Notes on Collagen

Notes on Collagen
by Eben van Tonder
20 July 2020



We are considering source material for fine emulation usages. Collagen has become my main area of interest. Here I post the relevant data related to collagen. It is my personal study notes if you will. 

“Collagen is a fibrous protein found in all multicellular animals (Voet et al., 2006). It is an important component in the support structures in vertebrates and invertebrates. It is the most abundant protein in mammals, corresponding to approximately 25% of the weight of all proteins (Ward and Courts, 1977; Voet et al., 2006), and is the major constituent protein of skin, tendons, cartilage, bones and tissues in general. In poultry and fish it plays a similar role to that of invertebrates and is an important component of the body wall (Ward and Courts, 1977).”

“Collagen molecules are about 280 nm long, with a molar mass of 360,000 Da; they are stabilized by hydrogen bonds and intermolecular bonds (Silva and Penna, 2012), which are composed of three helical polypeptide chains, each with about 1000 amino acids, which are called an α chain. The chains become entangled, forming a stable triple helix which is varied in size. The triple helix molecules have terminal globular domains and are called procollagen. These globular regions are cleaved in varying degrees to give a polymerized structure (tropocollagen), which is the basic unit of collagen. The tropocollagen molecules are stabilized by hydrophobic and electrostatic interactions (Nelson and Cox, 2004; Damoradan et al., 2010).” (Schmidt, et al., 2016)

“There are different kinds of collagen in vertebrates; they typically contain about 35% glycine (Gly), 11% alanine (Ala) and 21% proline (Pro) and hydroxyproline (Hyp). The amino acid sequence in collagen is generally a repetitive tripeptide unit (Gly-X-Y), where X is frequently Pro and Y is Hyp (Nelson and Cox, 2004).” (Schmidt, et al., 2016)

“At least 29 different types of collagen have been reported, which are classified according to their structure into: striatum (fibrous), non-fibrous (network forming), microfibrillar (filamentous) and those which are associated with fibril (Damoradan et al., 2010).” (Schmidt, et al., 2016)

“Type I collagen is the most common, primarily in connective tissue, in tissues such as skin, tendons and bones. It consists of three polypeptide chains, two of which are identical, which are called chain α1 (I) and α2 (I), and which are composed of different amino acids. Type II collagen occurs almost exclusively in cartilage tissue and it is believed that the α1 (II) subunit is similar to the α1 (I) subunit. Type III collagen is strongly dependent on age: very young skin can contain up to 50%, but with the passage of time that percentage can be reduced to 5-10%. Other types of collagen are only present in very small quantities, mainly in specific organs such as the basement membranes, cornea, heart muscle, lungs and intestinal mucosa (Schrieber and Gareis, 2007; Karim and Bhat, 2009).” (Schmidt, et al., 2016)

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. The table below shows how 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)

Collagen content of meat

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:

Tendons 95%
Skin 89%
Udder 42%
Nuchae 34%
Bone 24%
Stomach 23%
Aorta 23%
Lung 18%

Collagen Marker: Hydroxyproline (click on the link for a focused discussion on it)

Hydroxyproline becomes the market to indicate a high usage of collagen.  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.

Elastin is the main protein component of the elastic fibres, 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) Overcoming this is the main purpose behind my study!

“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. 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 maybe 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)

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 to determine a polymer’s physical, chemical, and biological behaviour. 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)

Application of Soluble Collagen Chemistry

“There is a growing interest in the extraction process of collagen and its derivatives due to the growing tendency to use this protein to replace synthetic agents in various industrial processes, which results in a greater appreciation of the by-products from animal slaughter. Collagen’s characteristics depend on the raw material and the extraction conditions, which subsequently determine its application. The most commonly used extraction methods are based on the solubility of collagen in neutral saline solutions, acid solutions, and acid solutions with added enzymes. Recently, the use of ultrasound, combined with these traditional processes, has proven effective in increasing the extraction yield.” (Schmidt, et al., 2016) Schmidt, et al., 2016 did a mini review of the “different collagen extraction processes, from raw materials to the use of combinations of chemical and enzymatic processes, as well as the use of ultrasound.” The information outlined in their review has been collected from different national and international journals in Agricultural Sciences and Science and Food Technology. They studied the different extraction processes, using four bibliographic databases and also some books of renowned authors, and selected articles published between 2000 and 2015. (Schmidt, et al., 2016)

Raw materials for collagen extraction

“Meat is the main product derived from the slaughter of animals, while all other entrails and offal are classed as by-products (Bhaskar et al., 2007), including bones, tendons, skin, fatty tissues, horns, hooves, feet, blood and internal organs. The yield of by-products that is generated depends, among other factors, on the species, sex, age and body weight of the animal. The yield varies from 10% – 30% in cattle, pigs and sheep and from 5% – 6% in poultry (Nollet and Toldrá, 2011). According to Bhaskar et al. (2007) about 40% of these by-products are edible and 20% are inedible.” (Schmidt, et al., 2016)

“Depending on the culture and the country, edible by-products can be considered as waste or as delicacies that command high prices (Toldrá et al., 2012). However, the majority of by-products are not suitable for human consumption due to their characteristics. As a result, a potential source of income is lost, and the cost of disposal of these products has become increasingly high (Jayathilakan et al., 2012). Nevertheless, there is a growing awareness that these by-products can represent valuable resources if they are used properly.” (Schmidt, et al., 2016)

“Generally, inedible by-products are used in the manufacture of fertilizers, animal feed and fuel but there is also a growing market in using them to obtain minerals, fatty acids, and vitamins and to obtain protein hydrolysates and collagen. Obtaining those products, which have high added value, is a better alternative to use these by-products, which would otherwise be discarded.” (Schmidt, et al., 2016)

