Notes on Collagen

Notes on Collagen
by Eben van Tonder
20 July 2020

Introduction

Reference: http://www.aionaalive.com/blogs/news/83482631-what-is-collagen

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)

Denaturation

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

“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)

Swelling

“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)

Pre-treatment

“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.

Conclusion

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.

Reference

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: http://www.ifrj.upm.edu.my

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. https://doi.org/10.1111/j.1365-2621.1973.tb01402.

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

Hot Boning in America

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Hot Boning In America

By Eben van Tonder
20 April 2020

Introduction

butcher 2

When we formulate recipes, we formulate for:

  1. Protein content (according to legislation);
  2. Functionality of protein sources and gelling properties;
  3. Taste;
  4. Water Holding Capacity (which speaks to affordability);
  5. Mouth-feel, bite and firmness / tenderness;
  6. Freeze/ thaw stability where required;
  7. Visual appeal;
  8. Shelf life;
  9. Emulsion stability.

Hot boning is a technique where practitioners claim that water holding capacity is high, without the need to use phosphates.  In emulsions made from such meat there is no need for non-meat extenders, emulsifiers and stabilisers.  The processing is also achieved without the need for expensive and unnecessary refrigeration. It can have a material impact on shelf life by extending it and renders the end product firmer with a better visual appearance.  It is therefore worth a proper consideration.

Hot boning is when bones and fat are removed from the animal carcass within a few hours after slaughter, before chilling. Some researchers distinguish between hot and warm boning.  We will get into these differences at a later stage.

A short and clear description of hot boning is given by Dr. Lynn Knipe, who is, amongst other things, responsible for the processed meats extension programs at Ohio State University and conducts research related to the quality and safety of processed meat products.

Dr. Knipe writes that “the fresh, “bloom” color of meat is enhanced with rapid chilling (using CO2) of pre-rigor meat, as soon after hot boning as possible.  This improvement in color can be reflected in a sharper particle definition (less smeared look), as well as a leaner appearance.  While there are other functional advantages to hot boning of meat, currently, the main commercial reason for pre-rigor boning of pork is to extend the shelf life (time until the lean loses color) of the fresh color.  Other advantages to pre-rigor processing include a firmer texture to the final cooked sausage, with less cooking loss.” (Knipe)

Schematically, the difference between hot-boning and cold-boning is represented as follows:

Cold vs hot boning
By Fung, et al, 1981

A German friend who is a 3rd generation Master Butcher tells me that his dad never used emulsifiers or stabilisers in his fine meat emulsion, and his secret was hot boning.  Well, it was not really a secret – it was practiced throughout Germany.

Let’s briefly look at the ingredients normally used in sausage production.  We will consider them by listing protein content and the relative price of the different proteins.  This will show that when formulating products, a proper evaluation of the different ingredients is required.

Protein options in formulating recipes

In the table below I give the relative protein %’s of different functional ingredients and the Rand price as it was in April 2020.  The links attached to this paragraph title and title below in the table are live and you can download the spreadsheet and insert the price of these protein sources in your own currency.   You can also adjust the protein % of the particular product you use.  The manufacturer must be consulted to get this information.  The final protein % will depend on the particular product blend and the production method used.

Protein options in formulating recipes (Mellett)
Protein Source  Price in SA (Rand) % Protein  Rand/ kg Protein
Soya / TVP         14.00 48%       29.17
Soya Isolate         39.00 90%       43.33
MDM/ MRM         10.00 10%     100.00
Pork 80/20         36.00 16.70%     215.57
Beef 90/10         55.00 19%     293.33
Skin           8.00 29%       27.59
Offal         15.00 18%       83.33

If you do not know that pork loin typically contains 20.85g of protein per 100g of meat (20.85%), you can calculate it as 0.8kg of lean meat (in a 80/20 trim ratio) to get to the % lean that is 0.8 (% lean) / 4.8 = 16.7% protein.  It follows from the formulas below.

-> Remember the key equations:

%N  x 6.25 =       % Protein

% Protein x 4.8 = % lean

6.25 x 4.8 = 30

So, %N x 30 = % lean  (Mellett)

The red and blue raw materials show the difference between high and low-end products.

-> High-End and Low-End Products in South Africa

Local food legislation invariably calls for a minimum protein percentage and usually specifies what the source of the proteins must be.  The hybrid meat formulations in South Africa usually contain a mixture of the ingredients listed in blue.  High quality sausages or loaves or hams are produced in South Africa from either primal cuts (whole muscle) or from the ingredients listed in red.  The question is if hot boning is used and all the costs are taken into account, including labour, energy (refrigeration and cooking), is it possible to come close to the price point when low-end products are produced, again, taking every input cost into account.

