Collagen, Reticular and Elastic: A Closer Look 17 January 2022 Eben van Tonder
Dedicated to Dawie and Zelda
I’ve been working with animal parts high in collagen for the last few years, being bones, skin, tendons and organs. It is part of broader work aimed at using the complete animal and by-products for human food and animal feed, respectively. On the plant matter side, similar work is undertaken to use the entire plant to eliminate waste and achieve greater bioavailability from both for its functional role in food ingredients perspective as well as nutrition.
On 6 to 9 January 2022, I had an extremely productive weekend with Dawie Hyman and his twin sister Zelda at Boggomsbaai on the South African southern Cape coast. Inspired by them I decided to take a closer look at collagen to continue my investigation of skin/ hide/ tendons for an increased role in providing structure in Frankfurter style sausages both hot and cold. I dedicate this set of personal notes to Dawie and Zelda for their inspiration, motivation, and great friendship.
The structure of my investigation is based on the Ushiki (2002). These are my private notes to review the available scientific data which invariably leads to an application in food processing. I have been challenged to look at the role of collagen networks in providing structural support.
The overview of a few key papers written on the subject of extracellular networks and reviewing how collagen strands are formed in vitro and how the casings industry process collagen into edible casings, provided me with a completely new set of tools for further investigation into manipulating
One of my projects for 2022 will be focused on a production facility being set up in West Africa. Being back in such an environment is very exciting for me because it allows the slow progression of existing methods as opposed to the relentless pressure of a purely R&D environment where there is often unrealistic expectation of progress that must be turned into profit for the project to continue. I have been in such an environment for almost two years now. In contrast to this, a traditional meat processing plant’s first priority is to stick to time tested processes and incremental, almost unnoticeable changes. The relentless nature of the R&D environment taught me much and provided a firm basis for future work, but I believe this to be best done in a factory environment where such work is no more than 10% of daily tasks, giving ample time for careful thought and theoretical work before progressions are attempted.
When I had the opportunity to re-look the matter of bacon production, it was done in such an environment at Woodys Consumer Brands. Best Bacon and Rib System on Earth developed from this.
Apart from my own conclusions, reviewing the structures under discussion is rewarding, enriching and by itself provides great insight into the work of the meat/ plant processor. I prefer giving the interesting sections by quoting the authors verbatim allowing me the luxury of reviewing their work from time to time and discovering new elements for application that I have previously missed.
Philosophically, the work is very important to me as it goes to a fundamental requirement I have in meat and plant processing that it must be done with great respect as it involves the ending of life for the sake of survival. This is more obviously related to animals, but it is a faulty perspective that causes us not to see plants in the same light. Waste is an act of disrespect. Animal and plant waste is at the heart, I believe, of mainly Western disease. I made it a mission to investigate the entire animal carcass and the entire plant and find the great value that nature bestowed on every part of the animal and plant.
With these preliminary thoughts, let’s delve into the subject.
A. Relook at Collagen
Previous notes on collagen and gelatin I made are:
Collagen Marker: Hydroxyproline
I looked at unprocessed beef tendons for inclusion in recipes:
Regarding Meals with Beef Tendons
Every application is in-line with the landmark work on meat emulsions which I feature in: Review of comminuted and cooked meat product properties from a sol, gel and polymer viewpoint
Most of the experimental work was done where I also considered Cell Disruption Technology:
The End of the Rendering Plant
The Power of Microparticles: Disruptor (DCD) Technology
“Collagen fibres present a cord- or tape-shaped 1-20 μm [10-6; millionth] wide and run a wavy course in tissues. These fibres consist of closely packed thin collagen fibrils (30-100 nm [10-9; billionth] thick in ordinary tissues of mammals), and exhibit splitting and joining in altering the number of the fibrils to form a three-dimensional network. Individual collagen fibrils (i.e., unit fibrils) in collagen fibres have a characteristic D-banding pattern whose length ranges from 64 to 67 nm [10-9; billionth], depending on tissues and organs. During fibrogenesis (mechanism of wound healing and repair), collagen fibrils are considered to be produced by fusing short and thin fibrils with tapered ends.” (Ushiki, 2002)
In vertebrates, there are 28 collagen types, and these are classified “according to domain structure, function and supramolecular assembly [for a review, see Mienaltowskiand Birk (2014)]. The most abundant are the fibrillar collagens that form the basis of the fibrils in bony, cartilaginous,fibrous and tubular structures.” (Kadler, 2017)
“Collagen fibrils are complex macromolecular assemblies that comprise different fibrillar collagen types (Hansen &Bruckner 2003). The fibrils are either ‘predominately type I collagen’ or ‘predominately type II collagen’. Predominatelytype I collagen fibrils occur in bony, tubular and fibrous tissues, whereas cartilaginous tissues contain predominately type II collagen fibrils. Collagen fibrils range in length from a few microns to centimetres (Craiget al.1989) and therefore have molecular weights in the tera Dalton range [based on calculations described by Chapman (1989)]. The fibrils provide attachment sites for a broad range of macro-molecules including fibronectin, proteoglycans and cell surface receptors such as integrins, discoidin domain-containing receptors and mannose receptors (Di Lulloet al.2002; Joki-nenet al.2004; Sweeneyet al.2008; Orgelet al.2011). Furthermore, the fibrils vary in diameter depending on species, tissue and stage of development (Parryet al.1978;Craiget al.1989) and in response to injury and repair (Pin-gelet al. 2014). Collagen fibrils are arranged in exquisite three-dimensional architectures in vivo including parallel bundles in tendon and ligament, orthogonal lattices in cornea, concentric weaves in bone and blood vessels and basketweaves in skin.” (Kadelr, 2017)
“Reticular fibers are usually observed as a delicate meshwork of fine fibrils stained black by the silver impregnation method. They usually underlie the epithelium.” (Ushiki, 2002) “The epithelium is a type of body tissue that forms the covering on all internal and external surfaces of your body, lines body cavities and hollow organs and is the major tissue in glands.” (Cleveland Clinic)
“Epithelial tissue has a variety of functions depending on where it’s located in your body, including protection, secretion and absorption.
The organs in your body are composed of four basic types of tissue, including:
All substances that enter or leave an organ must cross the epithelial tissue first.
