Three essential features: (1) cell, (2) fibres, and (3) matrix.
The fibrous connective tissues in meat form a continuous mesh, as shown in the image to the left, from the microscopic strands of endomysium around individual muscle fibers, to the larger layers of perimysium that delineate bundles of muscle fibers, all being gathered and connected to the thick, strong epimysium on the surfaces of individual muscles.
The image below shows a thick layer of perimysium.
The endomysium, perimysium and epimysium contain two types of protein fibers, collagen and elastin, which now we will consider in detail.
Collagen is the most abundant protein in the animal body, and the collagen
which occurs in meat may be an important source of meat toughness. Beef
carcasses have to be graded by age mainly because of age-related changes
in collagen that cause meat from older cattle to be tough.
Large amounts of collagen are found in animal skin. In pig skin, for example, collagen fibers are tightly woven from two directions to form a tightly woven meshwork. Collagen is a raw material for major industries in leather, glue and cosmetics.
Under a light microscope, collagen fibers in the connective tissue framework
of meat range in diameter from 1 to 12 micrometres (0.001 millimetre =
1 micrometre). They do not often branch and, when branches are found, they
usually diverge at an acute angle. Collagen fibers from fresh meat are
white, but usually they are stained in histological sections for examination
under a microscope. The most common stain for light microscopy is eosin,
which stains collagen fibers pink. Unstained collagen fibers may be seen
by polarized light since they are birefringent (light that comes through
Polaroid sunglasses is polarized, with all its waves in one plane; birefringent
means having two refractive indices). By rotating the plane of polarized
light, collagen fibers appear bright against an otherwise dark background
(when two Polaroid lenses are perpendicular they block most of the light,
but collagen fibers can rotate the light so that they appear bright). The
birefringence of collagen fibers in meat is lost at the point during heating
when gelatinization occurs. Collagen fibers have a wavy or crimped appearance
which disappears when they are placed under tension.
Collagen fibers fluoresce with a blue-white light when excited with UV light so that the amount of connective tissue on a cut meat surface may be measured very rapidly. Peak excitation is around 370 nm so that the prominent 365 nm peak emission of a mercury arc lamp may be used. Some indication of collagen fiber diameter may be obtained by spectrofluorometry (measuring the wavelengths of fluorescence) because the fluorescence is quenched (fades) fairly rapidly. Thus, large collagen fibers retain a central core with a pre-quenching emission spectrum for longer than small fibers. Fat only fluoresces weakly, to about the same extent as areas of muscle with a low connective tissue content. This is how the connective tissue content of meat may be measured with a fiber-optic probe for the on-line detection of tough beef. Collagen fluorescence increases with animal age so that probe measurements may have a promising future in beef grading, and fiber-optic probe measurements now have been correlated with consumer taste panel evaluation of chewiness in beef . It is important remember, however, that a probe for connective tissue cannot account for toughness caused by short sarcomeres or inadequate aging of the meat!
Electron microscopy reveals that collagen fibers are composed of parallel bundles of small fibrils with diameters ranging from 20 to 100 nm (0.001 micrometre = 1 nanometre). Collagen fibrils typically have diameters which are multiples of 8 nm that may show the manner in which they grow radially. Collagen microfibrils (even smaller structures that make up fibrils) may appear to have a tubular structure with an electron-lucent lumen (appearing empty under the electron microscope).
Collagen fibrils are formed from long tropocollagen molecules which are staggered in arrangement but tightly bound laterally by covalent chemical bonds. For electron microscopy, when negatively stained with heavy metals that spread into the spaces between the ends of molecules, collagen fibrils appear to be transversely striated. The periodicity of these striations is 67 nm but often shrinks to 64 nm as samples are processed for examination. Although collagen fibers are located outside the cell, the initial stages of collagen fibril assembly may be within the cell, with fibril morphology being regulated by a special site on the fibroblast membrane (cells that form connective tissue fibers are called fibroblasts).