“The main sources for collagen extraction are byproducts from the slaughter of pork and beef (Jia et al., 2010; Silva and Penna, 2012). Several of these by-products have been studied, including the Achilles tendon (Li et al., 2009), pericardium (Santos et al., 2013), bovine inner layer of skin (Moraes and Cunha, 2013) and bovine bones (Paschalis et al., 2001), porcine skin (Yang and Shu, 2014) and porcine lung (Lin et al., 2011).” (Schmidt, et al., 2016)

“Recent research has examined alternative sources for the extraction of collagen, with particular emphasis on fish by-products (Muralidharan et al., 2013; Kaewdang et al., 2014; Ninan et al., 2014; Wang et al., 2014; Mahboob, 2015; Tang et al., 2015). This is mainly due to religious restrictions, regarding the non-consumption of pork by Muslims and Jews, and also the risk of bovine spongiform encephalopathy (BSE) (Kaewdang et al., 2014). The latter belongs to a family of diseases known as transmissible spongiform encephalopathies, which are caused by the accumulation of the pathological prion protein (PrPSc) in the brain and central nervous system, which affects adult bovines (Callado and Teixeira, 1998; Toldrá et al., 2012).” (Schmidt, et al., 2016)

“The extraction of collagen from fish has been carried out in several species using different byproducts, such as Japanese sea bass skin (Lateolabrax japonicus) (Kim et al., 2012), skin of clown featherback (Chitala ornata) (Kittiphattanabawon et al., 2015), bladder of yellow fin tuna (Thunnus albacares) (Kaewdang et al., 2014), skin and bone from Japanese seerfish (Scomberomorous niphonius) (Li et al., 2013), cartilage from Japanese sturgeon (Acipenser schrenckii) (Liang et al., 2014), and the fins, scales, skins, bones and swim bladders from bighead carp (Hypophthalmichthys nobilis) (Liu et al., 2012). Despite the extraction of marine collagen is easy and safe this collagen presents some limitations in their application, due to its low denaturation temperature (Subhan et al., 2015).” (Schmidt, et al., 2016)

“The extraction of collagen from poultry slaughter waste has also been researched, but with less emphasis because of the risk of the transmission of avian influenza (Saito et al., 2009). Studies have been performed regarding emu skin (Dromaius novaehollandiae) (Nagai et al., 2015), and chicken feet (Saiga et al., 2008; Almeida et al., 2012a; Hashim et al., 2014), chicken sternal cartilage (Cao and Xu, 2008), chicken skin (Cliche et al., 2003; Munasinghe et al., 2015) and chicken tarsus (Almeida et al., 2012b) etc.” (Schmidt, et al., 2016)

“The processing of by-products can convert a product with low value, or one that requires costly disposal, into a product that is able to cover all the costs of processing and disposal, with consequent higher added value and reduced environmental damage (Toldrá et al., 2012).” (Schmidt, et al., 2016)

Collagen extraction process

“Collagen can be basically obtained by chemical hydrolysis and enzymatic hydrolysis (Zavareze et al., 2009). Chemical hydrolysis is more commonly used in industry, but biological processes that use the addition of enzymes are more promising when products with high nutritional value and improved functionality are required (Martins et al., 2009). Moreover, enzymatic processes generate less waste and may reduce the processing time, but they are more expensive. To extract collagen it is necessary to remove numerous covalent intra- and intermolecular cross-links, which primarily involves residues of lysine and hydroxy-lysine, ester bonds and other bonds with saccharides, all of which makes the process quite complex (Ran and Wang, 2014).” (Schmidt, et al., 2016)

“Before the collagen can be extracted a pretreatment is performed using an acid or alkaline process, which varies according to the origin of the raw material. The pre-treatment is used to remove non-collagenous substances and to obtain higher yields in the process. The most commonly used extraction methods are based on the solubility of collagen in neutral saline solutions, acidic solutions, and acidic solutions with added enzymes. The table below presents a summary of the procedures employed in the extraction of collagen from animal by-products.” (Schmidt, et al., 2016)


“Due to the nature of the cross-linked collagen that is present in the connective tissue of animals, it dissolves very slowly, even in boiling water. As a result, a mild chemical treatment is necessary to break these cross-links before extraction (Schreiber and Gareis, 2007). To this end, diluted acids and bases are employed, and the collagen is subjected to partial hydrolysis, which maintains the collagen chains intact but the cross-links are cleaved (Prestes, 2013).” (Schmidt, et al., 2016)

“In the acidic form of pre-treatment, the raw material is immersed in acidic solution until the solution penetrates throughout the material. As the solution penetrates the structure of the skin at a controlled temperature it swells to two or three times its initial volume and the cleavage of the non-covalent inter- and intramolecular bonds occurs (Ledward, 2000). The acidic process is more suitable for more fragile raw materials with less intertwined collagen fibres, such as porcine and fish skins (Almeida, 2012b).” (Schmidt, et al., 2016)

“The alkaline process consists of treating the raw material with a basic solution, typically sodium hydroxide (NaOH), for a period that can take from a few days to several weeks (Prestes, 2013). This process is used for thicker materials that require a more aggressive penetration by the treatment agents, such as bovine ossein or shavings (Ledward, 2000). NaOH and Ca (OH)2 are often used for pre-treatment, but NaOH is better for pre-treating skins because it causes significant swelling, which facilitates the extraction of collagen by increasing the transfer rate of the mass in the tissue matrix (Liu et al., 2015).” (Schmidt, et al., 2016)