You have many different kinds of epithelial tissue throughout your body. Some examples of epithelial tissue include:
- The outer layer of your skin (epidermis).
- The lining of your intestines.
- The lining of your respiratory tract.
- The lining of your abdominal cavity.
- Your sweat glands.”
Reticular fibres “cover the surface of such cells of muscle cells, adipose cells or fat cells, connective-tissue cells specialized to synthesize and contain large globules of fat and Schwann cells which are the main glial cells of the peripheral nervous system which wrap around axons of motor and sensory neurons to form the myelin sheath. “Electron-microscopically, reticular fibres are observed as individual collagen fibrils or a small bundle of the fibrils, although the diameter of the fibrils is thin (about 30 nm [10-9; billionth]) and uniform. Reticular fibres are continuous with collagen fibres through the exchange of these collagen fibrils. In silver-impregnated specimens, individual fibrils in reticular fibres are densely coated with coarse metal particles, probably due to the high content of glycoproteins around the fibrils.” (Ushiki, 2002)
“Elastic fibres and laminae (a thin layer or scale of organic tissue) are composed of micro-fibrils and elastin components. Observations of the extracted elastin have revealed that elastin components are comprised of elastin fibrils about 0.1-0.2 μm [10-6; millionth] thick. Elastic fibres and laminae are continuous with networks and/or bundles of microfibrils (or oxytalan fibres) and form an elastic network specific to individual tissues.” (Ushiki, 2002)
Two Systems but Three Types
> Two Systems
“The fibrous components of the extracellular matrix are thereby morphologically categorized into two systems:
a. the collagen fibrillar system (constituents of tendons) as a supporting framework of tissues and cells, and
b. the microfibril-elastin system for uniformly distributing stress to maintain the resilience adapted to local tissue requirements.” (Ushiki, 2002) “Fibrillin microfibrils are extensible polymers that endow connective tissues with long-range elasticity and have widespread distributions in both elastic and non-elastic tissues. They act as a template for elastin deposition during elastic fibre formation and are essential for maintaining the integrity of tissues such as blood vessels, lung, skin and ocular ligaments.” (Thomson, 2019)
> Three Types of Fibres
“Fibrous components of the extracellular matrix are classically divided into three types of fibres: collagen, reticular and elastic. This classification is based on the light microscopic findings (e.g., their shapes, staining properties and arrangements) and chemical properties of these fibres (e.g., MALL, 1896; FOOT, 1928; HAS, 1942); collagen fibres appear as thick and wavy strands stained pink with eosin, while reticular fibres are fine fibres stained dark with the silver impregnation method. Elastic fibres, on the other hand, are observed as a cord or sheet stained purple with resorcin-fuchsin or aldehyde-fuchsin staining, and are highly resistant to boiling water, in contrast with collagen fibres which are easily gelatinized in hot water.” (Ushiki, 2002)
“Electron microscopy has also revealed the ultrastructures of these fibrous components, namely that the collagen and reticular fibres are composed of fibrils with a unique banding pattern (ScHmirr et al., 1942), and elastic fibres comprise both fibrous and amorphous elements (GREENLEE et al., 1966). Advances in biochemistry and immunohistochemistry have also provided detailed information on the nature of these fibrous components, and a number of reviews are available, especially in consideration of the biochemical properties of the fibrous components (e.g., Ross, 1973; SANDBERG et al., 1981; KUHN, 1987; also see books edited by HAY, 1991; YURCHENCO et al., 1994)” (Ushiki, 2002)
a. Collagen Fibres – Basic Structure
“Fresh collagen fibres are colourless strands 1 to 100 μm thick that usually follow a wavy course without branching in tissues. These fibres are stained pink with eosin and green with the Masson trichrome staining method (Fig. la).” (Ushiki, 2002)
“Electron microscopy shows collagen fibres to be a bundle of closely packed thin fibrils with periodical cross striations (SCHMITT et al., 1942) (Fig. lb, c); these unit fibrils are called “collagen fibrils.” ” (Ushiki, 2002)
“In specimens stained with a cationic dye such as Alcian blue and Cupromeronic blue, very thin filaments (less than 10 nm thick) are visible within the bundle of collagen fibrils (Fig. Id) (Scum’, 1980, 1988, 1995; SCOTT and ORFORD, 1981; RUGGERI and BENAZZO, 1984, RASPANTI et al., 2002). These filamentous structures have been considered proteoglycans, including large dermatan sulfate proteoglycans and such small molecules as decorin (FLEISCHMAJER et al., 1991). The proteoglycan filaments appear to connect neighbouring collagen fibrils by transversely and periodically attaching to a specific site of the fibrils.” (Ushiki, 2002)
“These findings indicate that proteoglycans play a role in synchronizing the position of bands in neighbouring fibrils and determine the distance of two neighbouring fibrils to fasten themselves into a bundle.” (Ushiki, 2002)
“Thus, the structure of collagen fibres in which parallel fibrils are bundled with flexible proteoglycans is in accordance with their mechanical properties, since collagen fibres are flexible but offer great resistance to a pulling force.” (Ushiki, 2002)
b. Collagen Fibres – Arrangement
“The size and shape of collagen fibres (i.e., bundles of collagen fibrils) vary depending on tissues and organs, even within the same species. They are usually of a cord- or tape-shape with a width of 1-20 μm, and take a wavy course (Fig. 2a, b), even if they form dense fibrous connective tissues such as the tendon (ROwE, 1985). The wavy arrangement of these fibres probably provides resilience to the fibres themselves, which also serves as a cushion against the direct tension to collagen fibres.” (Ushiki, 2002)
“In loose connective tissues, collagen bundles sometimes run parallel to each other to be twined into a larger bundle, while they come to split and join by changing the number of collagen fibrils, thus forming a three-dimensional meshwork throughout the tissues. Much thinner fibres also often participate in the collagen fibre network (ORBERG et al., 1982; USHIKI and IDE, 1990); these fibres are composed of single or several collagen fibrils, which are produced by leaving the thicker fibers to rejoin them in another portion. This fibrillar network is similar to the network formed by reticular fibres, but does not have argyrophilic properties (i.e. do not readily stained black by silver salts).” (Ushiki, 2002)
This fibrillar network probably plays a role in maintaining a specific arrangement of collagen fibres in each tissue and organ.
