Extraction of Natural Nanostructured Hydroxyapatite from Pacific Cod ( Gadus macrocephalus ) Bone with a Thermostable Collagenolytic Protease and Its ex vivo Intestinal Bioavailability

Natural nano-hydroxyapatite (HA) was extracted from Pacific cod ( Gadus macrocephalus ) bone with a thermostable collagenolytic protease in the present study. Conditions for the enzymatic reaction were optimized to be 60 °C and pH 7.0, and a desirable extraction efficiency was achieved by using the crude collagenolytic protease. Dynamic light scattering, transmission electron microscopy and energy-dispersive X-ray analysis revealed that nano-HA are anionic spherical (about 110 nm) particles mainly comprised of calcium and phosphorus at an approximate ratio of 5:3. As evaluated with the mouse ex vivo intestinal segments, the extracted nano-HA displayed comparable level of intestinal bioavailability to the positive control CaCl 2 . By treating with inhibitors (NaN 3 , amiloride) and low temperature (4°C), clathrin-mediated endocytosis was assumed to involve the intestinal absorption of nano-HA. Overall, the application of thermostable collagenolytic protease is proved to be a promising alternative method for nano-HA extraction from natural resource with improved ecological and biological value.