Abstract:

Fresh bovine bones were used as raw materials to prepare bone powder. Neutral protease from Bacillus subtilis and trypsin were chosen to hydrolyze bovine protein in a two-step mode. The optimal dual-enzyme hydrolysis conditions of bone proteins were explored for maximizing degree of hydrolysis using L9(34) orthogonal array design. Results indicated that the content of proteins in bone powder was 30.72%, which was 3 times as high as that in fresh bovine bones. The sequential order for enzymatic hydrolysis of bone proteins exhibited an obvious effect on degree of hydrolysis of bone proteins. Sequential hydrolysis of bone proteins by trypsin first and then neutral protease exhibited a higher efficiency when compared with simultaneous dual-enzyme hydrolysis or sequential hydrolysis with neutral protease first. The optimal hydrolysis procedure of bone proteins was based on trypsin hydrolysis for 3 h on a 150 r/min shaking table with a material/liquid ratio of 1:2 and an enzyme dose of 2000 U/g protein at 45 ℃ and pH 8.0 followed by neutral protease hydrolysis for 3 h on a 150 r/min shaking table with an enzyme dose of 2000 U/g protein at 45 ℃. Under such conditions, the overall degree of hydrolysis reached 18.34%. Moreover, a better effect on debittering, decolorization and deodorization was obtained using activated carbon with smaller size.

Key words: bone proteins, Bacillus subtilis, neutral protease, trypsin, degree of hydrolysis