**6. Possible collagen extraction methods**

There are various methods to extract collagen from different animal tissues. The methods used to extract collagen from bovine or any other tissue such as fish skin; pigskin, rat tail, tendons etc. vary slightly, differing in enzyme concentration, acid concentration, salt concentration or pre-treatment period [6]. These variations can be studied and the most optimal method for bovine hide extraction can be obtained. However, acid extraction which results in acid-soluble collagen (ASC), pepsin extraction that gives pepsin solubilised collagen (PSC) and salt extractions. Some of the main extraction procedures found in literature are discussed in detail below [80].

#### **6.1 The salting-out method**

This method is seen as the least favourable method of collagen extraction. Collagen proteins, like general proteins have the property of being salt soluble. Different types of collagen proteins can be separated using the relationship between different collagen sources and salt concentrations. Neutral salt solutions are usually used, such as NaCl, Tris–HCl, phosphate, or citrate. In the salting-out method, the concentration of salt is the key factor to control, if for example, the concentration of NaCl is less than 1 mol/L in the neutral solution, its suitable for dissolution of type I collagen, however, if the concentration is bigger than 1 mol/L, it will precipitate the type I collagen. Since mature sources of collagen are less soluble because most collagen protein molecules have cross-linked, the salting-out method is not an efficient method alone to extract collagen [80].

#### **6.2 The alkali method**

The main chemicals used in the alkali method of collagen extraction are sodium hydroxide and monomethylamine [81]. This extraction method is not favoured as the main extraction method due to similar reasons as the salting-out method.

Hattori et al. [81] prepared collagen from bovine hides by alkaline solubilisation with 3.0% NaOH and 1.9% monomethylamine. The study also extracted bovine hide collagen by acid and enzymatic methods for comparison. These methods were carried out on animals of different ages. The amount of collagen extracted through this method was estimated by comparing the hydroxyproline content in the whole hide with that in the extracted collagen.

#### **6.3 The alkali-enzyme method**

The alkali-enzyme method is not as effective as the acid-enzyme method. This is because alkali is such as NaOH does not have the ability to fully solubilise collagen

and disrupt the cross-linking in a collagen molecule. This method is more preferred for gelatine production [80].