-> Hot Boning – A way to Make High-End Products Affordable

Hot boning is of interest for its Water Holding Capacity and its ability to form stable emulsions without the need to add non-meat fillers, stabilisers and extenders and the firmer texture and visual appeal. Due to the availability of data from the USA, it makes it easier to trace the history of the development of the technique from there.

Early work on Hot Boning in America

The Des Moines Register reported in 1974 on the work of Dr. R. L. Henrickson of the Oklahoma Agricultural Experimental Station where he had been working on hot boned meat since 1965. His initial work was on pork, and later he included beef in his research.  Henrickson says that the concept was conceived by his research team in 1957. He is quoted as saying that pork from this process is “equal or better” in quality compared to conventional methods. It is interesting when he says that “we are fast approaching a time when social and economic pressures will force the implementation of new meat processing procedures.” (Des Moines Register, 1974) Such conditions have existed in many parts of the world for a long time.

Status of Hot Processing of Meat in the United States

Arguably one of the foremost authorities on hot boning, Dr. Henrickson writes that “there appears to be very little direct industry application of hot processing of primal cuts in the United States, even though most research evidence points to many advantages for the various available processing systems.”  In contrast to this, “the success of the pork sausage industry can be attributed directly to the short processing period from slaughter to the chilled or frozen package. The system makes raw seasoned sausage available to the consumer in less than 90 minutes after slaughter. This process not only takes advantage of economics in processing and chilling, but provides the consumer with a sanitary, longer shelf-life product. The major bulk of the raw pork sausage industry now uses pre-rigor pork.” (Henrickson, 1983)

“The raw pork sausage industry uses young sows with the proper ratio of fat to lean. This careful selection of the animal makes it possible to blend a product without a great amount of excess fat.”  If sausages are made, the following steps are followed.

-> Sausage Production

  • Separate the lean meat and fat from the bone;
  • Chopped into uniform pieces;
  • Cool it, partially;
  • Add spices / seasoning;
  • Grind;
  • Stuff into one and two pound grease-proof casings.
  • Cool down “using an ethylene glycol bath system.
  • Another option is to extrude the pork sausage links with or without casing directly onto a liquid nitrogen enclosed endless belt. “By the time each link reaches the end of the belt it has absorbed sufficient refrigeration to be case frozen.”
  • Packaged and tempered to 0 deg F / -18 deg C for marketing.

(Henrickson, 1983)

-> If Not All the Meat is Used Immediately

Pork tissue (lean and fat) which can not all be used for sausage production immediately are handled as follows.

  • Salted (2-4 percent) during the following procedure of . . .
  • Coarse chopping of the meat
  • Place in 50-60 pound / 20-25kg boxes and freeze.
  • The pre-salted meat is used in sausage manufacture because of its ability to yield myosin for binding.

(Henrickson, 1983)

There is a variation on the above system which is commercially appealing, namely to produce the slabs of coarse chopped meat with spices and fat or rind emulsion already blended in.  I have seen this widely in use in India and Nepal and my intention is to test these methods and create a product which can be exported to small scale butchers who lack the equipment or experience to create the emulsions.

Hot Boning and Some Chilling

butcher 4

Pre-rigor pork has been demonstrated to have many benefits.  In America it is a matter of preference. Dr. Henrickson writes that “the prospect of cutting hog carcasses directly from the dressing line prior to chilling makes the average packing house worker shudder. The reason most often given is that one cannot trim hot cuts to presentable standards of appearance.”

Dr. Henrickson argues that the attitude in the US against hot-boning due to appearance is invalid “since most of the primal cuts do not require a high standard appearance value. All pork cuts except the loin and spare rib are subjected to some manner of forming either by can, package, stockinette, casing or press. Therefore, the only primal cut which may require some form of smoothness is the loin. Smoothness of the loin can even be attained by leaving the back fat intact, conveyorizing the loin through a blast chill and then trimming. A few minutes in a blast chill at -50°F / -45 deg C should provide ample firmness for the necessary trim. An alternative would be to market a completely boneless loin, since the consumer is now discriminating against fat and bone. The whole concept of hot processing not only requires converting practices of plant and market, but the thinking of personnel.” (Henrickson, 1983)