“A study by Liu et al. (2015) evaluated the effect of alkaline pre-treatment on the extraction of acid-soluble collagen (ASC) from the skin of grass carp (Ctenopharyngodon Idella). Concentrations of NaOH from 0.05 to 0.1 M were effective in removing non-collagenous proteins without losing the ASC and structural modifications at temperatures of 4, 10, 15 and 20°C. However, 0.2 and 0.5 NaOH M caused a significant loss of ASC, and 0.5 M NaOH resulted in structural modification in the collagen at 15 and 20°C. In addition to the use of acids and bases, enzymes or chemicals may also be used to cleave the cross-linked bonds to obtain products with different characteristics (Schrieber and Gareis, 2007).” (Schmidt, et al., 2016)

Collagen Extraction 1

Collagen Extraction 2
By Schmidt, et al. (2016)

Chemical hydrolysis

“In the extraction of collagen which is soluble in salt, neutral saline solutions are used, such as sodium chloride (NaCl), Tris-HCl (Tris (hydroxymethyl) aminomethane hydrochloride), phosphates or citrates. Caution is required in this process in order to control the concentration of salt, but considering that the majority of collagen molecules are cross-linked, the use of this method is limited (Yang and Shu, 2014).” (Schmidt, et al., 2016)

“Acid hydrolysis can be performed by using organic acids such as acetic acid, citric acid and lactic acid, and inorganic acids such as hydrochloric acid. However, organic acids are more efficient than inorganic acids (Skierka and Sadowska, 2007; Wang et al., 2008). Organic acids are capable of solubilizing non-crosslinked collagens and also of breaking some of the inter-strand cross-links in collagen, which leads to a higher solubility of collagen during the extraction process (Liu et al., 2015). Therefore, acidic solutions, especially acetic acid, are commonly used to extract collagen.” (Schmidt, et al., 2016)

“For the extraction of acid-soluble collagen, the pre-treated material is added to the acid solution, usually 0.5 M acetic acid, and maintained for 24 to 72 hours under constant stirring at 4°C, depending on the raw material (Wang et al., 2014; Nagai et al., 2015; Kaewdang et al., 2014).” (Schmidt, et al., 2016)

“After the extraction stage, a filtering is performed to separate the supernatant (residue) from the collagen, which is in the liquid phase. To obtain collagen powder, the filtrate is usually subjected to precipitation with NaCl. The precipitate is then collected by centrifugation and subsequently redissolved in a minimum volume of 0.5 M acetic acid and then dialyzed in 0.1 acetic acid for 2 days, and distilled water for 2 days, with replacement of the solution on average every 12 hours.” (Schmidt, et al., 2016)

“Moraes and Cunha (2013) analyzed collagen from the inner layer of bovine hide that was extracted under different temperature conditions (50, 60 or 80°C) and pH (3, 5, 7 or 10) under stirring for 6 hours. The hydrolysates that were produced in different conditions showed distinct properties. The highest levels of soluble proteins were obtained from treatments at a temperature of 80°C and a pH below the isoelectric point. The products obtained in conditions of extreme pH (3 and 10) or high temperatures (60 and 80°C) were completely denatured. The extractions with acidic pH and high temperature produced collagen with reduced molar mass. In general, the hydrolysates obtained with acidic pH formed firmer gels. The water retention capacity of the gels was approximately 100%, except for the hydrolysates that were obtained at high pH (7 and 10) and above the denaturation temperature (80°C).” (Schmidt, et al., 2016)

“Wang et al. (2008) optimized the conditions for extraction of acid-soluble collagen in skin from grass carp (Ctenopharyngodon Idella), having evaluated the effects of the concentration of acetic acid (0.3, 0.5 and 0.8 M), temperature (10, 20 and 30°C) and extraction time (12, 24 and 36 hours). The three tested variables showed a significant effect on collagen extraction and a positive relationship was found between time and the collagen yield. Increased temperature and concentration of acetic acid increased the yield to a certain value, which then decreased. The optimal conditions to obtain the highest yield of acid-soluble collagen in skin from grass carp were: an acetic acid concentration of 0.54 M at a temperature of 24.7°C for 32.1 hours.” (Schmidt, et al., 2016)

“Acid-soluble collagen from the skin and swim bladder of barramundi (Lates calcarifer) was extracted by Sinthusamran et al. (2013). The pretreated raw materials were extracted with 0.5 M acetic acid for 48 hours at 4°C. The acid-soluble collagen from the swim bladder showed a higher yield (28.5%) compared to that which was obtained from the skin (15.8%). In both cases, the collagen was identified as type I, with some differences in the primary structure. Both the skin and the swim bladder of barramundi showed potential for collagen extraction.” (Schmidt, et al., 2016)

“In general, chemical hydrolysis processes seek optimum conditions for obtaining higher yields by controlling process variables such as concentration, pH, temperature, and process time.” (Schmidt, et al., 2016)

Additional Notes on Salt-Extracted Collagen

Fisherman adds the following notes on salt extracted collagen.  “Various buffers have been used to obtain salt-extracted collagen. Highberger et al. (1951) used an alkaline disodium phosphate buffer. Using isotopes Harkness et al., (1954) was able to determine that this fraction was a precursor to insoluble collagen. Jackson and Fessler (1955) and Gross et al. (1955) soon discovered that neutral salt probably extracts the same collagen as does the alkaline buffer and that both represent the most resent formed collagen. Increased amounts of collagen have been solubilized by increasing the concentration of NaCl. Perhaps no more than 10% of the total collagen can be extracted with salt, and generally much less than this is extractable. Less collagen can be extracted from skins of aged animals than from young animals and less is extracted from tendons than from skin. An amount of 0.5M NaCl in 0.5M tris buffer, pH 7.5 at 4 deg C for 2 – 4 days is currently used in this laboratory to obtain salt-extracted collagen. (Fishman (Ed), 1970)

