Basic Science for Carcass Grading
University of Guelph, Guelph, Ontario, Canada
Carcass grading is dominated by subjective evaluation augmented by a few technologies such as probe fat-depth measurements, ultrasonics and video image analysis (VIA) to estimate meat yield. Little or no progress has been made in predicting meat quality, which is a problem when sorting carcasses from heterogeneous populations. Many aspects of meat quality may be measured on excised meat samples in the laboratory – the challenge is to make measurements rapidly and non-destructively when carcasses are graded. Optical measurements offer the most promise, particularly polarized light. Aspects of the basic science relating to meat microstructure are reviewed to support this argument.
Key words: Carcass grading, Meat quality, On-line measurement, Polarized light.
Basic science is one of the highest achievements of the human intellect, whereas carcass grading is an imperfect agricultural process. How can basic science be involved with carcass grading? Take one of many possible examples. Quantum mechanics is at the leading edge of the basic sciences, quantum effects may be involved in our sense of smell (FRANCO et al., 2011), and olfaction is a major factor in consumer evaluation of meat – it is already being measured objectively (GHASEMI-VARNAMKHASTI et al., 2009). Thus, progress in understanding the biophysics of olfaction could lead to an on-line method for carcass grading. But why do we need on-line meat evaluation when most countries use subjective grading? Because subjective evaluation is based on guesswork, it is difficult to standardize and automate, and it is not very reliable. Surely we can improve on this by developing new technology. To develop new technology we must first improve our scientific understanding of meat quality.
Available on-line systems are limited to predicting meat yield and fatness (PASCOAL et al., 2010). Conventional laboratory apparatus is useless in an abattoir – it cannot withstand high humidity and rugged treatment and, worst of all, conventional apparatus requires destructive removal of meat samples and laborious sample preparation. All the available on-line systems are derivatives of other highly developed technologies such as VIA (TEIRA et al., 2003) and ultrasonics (YOKOO et al., 2008). Applications of existing technologies have been pushed to near their limit – and there is not much new to review. Thus, we need to consider how improving our biophysical understanding of meat quality might help us improve carcass grading systems. The biophysics of meat quality lags far behind the biochemistry of meat quality because most meat scientists are trained as biochemists. But biophysical systems offer the best hope of developing new optical and electronic sensors for rapid, non-destructive assessment of meat quality (SWATLAND, 1995).
On-line evaluation for meat quality is not a new idea - it originated in the 1930s (SWATLAND, 2003a). But developing commercial apparatus from the research of government or university scientists is difficult. The basic problem is how to transfer technology developed at the public expense to the private sector for commercial development – how should the profits from successful methods and the losses from unsuccessful methods be shared? Case histories in technology transfer show many examples where serious ethical and legal problems have occurred (MATKIN, 1996). Often the role of the meat scientist degenerates into merely testing or comparing privately developed commercial systems. Conventional laboratory apparatus can be evaluated against known standards, but testing predictions concerning meat quality is like testing predictions of tomorrow’s weather. The operating principles and software of commercial systems are secret. Sometimes nobody understands a system scientifically, and the outcome is strictly empirical – based on a multivariate correlation or a neural network.
Going beyond established methods of on-line grading such as fat-depth probes and VIA, the idea considered here is that an improvement of our biophysical understanding of meat quality is the best route for improving carcass grading. UV fiber-optic probes for elastin, collagen and pyridinoline cross-linking are quite reliable when used properly and can even detect seasonal changes in connective tissue (SWATLAND, 2003c). However, industrial trials using UV probes to predict beef toughness have failed because of two main reasons: (1) application to beef populations with uncontrolled sarcomere length from cold-shortening, and (2) inappropriate, uncontrolled anatomical placement of the probe. Thus, these are the main problems to be solved before UV probes can be used for beef carcass grading. Sarcomere length may be accessible using polarized light, as explained below, but light scattering must also be taken into account, both for its problematic interaction with polarized light and for the opportunites it offers to assess water-holding capacity, meat paleness and pH-related tenderization.
