**5. Methods used to extract collagen from bovine hides**

### **5.1 General extraction procedure requirements**

Every bovine collagen extraction procedure is restricted to the following four variable conditions:


Prior to collagen extraction, the sample is chopped to increase the extraction surface area and to speed up the extraction process. However, the temperature of the sample needs to be monitored, as high temperatures will unravel the tropocollagen making it soluble in solution, resulting in gelatine (denatured collagen). This greatly reduces the value of the protein, thus if native collagen is desired, any heating or denaturation of collagen should be avoided at every step of the process. Bovine collagen extraction is mostly carried out at temperatures of approximately 4°C to prevent bacterial contamination [9].

Collagen from juvenile sources (e.g. new-born calves or chicken embryos) will readily swell and dissolve in a low concentration of acetic acid solution and can be recovered by precipitating out the collagen by adding 1 to 5 M NaCl. However different types of collagen from different tissues will precipitate at different NaCl concentrations [59].

The older the animal/tissue sample, the greater the amount of lysinehydroxylysine covalent cross-links that form between tropocollagen units. These cross-links typically form between the unwound part of a tropocollagen strand and another part of another tropocollagen unit, improving structural strength and chemical resistance of collagen, making the sample largely insoluble in acetic acid. The amount of cross-linking depends on the type of tissue (i.e. tendons are highly crosslinked to give strength) and age of the tissue (i.e. mature sources, such as bull-hides have high cross-linking in comparison to younger sources such as calf-hides) [60].

#### *Collagen: From Waste to Gold DOI: http://dx.doi.org/10.5772/intechopen.94266*

In order to dissolve mature collagen, pepsin enzyme can be added to the acetic acid solution, which attacks and cleaves the unwounded part of tropocollagen, allowing the tropocollagen units to separate and dissolve [59].

The following sub-sections discuss the main extraction steps/parameters or variables in more detail.