c. Collagen Fibres – Structure of the collagen fibrils
“As described above, collagen fibrils are unit fibrils which can be observed in individual collagen fibers by electron microscopy (Fig. 3a, b). These fibrils are cylindrical in shape with a diameter ranging from 10 to over 500 nm (mean diameter about 40-80 nm) in mammals (PARRY and CRAIG, 1984). They show periodical striations (which are alphabetically named the A-E bands) in positively stained sections (ScHmiTT and GROSS, 1948; BRUNS and GROSS, 1974), while the characteristic alternation of dark and light zones is found along the negatively stained fibrils by TEM (TROMANS et al., 1963, OLSEN, 1963). The periodicity of these structures is determined by the length of the two closest D-bands in positively stained fibrils and called D-periodicity. The surface morphology of the collagen fibrils has also been studied by TEM of shadowed materials (GROSS and SCHMITT, 1948) and freeze-fractured replica (MARCHINI and RUGGERI, 1984; RASPANTI et al., 1989), SEM (RASPANTI et al., 1996) and AFM (Fig. 3c, d) (BASELY et al., 1993; USHIKI et al., 1996; YAMAMOTO et al., 1997). These studies revealed the presence of periodical grooves and ridges on the surface of collagen fibrils, which correspond to dark and light zones of negatively stained fibrils, respectively.” (Ushiki, 2002)
Numbers of studies have been devoted to the arrangement of collagen molecules in each fibril (e.g., see review of CHAPMAN and HULMES, 1984). They established that the periodic structure in collagen fibrils arises because the molecules about 300 nm long are assembled in parallel array and are mutually staggered by integral multiples of a D-period. The D periodicity has been estimated by electron microscopy and low-angle X-ray diffraction methods. Low angle X-ray diffraction of collagen fibrils showed that D is close to 67 nm in wet samples, and around 64 nm in air-dried samples (BEAR, 1944; BRODSKY and EIKENBERRY, 1982). By electron microscopy, D varies from 64-70 nm in ultrathin sections, and the variability has been interpreted as the effect of various degrees of shrinkage caused by the dehydration and embedment of samples. In contrast, some authors claim that the D-periodicity differs among collagen fibrils in different organs; for example, the D-periodicity of bovine corneal collagen fibrils was reported by X-ray diffraction to be shorter than that of rat tail tendon collagen fibrils (MARcBINI, et al., 1986).” (Ushiki, 2002)
“On the other hand, the presence of subfibrils in collagen fibrils has been reported by previous investigators using TEM of such samples as glycerinated or denatured tissues (Fig. 4a) (e.g., BOUTEILLE and PEASE, 1971; RAYNS, 1974; LILLIE et al., 1977). These studies indicate that right-turning subfibrils are tightly packed in individual collagen fibrils. RUGGERI et al. (1979) also noticed that the subfibrils have a straight or helicoidal arrangement depending on the types of tissue located. Ushiki (2002) investigated collagen fibrils of the cornea and sclera by AFM, and found a difference in D-periodicity between corneal and scleral fibrils in relation to the inclination angle of the subfibrils (YAMAMOTO et al., 2000a). More precisely, the corneal collagen fibrils (with a D-periodicity of 63 nm) show a helicoidal arrangement of right-turning subfibrils with a 15° spiral angle, while subfibrils in the scleral collagen fibrils (with a periodicity of 67 nm) run almost longitudinally along the fibrillar axis. The relationship between these subfibrils and collagen molecules is still an open question (CHAPN4AN and HULMES, 1984), although several authors consider the subfibrils to be aggregations of small numbers of collagen molecules (VEis, et al., 1967; SMITH, 1968; BOUTEILLE and PEASE, 1971).” (Ushiki, 2002)
“The diameter of collagen fibrils varies from 10-500 nm, depending on the locations of the tissues as well as the age and species of animal (PARRY and CRAIG, 1984). For example, the cornea has collagen fibrils with a regular diameter of about 30 nm, while the diameter of scleral collagen fibrils variously ranges from 25-230 nm (KomAi and USHIKI, 1991, YAMAMOTO et al.,1997). Collagen fibrils in tendons and ligaments show differing diameters with a peak one of 100-200 nm (PARRY et al., 1978). Another example is the diameter of collagen fibrils in the peripheral nerves (UsHIKI and IDE, 1990), where fibrils are thicker in the epineurium than in the endoneurium in various mammals (Fig. 4d). What determines the shape and size of collagen fibrils is an interesting question. Some investigators believe that the copolymeration of collagen molecules with other components of the extracellular matrix may influence the diameter of the fibrils formed (see review of CHAPMAN, 1989), while others have stated the importance of the copolymerization of different kinds of collagen molecules in one fibril (LAPIERE et al., 1977, FLEISCHMAJER et al., 1985, also see review of PROCKOP and HULNIES, 1994).” (Ushiki, 2002)
d. Collagen Fibres – Collagen molecule and its assembly
“Chemical studies have revealed that the type I, II, and III collagen molecules self-assemble into banded fibrils. The shape of these collagen molecules has been studied previously by TEM using shadowing techniques (e.g., SILVER and BIRK, 1984; see also a book edited by MAYNE and BURGESON, 1987), and recently by AFM (SHATTUCK et al., 1994; LIN et al., 1999; YAMAMOTO et al., 2000b). As for the type I molecules, they are thin and flexible threads about 300 nm in length in contrast with type I procollagen molecules with a globular C-terminal propeptide and fuzzy N-terminal propeptide in either end (Fig. 4h, c). The individual collagen fibrils are generally considered to be formed from collagen molecules by their self-assembly process in the extracellular environment (BIRK et al., 1995; KADLER et al., 1996). Our SEM studies showed a process of collagen fibril assembly in cultures of human osteosarcoma cells (HASHIZUME et al., 1999); the findings clearly showed that short and thin collagen fibrils (about 1 μm long and 30 nm thick) with tapered ends fused with each other in a helical direction with their periodicity synchronized with each other, forming longer and thicker collagen fibrils. During this process, the banding pattern from end to end in the fibrils is unidirectional, indicating that the directions of the collagen molecules are uniform throughout the length of the individual fibrils.” (Ushiki, 2002)
>> RETICULAR FIBERS
