**6.4 The acid-alkali oxidation method**

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 acid and 85% for HCl [82].