2. Optical properties of bulk meat
Any analytical optical system such as a probe or VIA involves three major pathways. The incident light (Fig. 1, A) may be directly reflected from the meat surface (Fig. 1, B). This specular (mirror-like) reflectance is partly polarized and has no selective absorbance by myoglobin. Thus, if the incident illumination is white light, specular reflectance is white. It is important to minimize specular reflectance using a polarizer when attempting to quantify marbling fat with VIA, otherwise bright spots of reflected light appear as flecks of marbling fat. Specular reflectance follows Fresnel equations and has a strong angular dependency, but unwrapped meat surfaces are never smooth and the angular dependency is extremely complex. Light that is not reflected enters the meat and some of it scatters back to escape as diffuse light from the meat surface (Fig. 1, C and D). Diffuse scattered light from meat appears the same at all viewing and illumination angles, mostly following Lambert’s law,
I = I0 cos Θ
where I is the luminous intensity, I0 is the luminous intensity in the normal direction, and Θ is the angle from the normal. But all diffuse light scattered from meat is not truly Lambertian. This does not affect the performance of typical apparatus such as a colorimeter, but is likely to affect the results from systems using coherent light from a laser.
Pathway C in Fig. 1 is short, because of a high degree of scattering from meat microstructure. The short pathway minimizes selective absorbance by myoglobin and the meat appears pale. Pathway D in Fig. 1 is long, because of a low degree of scattering from meat microstructure. The long pathway maximizes selective absorbance by myoglobin and the meat appears dark. Pathway C in Fig. 1 shows what happens with pale, soft, exudative (PSE) pork and poultry meat. Pathway D in Fig. 1 shows what happens with dark, firm, dry (DFD) pork, beef and poultry meat. But changes between pathways also may occur during post-mortem metabolism. Immediately after stunning and exsanguination, pathway D determines the appearance of the meat – it is dark. If normal post-mortem glycolysis occurs and the pH of the meat declines, then pathways change in the direction from D to C, thus beef will develop a normal bright red appearance. But if glycolysis is unusually rapid (fast pH decline) or extensive (low ultimate pH), then pathway C becomes dominant and meat appears unusually pale (PSE).
A key point that many researchers fail to grasp is that the apparatus itself interacts with the sample. For example, a fiber-optic probe may use either a short or a long pathway through the meat, depending on the separation of its illumination and receiving apertures. And the apertures themselves may change depending on the refractive index of meat fluids post-mortem – refractive index first increases (osmotic loss of interstitial fluid) and then decreases (release of water from fibrils). Everything depends on the nature of the apparatus. Different methods give different results. Also, it is difficult or impossible to escape from the problems outlined here. For example, take the case of a researcher who places a sample of bulk meat in the window of some type of complex optical apparatus – doubtless expensive, well-engineered and with high performance characteristics – but originally designed for an optically isotropic sample. From the UV to IR, with all planes of polarization, and with or without fluorescence – the penetration of the incident light must follow pathways similar to those shown in Fig. 1. A signal such as fluorescence polarization may change in some way or after some treatment, but the root cause may simply be a pH change affecting the different light pathways, not a fundamental change in the fluorophore. Other methods such as hyperspectral or multispectral imaging are equally prone to misinterpretation. In fact, one might argue that the optical complexity of apparatus is inversely related to the certainty of what is being measured.
Figure 1. Light incident on bulk meat (A) may be reflected (B) and scattered through short (C) or long (D) pathways.
In Figure 1, the microstructural sources of scattering along pathways C and D have been simplified to random dots but, in reality, scattering originates mostly from fibrils within muscle fibers. There are many possibilities, but the main features are shown in Figure 2. At point A in Fig. 2 the incident light may be scattered in a truly random manner if sarcoplasmic proteins have precipitated on the fibril surface. At point B in Fig. 2, Fresnel reflectance may occur on the surface of the fibril where there is an optical boundary between the filament lattice (high refractive index) and the sarcoplasm (low refractive index). Fibrils are very small and very numerous, so the reflected light is randomized in bulk meat. But, on individual fibrils seen in a polarizing microscope, the mirror-like nature of specular Fresnel reflectance is detectable. At point C in Fig. 2, incident light is refracted to pass through the fibril. The angle of refraction depends on changes in refractive index caused by post-mortem glycolysis. As pH decreases, filaments move close together laterally and refractive index increases. Thus, at a low ultimate pH, scattering is high and meat appears pale, as in pathway C of Fig. 1. Another source of scattering recently discovered is by reflectance from sarcomere disks, as at point D in Fig. 2. If scattering is minimal, multilayer interference from sarcomere disks causes the iridescence sometimes seen on meat (SWATLAND, 2011).
Figure 2. Muscle fibril scattering: from precipitated sarcoplasmic proteins (A), surface reflectance (B), refraction through the fibril (C), and reflectance from sarcomere disks (D).
Refractive scattering may need a little more explanation. In Fig. 2 it is obvious how light at points A, B and D can be scattered back to the meat surface, but what about the refracted light at point C which still looks as if it is passing deeper into the meat? To answer this question we must take into account that refracted light passes through large numbers of fibrils. As seen in Fig. 3, an increase in the angle of refraction may send incident light back to the meat surface.
Figure 3. Fibrils seen in cross section, showing how refracted light may change direction to cause incident light to be sent back to the meat surface as Lambertian scattering.
If Fig. 3 does not resemble anything familiar in bulk meat, consider Fig. 4. This shows a transverse section of beef immediately after stunning and exsanguination. The glycogen in the sarcoplasm between fibrils has been stained by the periodic-acid Schiff reaction so that fibrils appear as clear spaces like the fibrils seen in cross section in Figure 3. As post-mortem glycolysis proceeds, glycogen disappears and the fibrils decrease their diameter.
Figure 4. Transverse section of beef muscle fibers immediately after stunning and exsanguination stained for glycogen by the periodic-acid Schiff reaction and seen by light microscopy.
As muscle is converted to meat, a major physical change is the rigor mortis bonding of actomyosin locking thick and thin filaments together, best explained with a longitudinal section using electron microscopy (Fig. 5). Filament sliding is prevented and sarcomere length from Z line to Z line becomes fixed , although it may appear to increase if tensile forces disrupt Z lines as a sample is removed for examination. Long sarcomeres give tender meat with high water-holding capacity (WHC), while short sarcomeres give tough meat with low WHC.
Figure 5. Features of beef muscle fibers immediately after stunning and exsanguination seen in longitudinal section by electron microscopy. Glycogen appears as individual granules between tightly packed fibrils.
WHC determines how much fluid will be released from meat, often at great economic cost during refrigeration and meat processing, and is certainly a parameter we might wish to incorporate into grading, especially for pork. Fig. 6 shows a transverse section of pork after the completion of post-mortem glycolysis. Hence, glycogen granules have been lost, fibrils have decreased in diameter, and much of the water from between filaments has been released. Restraints between shrunken fibrils are visible (Desmin? in Fig. 6), giving some indication of the complexity of WHC. Not only is the system affected by pH acting directly on the lateral negative electrostatic repulsion between filaments, but also by post-mortem proteolysis of proteins linking fibrils and, thus, affecting the separation of fibrils as they shrink. Why the question mark for desmin? Because the presence of both thick and thin filaments in Fig. 6 shows the section is not through the Z line where most desmin is located. In longitudinal sections, the evidence for desmin limiting water loss from fibrils is more convincing (SWATLAND, 1985).
Figure 6. Electron micrograph of pork in transverse section.
To follow the release of water from fibrils, we may use x-ray diffraction, as seen in Fig.7. This shows that, as the pH approaches the isoelectric point of muscle proteins, a decrease in negative electrostatic repulsion reduces the lateral separation of filaments. This allows water to move from the filament lattice, into the sarcoplasm, through the muscle fiber membrane, into the extracellular space, and then to be lost from the meat as drip or evaporation - often at great commercial loss, especially in pork. Fluid movements as muscle is converted to meat can be detected electrically, which also offers a possibility for on-line grading. At the top of Fig. 5 can be seen traces of the plasma membrane on the muscle fiber surface, through which fluid released from the fiber will move into the interstitial (intercellular) space. Membranes in living muscle have a high dielectric constant which can be measured as capacitance using an alternating current. As post-mortem metabolism proceeds, and the membranes leaks fluid into the interstitial space, the capacitance is lost. Electrical resistance also decreases at this time.
Figure 7. The unit cell detected by x-ray diffraction shows the decreasing lateral separation of thick and thin filaments during post-mortem metabolism. From Bragg’s equation, the d1,0 spacing of the unit cell is closer to the x-ray axis than the d1,1 spacing.