“Basic structure of reticular fibers in relation to their staining properties. Reticular fibers are fine fibers forming an extensive network in certain organs. By light microscopy, these fibers are not visible in conventional stains such as hematoxylin and eosin, but are stained dark with a silver impregnation method (Fig. 5a) (MALLORY and PARKER, 1927; FOOT, 1928; NAGEOTTE and GUYON 1930). Thus, reticular fibers are also called argyrophilic fibers. The distribution of reticular fibers is rather restricted: they are usually found mainly in the basement of epithelial tissues, the surface of adipose cells, muscle cells and Schwann cells, outside the endothelium of the hepatic sinusoid, and the fibrous reticulum of lymphoid tissues. These fibers have a diameter of less than 2 μm. Although there are several modifications of BinscHowsKY’s impregnation method (MAREscH, 1905), a method reported by IsHII and ISHII (1965) yields specimens with suitable representation showing the fine structure of reticular fibers. In these specimens, reticular fibers are meshworks of very fine, dark fibrils, and are continuous with thin and reddish collagen fibers (Fig. 5a).” (Ushiki, 2002)
“Electron microscopy shows reticular fibers as individual collagen fibrils or a small bundle of collagen fibrils (Fig. 5h). These collagen fibrils have striations with a characteristic D-banding pattern similar to fibrils in collagen fibers, but their diameter is rather thin and uniform, ranging from 20-40 nm. Observations of silver-impregnated sections by TEM and SEMI (using backscattered imaging) show that individual collagen fibrils in reticular fibers are densely coated with coarse metal particles, while fine granular particles are sparsely found on fibrils in collagen fibres (Fig. 5c-e) (ScHwARTz, 1953; USHIKI, 1992b). This indicates that the size and density of metal precipitation particles determine the difference in tone between reticular fibers and collagen fibrils light-microscopically.” (Ushiki, 2002)
“Reticular fibers are also PAS-positive and have an affinity to cationic stains such as Ruthenium Red (IDE et al., 1989). These findings suggest that the surface of the individual fibrils in reticular fibers is embedded in an abundance of glycoproteins, which produce the stainability of fibers described above.” (Ushiki, 2002)
“Chemical and immunohistochemical studies, on the other hand, have revealed that reticular fibers, in contrast to collagen fibers composed of collagen type I, comprise mainly collagen type III (FLEISCHIVIAJER et al., 1980; MONTES et al., 1980) in association with other types of collagen (e.g., collagen type V), glycoproteins (STENNIAN and VAHERI, 1978), and proteoglycans/ glycosaminoglycans (MONTES et al., 1980; NISHIMURA et al., 1996). The difference in collagen type between collagen fibers and reticular fibers might be related to the diameter of the fibrils in the two fibers, although further studies will be needed in this point.” (Ushiki, 2002)
a. Reticular Fibers – Arrangement
“The arrangement of reticular fibers is important for understanding the functional role of the fibers in tissues and organs, and was first studies mainly by light microscopy (PLENK, 1927; NAGEOTTE and GUYON, 1930). SEM further revealed the threedimensional architecture of reticular fibers in relation to the surrounding components (e. g., MOTTA, 1975; SAWADA 1981; USHIKI and IDE, 1986). The method introduced by OHTANI (1987) is also useful for visualizing the fibrillar arrangement more directly and precisely by SEM, since it successfully removes cellular elements, elastic fibers, and basal laminae without any severe damage to the collagen fibrils.” (Ushiki, 2002)
“These findings show that reticular fibers form a delicate network of fine fibrils which underlie the basal lamina of such cells as epithelial, muscle and Schwann cells (OHTANI, 198.8, OHTANI et al, 1988, 1991, USHIKI and IDE, 1990, MURAKUMO et al., 1993). The firm attachment of the individual fibrils with the basal lamina indicates that the collagen fibril meshwork and the basal lamina, as a whole, form a distinct structural unit for the demarcation and support of cellular components (USHIKI and IDE, 1986; USHIKI et al., 1990).” (Ushiki, 2002)
“The reticular arrangement of the fibrils is also suitable for providing a space for molecular movement in the extracellular fluid. Concerning lymphoid tissues, reticular fibers act as a skeletal framework and support vessels and lymphatic sinuses within the tissues (USHIKI et al., 1995).” (Ushiki, 2002)
“Reticular fibers thus differ in structure, arrangement, and function from collagen fibrils, but are continuous with collagen fibers. In this sense, the two fibrous components are considered to form an extensive network of collagen fibrils as the collagen fibrillar system (USHIKI, 1992b).” (Ushiki, 2002)
>> Elastic Fibres
“Elastic tissues of the body owe their mechanical properties to the protein elastin. In complete contrast to the highly orientated, inextensible collagen fibre the elastin fibre occurs naturally in a contracted state and is capable of reversible extension to about double its length. Elastin is therefore generally found in the form of fibres. It is also found as membranes in the elastic ligaments, elastic blood vessels, and other compliant tissues such as lung and skin. The elastic arteries contain concentric layers of elastic fibres, and the ligaments have parallel fibres (Partridge, 1962).” (Bailey, 1878)
“Elastin was at first defined solely by its histological appearance. Largely through the work of Partridge and his colleagues, a precise chemical definition of elastin was reported in 1958. However, it was not until the cross-links were identified by this group in 1963 (Thomas et al., 1963) that the field opened up and a significant understanding of the relationship of structure to function began to emerge.” (Bailey, 1978)
“Like collagen, elastin is an extracellular insoluble polymeric protein; hence its intracellular biosynthesis as a soluble monomer, its extracellular aggregation and subsequent stabilisation by crosslinking considerably resemble the biosynthesis of collagen fibres.” (Bailey, 1978)
a. Elastic Fibres – Basic Structure
“Elastic fibers are generally twisted or straight strands stained by a resorcin-fuchsin or aldehyde-fuchsin method (Fig. 7a); these fibers are about 0.2 – 1.5 μm and sometimes branch to form a coarse network in loose connective tissues. In dense elastic tissues such as the aorta, elastic fibers fuse to form flattened sheets, or elastic laminae. Biochemically, elastic fibers are highly resistant to boiling water, in contrast with collagen fibrils which are easily gelatinized in hot water (RICHARDS and GIES, 1902).” (Ushiki, 2002)