Henrickson reports that there has been progress during the past thirty years and expresses the hope that the process will be widely adapted in the future.  He says that “even though the pork industry has been reluctant to adopt hot processing for primal cuts, it has reduced the period from kill to package. High volume (880 hogs per hour) ham production (kill to can in three days) has been practiced since 1965. Pickle solution is automatically injected into the meat and the cure is equalized in a matter of hours. A flexible vacuum wrapper makes the product ready for shipment and distribution in less than three days.  Hot processing could reduce this time by an additional day.” (Henrickson, 1983)

QC Perception

There is a widely held belief that microbial problems are a major drawback to the system of hot boning.  There is evidence that hot processing could provide a more sanitary products. (Henrickson, 1983)

These claims were further investigated by Fung, et al. (1981) who found that if “hot-boned meat is chilled adequately (from carcass temperature to 21 C with 9 h) during the first 24 h, the hot-boned meat is acceptable in color and odor and bacterial quality after 14 days of storage and 3 additional days of display. When meat is not chilled adequately (from carcass temperature to 21 C at 12 h), the shelf-life and storage life will not be acceptable.”

Their research showed the need for adequate chilling after boning the hot meat “at a rate sufficient to produce a bacteriologically acceptable product.”  Boxing the meat before chilling is, according to their data, doable, but should be approached with great care. They caution against too-rapid cooling rates of hot-boned meat which can lead to cold-induced muscle shortening, which, in turn, causes toughening of the meat. (Fung, 1981)

They claim that faster chilling rates of up to 3-9 h after fabrication can be used as an additional insurance for better microbial quality and still the processor will be able to avoid cold-induced toughening.  They also add that electrical stimulation can very successfully be used in conjunction with hot-boning, as an extra measure to prevent muscle toughening.  They therefore recommend “chilling hot-boned meat to 21 C within 3-9 h after fabrication, and with continuous chilling, to below 10 C within 24 h.” (Fung, 1981)

Another way to prevent cold shortening is to select bigger carcasses with more fat.  The smaller and leaner carcasses are more susceptible to cold shortening due to the reduced fat cover, which results in the deep areas chilling faster.   This results in tougher products.  Apart from carcass selection, this can be overcome by introducing a conditioning step (semi-hot boning) of 4 hours more until rigor has occurred (in beef it can take 24 hours or even longer). To reduce the time for rigor to occur, electric stimulation is used immediately after slaughter.  It must however be reminded that in pork, cold shortening is not such a big problem because postmortem metabolism in pork occurs faster.

Warm Chilling
Dutch semi-hot boning; Dikeman, 2014

Generally speaking, hot boning can even double microbiological shelf life due to the fact that surface bacteria have not had time to grow before antimicrobial salts are added.  Even if the meat is slightly tougher, in comminuted meats this is not a problem because a higher ultimate pH is achieved (what we achieve with phosphates in South Africa).  Because of the higher pH there is an increased water holding and emulsifying capacity, which will yield a product that is juicy and of superior quality.  Pre-rigor meat also acts as an oxygen-scavenger.  It removes residual oxygen from inside the package after closure, resulting in a long shelf-life.

This does not mean that micro should not remain a major concern in hot or semi-hot boning.  There will be an increase in moisture on cutting surfaces and great care must be exercised to prevent this becoming a vector for microbial contamination and growth.

Fat trim

In hot boning, it is easier to remove fat from the warm cut. Care must be taken to maintain a juicy product with flavour, brought out by the fat. It will be important to reduce fat variability rather than cause it to increase.  (Fung, 1981)

Summary of Benefits of Hot Boning Compared to Cold Boning

butcher 3

Ockerman and Basu from Ohio State University reported the following benefits of Hot Boning compared to cold boning.

->  Benefits

  • Higher meat yield (1.4%)
  • Labour savings (20%, faster – 4 mins / carcass) (with the right equipment to hold carcass still and pull muscles downwards)
  • Less weight loss during chilling (1.5% less)
  • Less purge in a vacuum package (0.1 – 0.6%)
  • More uniform products
  • Darker colour
  • Reduced refrigeration space (50 – 55%)
  • Lower refrigeration cost (40 – 50%)
  • Shorter processing time (40 – 50%)
  • Lower transport cost (primals vs carcasses)
  • Superior water holding capacity
  • Higher emulsifying capacity

(Dikeman and Devine, 2014)

-> Challenges

  • Shape distortion of cuts because the bone is removed;
  • Reduced flexibility in production;
  • Stricter hygiene requirements;
  • Increased temperature control;
  • New cutting procedure;
  • Retrofitting of traditional cold boning area;
  • Retraining or hiring new cutting personnel;
  • Possible reduced tenderness because of cold and rigor shortening;
  • Alteration of colour;
  • Accelerated micro growth.