Additional Notes on Acid Extracted Collagen

Fisherman adds the following notes on acid extracted collagen.  “After collagen-containing tissue has been extracted with salt solutions additional collagen can be extracted by employing cold weak acids. Under the best of conditions, as much as 20% of of the total collagen may be extracted with cold acids. Ground-up tissue containing collagen may be placed directly in cold acids (after thorough washing with water) for extraction of soluble collagen without an intermediate salt extraction. In other words, weak acids will extract both the acid and salt soluble fractions. 0.5 M acetic acid is generally used in our laboratory to obtain acid soluble-collagen (Piez et al. 1961). Other acids have been advocated for example 0.1M citric acid and 0.1M sodium citrate pH4.3 (Gallop, 1955) 0.5M dihydrogen phosphate (Dumitru and Garrett, 1957) and 0.15M citrate buffer pH 3.8 (Mazurov and Orekhovich, 1959). The amount of collagen obtained varies with several factors including the pH of the acid (more being extracted at low pH), the age of the animal (more being extracted from younger animals) and the type of collagen-containing tissue (more is being extracted from skin than from tendons).”    (Fishman (Ed), 1970)

Enzymatic Hydrolysis

“For the extraction of collagen by enzymatic hydrolysis, the raw material, which can be the residue of acidic extraction, is added to 0.5 M acetic acid solution containing selected enzymes such as pepsin, Alcalase® and Flavourzyme® (Novozymes®, Araucária PR, Brazil). The mixture is continuously stirred for about 48 hours at 4°C followed by filtration (Li et al., 2009; Li et al., 2013; Wang et al., 2014). The filtrate is subjected to precipitation and dialysis under the same conditions as for obtaining acid-soluble collagen.” (Schmidt, et al., 2016)

“Woo et al. (2008) optimized the extraction of collagen from the skin of yellowfin tuna (Thunnus albacares). Pre-treatment was performed with NaOH (0.5 to 1.3 N) at 9°C for 12 to 36 hours for the removal of non-collagenous protein. Subsequently, digestion with pepsin (0.6 to 1.4% (w/v) was performed in hydrochloric acid (HCl) solution (pH 2.0) at 9°C for 12 to 36 hours. The optimal extraction conditions were obtained with a pre-treatment of 0.92 N NaOH for 24 hours and digestion with pepsin at a concentration of 0.98% (w/v) for 23.5 hours.” (Schmidt, et al., 2016)

“Wang et al. (2014) isolated and characterized collagen from the skin of Japanese sturgeon (Acipenser schrenckii) using NaCl, acetic acid and pepsin for extraction. Initially, the skin was pretreated with NaCl and Tris-HCI and then the saline soluble collagen was extracted (SSC) in 0.45 M NaCl at pH 7.5 for 24 h with continuous stirring; this was performed six times. After the extraction with salt, the residue was suspended in 0.5 M acetic acid for the extraction of acid-soluble collagen (ASC); the procedure was carried out for 24 hours, twice. The material that was insoluble in acetic acid was used to extract pepsin-solubilized collagen (PSC) by using 0.1% (w/v) pepsin in 0.01 M HCl for 48 hours. The yields of SSC, ASC and PSC were 4.55%, 37.42% and 52.80%, respectively. All the isolated collagens maintained a triple helix structure and were mainly type 1 collagen, with similar morphology and amino acid profiles. The spectroscopic analysis in the midinfrared region using Fourier transform spectroscopy (FTIR) showed more hydrogen bonds in the PSC and more intermolecular cross-linking in the ASC. The different collagens also showed some differences in terms of thermal stability, which could have been due to the hydration level, as well as the number and type of covalent cross-links.” (Schmidt, et al., 2016)

“Kittiphattanabawon et al. (2010) extracted collagen from the cartilage of brown-banded shark (Chiloscyllium punctatum) and blacktip shark (Carcharhinus limbatus). Pre-treatment was performed using NaOH and ethylenediamine tetraacetic acid (EDTA). The extraction was initially performed with acetic acid for 48 hours at 4°C. Thereafter, the residue that was not dissolved by the acidic extraction was extracted with porcine pepsin in acetic acid for 48 hours at 4°C. The collagen extracted by pepsin had a much higher yield than the acid-extracted collagen. Furthermore, the spectra of both collagens that were obtained by FTIR were very similar; suggesting that hydrolysis with pepsin does not affect the secondary structure of collagen, especially the triple helix structure.” (Schmidt, et al., 2016)

“The method of extraction can influence the length of the polypeptide chains and the functional properties of collagen, such as viscosity, solubility, as well as water retention and emulsification capacity. This varies according to the processing parameters (enzyme, temperature, time and pH), the pretreatment, method of storage and the properties of the initial raw material (Karim and Bhat, 2009).” (Schmidt, et al., 2016)

“Thus it is necessary to perform a partially controlled hydrolysis of the cross-linked bonds and the peptide bonds of the original structure of the collagen in order to obtain the ideal distribution of molar mass for a given application (Schreiber and Gareis, 2007). This factor has emphasized the use of selected animal or vegetable proteolytic enzymes, such as trypsin, chymotrypsin, pepsin, pronase, alcalase, collagenases, bromelain and papain (GómezGuillén et al., 2011; Khan et al., 2011) because these permit the control of the degree of cleavage of the substrate protein. In addition, enzymatic hydrolysis presents some advantages compared with chemical hydrolysis, such as specificity, the control of the degree of hydrolysis, moderate conditions of action, and lower salt content in the final hydrolysate. Furthermore, enzymes can be generally employed at very low concentrations and it is not necessary to remove them from the medium (Zavareze et al., 2009). Despite the high cost of enzymatic hydrolysis, the fact that it results in lower levels of waste, better control of the process and higher yield justifies the use of enzymes.” (Schmidt, et al., 2016)

