## 3 Results and Discussions  
Total Soluble Protein and Hydroxyproline Content of Salmon Bone Leached out during Alkaline Treatment  
The total soluble protein content liberated into alkaline solution used for the treatment of salmon bone, a leftover from the hydrolysis process, was monitored as a function of time (Figure 1a). A sharp increase in extractable protein from salmon bones was observed up to 50 min of the alkaline treatment. Thereafter, a slight decrease in protein content was noticeable up to 120 min. Alkali was able to solubilize the protein attached to the bone that remained after the enzymatic hydrolysis. This could lead to the removal of proteinaceous substances from the aforementioned bones. With continuous stirring at an operating temperature of 50 \({}^{\circ}\)C, proteins were likely to undergo denaturation or unfolding. This resulted in an increase in mass diffusivity, which in turn accelerated the mass transfer and solubilization of denatured proteins from the bone matrix. The result was in line with [PERSON] et al. [19], who reported the positive impact of high temperature on the alkaline extraction of protein from chicken bone waste. Alkaline solutions were reported to be effective in the removal of non-collogenous proteins from the starting materials used for collagen or gelatin production [20]. After 50 min, no further increase in the total soluble protein was found. This could be a result of less availability of soluble proteins in the bone. A further degradation of peptides to free amino acids or di-peptides could result in a lower content of proteins detected by the biuret method [21]. The content of hydroxyproline, a distinct amino acid presented in collagen, was determined during alkaline treatment as shown in Figure 1b. Hydroxyproline in the bone matrix represents collagenous proteins. Collagenous proteins were released with continuous stirring as a result of softening and rupturing of the bones induced by the alkaline condition at high temperature. The release of these collagenous proteins occurred continuously up to 100 min. A decline in hydroxyproline content was found at 120 min. In the present study, the alkaline treatment for 40 min was selected to remove non-collogenous proteins in the bone. Chemical Compositions of Bio-Calcium and Calcined Bone from Salmon Frame with and without Alkaline Treatment  
Chemical compositions of the bio-calcium and calcined bone are shown in Table 1. On a dry basis, bones that were calcined for 6 h had very low moisture content, of which those calcined for 9 h possessed no moisture as a result of the high temperature treatment on bones during calcination, which removed all the organic compounds and water molecules. For bio-calcium, Bio-cal-H had a higher moisture content than Bio-cal-A (\(p<0.05\)). Fluids or other proteinaceous residues might bind or form a complex with water in the bone matrix as indicated by a higher moisture content. The ash content of calcined powders was higher than that in their bio-calcium counterparts (\(p<0.05\)). During calcination, all organic components were combusted. It is noted that calcination for a longer time yielded a powder with higher ash content as a result of a more complete decomposition of the remaining organic matters. Overall, a high ash content of calcined powders (99.55-99.99%) was obtained. The Ca/P mole ratios of Bio-cal-A and Bio-cal-H were 1.66 and 1.60, respectively. A slight increase in the Ca/P ratio of the bone sample with alkaline treatment could be due to the removal of non-collagenous phosphoproteins presented in the bone matrix [22]. Calcination at an elevated temperature for a longer time also provided the calcined bone with increases in calcium and phosphorus contents, especially for the non-alkaline treatment samples. The result suggests that the removal of non-collagenous protein with alkaline treatment could have led to a higher ash content of Bio-cal-A.