(Dikeman and Devine, 2014)

In the USA, hot boning is used mostly by whole-pig fresh sausage processors who use hot boning and rapid salting.

Rigor Complex Formation of Actomyosin

With the onset of rigor mortis, ATP disappears from the muscle.  In the absence of ATP, actin and myosin combine to form rigor complex of actomyosin (Kamejima, et al., 1982) Willi Wurm, Master of Meat Science and Processing put it in terms that I can understand.  In private communication he said that “actomyosin has to be separated again during a sausage emulsion process, by adding phosphate. Only separated Actin and Myosin have the capability to make an emulsion with fat and water. With hot boning methods you can keep the Actin and Myosin separate, when you grind the deboned meat and add salt. After that you cool or freeze the meat or process. The Actin and Myosin remain separate, and you can process without phosphate. You can also vacuum pack whole muscle pieces before the postmortem process and wet-age it. It will be classified better than normal wet-aged beef meat. Be careful to store the warm packed meat for the first night outside the fridge on tables and then refrigerate the next morning for 4 weeks.

Commercial Buy-In

Oscar Mayer was the first to apply hot boning to a large commercial operation.  They used it to process packer sow hams to be used in sausage manufacturing. (Dikeman and Devine, 2014)  The weight of these sows, which is “owned by a packing plant”, therefore packer sows, is between 110 and 140kg.

After the initial publication of this article I received fascinating comments from around the world.

Gary Hendrix from NSC BEEF PROCESSING  sent me the following communication.  “We do hot beef boning, breaking primals down. The good thing about this is once broken down you can cryovac for aging. Reducing your shrinkage greatly. Getting a great bloom on your product as well. Reduce fatigue to your boners, difference between cutting a hot stick of butter or cold. Greater yield, easier to clean the carcass. Faster, reduces labor. Cooling down time is faster, less chance for pathogens to grow. These are only a few advantages.  We also have developed and patented the technology with which to process beef without exposing the spinal cord. A huge advantage for BSE. NO BONE MEAL AS WELL. Labor savings as much as 30-50%. We will soon be taking a 3 day industry standard of kill floor to truck down to 1 day.”  For those of you who are interested, Gary can be contacted at NSCbeef@yhaoo.com, 117 Land Grant Lane Baird, Tx 79504 325-665-0602 Cell 325-518-5038.

Another person (still awaiting permission to use his name) recalled that “all the American processors were using hot boned meat, also went to a company called Marjacks who were producing a lot of further processed products, not sure if they are still going but would be a good source of information as would Wayne Poultry as they had an incredible set up for hot deboning.”

Not everybody had such a positive experience with hot boning of beef.  Someone (awaiting aproval to use his name) said, “I used to do a bit back in the 80s not great for presentation or yield. Hot Beef Boning, selling vac packed into wholesale. Very fast, but poor yields and doesn’t do much for cutting quality. We soon stopped it.”

Conclusion

I have never been exposed to hot boning.  The South African Meat Safety Act of 2000 (ACT No. 40 OF 2000) stipulates that meat must be cooled to a core temperature of 7 deg C before dispatch.  Paragraph 40 (1) reads as follows.  “A chiller used for chilling warm carcasses, sides, quarters or portions must be capable of providing uninterrupted cooling to reduce the core temperature of the meat to 7 def C before dispatching.”  According to this definition, it seems as if hot boning can be done as long as the bond meat reaches the required 7 deg C temperature before dispatch. I will take the matter up with a meat inspector.

In Germany, hot boning was widely used.  Gero Lutge, the third-generation Master Butcher I was talking about in the introduction, sent me the following account of his dad’s use of hot boning.  he writes, “in the earlier years my dad went to the local abattoir on Monday morning to slaughter the amount of pigs he pre-ordered. Then he loaded it onto his bakkie (pick up) in half pigs. Had a Schnapps and a beer at the tavern on the abattoir premises and went back to his butchery to immediately brake the pigs, debone them and prepare them for the week ahead. The meat trimmed for emulsion processing was immediately processed with a lot of ice so still not cooled down. The only additive he added was curing salt and spice. Even if he filled the emulsion a day later, the water intake and binding was tremendously higher than with phosphate when the pH level of the meat decreased overnight in the chiller.”

