Notes on Collagen and Gelatin
by Eben van Tonder
20 July 2020
We are considering source material for fine emulation usages. Here we introduce collagen protein and gelatin. “Collagen is a family of closely related, although chemically distinct, extracellular matrix molecules. All collagen molecules consist of three polypeptide chains called α-chains. Each of these is coiled into a right hand triple helix. In some collagen types all three α-chains of the molecule are identical, while in others the molecule contains two, and in some cases even three different α-chains. In all collagen types, the polypeptide chains also have non-collagenous domains of various sizes. The triple-helix regions of the molecule characteristically have a repeating triplet amino acid sequence -Gly-X-Y-, where X and Y denote amino acids other than glycine.” (Harding, et al.,1992)
Irrespective of the animal species, collagen fibres have an amino acid composition in which glycine makes up around one-third of the total residues and the amino acids proline and hydroxyproline a further 15-30%. “Hydroxyproline is of very limited occurrence in proteins, the only other mammalian protein in which it occurs being elastin (2 %). Collagen is also the only protein reported to contain more than about 0.1 % hydroxy-lysine.” (Courtis and Ward, 1977) The presence of hydroxyproline is the marker used to determine the approximate inclusion of collagen into meat products. “Hydroxyproline is a part of collagen and occurs only in sinews, bones, gristle and skin.” (Buchi) It is therefore taken that a high percentage of hydroxyproline is indicative that a large percentage of collagen was added to the meat formulation.
Elast is the main protein component of the elastic fibers, and differs from proteins in that it has no triple-helical collagen-like domain. Nevertheless, the polypeptide chain has repeated -Gly-X-Y sequences, which contain 4-hydroxyproline but no hydroxylysine. The 4-hydroxyproline content of elastin may vary greatly, usually being about 10 to 15 residues per 1000 amino acids, but ranging up to about 50 residues per 1000 in special situations.
When solutions of collagen are heated at about 40°C or above, denaturation occurs and the helical structure is lost. (Courtis and Ward, 1977)
Reactivity of Collagen
“The reactive amino acid side chains all project outwards from the main body of the triple helix and in soluble collagens should, therefore, be accessible to all chemical reagents. In the compact fibrous forms of collagen, however, there is no guarantee that this will be so.” (Ward and Courtis, 1977)
“Native collagens, even the soluble forms, are very resistant both to the action of enzymes and chemicals, a property almost certainly related to the stable helical conformation of the molecule and the protection this affords to the peptide bonds of the individual chains.” (Ward and Courtis, 1977)
“Dilute acids lead to solubilization of varying amounts of collagen and on the basis of current hypotheses this would appear to be due to the action of the acid on labile intermolecular links of the Schiff’s base type (see p. 19). Attack on the collagen molecule itself appears to be negligible even at low pH values provided the temperature is below 20°C.” (Ward and Courtis, 1977)
“A long treatment in alkali is the traditional prelude to the conversion of collagen to gelatin. Complete breakdown of native collagen to small peptides can only be achieved by the action of a group of bacterial enzymes, the collagenases, the best documented being that isolated from Cl. histolyticum (see Mandl, 1961). These enzymes are specific for the -Gly-Pro-X-Gly-(Pro or Hypro-) sequence, cleavage occurring to give an N-terminal glycine. Even with such enzymes however, complete solubilization and breakdown of many collagenous tissues, e.g. mature ox hide collagen is difficult. Tadpole collagenase is even more specific in its action.” (Ward and Courtis, 1977)
Chang et al. (2011), investigated the effects of heat-induced changes in intramuscular connective tissue (IMCT) and collagen on meat texture properties of beef Semitendinosus (ST) muscle. Their conclusions are instructive. They compared heating in a water bath and microwave heating.