In the polypeptide strands, the small amino acid glycine occurs at every third position, and proline and hydroxyproline account for 23% of the total residues. The regular distribution of glycine is required for the packing of tropocollagen molecules and has been claimed as evidence that all animals are derived by evolution from a single ancestral stock, since the chance development of this unique regularity in unrelated animals is thought unlikely. Hydroxyproline is quite rare in other proteins of the body, and an assay for this imino acid (an imino acid is chemically similar, but not the same as an amino acid) provides a measure of the collagen or connective tissue content in a meat sample. Tropocollagen also contains a fairly high proportion of glutamic acid and alanine as well as some hydroxylysine.
(1) molecules with a long (about 300 nm) uninterrupted helical domain,
(2) molecules with a long (300 nm or greater) interrupted helical domain,
(3) short molecules with either a continuous or an interrupted helical
The various types of collagen of interest in understanding the structure
of meat are as follows.
Small diameter type III collagen fibers are called reticular fibers since, when stained with silver for light microscopy, they often appear as a network or reticulum of fine fibers. The larger diameter collagen fibers formed from Type I collagen are not blackened by silver.
Collagen fibers shrink when they are placed in hot water, and ultimately
they may be converted to gelatin. Around 65oC, the triple helix
is disrupted and the alpha chains fall into a random arrangement. The importance
of this change is that it tenderizes meat with a high connective tissue
content. Tropocollagen molecules from older animals are more resistant
to heat disruption than those from younger animals. In early studies, it
was suggested that reticular fibers, unlike collagen fibers, did not yield
gelatin when treated with moist heat. The original suggestion that reticular
fibers survive unchanged after cooking is wrong, but a modification of
the idea is plausible. Since a piece of meat may contain different types
of collagen, and since these types may differ in the thermal stability
of their cross links, it is possible that, at an intermediate level of
cooking around 65oC, endomysial collagen and perimysial collagen
may differ in the extent to which they are affected by the cooking treatment.
Heat-induced solubilization of Type I collagen is more important in improving
meat tenderness by cooking than is the effect of heat on Type III collagen.
Outside the cell, collagen molecules become aligned in parallel formations, and then they link up laterally to form fibrils. It is likely that tropocollagen monomers are partially assembled together in groups before they are added to an existing collagen fibril. Firstly, vacuoles containing procollagen fuse to form a fibril-containing compartment. Then the cytoplasmic extensions withdraw from between several fibril-forming compartments to create a bundle-forming compartment. Sometimes collagen fibrils occur intracellularly, but it is not clear whether this is collagen taken up by phagocytosis (engulfed by the cell) or a surplus of newly synthesized collagen.
The characteristic parallel staggered arrangement of tropocollagen molecules
in a collagen fibril is caused by the 67 nm repeating pattern of oppositely
charged amino acids along the length of the tropocollagen molecule. The
degree of overlapping of adjacent molecules and the gaps left between the
ends of molecules cause the striated appearance of collagen fibers seen
by electron microscopy. The fibroblasts of young animals are metabolically
more active than those of older animals, particularly for aerobic metabolism.
Accumulation of collagen in meat
Although the relative proportions of Types I and III collagen in a muscle
may be related to meat tenderness, the overall amount of collagen and its
degree of crosslinking also are important. The absolute amount of collagen
in an animal may increase as animals become older, and this may have an
effect on meat toughness, but rapid growth of muscle fibers also may dilute
the relative amounts of collagen in meat. Considering the supposed importance
of collagen in meat toughness, the absence of overwhelming evidence from
taste panel studies is rather curious. It seems reasonable that stewing
beef is tougher than prime steak because it has more collagen, but is collagen
responsible for differences in tenderness between the same cut of steak
from different carcasses? Recent studies with UV fiber-optics suggest that
it is, because this new technology allows us to see overall trends that
are difficult to identify by chemical analysis of small samples of meat.