The use of ultrasound in the collagen extraction process

“Ultrasound is widely used to improve the transfer of mass by wet processes, which are of importance in terms of mixture, extraction and drying (Li et al., 2009). Ultrasound has been used successfully in collagen extraction by reducing the processing time and increasing the yield (Kim et al., 2012; Kim et al., 2013; Ran and Wang, 2014; Tu et al., 2015).” (Schmidt, et al., 2016)

“Ultrasound is a process that uses the energy of sound waves which are generated at a higher frequency than the hearing capacity of human beings (higher than 16 kHz) (Chemat and Khan, 2011). The effects of ultrasound in liquid systems are mainly due to the phenomenon known as cavitation (Hu et al., 2013). During sonication, cavitation bubbles are quickly formed, which then suffer a violent collapse, resulting in extreme temperatures and pressures. This leads to turbulence and shearing in the cavitation zone (Chemat and Khan, 2011).” (Schmidt, et al., 2016)

“In a study by Kim et al. (2012), the extraction of acid-soluble collagen from the skin of Japanese sea bass (Lateolabrax japonicus) showed increased yield and reduced extraction time after ultrasonic treatment at a frequency of 20 kHz in 0.5 M acetic acid. Extraction with ultrasound did not alter the major components of the collagen, more specifically the α1, α2 and β chains.” (Schmidt, et al., 2016)

“Ran and Wang (2014) compared the extraction of collagen from bovine tendon with and without the use of ultrasound (20 kHz pulsed 20/20 seconds). Conventional extraction was performed with pepsin (50 Umg-1 of sample) in acetic acid for 48 hours. For the extraction with ultrasound the same conditions were used, but the times of extraction with ultrasound (3 to 24 h) and pepsin (24 to 45 hours) were varied, resulting in a total of 48 hours of treatment. The combination of ultrasound with pepsin resulted in a greater efficiency of collagen extraction, reaching a yield of 6.2%, when the conventional extraction yield was 2.4%. The adequate time for extraction using ultrasonic treatment was 18 h. The collagen that was extracted from bovine tendon showed a continuous helical structure, as well as good solubility and fairly high thermal stability. The use of ultrasound in conjunction with pepsin improved the efficiency of the extraction of natural collagen without damaging the quality of the resulting collagen.” (Schmidt, et al., 2016)

“Li et al. (2009) utilized ultrasound (40 kHz, 120 W) to extract collagen from bovine tendon using the enzyme pepsin. The results showed that ultrasound increased extraction by up to 124% and reduced the process time. These results were explained by the increased activity and dissolution of the substrate because irradiation allows for a greater dispersion of pepsin and opening of collagen fibrils, which facilitates the action of the enzyme. The use of circular dichroism analysis, atomic force microscopy and FTIR showed that the triple helix structure of the collagen remained intact, even after the ultrasonic treatment.” (Schmidt, et al., 2016)

“According to Kim et al. (2013) the use of ultrasound in the extraction of collagen generated a higher rate of yield than the conventional extraction method with 0.5 M acetic acid, even when using a low concentration of acid (0.01 M). In addition, the yield of collagen from the skin of Japanese sea bass (Lateolabrax japonicus) increased greatly with increased treatment time and amplitude of ultrasound.” (Schmidt, et al., 2016)

“However, studies of the effect of ultrasound on enzyme activity are still very limited (Li et al., 2009; Yu et al., 2014). Yu et al. (2014) suggested that the activity of the enzymes papain and pepsin can be modified by ultrasound treatment, mainly due to changes in their secondary and tertiary structures. The activity of papain was inhibited, and the activity of pepsin was activated by the ultrasound treatment that was tested.” (Schmidt, et al., 2016)

“The application of ultrasound for a long period of time may give rise to elevated temperatures and shear strength, as well as high pressures within the medium because of cavitation. It can also break the hydrogen bonds and van der Waals forces in polypeptide chains, leading to the denaturation of the protein/ enzyme (Ran and Wang 2014).” (Schmidt, et al., 2016)

Inclusion Rate of Collagen in Sausages

Notes from Wenther (2003):

Henrickson (1980) – beef hide protein, collagen, is a useful extender, moisturizer, texturizer, or emulsifer in different food systems.

Bailey and Light (1988) – Non-detrimental effects to coarse-ground sausages were observed with levels up to 30 percent of collagen from the corium layer of hides.

Wiley and others (1979) – as a “rule of thumb,” use of high collagen meats should be limited to 15 percent of the meat block.

Rust (1987) – collagen should be limited to 25 percent of the total protein content in a sausage.

Millier and Wagner (1985) – an addition of rind and sinew should be limited to 5 percent of frankfurters to prevent undesirable sensory characteristics.