-> Collagen Content
They found that “the mean content for total collagen of the raw meat was 0.66 ± 0.09 % (wet basis) and was within the normal range (lower than 1% wet weight). Total collagen content of microwave treated sample was higher compared to water bath treatment before heating temperature up to 80◦C, and showed significant differences (p 0.05) were found for soluble collagen content between water bath and microwave treated samples, and the same changes tendency were presented as total collagen content with increase in the temperature during water bath and microwave heating.” (Chang et al., 2011)
-> Collagen Solubility
“Changes of collagen solubility of water bath and microwave treated samples were irregular. There was an unaccountable variation in collagen solubility with a maximum at 75◦C for water bath heated meat. Collagen solubility changed unaccountably throughout heating due to juice loss and collagen solubilization. For microwave heating, the highest collagen solubility was found when heated to 90◦C, and could be attributed to the conversion of collagen to gelatin occurs at this temperature range. At 65◦C, collagen solubility of water bath and microwave treated samples were relatively higher simultaneously, partly because of the shrinkage effect of perimysial and endomysial collagen at about 65◦C (proved in DSC analysis, data not shown). According to the reports of Li et al., low correlations were found between meat-Warner-Bratzler shear force (WBSF) values and total collagen and collagen solubility, although previous data indicated a high relationship between peak shear force and collagen content for beef.” (Chang et al., 2011)
We will return to the solubility of collagen when we look at soluble collagen chemistry.
-> Instrumental Texture Profile Analysis (TPA)
“TPA provides textural change of meat during thermal treatment. It was found that the thermal conditions (internal temperature) and heating modes had significant effects on all the TPA parameters of meat except for resilience (Fig. 1G). Hardness, as a measure of force necessary to attain a given deformation, gave a different response to the different heating methods and temperatures applied. Hardness (Fig. 1A) of microwave treated sample was higher compared to water bath treatment at 65◦C, and showed significant differences (p < 0.05) in the temperature range from 75◦C to 90◦C. Hardness of water bath heated meat showed a maximum at 60◦C. Changes of adhesiveness (Fig. 1B) for water bath and microwave treated sample were irregular with increase in internal core temperatures, and there were no significant differences except for 75◦C between both thermal treatments.” (Chang et al., 2011)
“Springiness is an important TPA parameter and the date on springiness (Fig. 1C) for microwave heated meat had a changing point at 65◦C, and the meat springiness of water bath heated was higher compared to microwave treatment after 65◦C. This parameter seems to be affected by myosin and α-actinin denaturation, which occurs in this temperature range. Springiness of meat is likely related to the degree of fiber swelling which in turn should be reflected in the fiber diameter. As discussed above, the main changes of springiness during heating were consistent with the thermal shrinkage of intramuscular collagen at around 65◦C.” (Chang et al., 2011)
“Cohesiveness contributes to the comprehensive understanding of viscoelastic properties including tensile strength. Chewiness is the energy required to masticate a solid food product to a state ready for swallowing. Therefore, it is considered as an important parameter since the final phase of mouth feels and the ease in swallowing depends on the chewiness of meat. Cohesiveness (Fig. 1D), gumminess (Fig. 1E), chewiness (Fig. 1F),
and resilience (Fig. 1G) were all showed a maximum at 65◦C in microwave treated sample, however, gumminess and chewiness of water bath treatment reached the maximum at 60◦C. Changes of cohesiveness and resilience for water bath treated sample were irregular with the increase in heating temperature, it was may be result from the intercorrelation effects among TPA parameters during the long time heating for water bath compared with the microwave. Resilience was the only TPA parameter that presented no significant differences between two thermal treatments.” (Chang et al., 2011)
“Changes of TPA parameters in this study suggested that internal core temperature of
60◦C and 65◦C were the critical heating temperatures which affect meat texture for water