3. Polarized light
Polarized light has tremendous potential and, in the laboratory, can reveal many commercially important features of meat. Many of the components of meat, such as fibrils and connective tissue fibres, have a precise longitudinal arrangement of proteins. Thus, they are birefringent - having different refractive indices along and across their structure (Fig. 8). Polarimetry involves measuring the rotation of plane polarized light. Starting with the effect of pH on fibrillar birefringence, as reduced electrostatic repulsion brings filaments closer together, electron microscopy shows fibrils decrease in diameter (Fig. 6). Thus, the velocity of light changes across but not along the fibrils, and increases the optical path difference (along versus across fibrils). For polarimetry of meat, instead of measuring the rotation of plane polarized light, the intensity of light retaining its original plane of polarisation may be measured. Reflective surfaces at refractive index boundaries may maintain polarisation, but polarization can be lost by scattering.
Refractive index is defined by c, the velocity of light in a vacuum and v, its velocity in various components of meat, n = c/v. Passing through fibrils, light splits into two components at different velocities, the ordinary ray (O) and the extraordinary ray (E), with O ┴ E. Birefringence, nE - nO, may be either negative or positive in sign.
Figure 8. Birefringence from a single sarcomere measured by scanning with a polarizing microscope.
Retardation is the decrease in velocity of light passing through a medium and may be detected as a phase retardation, the interference caused by path difference E ¹ O. The path difference through a depth of meat (Γm) may be measured in the laboratory by ellipsometry using a de Sénarmont compensator,
Γm nm = Kλ nm/degree . uo
where u is the angle in degrees required for compensation, and Kλ is the de Sénarmont constant or path difference for 1o of rotation (Fig. 9).
Figure 9. Measurement of birefringence through a single muscle fiber using a De Sénarmont compensator in a polarizing microscope.
The path difference increases as pH decreases post-mortem towards the isoelectric point (Fig. 10). This is why polarized light is being given so much emphasis in this presentation. There is no hope of using electron microscopy or x-ray diffraction in on-line carcass grading, but the changes they detect can also be detected with polarized light.
Figure 10. As pH decreases post-mortem, the fibrillar path difference increases so that more light is refracted, scattering increases and the meat becomes more pale.
The change in fibrillar path difference with pH follows a similar pattern to that of the increase in paleness occurring post-mortem in meat, and refractive scattering is probably the major source of this paleness. One would normally expect extremes of post-mortem glycolysis to be curtailed by deactivation of glycolytic enzymes. However, if the pH decreases below the isoelectric point, then the path difference through fibrils decreases, refractive scattering through fibrils decreases, and the meat appears darker again. This may occur in processed meats where fermentation increases the normal decline in pH. At very low pH values for meat, precipitation of sarcoplasmic proteins occurs. But acid-denaturation of proteins is likely to be temperature dependent and particularly severe in a hot carcass immediately post-mortem because glycolysis is exothermic. Thus, a carcass with a slow but extended decline in pH might not have such pale meat as would be expected at its low ultimate pH. This explains why ultimate pH is not as useful as pH at 45 minutes post-mortem for the industrial prediction of pork colour. Thus, grading pork carcasses for meat quality as they leave the kill floor is quite feasible.
Along the length of muscle fibers, there are A (anisotropic) and I (isotropic) bands matching the distribution of thick and thin filaments, respectively (Fig. 5). At the midlength of the A band in a relaxed fiber, the otherwise strong birefringence of the A band is slightly weaker in the H zone, between the ends of thin filaments. Similarly, the otherwise weak birefringence of the I band has a slightly stronger region at the midlength of the I band caused by the Z line. Birefringence of the Z line indicates a high degree of protein filament alignment, and weakening and structural alteration of the Z line contributes to the tenderisation of beef during conditioning. Given that short sarcomeres and strong Z lines are classical sources of meat toughness, the challenge is to make similar measurements in bulk meat. At present, this is very difficult. The main problem is depolarisation by scattering - hence the interest here in the balance between polarization and scattering.
4. Measurements at a meat surface
From Brewster's law, the polarization angle (Θ) of reflected rays is related to refractive index, n = tan Θ, but the refractive index of meat fluids is quite variable. One might expect the first fluid lost from meat post-mortem to be sarcoplasmic in origin with a high protein content, and fluid released later from the filament lattice to have a lower protein content and refractive index. However, this is difficult to demonstrate experimentally because different physiological types of muscle fibers are at different stages of fluid release.