I am sufficiently intrigued to at least test pre-rigor meat for sausage production and legally there may be a way to do it even in South Africa.  The motivation will be to simplify the process by removing the need of the 2-4% addition of extenders, stabilisers and emulsifiers. I am motivated by the comparison made by Ockerman and Basu from Ohio State University between cold and hot boning where they clearly and persuasively show the economic advantages of hot boning.

There is every reason to look into this very carefully!

References

Dikeman, M., Devine, C.. 2014. Encyclopedia of Meat Sciences. Second Edition. Academic Press.

The Des Moines Register Sun Mar 10, 1974 (Active link to article)

Fung D. C., Kastner C. L., Lee C-Y., Hunt M. C., Dikeman M. E., Kropf D. H..  1981. Initial Chilling Rate Effects on Bacterial Growth on Hot-Boned Beef.  Journal of Food Protection, Vol 44, July 1981.

Henrickson, R. L.. 1983. Status of Hot Processing of Meat in the United States. Oklahoma Agricultural Experiment Station, Animal Science Research Report.

Kamejima, S., Ishioroshi, M., Yasui, T.. (1982) Heat Induced Gelling Properties of Actomyosin: Effect of Tropomyosin and Troponin, Agricultural and Biological Chemistry, 46:2, 535-540, DOI: 10.1080/00021369.1982.10865074

Knipe, L.  https://meatsci.osu.edu/node/127

Mellett, F. Private conversations.

Protein Functionality, the Bind Index and the Early History of Meat Extenders in America

Bacon & the Art of Living 1

Protein Functionality, the Bind Index and the Early History of Meat Extenders in America

Eben van Tonder
10 April 2020

bowl cutter

Introduction

In the meat industry in most parts of the world, it is customary to use non-meat ingredients in meat products, especially in comminuted sausages and lunch loaves. I know that here in Southern Africa, the indigenous tribes have been using ground peanuts (and presumably other groundnuts) as meat extenders for millennia before any European settler arrived here.

I can only imagine that this must have been the case with primitive people around the world wherever there was a shortage of meat.

Who popularised this in the West is a question that intrigued me. Off the bat, as one can imagine, these non-meat ingredients were probably introduced in countries where food scarcity was common or in times when food shortages forced humans to “stretch” the little meat they could get their hands on, such as during times of war.  In this article we briefly introduce the functionality of meat protein and ask if we can identify such a movement with the inclusion of meat extenders or replacers to pure meat in America during one of the major wars they were involved in.  The two prime candidates must surely be the two world wars and especially the second when huge food shortages were experienced in America and around the world.

The Functionality of Meat Proteins

The first question is if meat protein on its own is not sufficient to bind comminuted meat in sausages and lunch loaves.  Can a stable emulsion be formed without the use of non-meat additives such as soya isolates and concentrates and the use of different stratches either as emulsifiers or stabilisers? This includes the use of bulking agents such as rusk, which is in reality a meat extender.  This is a level of detail that I was hoping to get into a bit later in a subsequent article, but it explains my point, namely that meat proteins on their own, they have the ability to bind meat extremely well, depending on the muscle and the animal species.

Generally speaking, you will see from what follows that beef meat protein in general provides the best bind and pork, less so due to the higher fat percentage which interferes in binding, especially in emulsions.

There is a major difference between the functionality of different muscle groups in pork and even between different animals.  The sausage producer is interested in how these different proteins bind.  We therefore present the concept of a “bind constant” (functionality coefficient) that was developed to measure this and a “least-cost formulation” (linear programming) computer program to manipulate the model and minimize cost.

The man who pioneered the large-scale use of these technologies is Robert L. Saffle, during his tenure at the University of Georgia.  He did not invent any of the techniques, but was the one man responsible for propagating its use.  He also standardized their use, documented their workability and educated and encouraged processors to use it.

He was very successful at this and largely due to his work,  meat processors throughout the world recognize the word “bind” as having the basic meaning of the capability of meat to bind the sausage together. The value is referred to as the “bind constant,” “bind value” or “bind index.”

Proximate Analysis and Functional Indices of Various Meat Materials

What follows is a compilation of all meats tested by Saffle and his co-workers, in particular John A. Carpenter at the University of Georgia.  It gives the proximate analyses and average measured bind/colour indices. I included the bind index values in the first column because I wanted to show them in descending order and I separated it for different species.

Compiled by J. Carpenter, R. Saffle, H. Ockerman, Anderson & Bell and slightly modified by myself.

Bind values
When you look at pork, the highest bind value is from the shoulder muscle.   The blade is from the lower shoulder.