bath and microwave heating respectively. Furthermore, according to our previous studies, the maximal shrinkage temperature of IMCT collagen was within this range, this can give a full relationship of heat-induced change of collagen to meat quality, especially the meat texture; the results were also clearly showed in the SEM photographs.” (Chang et al., 2011)
“The swelling of collagen fibres in tissues such as tendon or skin is of two types: osmotic and lyotropic (see Gustavson, 1956). The first occurs in acid or alkaline solutions and is related to the positive or negative charge on the protein reaching a maximum at pH 2-0 and 12-0 and then decreasing again at more extreme pH values at the rising ion concentration reduces the change effect. The fibres swell laterally, contract in length and become glassy and translucent in appearance. The swelling is reversed by neutralization, by the addition of salts which reduce the effect of charge or by the presence of anions (or cations) having a specific affinity for the charged groups. This type of swelling has been considered in terms of the Donnan equilibrium (Procter and Wilson, 1916) which provides a satisfactory explanation in practical terms. X-ray diffraction studies (Burge et al., 1958) showed that the lateral spacing of about 11 Å, attributed to the distance between the molecules, was increased to 13-5 Å in salt free water in the pH range of minimum swelling but increased to 15Å at pH 2-0. Structural stability, as indicated by fall in shrinkage temperature, is also affected, suggesting that water actually penetrates into the tropocollagen molecule, but it is difficult to disentangle the effects of swelling, pH and ion concentration.” (Courtis and Ward, 1977)
“Swelling in neutral salt solutions has rather different effects. The fibres become opaque and flaccid, length is relatively unaffected and cohesion between fibrils is reduced. The uptake of water varies greatly with the salt, increasing with its tendency to disrupt hydrogen bonds. Dimensional changes probably first occur in the less ordered polar areas of the molecule leading to more general disruption under favourable conditions, i.e. rise of temperature. (For fuller discussion of the effect of salts on the collagen triple helix see von Hippel, 1967.)” (Courtis and Ward, 1977)
Collagen as a Constituent of Mammalian Tissue
“Mammalian tissues have many things in common. For example, they usually consist of cells embedded in a matrix consisting of collagen, elastin, and mucopolysaccharides. The interactions of these components give the tissue its structural properties, while the cells embedded in the matrix give the tissue its metabolic properties. The proportion of matrix present depends on the tissue function so that structural tissue (e.g. skin, bone or tendon) consists mainly of connective tissue, while tissues with a major metabolic function (e.g. liver or brain) contain little connective tissue.” (Courtis and Ward, 1977)
“Neuman and Logan (1950) based the collagen and elastin contents on hydroxyproline determinations of preparations similar to those of Lowry, assuming collagen contains 13.4% hydroxyproline and elastin contains 2% hydroxyproline. While the former assumption is substantially true (modern literature favours a hydroxyproline value of 14.4% in collagen) the existence of hydroxyproline in elastin is not now accepted with certainty.” (Courtis and Ward, 1977)
“More recently Dahl and Persson (1963) have estimated the hydroxyproline content of several tissues by direct tissue hydrolysis, and their results can be converted to values of collagen content if one assumes that all the hydroxy-proline is derived from collagen. In Table II some of these results have been collected in order to indicate what may be regarded as typical values. Since in no case did the author give precise details of the tissues used, the table should be considered only as a guide to collagen-rich tissues.” (Courtis and Ward, 1977)
The table above is very interesting as it gives potential sources of collagen which suppliers can rank in price in order to determine the cost of collagen. In bovine, collagen containing material can therefore be ranked as follows:
Soluble Collagen Chemistry
“Varying amounts of fibrous collagen dissolve in cold acidic or near neutral buffers or even water. This material is referred to as soluble collagen and usually represents only a small fraction of total collagen present in any tissue. However, soluble collagen has provided the sample used for most studies concerned with the chemistry of collagen.”