“By TEM of ultrathin sections stained with uranyl acetate and lead citerate, elastic fibers are seen to consist of amorphous and fibrous components (Fig. 7b) (GREENLEE et al., 1966; Ross and BORNSTEIN, 1969). Amorphous components are densely stained with tannic acid treatment by TEM (Fig. 7c) (MIZUHIRA and FUTAESAKU, 1972) and are composed of substances which can be purified in boiling water and are recognized biochemically as the protein named elastin (e. g., see a review of Ross, 1973). Elastin endows elastic fibers with the characteristic property of elastic recoil. Fibrous components, on the other hand, correspond to the microfibrils which were recognized by TEM in various tissues and organs by Low (1962). Microfibrils are 10 nm in diameter and composed of various glycoproteins, including fibrillin (SAKAI, et al., 1986) and the amyloid P component (INouE and LEBLOND, 1986; INOUE et al., 1986).” (Ushiki, 2002)
“By conventional SEM, elastic fibers are observed as cobwebbed cords entangled with microfibrils (Figs. 8a, b, 11b) (USHIKI, 1992b).” (Ushiki, 2002)
b. Elastin Fibres – Arrangement
“Since elastic fibres are intermingled with collagen fibrils and cellular elements in tissues, it is usually difficult to demonstrate their arrangement both extensively and three-dimensionally. For this reason, previous SEM investigators have attempted to extract elastic fibres by autoclaving tissues (GRu’r et al., 1977), or by utilizing treatments with chemical agents and enzymes: e.g., guanizinium chloride, collagenase, sodium hydroxide, and formic acid (KUHN, 1974; KEWLEY et al., 1977; WASANO and YAMAMOTO, 1983; SONG and ROACH, 1985; CRISSMAN, 1987). These methods selectively remove non-elastin components including microfibrils, collagen fibrils and cellular elements, and are effective for observing the architecture of elastin components in tissues by SEM (Figs. 8c, d, 9c, d). On the other hand, the treatment of tissues with a KOH method is effective for observing the special relationship between elastin components and cellular elements by SEM, since this method removes collagen fibrils and basal laminae while leaving cellular and elastin elements unchanged at their original shapes and locations (Fig. 9a, b) (USHIKI and MURAKUMO, 1991).” (Ushiki, 2002)
“Through these studies, several investigators have demonstrated that elastin components form a continuous network or sheet with a smooth surface (KuHN, 1974; WASANO and YAMAMOTO, 1983), while others have considered them as a fibrous network or sheet composed of fibrils about 0.1-0.2 μm (KEWLEY et al., 1977; HART et al., 1978). Our previous studies revealed that the surface structure and organization of elastin components are changeable depending on the procedures after extraction, and yielded evidence that the elastin component including aortic laminae are fibrous when extracted tissues are adequately treated (UsHIKI and MURAKUMO, 1991; USHIKI, 1992a).” (Ushiki, 2002)
“Elastin components show morphological features specific to individual tissues and organs (UsHIKI and MURAKUMO, 1991). A typical organization of elastin fibers in the loose connective tissue is a loose network of elastin fibers about 0.2-1.5 μm thick (Fig. 8c). An elastin sheet lining the serosal covering of the mesothelium consists of fine fibers ranging from 0.1- 1.0 μm thick, which run in various directions two-dimensionally and are elaborately interwoven, forming a delicate lacework-pattern (Fig. 9a, b). Elastic laminae in the aorta appear as a solid sheet about 2 μm thick with numerous oval fenestrations of varying diameters from 1-10 μm (Figs. 8d, 9d). These laminae appear to be composed of fibrous structures about 0.1-0.2 μm thick. It is therefore evident that extracted elastin components are basically composed of thinner fibrils about 0.1-0.2 μm thick (Fig. 9h), even though some investigators further recognized very thin (3-4 nm thick) elastin filaments by TEM of negative-stained or freeze-etched specimens (GOTTE et al., 1974; FORNIERI et al., 1982). The elastin fibrils are present individually or in bundles, and so form elastin fibrils, fibers and/or laminae in individual tissues (Fig. 10) (UsHIKI and MURAKUMO, 1991).” (Ushiki, 2002)
“The organization of elastin fibers and laminae apparently influences the resilience of tissues suitable for their mechanical properties. Concentric elastic laminae with connecting interlaminar fibers are suitable for distributing blood pressure uniformly and effectively to the vascular wall. The elastic sheet lining the mesothelium is believed to give elasticity to the serous membrane and protect the mesothelium against any distention and contraction of such organs as the lung and urinary bladder.” (Ushiki, 2002)
> Microfibril-elastin network system
“Microfibrils are usually present in and around elastin fibers, where they appear to be arranged in random directions to the elastin fibers (Fig. 8b) 1992b). In stretched fibers, the microfibrils change in their direction along the fibers, in response to stretching of the elastin fibers. Microfibrils often leave elastin fibers to form a bundle or cobwebby meshwork in various tissues (Figs. 8a, 11b).” (Ushiki, 2002)
“Light-microscopically, characterized fibrous structures are observed when sections are treated with peracetic acid before aldehyde-fuchsin staining (Fig. 11a) (FunmER and LILLIE, 1958). These fibrous structures are continuous with clastic fibres and are called oxytalan fibres. By TEM, the oxytalan fibres are observed as a bundle of microfibrils (Fig. 11b) (COTTA-PEREIRA et al., 1976). The oxytalan fibres can be also found in the zonule fibres of the eye and in the dermis where it connects the elastic fibres to the basal lamina. As far lymphatic vessels, oxytalan fibres act as anchoring fibres which connect the elastic fibres and lymphatic endothelium, thus preventing the collapse of initial lymphatics in tissues (Gum et al., 1990). In addition, so-called elaunin fibres (GAw-LIK, 1965) have intermediate characteristics between oxytalan fibres and elastic fibres by TEM (COTTA-PEREIRA et al., 1976). These findings indicate that microfibrils and elastin fibrils produce oxytalan fibres, elaunin fibres, and elastic fibres to form, as a whole, the microfibril-elastin fibre system which plays a role in maintaining the resilience adapted to local tissue requirements.” (Ushiki, 2002)
> Fibrillar System
What is the fibrilar system? Fibrils are structural biological materials found in nearly all living organisms. It differs from fibres which are longer than it is wide and filaments which are long-chain protein monomers as found in muscles and hair. Fibrils tend to have diameters ranging from 10-100 nanometers (whereas fibres are micro to milli-scale structures and filaments have diameters approximately 10-50 nanometers in size). Fibrils are not usually found alone but rather are parts of greater hierarchical structures commonly found in biological systems.