Figure 11. Planes of polarization of light reflected from the meat surface versus light transmitted into the meat.
For an area of meat that is illuminated to produce an ellipse (Fig. 11, ABCD), if the light from the illuminator is unpolarized and the central axis of the cone of illumination is at a fixed angle of 45° to the meat, the reflected rays tend to be polarized primarily at 90° to the plane of incidence, while the rays refracted and transmitted into the sample tend to be polarized primarily in the plane of incidence. The efficiency of polarizers and analyzers may be evaluated by measuring their extinction coefficients at different wavelengths,
k = log10 (T0 / T90)
where T0 is with the analyzer parallel to polarizer, and T90 is with the analyzer perpendicular to the polarizer. A similar approach may be used to find the degree to which light reflected from meat retains its initial polarization, using R rather than T, and replacing the polarizer by the sample. Thus,
k = log10 (R0 / R90)
so that a high extinction coefficient indicates a strong polarizer, and vice versa. A subscript denotes the angle of tilt at which k is measured. In the laboratory, with light passing through excised samples of meat, there is no limit to the information that can extracted on key parameters we might wish to exploit in on-line carcass grading. But there are major obstacles for adapting these methods for on-line use in grading.
· How can we get a defined light path through the meat if we are limited to a meat surface, either from ribbing a carcass or by inserting a probe with an optical window?
· If we use a probe, how can we maintain polarization (it is lost in ordinary optical fibers and polarization-preserving fibers require precise optical alignment and are monochromatic)?
· If we use front-face polarizers on optical fibers, how can we protect them against abrasion with repeated use?
There are some solutions – yet to be fully exploited. A graded-index lens may be used to make the final connection with the carcass. These lenses are small cylinders of glass with a radial gradient in refractive index. Polarization is preserved. Parallel probes may be used, so that the optical path length through the meat is fixed. But there are also basic problems – yet to be fully elucidated. The birefringence signal (Fig. 8) behaves in a logical manner over a certain range – birefringence increases as sarcomeres get shorter. But, as often happens in severely cold-shortened beef, once the thick filament reaches the Z line it can pass through it. Then the neat lateral arrangement of filaments is lost, and birefringence starts to decrease. Thus, both seriously cold-shortened sarcomeres (very tough meat) and sarcomeres stretched to their maximum (very tender meat) give a weak birefringence signal. And there is another contradictory response from sarcomere length versus scattering.
5. Sarcomere length versus scattering
Sarcomere length has a strong effect on meat toughness and any method for on-line measurement would be a tremendous asset in carcass grading. However, the problems involved in using light scattering to find sarcomere length are formidable. Rapid post-mortem refrigeration causes unrestrained muscles to contract, and the increased overlap of thick and thin filaments when rigor mortis develops causes toughness. Muscles cooled slowly or stretched have little filament overlap and are tender. Carcasses lacking adipose insulation, with a high surface to volume ratio, or first into an empty meat cooler have the highest risk of cold shortening. Meat with short sarcomeres tends to have a high degree of scattering and, hence, may appear pale (SWATLAND, 2003b). Figure 12 shows a correlation of reflectance with sarcomere length in beef – short sarcomeres increase birefringence and light scattering so the correlation is negative. A major problem is that beef with a rapid rate of post-mortem glycolysis tends to be tender because of enhanced proteolysis – and rapid glycolysis also tends to make meat pale by increasing light scattering. Thus, paleness or a high degree of light scattering may indicate toughness if it is caused by short sarcomeres but also tenderness if it is caused by rapid glycolysis. This has caused considerable confusion in the scientific literature. Researchers who have avoided any cold shortening but who have high variance in rates of glycolysis in their experimental samples have found that tender meat tends to be pale, researchers who have allowed cold shortening but who have uniform rates of glycolysis in their samples have found tough meat tends to be pale, and researchers who have samples with a high variance in both sarcomere length and rate of glycolysis have found no relationship between paleness or light scattering and meat tenderness. Everything depends on the nature of the samples and when they are tested because both cold-shortening and glycolysis may show both diurnal and seasonal cycles. The problem occurs regardless of the level of instrumental complexity, and may appear with the simplest hand-held colorimeter as well as with complex methods of VIA.