“It has been known for some time that a part of mammalian collagen from tendon and from many other tissues can be extracted by dilute aqueous solutions of organic acids or buffered citrate of pH 3–4, while most of the collagen remains insoluble. There is in addition a quantitatively minute fraction which can be extracted at neutral or slightly alkaline pH by salt solutions and this has been called “neutral-salt-soluble collagen.” It has been suggested (Green and Lowther, 1959; Jackson and Bentley, 1960) that there are no sharp divisions between these different soluble fractions and that there is a continuous spectrum of molecular species varying in degree of aggregation and cross-linking.” (Munro (Ed), 1964)
“Soluble collagen chemistry dates back to the studies by Zachariades (1900) who observed swelling of tendons immersed in weak acid solutions.” (Fishman, 1970) Let’s look into the significance of swelling. Collagen is classed as “a naturally occurring matrix polymer.” (Cheema, 2011) When a polymer dissolves, the first step is a slow swelling process called solvation in which the polymer molecule swells by a factor 𝛿, which is related to CED. Linear and branched polymers dissolve in a second step, but network polymers remain in a swollen condition. (Carraher, 2003)
Polymer mobility is an important aspect helping determine a polymer’s physical, chemical, and biological behavior. Lack of mobility, either because of interactions that are too swift to allow the units within the polymer chain some mobility or because there is not enough energy (often a high enough temperature) available to create mobility, results in a brittle material. Many processing techniques require the polymer to have some mobility. This mobility can be achieved through application of heat and/or pressure, or by having the polymer in solution. Because of its size, the usual driving force for the mixing and dissolving of materials is much smaller for polymers in comparison with smaller molecules. Here we will look at some of the factors that affect polymer solubility. The physical properties of polymers . . . are related to the strength of the covalent bonds, the stiffness of the segments in the polymer backbone, and the strength of the intermolecular forces between the polymer molecules. The strength of the intermolecular forces is equal to the CED, which is the molar energy of vaporization per unit volume. Since intermolecular attractions of solvent and solute must be overcome when a solute dissolves, CED values may be used to predict solubility. (Carraher, 2003)
“A polymer dissolves by a swelling process followed by a dispersion process or disintegration of the swollen particles. This process may occur if there is a decrease in free energy. Since the second step in the solution process involves an increase in entropy, it is essential that the change in enthalpy be negligible or negative to assure a negative value for the change in free energy.” (Carraher, 2003)
“Soluble collagen chemistry was taken up again (following Zachariades, 1900), principally by Nageotte (1927a – e, 1928, 1930, 1933), Nageotte and Guyon (1933 and 34), Huzella (1932), Leplat (133a, b), Faure-Fremiet (1933a, b) and Guyon (1934). These authors worked with the dilute acid extracts and demonstrated a protein content.” (Fishman, 1970)
“Much of the knowledge of soluble collagen chemistry derives from initial papers by Tustanowski (1947) and Oreskovich, et al. (1948a, b) who demonstrated a revisable solubility of collagen fibrils that had dissolved in citric acid buffer (ph 3 – 4.5) and underwent reformation into collagen fibrils upon dialysis against water.” (Fishman, 1970)
The Science and Technology of Gelatin, 1977; Edited by A. G. Ward, A. Courtis,
Imperial College of Science and Technology, London, England. Academic Press
Maria Cristina Messia and Emanuele Marconi. 2012. Innovative and Rapid Procedure for 4-Hydroxyproline Determination in Meat-Based Foods. Article in Methods in molecular biology (Clifton, N.J.), January 2012, DOI: 10.1007/978-1-61779-445-2_22 · Source: PubMed
Cheema, U., Anata, M., Mudera, V.. 2011. Collagen: Applications of a Natural Polymer, Submitted: December 2nd 2010; Reviewed: June 29th 2011, Published: August 29th 2011, Researchgate.
Courtis, A. and Ward, A. G.. 1977. The Science and Technology of Gelatin, 1977;
Imperial College of Science and Technology, London, England. Academic Press
Charles E. Carraher, Jr.. 2003. Seymour-Carraher’s Polymer Chemistry. Sixth Edition. Marcel Dekker Inc.
Fishman, W. (Editor). 1970. Metabolic Conjugation and Metabolic Hydrolysis. Volume 2. Academic Press.
Haijun Chang , Qiang Wang , Xinglian Xu , Chunbao Li , Ming Huang ,
Guanghong Zhou & Yan Dai (2011) Effect of Heat-Induced Changes of Connective Tissue and Collagen on Meat Texture Properties of Beef Semitendinous Muscle, International Journal of Food; Properties, 14:2, 381-396, DOI: 10.1080/10942910903207728
Harding, J. J., James, M. and Crabbe, C. 1992. Post-translational Modifications of Proteins. CRC Press.
Munro, H. N., Allison, J. B. (Editors). 1964. Mammalian Protein Metabolism, 1st Edition, Volume I, eBook ISBN: 9781483272924, Academic Press, 1st January 1964