Wikipedia elaborates on the structure and mechanics of fibrils as follows. They “are composed of linear biopolymers and are characterized by rod-like structures with high length-to-diameter ratios.
Visualise length to diameter ratios. The bigger the number, the longer the strand. (Image from US Neodymium Magnets)
“They often spontaneously arrange into helical structures. In biomechanics problems, fibrils can be characterized as classical beams with a roughly circular cross-sectional area on the nanometer scale. As such, simple beam bending equations can be applied to calculate flexural strength of fibrils under ultra-low loading conditions. Like most biopolymers, stress-strain relationships of fibrils tend to show a characteristic toe-heel region before a linear, elastic region. Unlike biopolymers, fibrils do not behave like homogeneous materials, as yield strength has been shown to vary with volume, indicating structural dependencies. Hydration has been shown to produce a noticeable effect in the mechanical properties of fibrillar materials. The presence of water has been shown to decrease the stiffness of collagen fibrils, as well as increase their rate of stress relaxation and strength. From a biological standpoint, water content acts as a toughening mechanism for fibril structures, allowing for higher energy absorption and greater straining capabilities.
Fibrils mechanical strengthening properties originate at the molecular level. The forces distributed in the fiber are tensile load carried by the fibril and shear forces felt due to interaction with other fibril molecules. The fracture strength of individual collagen molecules is as a result controlled by covalent chemistry between molecules. The shear strength between two collagen molecules is controlled by weak dispersive and hydrogen bond interactions and by some molecular covalent crosslinks. Slip in the system occur when these intermolecular bonds face an applied stress greater than their interaction strength.”
“Intermolecular bonds breaking do not immediately lead to failure, in contrast they play an essential role in energy dissipation that lower the stress felt overall by the material and enable it to withstand fracture. These bonds, often hydrogen bonding and dispersive Van der Waals interactions, act as “sacrificial” bonds, existing for the purpose of lowering stress in the network. Molecular covalent crosslinks also play a key role in the formation of fibril networks. While crosslinking molecules can lead to strong structures, too much crosslinking in biopolymer networks are more likely to fracture as the network is not able to dissipate the energy, leading to a material that is strong but not tough. This is observed in dehydrated or aged collagen, explaining why with age human tissues become more brittle.
I quote two interesting comments from Gross (1958).
“Factors which may regulate the rate of fibril formation in systems in vitro are of interest from a physiological viewpoint for the clues they may give concerning mechanisms in viva, and from a physical chemical point of view for the light, they may shed on intermolecular reactions. Collagen soluble in cold neutral salt solutions has the interesting property of precipitating, on warming to body temperature,
as a rigid gel composed of fibrils with the characteristic axial periodicity of native collagen. It has been postulated that fibrils are formed under similar conditions in the extracellular tissues by spontaneous polymerization of collagen molecules secreted by the fibroblast into the ground substance.” (Gross, 1958)
In their paper Gross and Kirks (1958) describe some of the environmental factors which can influence the rate of fibril formation in neutral solutions of collagen. I made the paper available for download in the reference section below.
In their summary, they list the accelerators of fibril formation being SCN-, HCOB-, I-, Br-, F-, Cl-, in that order of effectiveness as measured by their relative ability to reverse the inhibitory effect of urea. Lysine
and Li+ were also strong accelerators of gelation.
Karl Kadler did a mini review of collagen fibrillogenesis in response to him receiving the Fell Muir Prize for 2016 by the British Societyof Matrix Biology. I have his article available for download in the referense section.
Fibrillogenesis “is the process by which triple helical collagen molecules assemble intocentimetre-long fibrils in the extracellular matrix of animals. The fibrils appeared abillion years ago at the dawn of multicellular animal life as the primary scaffold fortissue morphogenesis. The fibrils occur in exquisite three-dimensional architecturesthat match the physical demands of tissues, for example orthogonal lattices in cornea, basket weaves in skin and blood vessels, and parallel bundles in tendon, ligament and nerves.” (Kadler, 2016)
“Creating collagen vibrils in vitro is of the greatest interest for our study. Kadler refers to the work of Gross and mentions other researchers when he writes, “Gross (Gross & Kirk 1958), Wood & Keech (Wood & Keech 1960), Hodge & Petruska (Hodge 1989), Silver (Silver & Trelstad 1980) and Chapman (Bard & Chapman 1968), to name a few, showed that exposure of animal tissues (typically skin and tendon) to weak acidic solutions (typically acetic acid) or neutral salt buffers yielded a solution of collagen molecules that when neutralized and warmed to approximately 30°C, produced elongated fibrils that had the same alternating light and dark transmission electron microscope banding appearance as fibrils occurring in vivo (Holmes & Chapman 1979).” (Kadler, 2016)
“The characteristic banding pattern of the fibrils arises from D-stagger-ing of triple-helical collagen molecules that are 4.49Dinlength (where D is 67 nm, to a close approximation). The electron-dense stain used at neutral pH penetrates more readily into regions of least protein packing (the ‘gaps’) between the N- and C-termini of collagen molecules that are aligned head-to-tail along the long axis of the fibril. The fact that fibrils with D-periodic banding could be formed in vitro from purified collagen showed that all the information required to form a collagen fibril was contained within the amino acid sequence and triple helical structure of the collagen molecule (Hulmeset al.1973).” (Kadler, 2016)
“Previous TEM studies have demonstrated that bundles of microfibrils first appear during elastogenesis (ALBERT, 1972; SPICER et al., 1975). Elastin components are produced as the deposition of a small amount about 0.1 μm wide within the bundle. As the elastin components increase in number, they fuse together to become mature elastic fibers. According to our SEM studies on the extracted elastin components in the developing aorta, elastic laminae are first observed as a meshwork of fine elastin fibrils which increases in its density of elastin fibrils in the meshwork to become an elastic lamina with numerous fenestrations (Fig. 12a, b). These findings support the idea that microfibrils produce a fundamental framework of the microfibril-elastin system, which is added by the deposition of elastin fibrils about 0.1 μm thick, thus forming elastic fibers and laminae continuous with oxytalan fibers. Elaunin fibers are considered a transition form between oxytalan fibers and elastic fibers.” (Ushiki, 2002)
Conclusion by Ushiki (2002)
“The present review describes the features of three major fibrous components: collagen, reticular and elastic fibers. For a comprehensive understanding of the fibrous components in connective tissues, we propose categorizing them into two systems (Fig. 13): the collagen fibrillar system and microfibril-elastin system. The collagen fibrillar system acts as a supporting framework of tissues and cells, where reticular fibers connect collagen fibers with the basal laminae of such cells as epithelial, muscle, adipose and Schwann cells. The microfibril-elastin system is composed of microfibrils and elastin fibrils, which use different proportions of the two components to produce elastic fibers, elastin fibers and oxytalan fibers. The microfibril-elastin system thus plays a role in distributing stress forces uniformly in tissues.” (Ushiki, 2002)
The above discussion has several applications in the sausage-making industry. When one has raw material available such as hides/ skin, one must consider the options what the material can be used for:
- Skin or tendon emulsion can be made where the object is water binding;
- Collagen or skin chunks can be made, not to retain water but to provide firmness to the sausage matrix. In this instance it will be advisable not to hydrate the collagen or skins.