Within a carcass, there may be considerable differences in collagen content between different muscles and this is reflected in their retail price. Collagen content also may differ between sexes. For example, the hydroxyproline content is higher in pork from females than castrated males. However, the amount of collagen in meat, when expressed as a proportion of wet sample weight, also is affected by fat content. In steaks from a veal carcass, for example, the collagen content might exceed 0.5%, but could be much less in the same region from a steer carcass in which fat had accumulated to "dilute" the collagen content.
Collagen in meat may be studied by measuring collagen fibril diameters
in electron micrographs. In tendons, fibril diameters in the fetus are
unimodal but become bimodal in the adult. Large diameter fibrils may have
more intrafibrillar covalent cross-links, while small diameter fibrils
may have more interfibrillar non-covalent cross links. Thus, fibril diameter
may be related to fibril strength and elasticity. Meat with large diameter
collagen fibers tends to be tougher than meat with thinner collagen fibers.
Little is known about the mechanisms by which collagen fibers become arranged in a muscle, or about the interactions which occur between fibroblasts and the fibers that they produce, although it is possible that glycosaminoglycans play some part in this interaction.
Collagen is very important in muscle development. Myoblasts, the cells
that form muscle fibers, develop a parallel alignment when cultured on
a substrate of Type I collagen, but they do not become elongated or aligned
on Type V basement membrane collagen. Myoblasts may themselves form Types
I, III and V collagen, while myotubes (immature muscle fibers) also may
form collagen, but only when associated with fibroblasts. The identification
of collagen in developing muscle is complicated by the fact that the tail
unit of the acetylcholinesterase molecule (involved in neural control of
muscle contraction) has a collagen-like sequence that contains hydroxyproline
Pyridinoline, a non-reducible cross-link, may be involved in the increased heat stability of epimysial connective tissues from older animals. Although changes in collagen solubility might be an important factor affecting the tenderness of beef from older animals, the effect in younger animals at a typical commercial slaughter weight may be relatively slight. However, relative to increasing maturity levels used in US beef grading, the pyridinoline content and thermal stability of intramuscular collagen both increase.
Differences in the degree of cross linking may occur between different
muscles of the same carcass, and between the same muscle in different species.
For example, collagen from the longissimus dorsi is less cross-linked than
collagen from the semimembranosus, and collagen from the longissimus dorsi
of a pork carcass is less cross-linked than collagen from the bovine longissimus
dorsi. Nutritional factors such as high-carbohydrate diet, fructose instead
of glucose in the diet, low protein, and pre-slaughter feed restriction
may reduce the proportion of stable cross links. Nonenzymic glycosylation
(a reaction between lysine and reducing sugars) may be involved in the
interaction between diet and collagen strength. In general, the turnover
rate of collagen is accelerated in cattle fed a high energy diet. The rate
of collagen turnover in skeletal muscle may be about 10% per day and the
turnover time for collagen may be inversely proportional to collagen fibril
Elastic fibers are made of the protein elastin.
Elastic fibers usually are pale yellow. When elastic fibers are stretched,
they may become visible in polarized light without staining, but this requires
careful attention to the refractive index of the mounting medium. In the
bovine ligamentum nuchae, the pattern of birefringence indicates that there
are two micellar structures, one arranged circularly on the outside and
the other arranged axially in the centers of the fibers. Elastic fibers
in meat have a small diameter (approximately 0.2 to 5 microns) although
they are much larger in the ligamentum nuchae. Elastic fibers in the connective
tissue framework of meat are usually branched.
Electron microscopy reveals that elastic fibers are composed of bundles of small fibrils approximately 11 nm in diameter embedded in an amorphous material. In the bovine ligamentum nuchae, fibrils may be constructed from smaller units or filaments approximately 2.5 nm in diameter. Elastin filaments are bound by non-covalent interactions to form a three-dimensional network and elastic fibers are assembled in grooves on the fibroblast surface where initially rope-like aggregations of fibrils become infiltrated with amorphous elastin. Unlike the situation in elastic ligaments, where elastin forms fibers, the elastin of the arterial system occurs in sheets that condense extracellularly in the absence of fibrils.
