Differences in light scattering between pale and dark beef longissimus thoracis muscles are primarily caused by differences in the myofilament lattice, myofibril and muscle fibre transverse spacings
Introduction
The appearance of meat is largely dependent on the colour of the muscle and is a key attribute of consumer acceptability. Within beef muscle, the red pigments, myoglobin and haemoglobin, are the primary absorbers of certain wavelengths of light and so give rise to red meat colour. In addition, structural elements of the muscle (myofibrillar, cytoskeletal and interaction with other sarcoplasmic proteins) that both absorb and scatter the light (Macdougall, 1970) affect the paleness/darkness of the muscle. Light scattering by diffusion or deflection by particles or interfaces is thought to occur between various structures of the muscle that have different refractive properties, such as changes in the optical density that occur in the gaps between adjacent myofibrils and/or at the A-I interfaces of the sarcomere (Offer et al., 1989), or even the intersection between the I-band and the Z-line, but the exact components responsible for light scattering are yet to be defined.
The structure and light scattering properties of dark and pale meat are very different and are associated with the pH of the muscle. Previously, beef longissimus muscles classified as dark, with a high pH (pH > 5.8), have been shown to have muscle fibres that are swollen, with less light scattering compared to light muscles with a low pH (pH = 5.4) (Hughes, Clarke, Purslow, & Warner, 2017). Dark, high pH meat has a ‘swollen’ structure (larger diameter muscle fibres) due to reduced shrinkage and is accompanied by more transmittance and less light scattering and reflectance (Irving, Swatland, & Millman, 1989; Swatland, 2008). These muscles also typically have a shorter sarcomere length compared to lighter muscles (Hughes et al., 2017; Irving et al., 1989; Warner, Kauffman, & Greaser, 1997), and in pork longissimus have been shown to have a larger spacing between the myofilaments (Irving et al., 1989). In comparison, pale, low pH meat, undergoes greater transverse shrinkage of the lattice, which is accompanied by greater muscle fibre shrinkage and greater fluid loss, classically giving rise to pale, soft, exudative (PSE) meat. These findings have mainly been observed in pork and chicken, and also in beef (Tarrant & Mothersill, 1977), although the opposite condition of dark, firm dry (DFD) meat with a high ultimate pH is more common.
Previously we have shown that reducing the pH environment of a muscle fibre induces fibre shrinkage and is associated with increased global brightness or more light scattering within that fibre (Hughes et al., 2017; Hughes, Clarke, Purslow, & Warner, 2018). However, not all of the changes in light scatter that occur in low pH meat can be reversed by increasing the pH post-rigor, which suggests that some irreversible change such as protein denaturation at low pH may either contribute to changes in myofilament lattice spacing (e.g. by denaturation of myosin heads) or induce further light scattering by precipitation of particles in the sarcoplasmic fluid, or increase scattering from certain structures by decorating them with aggregated protein. In pale pork, the shrinkage of the myofilament lattice has been linked to both myosin head and sarcoplasmic protein precipitation (Liu, Arner, Puolanne, & Ertbjerg, 2016). So, it is likely these events do not occur in dark meat and consequently there is less development of the light scattering. Therefore, we hypothesise that three mechanisms are responsible for decreased light scattering in dark meat, namely (i) a larger lateral separation of thick and thin myofilaments (ii) decreased optical protein density in the I-band and (iii) decreased denaturation of sarcoplasmic proteins.
The aim of this current investigation is to test this hypothesis. Whereas pH differences in previous work were assumed to drive changes in the myofilament lattice spacing, we aim to make a direct correlation between light scattering, colorimetric measurements and myofilament lattice dimensions measured by small angle X-ray diffraction (SAXS). In addition, as the optical protein density of the sarcomere could be impacted by the integrity and location of the sarcoplasmic proteins, the degree of denaturation or aggregation of some sarcoplasmic proteins will be monitored by measuring the activity of two of the most abundant sarcoplasmic proteins enzymes, namely aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Mass spectroscopy is also used to identify proteins or fragments produced by post-mortem proteolysis that are more prominent either in light or dark muscles.
Few visualisation techniques have been employed to identify the specific proteins, components or regions of the cell that are involved in light scattering. More recently, we have used reflectance confocal laser scanning microscopy (rCLSM) as a useful tool to visualise the overall global brightness as a quantitative measure of light scattering intensity (Hughes et al., 2017). By using rCLSM we can obtain measurements of the structural dimensions in the muscle cell, but also the periodic spacing (distance between global brightness peaks) of the scattering elements involved. This technique will be employed to facilitate the identification of these scattering elements and possible sites of increased or decreased optical density contrast in pale versus dark meat samples.
Section snippets
Carcass assessment, sample collection and storage
Beef longissimus thoracis muscles from the left side of the carcass were collected 72 to 96 h post-mortem (PM) from a commercial meat processor. Samples were collected 3 to 4 d PM to maximise the time from slaughter to grading, thus ensuring the final pH had been reached and meat colour compliance was more likely (Hughes, Kearney, & Warner, 2014). Carcass sides were quartered between the 10th and 11th ribs, and the exposed muscle surface colour was developed (bloomed) for 1 h at 10 °C. Muscles
Characterisation of muscle colour groups
Table 1 shows the colorimetric measurements on the three colour groups of muscle samples studied. Muscles that were classified as non-compliant by AUS-MEAT standards, had a dark colour in both orientations and lower lightness in the muscle. The dark colour group had a lower lightness, redness and yellowness value, regardless of measurements made longitudinal or transversely to the muscle fibre axis (P < .001). The confocal micrographs in Fig. 1 show the differences in the intensity of scattered
Discussion
In light muscles, the myofilament lattice had shorter spacings between myofilaments thus promoting an increased A-band protein density, which combined with the integrity of the Z-line, would increase the contrast or refractive index mismatch both along the sarcomere length and between adjacent myofibrils
Muscle lightness had a strong correlation with the transverse lattice spacing between myofilaments, with dark samples of beef longissimus thoracis having a longer distance between myosin-myosin
Conclusion
Increased light scattering in muscle fibres with lower ultimate pH post-rigor has contributions from mechanisms that are both reversible, on reverting the pH to higher values, and from irreversible mechanisms. Myofilament lattice spacing appears to be central not only to lightness but also to drip loss in post-rigor meat. Within the muscle cells of dark meat, the wider lateral spacing of myofilaments, shorter sarcomere lengths, lack of gaps between myofibrils, loss in integrity of the Z-line
Acknowledgements
The authors and CSIRO acknowledges funding provided by Australian Meat Processor Corporation (AMPC) and matching funds provided from the Australian Government, via Meat and Livestock Australia (MLA), to support the research and development detailed in this publication. The support of Griffith University, in particular the Imaging and Image Analysis Facility is also gratefully acknowledged. Peter Purslow acknowledges the support of FONCyT (PRH-PICT 2013-3292).
This research was undertaken on the
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