- If a fine emulsion is made with no show pieces, one can pre-prepare the fine skins/ tendons in the bowl chopper or micro-cutter seperately before the susage blend is made. Add the dry skin/ tendons towards the end of the blending process after all the water has been added.
- One must cook/ smoke with optimal crosslinking in mind.
- If unhydrated skin/ tendons are used, it should be possible to use a higher inclusion ratio than if its used in hydrated form.
- Hydration has an impact on the “solidness” of the sausage and its pastiness (resistance to bite). Amount used and hydrated or unhydrated are two of the most basic parameters which must be tested for when formulating a sausage with a high percentage of skin/ hide/ tendons included.
- I will incorporate the ingreadients tested by Gross (1958) and dealt with under Fibrilogenesis above, in my trails.
- Where resistance to boiling water is required (no gelatinization), I will be interested in, for example, the aorta which is rich in elastin fibers.
B. How are Collagen Casings made?
In this last section, I want to expose myself to more techniques used in the industry to manipulate skin or tendons. How do they do it? I first quote a traditional meat-man, explaining the basic process of producing collagen casings from beef hides.
Two Process overviews
The corium layer (splits) of USDA Approved cattle hides is extruded from between the grain (hair) layer and the fat and muscle layer. The hide consists essentially of collagen. Protein and water are chopped and mixed with lactic acid and cellulose fibers causing them to swell and form a slurry.
The acid-swollen slurry is de-aerated under vacuum and is then homogenized and filtered to tease the collagen fibers apart.
The resultant slurry is again de-aerated and stored in chilled tanks.
It is then extruded through a die with counter-rotating sleeves, which “weave” the collagen fibers together as they pass through the die. The slurry, which is now in the form of a casing, passes directly into a concentrated coagulating solution of an inorganic salt.
The casing is chemically treated in the process machine to cross-link the fibers and give the casing integrity. The casing is then washed and plasticized with glycerin.
The casing is then dried, partially re-humidified and wound on reels. The reels are taken to the shirring area where the collagen casing is shirred on machines similar to the type commonly used in the shirring of regenerated cellulose casings. (askthemeatman)
One of the many reasons why I think it’s counterproductive to take out a patent on certain inventions, especially chemical in nature, is because of what I am just about to do now. I refer you to US3413129A for Johnson and Johnson, invented by Emanuel R Lieberman. It is an invention of an alternative way to produce edible collagen casings. The invention was done in 1968 which makes this one of the first of its kind patents. I give the description next.
ABSTRACT OF THE DISCLOSURE
A collagen casing for sausages of the weiner or frankfurter type is manufactured by extruding a mass of acid-swollen collagen fibrils obtained from animal hide and cellulose fibers into an ammonium sulfate coagulating bath, hardening the extruded casing in an aqueous solution containing from about 0.15 percent to 10 percent by weight of ammonium hydroxide and a non-toxic ammonium salt, plasticizing the hardened casing, drying the casing. While inflated, heating the dried casing from C. to C. over a period of 8 to 12 hours, and then heating said casing for an additional 12 to 24 hours at about 80 C. This invention relates to an improved collagen casing and more particularly to extruded collagen casings that have been treated with an aqueous solution of ammonium hydroxide. While not limited thereto, the present invention is adapted to being utilized as a casing for sausages of the weiner or frankfurter type. Prior to the present invention, this type of sausage was either prepared by using expensive natural casings or inedible cellulose casing to contain the meat emulsion during the smoking and cooking process. The inedible cellulose casing must be removed by the manufacturer before the wieners are packaged for sale. The resulting product is known in the meat industry as a skinless Wiener.
There has long been a need for an extruded collagen casing that would be edible, non-toxic and sufficiently strong to stand up under stuffing, linking, smoking, washing and cooking. It is now known that edible casings for pork sausage may be prepared by extruding a tubular body from a fluid mass of swollen collagen fibrils, hardening this tubular body in the wet state and drying the collagen casing so produced. A method of producing such collagen casings is described in US. Patent No. 3,123,482.
Extruded collagen casings that are suitable for the manufacture of fresh pork sausages may not be entirely satisfactory for the production of sausages of the Wiener or frankfurter type. This is due to the differences in processing pork sausages and wieners. Thus, a meat emulsion of the pork sausage type may be stuffed, linked by twisting on a Famco linking machine, and packaged for sale without cooking. Sausages of the Wiener or frankfurter type, however, are linked on a Ty-linker, racked on a stick, smoked at temperatures from about F. to about F. or F. for several hours, rinsed with hot water at about 180 F. to F. for several minutes, and then rinsed with cold water for several minutes. The consumer may cook this product by deep fat frying, i.e., the frankfurter is plunged into a cooking oil that has been heated to 350 F. Sometimes such frankfurters have been chilled or even frozen prior to such cooking, so that the casings are subjected to great thermal stresses and pressures from steam or vapor generation. It will be appreciated, therefore, that a collagen casing used in the production of frankfurters must of necessity have a high wet strength to survive the more vigorous treatment in the linker.