    Blade Bone source: https://www.turnerandgeorge.co.uk/pork-blade.html

History of Meat Binding

Labudde and Lanier (1955) put a date to the recognition of when differences in binding quality between different meat cuts were recognised when they say, “It was well recognized by the 1950s that certain kinds of meats bound the comminuted sausage more tightly together than other kinds of meats.”  I wonder what my friends in Germany would say about this statement.  I believe it was recognised probably hundreds of years before the 1950s.

They accurately report on early classification of meat binding ability. “Cuts of meat were classified into gross categories, such as good binders (bull meat, cow meat), poor binders (hearts, cheeks, fat meat) and fillers (lips, tripe, stomachs)” They are correct when they state that “sufficient lean meat of good “bind” was known to be needed to make the meat paste hold together during cooking and to develop a minimum acceptable level of firmness at the end.” (Labudde and Lanier,  1995)   This is my main thesis!  The question is how and when did this change?

Dr. Francois Mellett, who was trained in Germany (did his doctorate in German) and who trained German butchers in the Master program, tells me they don’t work with startches in sausage making in Germany. At least, not when he studied there.  Another German Master Butcher, Gero Lutge tells me that his dad, who was also a master butcher, used no extenders and that it is not very common in Germany.  It was actually these two comments that set me on this journey to unravel what is going on.  The German, and I assume, Central, North and East European traditions all concur on this point in stark contrast to the rest of the world where it became the norm to use stabilizers and emulsifiers (extenders) in sausage production.

The matter becomes wonderfully complex because it addresses matters like affordability and the quality of raw material, but what a journey!

There is a personal preference that creeps in here.  I am personally not thrilled with non-meat additives to the meat I eat.  Using meat replacers and additives is something I do as a meat producer, but I am not happy about it and I try, wherever possible, to rely on equipment and its proper handling together with a thorough understanding of meat to drive our innovations and not, in the first place, reach for the handbook of non-meat extenders and substitutes.  This is a grave mistake.

 This is another personal reason for this study.  I want to be very clear in my mind on what is the best way to use equipment to allow the meat itself to do the bulk of the work.

I am a severe asthma sufferer.  A specialist asked me one day if I religiously use the best medication to keep the condition under control to which I responded in the affirmative.  To my surprise, he was not happy with that answer.  Any chemical you put in your body, no matter how serious a condition you are trying to manage, is always a bad thing.  He encouraged me to continually try and develop an alternative, more natural way of managing the condition.  He even suggested that I try to reduce my reliance on medication.  He suggested that I should determine when I can control the symptoms without medication and when I can no longer do that and I must rely more heavily on medication.  Over the years, I have headed his advice to great benefit.

Most of the additives we are talking to in the meat are natural products themselves, which is why it is allowed, but the principle remains the same.

Before anything became “industrial”, it was first used in the home and meat and meat production is a prime example.

-> Home use of Binders

As every major industry we have today, it all started in the home.  The following Q & A appeared in an American newspaper in 1950.  Mrs GRH wrote in with a question about her meat loaf that is not sticking together.

Courier_Post_Thu__Aug_17__1950_
Reference:  Courier Post (Camden, New Jersey), Thu, Aug, 17, 1950

The advice from the chef is that Mrs. Mrs GRH either did not use a binder or used too little of it. The binders they suggest she should have used are thick white sauce, bread crumbs with a liquid, cooked rice and/or mashed potatoes.  They suggested “good old fashion kneading.” Lean meat, 2 pounds, is suggested and add 4 tablespoons of flour, 1½ cups of milk and 1 cup of soft bread crumbs or mashed potatoes.  They suggest two kinds of ground meat for flavour (beef and pork).  As we have learned, beef added to the pork would also enhance the binding.  Dice and fry ¼ pound of mildly salted pork till it is crisp and light brown, and add it for flavour, as show-pieces and mouth feel. The celery, onions and other seasoning is cooked in the salt pork dropping to develop the flavour.

This “home-level-technology” of binders, how long has this been part of the human cultural and technological matrix?  One will have to survey its prevalence in cookbooks since the time of the writing of the first one. I had a look at references in the “First American Cookbook” published in 1796 by Amelia Simmons.

first american cookbook

Several interesting things catch your eye as you work through this historical document.  For starters, there are no sausages.  Second, the use of binders is used widely, especially grated bread, butter and eggs.  In her stew pie she uses a shoulder of veal, slices of raw salted pork and half a pound of butter.  It’s not our focus here but note the common use of veal.  I find the same in German cookbooks of this time.  Her turkey stuffing calls for grated wheat loaf, butter, finely chopped salted pork and eggs.  For meatballs she uses veal, grated bread, salted pork.

