**6.5 The acid method**

**6. Possible collagen extraction methods**

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

Bovine 0.5 M NaCl

*Biotechnological Applications of Biomass*

efficient method alone to extract collagen [80].

hide with that in the extracted collagen.

**6.3 The alkali-enzyme method**

**220**

**6.1 The salting-out method**

**Table 6.**

**6.2 The alkali method**

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].

**Collagen source Chemical Reference**

Fish 0.1 M NaOH [70–74]

1 M NaCl K2HPO4 – 0.1 M NaOH

[77] [78] [79] [65]

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

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

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

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 solubilisation efficiencies of each acid.

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 tissue and into solution [73].

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 product (**Table 7**).

## **6.6 The enzyme method**

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


tubes are utilised with different cut off molecular weights to separate pure collagen

Ultrafiltration can be applied to remove the non-collagenous material prior to lyophilisation of collagen. Ultrafiltration utilises positive pressure to force a liquid through a semi-permeable membrane to separate species in an aqueous solution by molecular size, shape or charge. Ultrafiltration has the advantages of having a high throughput, cost-effective and large-scale purification is possible without being

Ultrafiltration enables the removal of solvents and salts of lower molecular weight from a solution (permeate). Thus, this results in the enrichment of the retained molecule (pure collagen). Ultrafiltration membranes can retain molecules in the range of 10 kDa to 1 MDa, thus concentration and purification of collagen

• Crossflow/tangential flow filtration: the incoming feed passes parallel across the surface of a semi-permeable membrane. A permeate and a retentate stream are generated, where the permeate is the portion of the fluid that passes through the membrane and the remainder of the feed stream, which does not pass through the cross-flow membrane, is known as the retentate stream.

• Dead-end filtration: The feed moves towards the filter membrane. The

particles that can be filtered are settled on the filter surface, however, this type of filtration is not sustainable as the accumulated solids need to be removed

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

The popularity of collagen extraction continues to increase due to many reasons. It is a high strength protein, bio-derived, has excellent biocompatibility, biodegradability, and has weak antigenicity. Another main reason that relates to waste valorisation and sustainability is the fact that collagen can be extracted from almost any mammalian skin, bones, cartilage, fish skin and even chicken feet. Most often, the meat industry results in these by-products that can end up in landfill. These advantageous

Research to this day is being carried out to improve extraction methods in terms of efficiency and economics. In addition to improving extraction methodologies, research is being carried out on collagen to enhance its use in several industries (**Table 9**).

> • Bakerman [91] extracted human skin collagen with age via the acid-solubilisation method. No defatting or demineralization steps were carried out, citric acid was used as the solubilisation agent. There was no mention of methods of collagen content analysis,

(0.3–3%) to disinfect collagen after extraction especially from fish sources.

characteristics have made collagen one of the most useful biomaterials.

from other solvents, salts and enzymes and other impurities.

(300 kDa) can be successfully achieved through this process.

periodically or the filter needs to be replaced.

**9. Collagen extraction research and its applications**

**8. Collagen preservation**

**Period Collagen extraction research**

only extracted yield was reported.

**1960– 1969**

**223**

**7.2 Collagen purification via filtration**

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

*Collagen: From Waste to Gold*

limited to lab-scale purification by dialysis.

#### **Table 7.**

*Acids used for collagen extraction.*


#### **Table 8.**

*Enzyme extraction methods used for collagen extraction.*

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

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 extraction process (**Table 8**).

#### **6.7 The acid-enzyme method**

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

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

#### **7.1 Collagen purification via dialysis**

Dialysis is a preferred method of purification for collagen extraction, however, scaling up this technique for commercialisation has proven to be difficult. Dialysis tubes are utilised with different cut off molecular weights to separate pure collagen from other solvents, salts and enzymes and other impurities.