An additional requirement for the frankfurter casing is that the casing should not become wrinkled and lose “ice bonding to the meat during smoking or the hot and cold rinses that follow smoking. In other words, the casing must be sufficiently elastic (not permanently deformed) so that the stress does not relax during the smoking rising cycle. On chilling after smoking, the meat contracts slightly (becomes more dense) and the casing must also shrink or the finished product will have a poor appearance.It is an object of the present invention, therefore, to produce a new and improved extruded collagen casing adapted to be utilized as a casing for sausages of the weiner or frankfurter type.
It is another object of the present invention to produce an edible casing that is exceptionally tender when eaten, yet sufficiently strong to survive linking in the Ty linker.
It is a further object of this invention to produce an edible collagen casing that will retain a smooth symmetrical appearance after smoking.
Still another object of this invention is to provide an edible collagen casing suitable for use with pork sausages or frankfurters that will not burst or peel off during cooking.
In accordance with the present invention, it has been discovered that a much improved Wiener casing may be produced by the procedure described in US. Patent No. 3,123,482 if a dilute solution of ammonia is substituted for the alum hardening agent. The use of ammonia in place of alum produces a casing that is more fragile and difficult to process during the manufacturing process. Yet the difficulty in processing is more than compensated for by the improvement in appearance and in-use performance of the finished casing.
Numerous laboratory and field tests have demonstrated that when an ammonia solution is substituted for the alum hardening solution in the process identified above, the product obtained more closely resembles natural casing. This difference is particularly apparent after stuffing, linking, smoking and cooking.
The fluid mass of swollen collagen that is extruded to form the casings of the present invention may contain from about 3.2 percent to about 4 percent by weight of collagen (calculated on the basis of dry collagen Weight) a non-collagenous filler such as cellulose or starch. If a fibrous filler such as cellulose is employed the amount may vary from about 5% to about 42% of the total solids present in the extrusion mixture. Smaller amounts of starch may be substituted for a part of or all of the cellulose.
The ammonia hardening bath may contain from about 0.15% to 10% ammonium hydroxide and from about 1% to 10% of a salt such as ammonium sulfate or ammonium lactate. To improve the wet tensile strength and elasticity of the ammonia hardened casing it is desirable to add a small amount of reducing sugar to the casing as described in US. Patent No. 3,151,990. The amount of reducing sugar employed, however, is only about one tenth of the amount required to treat an alum hardened casing. Indeed, the sugar treatment may be eliminated entirely if the ammonia hardened casing is heat-cured for a prolonged period of time, i.e., about 24 hours at 80 F.
Suitable sugars for the treatment of ammonia hardened collagen casings are reducing sugars which have a free aldehyde or keto group that is not in glucoside combination with other molecules. Examples of such reducing sugars are erythrose, threose, arabinose, ribose, xylose, cyclose, fucose, mannose, glucose (dextrose), galactose, fructose (levulose), etc. These sugars may be most conveniently applied to the collagen casing in the form of dilute solutions. The amount of sugar present in solution is related to the dwell time of the casing in the solution and the reactivity of the sugar used, and may vary from about 0.005 percent to about 0.08%. It is preferred to add the reducing sugar to the plasticizing bath, which bath follows the washing step and is the last bath contacted before the casing is dried.
Alternatively, a smoke solution derived from wood smoke vapor may replace the reducing sugar in the plasticizing bath. Smoke flavoring solutions contain a large number of acetic, phenolic and carbonyl (aldehyde) compounds that will react with collagen and improve the physical properties of the ultimate casing. The chemical constituents of smoke flavoring are discussed in’an article by Hollenbeck and Marinelli, Proceedings of the Fifteenth Research Conference, sponsored by the Research Council of the American Meat Institute Foundation of the University of Chicago, page 67 (1936). The smoke products identified in that article have been found useful in processing the ammonia hardened casings of the present invention.
The casing after it leaves the plasticizing bath is inflated and dried in a rapid stream of air and then heat cured in a forced draft oven, raising the temperature slowly from 40 C. to 80 C. during an 8 to 12 hour period. The heat treatment at 80 C. is continued for an additional 12 to 16 hours.
It will be understood that the foregoing general description and the following detailed description, as well, are exemplary and explanatory but do not restrict the invention.
The process for the manufacture of ammonia-cured collagen casings of the present invention may be more fully understood from the following detailed description and examples taken in connection with the accompanying drawings, wherein:
I include the full patent below for download.
Conclusion to Collagen Casings
The two processing overviews is enough to arm the NPD specialist with an ample starting point for investigating the production of skins/ hides and tendons to fulfil various function in fine emulsions. I refer you to the basic understanding of these emulsion type sausages in Review of comminuted and cooked meat product properties from a sol, gel and polymer viewpoint
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Bailey, A. J. (1978) Collagen and elastin fibres. J. clin. Path., 31, Suppl. (Roy. Coll. Path.), 12, 49-58
Gross, J., Kirks, D. (1958) The Heat Precipitation of Collagen from Neutral Salt Solutions:
Some Rate-Regulating Factors”
Kadler, K. E. (2017) Fell Muir Lecture: Collagen fibril formation in vitro and in vivo. https://doi.org/10.1111/iep.12224
Jennifer Thomson, Mukti Singh, Alexander Eckersley, Stuart A. Cain, Michael J. Sherratt, Clair Baldock,
Fibrillin microfibrils and elastic fibre proteins: Functional interactions and extracellular regulation of growth factors, Seminars in Cell & Developmental Biology, Volume 89, 2019, Pages 109-117, ISSN 1084-9521, https://doi.org/10.1016/j.semcdb.2018.07.016. (https://www.sciencedirect.com/science/article/pii/S1084952118300181)
Ushiki, T.. (2002) Collagen Fibers, Reticular Fibers and Elastic Fibers. A Comprehensive Understanding from a Morphological Viewpoint. Division of Microscopic Anatomy and Bio-imaging, Department of Cellular Function, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan