The Mechanism of Colour Development in Beef Muscle- The Importance of Scattering within the Muscle Structure

Meat colour is important for consumer acceptability, with excessively dark, high pH often associated with consumer rejection and this can negatively impact the carcass value. Meat colour is determined chromatically by the pigment and achromatically by the microstructure of the meat. The chromatic attributes have greatest influence on the chroma and hue (including redness and yellowness) parameters and have been extensively researched, whereas the achromatic attributes are primarily related to the lightness, brightness or darkness of the muscle, rather than the hue and have had less research attention. Achromatic attributes are primarily determined by the quantity of light scattering in the muscle. This is the process in which light is diffused or deflected by collisions with particles of the medium (meat microstructure) that it transverses. The meat microstructure is any element of the muscle which would cause deflection of light. Dark, high pH muscles show tendency for lower levels of scattering, whereas light, low pH muscles have a tendency for more light scattering. To date, the specific individual components of the meat microstructure that cause these variations in light scattering are still unknown. Consequently, this thesis aims to identify the individual components of the meat microstructure that cause differences in achromaticity. This entails a literature review of the components of the microstructure which could be involved (chapter 1) and investigations at various length scales of the different structural components in the muscle. Firstly, at the macro level, the visual appearance of the muscle as observed by an AUS-MEAT qualified grader was assessed (chapter 3). Secondly, the dimensions of the muscle fibres and periodical light scattering elements within each fibre (chapter 5, 6 and 7) along with the myofilament lattice and myofibrils which compose these fibres (chapter 7) were assessed. Lastly, the sarcoplasm and extracellular fluid that surrounds these components (chapter 4 and 7) were also assessed. The hypothesis was that each of these components would contribute to light scattering in the muscle, with dark, high pH having lower levels of light scattering compared to the light, low pH muscles. In addition, in the thesis, a new microscopy imaging method for visualising and quantifying levels of light scattering in muscle was developed (chapter 2 and 5). This allowed for the determination of the effect of early post-mortem factors (pH, rigor temperature, stretching) on the light scattering properties of the meat. Using reflectance confocal laser scanning microscopy (RCLSM) the reflected light from either whole muscle or isolated muscle fibres was developed. The images captured were from the centre of the muscle fibres, rather than at the surface, and so were considered to be representative of the light scattered within the muscle fibre itself. After image capture, the mean intensity of the pixels on the image was assigned as the “global brightness” of the muscle fibre and used as an indicator of light scattering throughout the thesis. The periodical frequency of these pixels both in parallel (longitudinal) or perpendicular (transverse) to the fibre axis was also determined, and considered to be either the longitudinal periodicity or the transverse periodicity, respectively, of the light scattering pattern. These are discussed in detail in chapter 5, but also used in chapters 6 and 7. In a series of experiments, the structural and biochemical differences between light and dark muscles were investigated. Pale muscles with a higher colorimetric lightness (L*), more drip loss and low pH had muscle fibres with higher global brightness and a smaller diameter, compared to dark muscles which had a lower colorimetric lightness, lower drip and higher pH, with muscle fibres showing lower global brightness values and a larger diameter. Thus, dark muscle fibres had a swollen appearance which reduced the light scattering compared to light muscle fibres that had undergone more shrinkage, which promoted light scattering. The extent of the shrinkage appeared to be dependent upon the time post-mortem (longer times post-mortem reduced the incidence of dark meat) and upon the pH condition of the muscle. Various experiments (chapters 5 and 6) demonstrated that pH was central to the shrinkage of the muscle fibres and consequential to light scattering development. Lowering the pH surrounding the muscle fibres promoted shrinkage and increased light scattering, whereas increasing the pH promoted swelling and reduced light scattering. This occurred regardless if muscle fibres came from muscles of a low (light) or high (dark) ultimate pH (pHu) and was somewhat reversible (see supplementary movie in chapter 6). Muscle fibres from the light (low pHu) muscles did have some semi-permanent modifications which caused them to behave differently to dark (high pHu) muscle fibres and indicated some permanent structural modifications had occurred previously. In chapter 5, the transverse periodicity patterns (perpendicular to the muscle fibre axis) of the light scattering elements were observed to be in the region of 1-2 μm, and were likely the gaps that occur between myofibrils within a muscle fibre. In the longitudinal orientation, the periodicity pattern ranged from 1.2-2.5 μm and was more difficult to interpret, but thought to relate to the sarcomere length, the integrity of the Z-line and the optical protein density in the I-band. Regardless, the light scattering periodicity in transverse and longitudinal orientations were strongly correlated and indicated similar mechanisms were involved. Dark muscles also had shorter sarcomere lengths, longer myofilament separation and the appearance of more Z-line degradation compared to light muscles (chapter 7). Intensity ratios of lattice spacings from the synchrotron analysis, SDS-PAGE and proteomic analysis also indicated there was extra mass on the dark myofilaments, which was either from the Z-line degradation or from the modification of sarcoplasmic proteins. The sarcoplasmic protein glyceraldehyde 3- phosphate dehydrogenase (GAPDH) was implicated in the structural differences giving rise to light scattering, with more activity observed in dark muscles and also an increase in light scattering occurring when GAPDH was added back to washed muscle fibres. GAPDH also showed a less dense band in SDS-PAGE from drip samples from dark muscles (chapter 4) and indicated less of this protein is lost with lower drip loss. The similarities in correlation coefficients between lightness, drip loss and sarcoplasmic protein activities also suggests there are synergies between the light scattering mechanism and drip loss development, which should be explored further.