Preparation And Structure Characterization Of Cattle Bone Collagen Peptide Chelating Calcium

In this experiment, fresh bovine bone was used as raw material, after cleaning, degreasing and other pre-processing, and then made into powder. The best enzymatic hydrolysis process of collagen peptides, extraction of soluble bone calcium from bone residue, the best coordination process of collagen peptides chelate calcium were studied. Finally, after purifiied by ethanol, Fourier transform infrared spectroscopy, UV spectroscopy, Scanning electron microscopy, X diffraction spectroscopy were used to characterizate the structure of collagen peptide chelate calcium.Papain, A1398 neutral protease, pancreatic protease, and alkaline protease were used to hydrolyze bone powder respectively. Degree of hydrolysis (DH) and peptide concentration were used as indexs. Papain and alkaline protease were chosen to carry out the step by step enzymatic hydrolysis though the hydrolysis effect of bone power. The optimum compound enzymolysis technology is as following:time 2:2, E/S 2:2, temperature 55℃, the order of adding enzyme is as following:first papain and then alkaline protease. The collagen hydrolysis was divided into four components by Sephadex G-25, the molecular mass distribution from 117 to 81283 Da, the proportion of 4000 Da is at the most. Four components were collected and chelated with bone calcium, the result indicate that the peptides with a small molecular mass about 4000Da (mainlyⅡ,Ⅲ,Ⅳcomponent) was significantly easier to interacted with bone calcium. The amino acid analysis showed that proline, glycine which are characteristic amino acids of collagen have a higher content. The content of acidic amino acid such as threonine aspartic acid is aslo higher.These results indicates that bone protein hydrolyzate is a good peptide source to preparation peptide chelate calcium.Hydrochloric acid, acetic acid, lactic acid, citric acid were used to extract soluble bone calcium from bone residue. The results showed that the hydrochloric acid has the best effects,31.5mg bone calcium were extracted from per gram bone residue. Three-factor quadratic orthogonal test was aplicated to optimize the technology. The optimum conditions is as following:time (t) 95 min, temperature (T) 70℃, solid-liquid ratio (m:V) 1:20.32.34 mg were extracted from per gram bone residue under above conditions. Rotary evaporation were used to disacidify acid, the ratio of disacidify acid was 80%. Active carbon was used to decoloration, the optimum conditions is:carbon concentration 2%, bleaching temperature 20℃, Bleaching time 10 minutes, under this conditions the decolorization rate exceed 99.50%.The optimum chelation conditions is as following:pH 8.0, time(t) 60 min, mass ratio2:1, temperature 60℃. The chelation rate was up to 91.89% under above conditions. "Sodium sulfide method"was used to identify collagen peptides chelate calcium, the result showed that collagen peptides chelate calcium is really different from collagen peptide.Elemental analysis, solubility tests under different pH, IR, UV scanning, SEM, X diffraction were used to characterizate the structure of collagen peptides chelate calcium. Solubility tests under different pH showed that collagen peptides chelate calcium is a soluble substance. In infrared spectra experimental, after collagen peptides combined with calcium the amideⅠ,Ⅱwave number were displaced.In UV scan spectra, the characteristic absorption peak of carbonyl group and peptide linkage of collagen peptide are clearly shifted. In SEM spectra, a lot of white grains can been see "embedded" clearly in the surface of collagen peptide. After combined with calcium, the X ray diffraction spectra showed that the no rules non-crystal structure collagen peptides turned into rules crystal structure. According to the structure analysis that collagen peptide chelate calcium is a five-membered ring structure, calcium is in the center and combined strongly with amino-group and carboxyl-group.