A high rigor temperature, not sarcomere length, determines light scattering properties and muscle colour in beef M. sternomandibularis meat and muscle fibres
Introduction
Meat colour is determined not only by the quantity and oxidative status of myoglobin, but also by the structural opacity and light scattering properties of the muscle fibres from which they are composed (MacDougall, 1982). One of the areas of the muscle that may impact on the light scattering properties of the meat is the sarcomere length, as determined primarily by the lengths of the isotropic (I-band), rather than the anisotropic area or A-band (Periasamy, Burns, Holdren, Pollack, & Trombitas, 1990). Currently there is no research relating the sarcomere length microscopic structural characteristics to the light scattering properties of post-rigor beef muscle, after the ultimate pH has been reached.
Some preliminary studies indicate there is an inverse relationship between sarcomere length and light scattering within muscle fibres at a high pH (~pH 7) or physiological conditions (Bozler, 1958; Jeacocke, 1984). Bozler (1958) measured light scattering using a spectrophotometer on glycerol-extracted rabbit muscle fibres and a chemical induced contraction (via addition of either ATP or CaCl2), which caused an increase in light scattering by over 55%. R. Jeacocke (1984) used various optical set-ups to investigate light scattering in beef M. sternomandibularis single muscle fibres and in the whole muscle. Using a rigor temperature of 22 °C in the single muscle fibre preparation, Jeacocke (1984) observed that an increase in scatter associated with rigor was proportional to the degree of filament overlap. Both of the Bozler and Jeacocke experiments were conducted close to physiological pH conditions (pH 7 and 6.9 respectively), and Jeacocke (1984) noted the diminished effect of rigor-induced light scattering as the pH of the bathing medium was lowered. Therefore, this experiment aims to further explore the generation of light scattering at various sarcomere lengths (via stretching) after the muscle has passed through rigor and the consequential impact of the associated lower ultimate pH (pHu).
In contrast to the above studies on stretched sarcomere lengths, shorter sarcomeres have been observed in dark, high pH meat (both pork longissimus muscles and beef longissimus muscle fibres) compared to paler, low pH meat (Hughes, Clarke, Purslow, & Warner, 2017; Irving, Swatland, & Millman, 1989; Warner, Kauffman, & Greaser, 1997). This may be indicative of a positive relationship between sarcomere length and light scattering, although it is conflated with pH effects. Sarcomeres are isovolumetric in vivo (see Millman (1998) for a review) and the shorter sarcomeres in dark pork loins are known to have myofilaments which are further apart giving rise to a lower red reflectance (Irving et al., 1989). A larger fibre diameter with increased myofilament lattice spacing is thought to contribute to decreased light scattering (Hughes et al., 2017). In addition, decreasing the pH of the surrounding medium in post-rigor beef longissimus muscle fibre fragments promotes fibre shrinkage and increased light scattering properties, but the reversibility of these structural changes is still under question as decreased pH may cause sarcomeric proteins to denature. Therefore, we aim to visually quantify the effect of pH cycling on the reversibility of the swelling/shrinkage mechanism of muscle fibre fragments. Some of the above observations appear to conflict those reported at physiological pH and suggest either the muscle pHu and/or the rigor temperature to which the muscle is exposed, may override the increased light scattering during contraction at a physiological pH ~ 7.
A high rigor temperature (35–40 °C) is known to generate structural alterations in the muscle, which could increase light scattering (Hughes, Oiseth, Purslow, & Warner, 2014; Kim, Warner, & Rosenvold, 2014). Elevated rigor temperatures in the muscle are associated with increased myosin and sarcoplasmic protein denaturation, both of which contribute to shrinkage of the myofilament lattice spacing (Liu, Arner, Puolanne, & Ertbjerg, 2016). Together, this effect may translate to shrinkage at larger length scales, producing more shrinkage of the myofibrils and muscle fibres and generating more opportunity for light scattering. Alternatively, it is possible that denatured sarcomeric proteins may deposit on myofilaments and change their optical protein density.
Currently the exact mechanism of light scattering is still unknown, but at the cellular level, the length of the I-band, the lateral distance between adjacent A-bands of different myofibrils and the A-I interface junction, could be involved (Offer et al., 1989), which would translate to structural differences at the muscle fibre and meat surface level. We hypothesize that (a) stretching muscles during rigor will generate a structure which favours light scattering, by increasing the length of the I-band, via longer sarcomeres; (b) a high rigor temperature would facilitate the protein reconfiguration and structural alterations which would promote light scattering; and (c) reducing the pH of immersion buffer will shrink the muscle fibre fragment whilst increasing light scattering but after exposing muscle fibres to low pH conditions, some permanent, irreversible structural modifications would occur.
Section snippets
Sample collection
All muscles were collected from carcasses derived from female yearlings (zero permanent incisor teeth) within the same group of cattle. The mean subcutaneous rib fat depth over the loin, at the 10th/11th rib, was 5 ± 0.8 mm, with a mean hot carcass weight of 165 ± 11.1 kg (average ± s.e.). From 4 carcasses, M. sternomandibularis (tongue root) muscles were collected from both sides at 10 min post-mortem (PM) and placed in individual plastic bags for transportation. These muscles were selected
Results and discussion
Contrary to our hypothesis, the stretching treatment did not directly impact light scattering values. As shown in Table 1, Table 2, with graphical displays in Fig. 2, Fig. 3, stretching increased both the sarcomere length (P < 0.001) and the longitudinal peak distances (P < 0.05); however, no change in either lightness or global brightness was observed (P > 0.05). The stretching treatment was successful, as indicated by the increase in average sarcomere length from 2.06 for unstretched to 2.61
Conclusion
Manipulation of sarcomere length, rigor temperature and pH has provided tools to dissect the various sources of light scattering by muscle fibres. Sarcomere length alone did not impact on the light scattering properties of the muscle. A high rigor temperature increased lightness and global brightness of the muscle and was associated with alterations in the structural configuration of the muscle, creating increased light scattering. When stretched, the high rigor temperature promoted muscle
Acknowledgments
The authors and CSIRO acknowledges funding provided by Australian Meat Processor Corporation (AMPC) (2013/ 3005) 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, and in particular the Imaging and Image Analysis Facility, is also gratefully acknowledged. We also acknowledge the support of FONCyT (PRH-PICT 2013-3292). The authors have no
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