**8. Collagen preservation**

protein, resulting in a protein whose triple helix structure is better preserved. Thus, the extracted collagen will have a better purity, and retain stable physical and chemical properties. The enzyme method also provides mild reaction conditions that avoid equipment corrosion and less energy consumption. However, reaction

**Collagen source Enzyme type and conc. Reference**

Fish 1% (w/w) pepsin [62, 66, 69]

Pepsin

**Collagen source Acid type and concentration Reference**

10% acetic acid with 0.2% chlorhydric acid

[14, 62, 81, 84–87]

[62, 66, 67, 69, 70]

[90] [62, 86–88]

[88]

[73, 82] [73, 82]

Bovine 0.5 M acetic acid

*Biotechnological Applications of Biomass*

Fish 0.5 M acetic acid

Bovine 1% Trypsin

*Enzyme extraction methods used for collagen extraction.*

*Acids used for collagen extraction.*

**Table 7.**

**Table 8.**

0.15 M HCl Citric acid

The enzyme solubilisation method works by disrupting the cross-linking that occurs in collagen. The chosen enzyme cleaves to the amino telopeptides from the tropocollagen molecule thus disrupting the cross-linking and allowing solubilisation of the collagen molecule. Enzyme solubilisation is mostly required when extracting collagen from mature tissue, this is due to the cross-links forming keto-imines which are increasingly difficult to disrupt as they contain strong intermolecular bonds. However, the enzyme method has the disadvantage of not only breaking the collagen molecule but also resulting in the scission of other proteins may occur too, hence, causing protein contamination as a result [83]. Enzymes have been used in collagen extraction, McClain et al. [78] used papain at 0.1% in buffers containing 0.02 M phosphate and 0.003 M EDTA as a solubilisation method for collagen. The enzyme method is usually combined with the acid method to enhance the

The enzyme-acid solubilisation method is seen to be the most effective way to extract collagen. Both acids (citric acid, hydrochloric acid, acetic acid) and enzymes have the capability to disrupt the cross-links in a collagen molecule and make collagen soluble in solution. Addition of both an acid and an enzyme speed up the reaction time and results in a collagen protein well-kept in its triple helix structure [80]. Concentration and acid/

Dialysis is a preferred method of purification for collagen extraction, however, scaling up this technique for commercialisation has proven to be difficult. Dialysis

enzyme type greatly depend on the collagen tissue and method optimization.

**7. Collagen purification via dialysis and filtration**

time may be long, depending on the type of enzyme used [89].

extraction process (**Table 8**).

**6.7 The acid-enzyme method**

**7.1 Collagen purification via dialysis**

**222**

In order to preserve extracted collagen, it is usually freeze-dried and stored at conditions not exceeding 4°C. However, some researchers use hydrogen peroxide (0.3–3%) to disinfect collagen after extraction especially from fish sources.