Collagen Sources

-Beef Tripe
“Randall and others (1976) replaced the beef component in a meat emulsion system up to 80 percent with frozen honeycomb beef tripe. There were minimal changes in cooked yields at the 20 percent replacement level, but at 40 percent, the tripe caused adverse yield results. Drip losses paralleled the cooked yield results and at the 60 and 80 percent replacement levels, measurable lipid losses occurred with the tripe. Due to the nature of tripe (connective tissue protein), reduced-fat and water binding occurred by replacing the salt-soluble muscle protein. Firmness decreased at the 60 and 80 percent replacement levels and cohesiveness decreased at all replacement levels.” (Wenther, 2003)

“Jones and others (1982) conducted research in which beef tripe was used in 30 batches of bologna as a collagen source. Meat emulsions were prepared with five tripe levels (0, 10, 20, 30 and 40 percent of the formulation). Total collagen and insoluble collagen were significantly higher (P<0.05) for each increasing tripe level. Only minor differences were observed in the soluble collagen fractions. In comparison to lower tripe levels, the 40 percent tripe level had a lower smokehouse yield (P<0.05). The authors also concluded that the higher the collagen content in the formulation leads to a more “brittle” emulsion which was determined by lower hardness and chewiness scores. Furthermore, the authors reported decreased firmness and bind values in the cooked product and decreased visoelastic properties. In the raw batter in formulations that contained tripe levels greater than 10 percent.” (Wenther, 2003)

Tendons from Beef Hind Leg Muscles
“Sadler and Young (1993) replaced a portion of the lean in a conventional emulsion formulation with tendon from beef hind leg musdes. The tendons were homogenized and used either in a raw state or a preheated state. In the preheated treatment, the homogenized tendon was subjected to four temperature ranges (50, 60 70, 80 °C). In the first study, all treatments were observed by replacing 20 percent of the meat protein with 20 percent tendons (all treatments). Hardness doubled by replacement with raw tendon or tendon heated at 50 °C, but returned to approximately no-replacement levels at temperatures higher than 50 °C.” (Wenther, 2003)

“In the second study by Sadler and Young (1993), a portion of the lean meat was replaced with 0, 5,10,15, 20 or 25 percent tendon homogenate (raw and preheated at 70 °C). All attributes measured by the sensory evaluation decreased with increasing collagen content, but to a lesser extent with preheated tendon. By comparison of panel scores and texture profile analysis, it was determined that reduced fracturability was the texture parameter that panellists objected to when heated tendon replaced some of the lean. The authors concluded that a 60 °C preheated tendon homogenate at a 20 percent lean meat replacement can be effective for positive sensory attributes.” (Wenther, 2003)

Desinewed Connective Tissue
“Desinewed connective tissue has been obtained from cow meat and beef hind shank meat and utilized by many authors. Ladwig and others (1989) added two levels of collagen to meat emulsions to determine the effect of muscle collagen on emulsion stability. The authors revealed that adding additional collagen to meat emulsions shortened the total chopping time and decreased emulsion stability, but had no effect on protein solubility.” (Wenther, 2003)

“Eilert and Mandigo (1993), Eilert and others (1996ab), and Calhoun and others (1996ab) performed extensive research with desinewed connective tissue from beef hind shank meat. Eilert and Mandigo (1993) noted that thermal processing yield losses declined with increased modified connective tissue level (0, 10, 20, 30, 40 percent) and hypothesized that the addition of modified connective tissue may be effective for reducing processing yield losses in low-fat meat systems.” (Wenther, 2003)

“Eilert and others (1996ab) and Calhoun and others (1996ab) studied the relationship between phosphates and desinewed beef connective tissue. Collagen solubility was maximized with a 3.5 percent acidic phosphate solution, while hydration was optimized with a 3.5 percent alkaline phosphate solution (Eilert and others 1996a). The authors concluded that exposing connective tissue to high concentrations of phosphate will dramatically alter binding and solubility.” (Wenther, 2003)

“Calhoun and others (1996ab) expanded on the previous research with studies of preblending connective tissue with phosphates. While Calhoun and others (1996a) revealed that preblending sodium add pyrophosphate with modified beef connective tissue and subsequent addition of alkaline phosphate created a modified connective tissue product similar to the control product, Calhoun and others (1996b) determined that preblending modified connective tissue and sodium tripolyphosphate was not beneficial.” (Wenther, 2003)

“Osbum and other (1999) determined that the incorporation of desinewed beef connective tissue gels in reduced-fat bologna decreased (P<0.05) product hardness and increased juiciness, which indicated potential for the utilization of beef connective tissue gels as water-binders and texture-modlfiylng agents in reduced-fat comminuted meat products.” (Wenther, 2003)

Beef Hide (Skin)
“Although hamburger is not considered a processed product, hamburger is an intermediate particle-size product (Whiting 1989) and defined In the Code of Federal Regulations with section 319.15b (USDA 2002a) as: “Chopped fresh and/or frozen beef, with or without added beef fat and /or seasonings. Shall not contain more than 30 percent fat, and shall not contain added water, binders or extenders. Beef cheek meat may be used up to 25 percent of the meat formulation.” Chavez and others (1985) added bovine hide collagen as an extender to ground beef replacing lean meat at a level of 0, 10, or 20 percent. Beef patties with the collagen were found to be superior (P<0.05) in juiciness by the taste panel, while the flavor, texture, and overall acceptability decreased as the collagen level increased.” (Wenther, 2003)

“Asghar and Henrickson (1982) investigated the effect of the addition of food-grade bovine collagen at 10, 20, and 30 percent levels on other protein fractions in bologna. The authors revealed that the solubility of sarcoplasmic and myofibrillar proteins decreased, while percent solubility of collagen increased with increasing level of added hide collagen. Rao and Henrickson (1983) replaced 20 percent of the lean meat component in bologna with 20 percent beef hide collagen. The replacement did not alter the functional characters such as raw bologna emulsion stability and pH, cook yield, pH, water activity, and expressible moisture in the cooked bologna. The bologna with collagen had increased (P<0.05) shear force values compared to bologna with no collagen.” (Wenther, 2003)

Pork Skin
“Satterlee and others (1973) produced pork skin hydrolyzates and replaced non-fat dry milk in a sausage formulation. The utilization of pork skin hydrolzates produced sausage with a slightly better water and fat holding ability even though the emulsion capacity was slightly lower than the capacity of non-fat dry milk emulsions.” (Wenther, 2003)

