n e g a t i v e reaction.
= slightly
some reactivity was visible, but the binding of antibodies to actin was less distinct. Actin appeared to be a relatively heat-stable muscle protein in comparison with other muscle proteins. This observation agrees with the findings of Den Hartog et al. (1988). In all frozen sections this antibody failed to bind to actin. The frozen sections were fixed in acetone, a commonly used fixative in immunohistochemistry. Two different ways of acetone fixation were tested, but failed to be successful in improving the antibody binding. In the experiments with anti-desmin, this antibody did not react with desmin in paraffin or with desmin in frozen sections. Fixation in Carnoy's fluid did not improve the antibody-antigen binding. The exclusive appearance of desmin in muscle tissue makes this protein particularly suitable for microstructural studies ofcomminuted meat systems. However, another property of desmin may have induced the negative results in our experiment. Robson and Huiatt (1983) reported that due to its susceptibility to degradation, nearly all desmin in post-mortem meat had disappeared after 1 week ofstorage. Freezing, thawing, comminution and other processes which are commonly applied in the meat industry may easily have accelerated the denaturation of desmin. Thus, even very specific antibodies against bovine desmin would fail to recognize the desmin molecule. The polyclonal antibody anti-myoglobin reacted very specifically with myoglobin in the sections of beef muscle that had been comminuted to a limited extent. No reaction was observed with myoglobin in sections of beef muscle that had been comminuted intensively or comminuted in
lmmunohistochernical identification of muscle proteins
59
combination with additives. Few intact muscle fibre fragments showed a positive reaction. Myoglobin is evenly distributed within aerobic fibres of striated muscle. Despite its solubility, the in-vivo distribution of myoglobin is preserved in post-mortem meat (Swatland, 1984). However, when muscle tissue is comminuted myoglobin molecules are partly released and are probably dissolved easily in the water phase outside the muscle particles. The observed reactivity of the antibodies tested indicates that immunohistochemical examination of fresh and undamaged muscle tissue yielded better results than that of treated muscle samples. This was also observed by Manz (1985) and Den Hartog el al. (1988) in demonstrating specific muscle proteins in heated meats by serological and chemical methods, respectively. Because of the complexity of the immunohistochemical reactions it is not easy to explain the precise cause of failure of antigen recognition by the antibody. Possibly, the antigenic sites of the protein molecules may be destroyed by the fixation method and/or by the technological processes. It is likely that the same problem will occur with antibody labelling to electrondense compounds that are used to identify and localize meat proteins by electron microscopy. The utility of immunohistochemical techniques to study the microstructure of treated muscle samples and processed meat products seems to be limited due to the rapid denaturation of the specific muscle proteins. Proper specimen fixation and appropriate antibody labelling, conditions mentioned by Cassens et al. (1984), are not the only prerequisites to immunohistochemically localize specific muscle proteins in processed meat products. Undesirable cross reactions and false positive reactions are likely to occur in complex mixtures of several denatured animal proteins. So far only the immunohistochemical identification of soya protein in meat products seems to be successful (Groves, 1988). With respect to proteins of animal origin in these products, the development of highly specific antibodies against processed proteins, like, for instance heated actin or myosin, may provide new possibilities. Fluorescent staining with NBD-phallacidin resulted in positive reactions in frozen sections of those muscle samples that had been comminuted, as well as in sections of samples comminuted in the presence of water, salt and polyphosphate. In the latter sections, however, the intensity of the staining was less distinct. Specific staining of actin in the heated samples was unsuccessful, with the exception of the actin in the tunica media of bloodvessels. Another disadvantage of this technique was the rapid loss of fluorescence intensity during microscopy and photography. The NBDphallacidin technique does not appear to be a useful alternative for the innunohistochemical identification and localization of muscle proteins in comminuted meat products.
60
Monique H. G. Z(iderveld, Peter A. Koolmees
A C K N O W L E D G E M ENTS The authors thank Dr W. J. C. Amesz and Miss A. Hey of the D e p a r t m e n t of Molecular Cell Biology, Subfaculty of Biology, University of Utrecht, for providing the anti-actin and for skilful help. This study was supported by the C o m m o d i t y Board for Livestock and Meat at Rijswijk, The Netherlands.
REFERENCES Asghar, A. & Bhatti, A. R. (1987). Adv. Food Res., 31,343. Beesley, J. E. (1987). Int. Analyst, !, 20. Bourne, J. A. (1983). Handbook of Immunoperoxidase Staining Methods. Dako Corp., Santa Barbara, p. 11. Cassens, R. G., Carpenter, C. E. & Eddinger, T. J. (1984). Food Microstructure, 3, 1. Cassens, R. G., Sebranek, J. G. & Loveday, H. D. (1987). Rec. Meat Conf. Proc., 40, 29. DeLellis, R. A., Ed. (1988). Advances in lmmunochemistry. Raven Press, New York. Groves, K. (1988). Immunocytochemistry for food research. Paper presented at the Food Microstructure Conference, University of Reading, UK, 20--22 Sept. Hartog, J. M. P. den, Jonker, M. A., Roon, P. S. van, Haasnoot, W. (1988). Meat Sci., 22, 293. Hermansson, A.-M. (1987). Proe. 33rd International Congress Meat Science & Technology. Helsinki, Finland, p. 290. Lee, C. M. (1985). Food Microstructure, 4, 63. Manz, J. (1985). Fleischwirts., 65, 497. Ringkob, T. P., Marsh, B. B. & Greaser, M. L. (1988). J. Food Sci., 53, 276. Robson, R. M., Huiatt, T. W. (1983). Rec. Meat Conf. Proc., 36, 116. Schmidt, G. R., Acton, J. C. & Ray, F. K. (1985). Rec. Meat Conf. Proc., 38, 44. Swatland, H. J. (1984). Food Microstructure, 3, 9. Weber, A. (1984). Proc. 30th European Meeting Meat Research Workers, Bristol, UK, p. 135.