Ageing and cathepsin inhibition affect the shrinkage of fibre fragments of bovine semitendinosus, biceps femoris and psoas major during heating

Abstract

This study examines the effects of ageing (1, 14 days), cathepsin inhibition (No or Yes) and temperature (25-90 °C) on the shrinkage of fibre fragments from three bovine muscles (semitendinosus, biceps femoris and psoas major) during heating. Shrinkage was quantified using light microscopy images. Muscle fibres (except in psoas major) had greater transverse shrinkage, and less longitudinal shrinkage in aged than in unaged muscles at temperatures ≥60–75 °C. In addition, cathepsin inhibition during heating at ≥65–90 °C caused greater transverse shrinkage in semitendinosus fibres, and reduced longitudinal shrinkage for all muscles. At temperatures ≥75 °C, the longitudinal and transverse shrinkage of the fibres was correlated for all muscles. Ageing of biceps femoris increases volume shrinkage on a fibre level, and hence potentially cooking loss, while cathepsin activity in the semitendinosus reduces volume shrinkage. In conclusion, cathepsin activity and ageing influence the shrinkage that occurs during heating and these factors should be explored further to enable optimisation of thermal meat processing.

Introduction

Understanding meat shrinkage and water loss for different muscle types is important in optimisation of heating and other processing conditions. Shrinkage in meat and the loss of fluid of meat during cooking (cooking loss) are inter- related (Hostetler & Landmann, 1968; Offer & Trinick, 1983). Offer and Trinick (1983) classified water loss during cooking as a consequence of shrinkage of the filament lattice. On the other hand, Hostetler and Landmann (1968) stated that the cooking-induced shrinkage is partly due to the expulsion (dripping cooking loss) and partly due to evaporation (evaporative cooking loss) of water during cooking. However, shrinkage and cooking loss are both a consequence of the process of protein denaturation associated with heating of meat (Offer & Trinick, 1983). Heat-induced shrinkage of meat has been explored on different levels of structural organisation; muscle pieces including steaks, strips, blocks (Barbera & Tassone, 2006; Bendall & Restall, 1983; Bouton, Harris, & Shorthose, 1976; Purslow, Oiseth, Hughes, & Warner, 2016; Vaskoska, Ha, Batool Naqvi, White, & Warner, 2020), muscle bundles (Bendall & Restall, 1983), single dissected fibres (Bendall & Restall, 1983), fibre fragments (Hostetler & Landmann, 1968; Purslow et al., 2016) and myofibrils (Purslow et al., 2016). The key differences in connective tissues between the various levels of structural organization in a muscle are that: perimysium and endomysium are present in muscle pieces and bundles; only endomysium is present in dissected fibres and fibre fragments; while connective tissue is absent in myofibrils.

Fibre fragments have been used as a model system in previous studies to understand meat shrinkage and they can provide useful insights about the contribution of the muscle fibres to the process of shrinkage of a cut of meat (Bendall & Restall, 1983; Hostetler & Landmann, 1968). Heating of single fibres/fibre fragments on a microscope stage allows continuous observation under conditions of carefully controlled temperature and rates of heat transfer. Historically, heating of fibre fragments was initially attempted on flat microscope slides heated with a heating element and cooled with water (Hostetler & Landmann, 1968). The fibre fragments of longissimus thoracis et lumborum were obtained by homogenization in a saline solution and observed whilst the temperature was continuously increased up to 80 °C and by incubation at a constant temperature (37 °C, 45 °C, 53 °C, 61 °C, 69 °C, 77 °C) (Hostetler & Landmann, 1968). Later, Bendall and Restall (1983) isolated single myofibres of psoas major 1 cm in length and heated them in a Na citrate buffer in a well-slide on a microscope stage.

Proteolysis is a biochemical event that largely affects meat tenderness and water- holding capacity. Ageing of raw meat has been largely attributed to proteolysis by calpains (Koohmaraie & Geesink, 2006) and there is evidence that these processes primarily affect titin, nebulin and desmin (Lusby, Ridpath, Parrish, & Robson, 1983; Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995). A less researched aspect of the contribution of proteolysis to meat quality is the potential enzymatic proteolytic activity during cooking, particularly of a class of endogenous enzymes called cathepsins. Cathepsins are enzymes located in the lysosomes of the muscle cell and they are potentially released during ageing (Spanier, McMillin, & Miller, 1990) and cooking (Christensen, Ertbjerg, Aaslyng, & Christensen, 2011) of meat. Research has shown that cathepsins B, L and D retain 43–86% activity in meat at temperatures between 55 °C and 58 °C (Draper & Zeece, 1989;Kurth, 1986 ; Spanier et al., 1990), and 21–27% of cathepsin activity was still remaining at 70 °C (Kurth, 1986; Spanier et al., 1990). Wang et al. (2013) reported no reduction in cathepsin activity during cooking at temperatures lower than 70 °C and found a strong negative correlation between the activity of cathepsin D and the Warner Bratzler-shear force. Christensen et al. (2011) even noted an increase in the cathepsin B + L activity with heating of porcine longissimus thoracis et lumborum and semitendinosus to 63 °C by measuring the release of enzyme in the cooking loss. Enzyme inhibition is a useful approach for identifying the effect of an enzyme in an analysed process, and it has been used in meat science to determine the role of enzymes on tenderness and cooking loss (Hopkins & Thompson, 2001; Purslow et al., 2016). Enzyme activity during cooking of meat is particularly interesting since it is well known that heating leads to changes in the secondary, tertiary and quaternary structure of meat proteins as main constituents of meat. Therefore enzymes, if not denatured themselves, might favour the denatured condition of the proteins in order to act on the primary structure (Christensen et al., 2013; Schwartz & Bird, 1977).

Recently, Purslow et al. (2016) conducted a study of the effect of ageing and protease activity on the shrinkage of fibre fragments as models for the shrinkage of meat. They obtained fibre fragments of unaged and aged semitendinosus by homogenization and heated them in a mannitol buffer with or without protease inhibitor in concave slides on a microscope stage and observed the changes in dimensions using a confocal microscope. This resulted in the preliminary evidence that proteolysis during post- mortem ageing and cooking affects the dynamics of shrinkage of fibre fragments from bovine semitendinosus (Purslow et al., 2016). Therefore, it appears necessary to explore the effect of proteolysis during ageing as well as during cooking by cathepsins, as a relevant class of enzymes, in muscles other than semitendinosus and of distinctly different nature. Employing this approach, in the current study, we tested the effect of ageing period and cathepsin activity on the process of thermally induced shrinkage of fibre fragments from three muscle types. We investigated three muscles that according to previous research differ in their cooking loss (biceps femoris < psoas major < semitendinosus) (Rhee, Wheeler, Shackelford, & Koohmaraie, 2004) and their fibre type (psoas major being predominately type I and semitendinosus and biceps femoris being predominately type IIX (Hunt & Hedrick, 1977; Kirchofer, Calkins, & Gwartney, 2002)). Additionally, psoas major is known to have much greater sarcomere length than semitendinosus and biceps femoris (Herring, Cassens, & Rriskey, 1965).