“Sadowska and others (1980) and Sadowska (1987) utilized varying levels (5, 15, 20, or 25 percent) of raw and cooked (100 °C for 0-90 minutes) pork skin collagen to examine the rheological properties of sausage batters and cooked sausage, respectively. It was reported that replacing 20 percent of the meat protein with pork skin collagen decreased batter viscosity and cooked sausage elasticity. Incorporation of cooked skin (15 percent of the total protein) resulted in batter with higher viscosity and higher cooked sausage elasticity when compared to batter or cooked sausage not containing pork skin collagen. The authors concluded that the addition of greater than 2.5 percent pork skin collagen would result in altered cooked sausage texture and appearance. Puolanne and Ruusunen (1981) hypothesized that connective tissue may be important for the water binding capacity and firmness of cold sausage.” (Wenther, 2003)

Quint and others (1987) produced a loaf product that contained flaked pork skin and water that was pre-emulsified by passing it through an emulsion mill. The authors determined that the incorporation of the pre-emulsion improved bind of the emulsion and increased firmness, redness (a value), and yellowness (b value) colors of the loaf product. Delmore and Mandigo (1994) also used flaked pork skin sinew to low-fat, high-water added frankfurters at varying levels (0, 10, 20 percent of the formulation). Cooking yield, texture, and purge of the frankfurters were not altered by replacement levels of up to 20 percent pork connective tissue. There was no difference in juiciness, favor, texture, or overall acceptability detected by consumer sensory panelists between frankfurters containing 0 to 10 percent pork sinew. Fojtik (1997) incorporated flaked pork skin at levels of 10 and 20 percent into fresh pork sausage. The author reported that consumer panelists ranked lowfat sausage patties containing 10 percent pork skin higher for flavor, juiciness and overall acceptability than patties containing higher fat levels or pork skin levels. Fojtik concluded that the patties that contained 10 percent pork skin were more tender than those containing 20 percent pork skin (Fojtik 1997).” (Wenther, 2003)

Osbum and others (1997) produced gels from flaked pork skin with varying amounts of added water (100, 200, 300, 400, 500, 600 percent). These pork skin gels were utilized in reduced-fat bologna at levels of 10-30 percent addition. The greatest purge for any bologna occurred with the 600 percent added water, 30 percent addition treatment. Taste panel analysis revealed that juiciness scores increased as added water and percent gel addition increased. The overall acceptability of the pork connective tissue bologna tended to increase as added gel and added water increased. The authors summarized that the incorporation of pork connective tissue gels varied the functional, textural, and sensory attributes in reduced-fat bologna (Osbum and others 1997).” (Wenther, 2003)

“More recently, Prabhu and Doerscher (2000) utilized processed pork skin collagen in reduced-fat frankfurters to increase cooking yield and decrease purge in the final product. The authors also researched the effect of pork collagen in fat-free pork sausage formulations. The results indicated increased cooked yields with a reduction in cooked diameter shrink. The authors concluded that the addition of 1 percent hydrated collagen at a 1:4 ratio is a cost-effective (e.g improved yields), functional ingredient that can improve the quality (e.g. texture improvement) of various meat products.” (Wenther, 2003)

“Hoogenkamp (2001) cited the use of pork skin (rinds) in the production of preemulsions, which are another method to incorporate this raw material into emulsified meats. Pork skins are pre-blanched for about 20 minutes at 80 °C to soften the collagen tissue. The pork skins are added into the chopper prior to the addition of fat and chopped to a fine particle size which allows an increase in the pre-emulsion ratio utilized in the formulation.” (Wenther, 2003)

“Researchers have studied the use of skin in raw or cooked form. Sadowska and others (1980) and Sadowska (1987) utilized varying levels (5, 15, 20, or 25 percent) of raw and cooked (100 °C for 0-90 minutes) pork skin collagen to examine the rheological properties of sausage batters and cooked sausage, respectively. It was reported that replacing 20 percent of the meat protein with pork skin collagen decreased batter viscosity and cooked sausage elasticity. Incorporation of cooked skin (15 percent of the total protein) resulted in batter with higher viscosity and higher cooked sausage elasticity when compared to batter or cooked sausage without pork skin collagen. The authors concluded that the addition of greater than 2.5 percent pork skin collagen would result in altered cooked sausage texture and appearance. Puolanne and Ruusunen (1981) hypothesized that connective tissue may be important for the water binding capacity and firmness of cold sausage.” (Wenther, 2003)

-Poultry / Turkey Skin
“Poultry skin is also a source of collagen that may be used in comminuted meat systems. Campbell and Kenney (1994) listed poultry skin as generally being a filler ingredient in poultry or mixed-species batter sausages. The authors described that poultry skin may be listed on ingredient labels as “poultry by-products” and in other products skin cannot be added in higher proportion than occurs naturally.” (Wenther, 2003)

“Due to its high collagen content, broiler skin meat possessed inferior emulsifying capacity (Maurer and Baker 1966). Moreover, Hudspeth and May (1969) analyzed skin, heart, and gizzard tissues of turkeys, hens, broilers, and ducklings for emulsifying capacity of salt-soluble protein. The authors reported that skin was the least desirable tissue in emulsification properties and was not as effective in emulsifying ability as muscle tissue from the same class of poultry.” (Wenther, 2003)

“On the other hand, Prabhu (2003) reported that functional collagen proteins from chicken and turkey skins can bind three to four times their weight in water and can form a firm elastic “cold” gel producing texture characteristics that are similar to meat. Prabhu stated that this gel functions as a matrix stabilizer of finely comminuted and coarse-ground meat products such as frankfurters or sausages. The author suggested that collagens immobilize free water and prevent moisture loss during heat processing as well as improve texture while reducing purge loss.” (Wenther, 2003)

Heat Modified Collagen

Notes from Tarté, R. (Ed) (2009).