-> Meat Binders for Industry (presumably for sausages)

The article below testifies to the use of binders in making hamburgers

Battle_Creek_Enquirer_Fri__Jan_30__1948_
Battle Creek Enquirer (Battle Creek, Michigan), Fri, Jan 30, 1948

I am not sure exactly what the advertisement above is saying.  Is the Ground Beef Chuck the binder?  In which case they are advertising the use of a cheaper meat cut (chuck) to use for hamburger patties, which is better than using other binders (non-meat).  Either way, it shows the “hot topic” during World War II when severe food shortages impacted the world at large, including America.  More about this later.  (I assume Binders is not the surname of the well-known meat processor of this time, R. Binder Co., because as far as I can see he always spelled his name, when used in this way, with an apostrophe “s”. It could have been a typing error when the newspaper did the typesetting 🙂 )

-> List of Newspaper References with the word “meat binder”

The Second World War was from 1939 to 1945.  Severe food shortages occurred during the war, but especially towards the end.

From 1946

Marysville_Journal_Tribune_Mon__Aug_26__1946_
Reference: Marysville Journal Tribune Mon, Aug 26, 1946.

To ease the shortage of bread, they recommended housewives to substitute bread with potatoes.  This includes potatoes as binder.

The_Record_Thu__Jul_11__1946_
Reference:  The Record Thu, Jul 11, 1946

From 1944

The_Chillicothe_Constitution_Tribune_Thu__Dec_7__1944_
Reference:  The Chillicothe Constitution Tribune, Thu, Dec 7, 1944

From 1943 (two months before the start of the War)

The term “Meat Extenders” was used synonymously with “Binder”.

Chattanooga_Daily_Times_Fri__Jun_25__1943_
Reference:  Chattanooga, Daily, Times, Fri, Jun 25, 1943

Pre-1943 references to Binders

Abbeville_Progress_Sat__Feb_10__1940_
Reference: Abbeville Progress, Sat, Feb 10, 1940

There are several pre-1943 references to meat binders, but all of them refer to butchers’ twine.  The one I give above is the least clear, but it is easy to see how the reference is not to binders as we are discussing here.

From 1974

By the 1970s, meat binders were being discussed as part of the American meat landscape.  The article below is a good case in point.

Fort_Worth_Star_Telegram_Thu__Aug_22__1974_
Reference:  Fort Worth Star Telegram, Thu, Aug, 22, 1974

The Crucial Year of 1943

The watershed year for the introduction of meat binders and extenders into the USA seems to have been 1943.  Here is an article from that year when a group of women belonged to the Matoy Home Demonstration Club.  These clubs (also known as homemaker clubs, home bureaus or home advisory groups) were a program of the U.S. Department of Agriculture’s Cooperative Extension Service, which had the goal of teaching farm women in rural America better methods for getting their work done.  This meeting, crucially during the war, was probably arranged to introduce ways to deal with wartime food shortages.

Durant_Weekly_News_Fri__Jul_23__1943_
Reference:  Durant Weekly News (Durant, Oklahoma), Fri, Jul 23, 1943

Other clubs received training on meat substitutes and extenders during the same time.  Interesting – the fact that meat extenders and substitutes were used in the same sentence.

Durant_Weekly_News_Fri__Jul_23__1943_ (1)
Reference:  Duran, Weekly News, Fri, Jul 23, 1943

They held yet another club where Miss Pearl Winterveld was doing the demonstrations during this time.

Durant_Weekly_News_Fri__Jul_23__1943_ (2)
Reference:  Durant Weekly News, Fri, Jul 23, 1943

Another club where Miss Pearl was doing her magic reported on their training.

Durant_Weekly_News_Fri__Jul_23__1943_ (3)
Reference:  Durant Weekly News, Fri, Jul_23, 1943

Another two clubs reported demonstrations for meat extenders and meat substitutes in the same publication.  This is remarkable!  The photo below, courtesy of the Cornell University Library – shows a meat canning demonstration at a meeting of the Akron Home Economics Club on December 19, 1916.

Farm_and_Home_Bureaus-_Meat_canning_demonstration_at_meeting_of_the_Akron_Home_Economics_(cropped)_(3856810708)
Meat canning demonstration at a meeting of the Akron Home Economics Club on December 19, 1916 from the Cornell University Library.

