**5.4 Non-collagenous protein removal**

Contaminating proteins need to be removed after defatting and demineralization. Most collagen extraction methods utilise salt or alkali solutions to solubilise the contaminants. Collagen is a lot more chemically resistant than most other proteins therefore, it is much less likely to be degraded or solubilised by a weak salt (**Table 6**).


#### **Table 3.**

*Processing temperatures used to extract collagen.*


#### **Table 4.**

*Solvents used for de-fatting of collagenous tissue in literature.*


#### **Table 5.**

*Chemicals used for demineralisation in literature.*


and disrupt the cross-linking in a collagen molecule. This method is more preferred

A series of repetitive steps having acid then alkali soaking of samples for a long period can be used to extract collagen. However, this method requires a very long period and the reaction time is very slow. It does not work for mature tissues as it is near impossible for acid and alkali alone to disrupt the cross-linking developed in mature tissue, thus an enzyme is a must requirement. The collagen yield extracted decrease or increase for the same tissue type depending on the literature. These differences are due to denaturation of protein during the process of extraction, the difference in environmental temperature and the solubilisation method used to extract the collagen. The yield of collagen by the different acid (HCl, citric acid, acetic acid) is dependent on the reaction time. The longer the period of solubilisation, the greater the yield of collagen being extracted. For example, Skierka [82], concluded that during a 24 hour of collagen extraction in acid, about 33% of collagen was solubilised, and after 72 hours, about twice as much collagen was solubilised.

The solubility of collagen in acids depends up the enzyme concentration. A low concentration of enzyme with an acid can completely solubilise collagen; however, it will also depend on the type of acid. For example, enzyme concentration on the solubility of collagen in citric acid and HCl gave a maximum yield of 75% for citric

Acids such as acetic acid, citric acid and hydrochloric acid (HCl) of low concentration can be added to collagen-containing samples. Acids at a pH of 2–3 and a concentration of approximately 0.5 mol/L can be used to solubilise collagen. In acid extraction of collagen, the acids swell collagen, disrupting the hydrophobic and electrostatic interactions between the tropocollagen units, and release the acidsoluble collagen (ASC). Yang et al. [80] concluded that citric acid has the best effect to extract collagen, second being acetic acid and last being hydrochloric acid. However, according to Skierka [82] and Higham [83], the most effective acid for collagen solubilisation was acetic acid and the least effective solvent was HCl. In order to achieve a sound conclusion, experiments need to be carried out to investigate the

The acid molecules disrupt the collagen cross-linking in order to solubilise the collagen by allowing ligand substitution for each peptide side chain, causing disassociation of the cross-link. Thus, swelling the collagen and solubilising it out of the

The acid method is seen to be corrosive to the experimental equipment in terms of large-scale production. However, using a low concentration of acid in combination with an enzyme will avoid equipment corrosion and achieve a high yield

The enzyme method is seen to be as the ideal method of collagen extraction. The

three commonly used enzymes for collagen extraction are pepsin, papain and tryptase [80]. The enzyme acts on the non-helical peptide chains of the collagen protein, having no effect on the helix peptide chains of the collagen protein. The enzyme has better reaction selectivity and it is less destructive to the collagen

for gelatine production [80].

*Collagen: From Waste to Gold*

acid and 85% for HCl [82].

solubilisation efficiencies of each acid.

tissue and into solution [73].

product (**Table 7**).

**221**

**6.6 The enzyme method**

**6.5 The acid method**

**6.4 The acid-alkali oxidation method**

*DOI: http://dx.doi.org/10.5772/intechopen.94266*

**Table 6.**

*Chemicals used for non-collagenous protein removal in literature.*