“The functional properties of collagen can be modified collagen or collagen-rich raw material under different time/ temperature combinations some of which has been reported in the literature (eg 100deg for 30, 60 or 90 minutes; Sadowska et al; 1980) During processing of most processed meats native collagen generally melts and becomes gelatin too late in the process (i.e. at temperatures of between 75 and 80 deg C) to become part of the batters gel structure. Precooked collagen, on the other hand, solubilizes early during chopping and is, therefore, able to provide functionality to the meat batter. (Whiting, 1989) This was born out in a study that evaluated the effect of temperature on the water-binding ability of concentrated pork skin CT gels (Osburn, Mandogo, & Eskridge, 1997). Pork skin CT was first obtained by cutting pork skin into strips, followed by freezing, grinding, refreezing and flaking. It was then combined with varying amounts of water and heated at 50 deg C, 60, 70 and 80 deg for 30 minutes. Under these conditions, it was found that gels produced by heating to at least 70 deg C had the highest water-binding ability. After cooling, these 70 deg C gels were tested in reduced-fat (2%, 3.5%, 4,3%6.8% and 12% fat) bologna, resulting in decreased hardness and increased juiciness.”

See his notes on Enzyme Modified Collagen, p. 152.

Negatives of Using Collagen in Fine Emulsion Sausages

“The possibility in using collagen found in great abundance in beef hide has been discussed by Elias et al., 1970. They indicated that collagen fibres and granules could be isolated from beef hide and used as a possible binder extender in meat products. An early study has shown that collagen, the major protein of skin, bone and connective tissue was detrimental to the emulsifying capacity of poultry meat (Maurerand Baker, 1966). The inability of collagen to emulsify fat and its ability to convert to gelatin upon make it an undesirable ingredient in sausage formulations.” (Satterlee, et al., 1973)

“Another property of collagen its nutritional value should be discussed when collagen is to be considered as a food additive. Collagen is known to be deficient in the essential amino acid tryptophan and limiting in other essential amino acids such as lysine, threonine, and methionine. However, it has been shown (Ashley and Fisher, 1966) that chicks fed on a diet of 10% gelatin+ 3% casein had the body weight gains equal to those fed on a diet of 13% soy protein and 0.2% methionine. Erbersdobler, et al. (1970), using male rats as the experimental animal showed when collagen or gelatin was incorporated into the diet of levels of up to 5% of the total diet weight, there were slight improvements in daily gain and feed conversion. Therefore, collagen will not lower the nutritional quality, if used along with a balanced protein and maintained at a low level in the diet, such as would be the case when a collagen hydrolyzate is used as a binder or extender in meat emulsions.” (Satterlee, et al., 1973)

“Collagen in its native state is resistant to proteolytic action of most enzymes, but when heated the resultant gelation is easily enzyme degraded. Hydrolysis of a protein by means of an enzyme will also change the physical characteristic of the protein.” (Satterlee, et al., 1973)

Maurer and Baker (1966) found an inverse relationship between the collagen content in poultry meat and its emulsifying capability. They write that “the method used in this study provides comparative estimates of the capacity of individual parts of different classes of poultry to emulsify fat. It has been found that the collagen content of poultry meat is a reliable estimator of emulsifying capacity when dealing with meat and skin mixtures. Collagen can be detrimental to the process of making poultry meat emulsions because of the inability of collagen to dissolve and form stabilizing membranes necessary for emulsion formation. In general, the voluntary muscle meats such as breast and thigh have a higher emulsifying capacity than the gizzard, heart or skin of poultry meat. Light fowl total carcass was found to emulsify significantly less oil than any other class of poultry.”

The following graph illustrates their conclusions well.


That collagen on its own is not a silver bullet to cheaper or better sausage production is clear.  That it has very interesting characteristics is equally clear.  An understanding of the nature of collagen and the different techniques of manipulating it inform the meat processing professional in every respect. Understanding its limitations and potential is key before one begins test kitchen trails.

Further Reading

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

  • On making a tendon emulsion

Wenther, J. B.. 2003. The effect of preheated tendon as a lean meat replacement on the properties of fine emulsion sausages. Iowa State University.

Maurer, A. J., and Baker, R. C.. 1966. The Relationship Between Collagen Content andEmulsifying  Capacity of Poultry Meat. Cornell University.


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. DOI: 10.5772/24165

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

Schmidt, M. M., Dornelles, R. C. P., Mello, R. O., Kubota, E. H., Mazutti, M. A., Kempka, A. P. and Demiate, I. M.. 2016. Collagen extraction process. Mini Review. International Food Research Journal 23(3): 913-922 (2016) Journal homepage:

Satterlee, L. D., Zachariah, N. Y., Levin, E. 1973. Utilization of Beef and Pork Skin Hydrolyzates as a Binder or Extender in Sausage Emulsions.   First published: February 1973.

Tarté, R. (Ed). 2009. Ingredients in Meat Products: Properties, Functionality and Applications. Springer.

Wenther, J. B.. 2003. The effect of various protein ingredients utilized as a
lean meat replacement in a model emulsion system and frankfurters. Iowa State University.

Image Reference:

From Collagen Scaffolds for Orthopedic Regenerative Medicine, April 2011, JOM: the journal of the Minerals, Metals & Materials Society 63(4):66-73, DOI: 10.1007/s11837-011-0061-y. Gráinne Cunniffe and Fergal J. O’Brien

Leave a Reply