Figure 12. Spectral distribution of the simple correlation of fiber-optic reflectance with sarcomere length in beef.
In Fig. 12, the correlations are weak around the Soret absorbance band at 430 nm where small changes in hemoproteins are a source of variance, but fairly even across the remainder of the spectrum. But the relationship is not strong enough to be of any useful predictive value. In summary: two strong factors affecting beef tenderness act in opposite directions on light scattering, if the beef is tough because of a high pH it will be dark with low reflectance, but if beef is tough because of short sarcomeres it will be pale with high reflectance. Thus, if the range in pH is low while the range in sarcomere length is high, pale beef may be tougher than dark beef. But if the range in pH is high while the range in sarcomere length is low then pale beef may be more tender than dark beef.
Basic science might help us develop new methods for carcass grading, but we are still faced with the problem of applying the method in the very difficult working environment of the kill floor or meat cooler. There are many difficulties. With robotic equipment, stopping a moving carcass or travelling with it is difficult. Even with hand-held equipment (Fig. 13), the elastic nature of bulk meat causes problems. Thus, when a probe tip encounters resistant tissues it may pause or bounce which, respectively, may increase the width or double the peak of the signal produced from the target. Tissues in advance of the probe tip may be compressed or stretched as the probe is withdrawn, so that depth measurements are unreliable. This problem occurs even in the fat-depth probes now widely used for grading pork carcasses, and may be compounded with the problem of finding the separation between muscle and fat if the muscle has a very high degree of scattering (which is why infrared is used – to reduce scattering).
Figure 13. An optical fiber in a hand-held probe generating a signal from a target.
At the source of many engineering problems are anatomical problems. With the time available for carcass grading on a fast-moving line, it is difficult to make even a single measurement to assess the state of a whole carcass. Thus, considerable research is required to find the most reliable site to encompass intermuscular variation and this site must be located accurately on each carcass. But there is a high degree of biological variability in carcass structure, not just in muscularity and adiposity, but also in the skeleton. Numbers of ribs and vertebrae are far more variable than most butchers notice, and bone shapes vary from animal to animal. However, the only hope of accurately locating a grading point is to use skeletal reference points. For robotic grading, this calls for considerable sophistication in navigation relative to the skeleton, with ultrasonics as the most promising method (Fig. 14).
Figure 14. Ultrasonic navigation for a grading probe.
The general direction of research is determined by funding agencies and the politics of research support. Thus, applied scientists are mostly driven by short-term goals with a promised financial advantage, even though promised financial advantages are seldom achieved and amortization times are seldom specified. Basic scientists get major funding and mostly have an impressive grasp of science and technology, but are unlikely to ruin their career prospects by working on something like meat grading. The only hope of bridging this gap is the commercial development of new technology for industrial use in meat grading. But here there are different problems. The initial market for new technology is very small and cannot justify a major expenditure of venture capital, and there are major difficulties in patent protection and maintaining market penetration. Successful methods are most likely to be simple methods, and simple methods have many technical solutions, thus enabling patent avoidance. From within the meat industry, there are further obstacles. Even given an inexpensive, robust and reliable method of grading meat quality – how can this be turned to financial advantage? Sorting products for a premium market makes sense, but what happens to carcasses rejected because of tough meat or high drip loss? Until we solve these interrelated political and commercial problems reliable grading for meat quality may remain just a theoretical possibility.
Financial support from the Basic Research Funding programme of the Danish Bacon and Meat Council is gratefully acknowledged, one of the few organizations aware that meat scientists need to do their own basic research. In this overview of 40 years research, I am happy to acknowledge the contributions of my co-workers, especially Susan Belfry and Thayne Dutson in electron microscopy; Leo Diesbourg, Tom Irving and Barry Millman in x-ray diffraction; and Andrew Goldenberg and his team in robotics for the abattoir. Many of the technical staff at Carl Zeiss have helped me over the years, especially in finding calibrations and machine codes for opto-electronics. I was fortunate in having a famous meat scientist, James Bendall, to start my career, as well as another, Robert Cassens, as my supervisor when I was a student.
A full listing of original references for statements made in this short presentation may be obtained from http://www3.sympatico.ca/howard.swatland/retirement_page.htm
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