The Alexander City Outlook from Alabama reported in 1944 several demonstrations along the same line as listed above at Home Demonstration Clubs.  The Dadaville Record, also from Alabama, reported similarly on demonstrations of meat extenders and meat replacers in that same year at various club meetings.

By 1946 American soldiers started to return from Europe and clubs continued to spread the “gospel of meat extenders and meat replacers”.   In Alabama, the Wetumka Herald of 31 October 1846 reported along exactly the same as in 1943, 1944 and 1945 that demonstrations through the clubs were held at 6 locations.

What were these meat extenders and binders?

An article from 27 March 1943 gives us the detail of what was being demonstrated to the American housewife following that same year.

The_Salt_Lake_Tribune_Sat__Mar_27__1943_ (1)
Reference:  The Salt Lake Tribune Sat, Mar 27, 1943

The author emphasises the fact that knowledge is required to use these meat extenders.  He mentions that meat extenders were, at the time of writing, already a household name in America.  Still, I suspect that it did not extend much further back then, the beginning of the war, and it could not have been generally true if one takes into account the enormous effort that it took to spread the gospel of meat extenders following 1943.

Anyone wondering if the meat extenders included some magical products such as was developed by Carl Lindegren with his wife Gertrude Lindegren and reported on by the same newspaper in August of the same year when he boldly claimed that through yeast cell technology, they were able to produce “synthetic meat” – if you are expecting this, you are mistaken.  The meat extenders that was introduced to America was exactly what we still use today.  The key was vegetable sources of protein which included legumes, nuts, cereals, vegetables, and wheat.  Soya was identified as having the highest protein value.  To the housewife this gave them the option to use dried beans and peas, cooked rice, macaroni and other cooked pastes, nuts and nut butters, fresh or canned peas, corn or lima beans, potatoes, wheat flours, bread and crackers.

If the housewife used extenders with incomplete proteins, it was widely suggested in several newspaper reports to add to the diet elements with essential amino acids.  They suggest that they add eggs and milk products to their diet (which are binders in their own right).

The_Morning_News_Wed__Feb_17__1943_
Reference:  The Morning News Wed, Feb 17, 1943

The drive for meat extenders was directly related to the food shortages as a result of the war.  Brands such as Kellog’s All Bran which is a household name to this day, were marketed as meat extenders.

Council_Bluffs_Nonpareil_Fri__Mar_16__1945_
Reference: Council Bluffs Nonpareil, Fri Mar 16, 1945

Summary

The evangelists of meat extenders and replacers in the USA, from 1943 onwards, were the US Department of Agriculture through their program of Home Demonstration Clubs.  It is then because of the war that meat extenders are commonplace in a large part of the world, including South Africa.  I remember a story told by a South African meat master in his own right, Roy Oliver, whose memories goes back to the 1960s, that academics from meat science institutes in the USA regularly visited South Africa and encouraged industry to use meat binders, extenders and emulsifiers on an industrial scale.  They would send him various starches and soya products to work with and call him weekly to check on his progress, particularly taking note of the inclusion of these various emulsifiers and stabilisers.  He had to test this in meat emulsions made in the bowl cutter.

This in and off itself is an important historical clue as I suspect that South Africa was easier to access for many of these academics from the USA because of our historical close relationship with one country in the region I suspect was initially responsible for using serials, grains etc. in meat emulsions, namely Russia.

This sets up the subject of our next article!

References

Foegeding, A. A.. 1988.  Gelation In Meat Batters.  Paper presented at a conference.

Labudde, R. A., Lanier, T..  1995.  Protein Functionality and Development. American Meat Science Association.

Simmons, A..  1796. The first American Cookbook. Dover Publications.  New York.

Abbeville Progress, Sat, Feb 10, 1940

Battle Creek Enquirer (Battle Creek, Michigan), Fri, Jan 30, 1948

The Chillicothe Constitution Tribune, Thu, Dec 7, 1944

Chattanooga, Daily, Times, Fri, Jun 25, 1943

Council Bluffs Nonpareil, Fri Mar 16, 1945

Courier Post (Camden, New Jersey), Thu, Aug, 17, 1950

Durant Weekly News (Durant, Oklahoma), Fri, Jul 23, 1943

Fort Worth Star Telegram, Thu, Aug, 22, 1974

Marysville Journal Tribune Mon, Aug 26, 1946.

The Morning News Wed, Feb 17, 1943

The Record Thu, Jul 11, 1946

The Salt Lake Tribune Sat, Mar 27, 1943

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