Although elastin resembles tropocollagen in having a large amount of
glycine, it is distinguished by the presence of two unusual amino acids,
desmosine and isodesmosine. Like collagen, elastin contains hydroxyproline,
although it may not have the same function of stabilizing the molecule.
Tropoelastin, the soluble precursor molecule of elastin (molecular weight
70,000 to 75,000), is secreted by fibroblasts after it has been synthesized
by ribosomes of the rough endoplasmic reticulum and processed by the Golgi
apparatus. In the presence of copper, lysyl oxidase links together four
lysine molecules to form a desmosine molecule. Isodesmosine is the isomer
of desmosine. The aorta may be fatally weakened by a lack of mature elastin
in animals deprived of dietary copper. Elastin in the arterial system is
produced by smooth muscle cells instead of fibroblasts.
The functional properties of elastin in different tissues such as lung and aorta may be related to differences in the ratio of tropoelastin A to B. The elastin of elastic cartilage might be a different genetic type to that found in the vascular system but, overall, the diversity of different genetic types of elastin is far less than for collagen.
Cells from the vascular system may wander through connective tissues and even compact structures such as tendons have their own lymphatic and vascular supply, something that is not easily seen in an exsanguinated carcass. The vascular cells include a variety of lymphocytes and the plasma cells responsible for antibody production. Eosinophils are cells with bilobed nuclei and numerous cytoplasmic granules readily stained by eosin. The skeletal muscles of cattle, and sometimes sheep, may become inundated with eosinophils (eosinophilic myositis). The affected areas appear as irregular pale lesions and often are detected by meat inspectors looking for muscle parasites. Eosinophils may be attracted to areas of antibody activity and eosinophilic myositis may be an allergic response.
Located around the body are some very interesting cells called mast cells. They are involved in a variety of vital body functions, like resisting disease, but they might also have a special importance for the meat industry. Mast cells occur within the skeletal muscles of meat animals, mainly in the perimysium and epimysium. The numbers of mast cells may be increased in pathological situations and, in denervated muscle, mast cells may move from the central tendon into the belly of the muscle. The cytoplasm of mast cells contains large numbers of metachromatic granules (metachromasia is a color change of dyes such as methylene blue so that metachromatic granules are purple while the surrounding tissue is blue). Mast cells contain heparin and histamine. Heparin prevents the coagulation of blood and histamine increases the permeability of small blood vessels. Heparin also activates the enzyme lipoprotein lipase involved in the accumulation of triglyceride by adipose cells, so there could be some relationship between the distribution of mast cells and the availability of fatty acids for storage in marbling fat in meat. Mast cells also may release a substance that activates cell division in nearby cells. Thus, in both availability of fatty acids for storage and in the formation of new fat cells, the development of intramuscular marbling fat in meat may have some relationship to the distribution of mast cells. Mast cells sometimes come into close contact with skeletal muscle fibers, but most mast cells are located along fine branches of the lymphatic system in the perimysium and endomysium. Mast cells also have been implicated in the regulation of collagenase activity and, thus, may have part to play in the turnover of collagen and its cooking-resistant strength.
Why meat must be a natural food for us
My favourite gem of information about connective tissue concerns the digestibility of elastin. During the digestion of meat in the human gut, elastic fibers are broken down by elastase, an enzyme from the pancreas that would not be there if our evolutionary ancestors had not been at least partly carnivorous. In other words, I have never read of the occurrence of elastin in any human food except meat. So if we have evolved a highly specific enzyme, elastase, to deal with elastin in our food, this can only mean that we are the descendants of meat eaters.
The meat industry always seems to be under attack from the popular press with a stream of bad news stories questioning meat in the human diet. Thus, I derive great peace of mind from knowing scientifically that meat must be a natural component of my diet. This fits nicely with my intuitive belief that, thousands of years ago, my ancestors worked hard all day running down something tasty to bring back to the family, and that the best part of the day was sitting around a camp fire gnawing on a chunk of partly burnt meat, chewing the fat, and washing it down with home-brew.