Enzymatic Preparation Of Ox Bone Collagen Peptide And Its Free Radicals Scavenging Activity

Recently, preparation of natural food antioxidants with high activity and low toxicity has attracted more attentions due to the undesirable safety of chemosynthetic antioxidants. Collagen peptide is the degradation product of collagen with lots of physical activities. However, till now, the research of collagen peptide with antioxidant activity was mainly focused on aquatic and animal skin, but less about the collagen peptide from animal bone, especially ox bone. In this dissertation, enzymatic extraction of collegen from ox bone was studied and the feasibility of enzymatic hydrolysis of collagen of ox bone was explored and the reaction conditions were optimized. The antioxidant activity of collagen peptides was determined and the effect of hydrolysis degree on the in vitro antioxidant activity of the products was investigated. Besides, the characterization of the product collagen peptides was also studied.An enzymatic extraction of bone collagen from ox bone powder was studied and the best pretreatment procedure of the raw materials was determined as follows: using hexane to exclude tallow, using 5 times weight 0.25 mol/L EDTA to exclude Calcium 2 days. The best enzyme extraction conditions were as follows: the enzyme dosage of 30 mg/g, the enzymatic reaction time of 120min, pH 1.7, the reaction temperature of 37℃, liquid-solid ratio of 6:1, and extraction time 5 hours. Under these conditions, the extraction rate of ox bone collagen reached 9.93%. The mean molecular weight of the achieved product was shown to be10250,containing 75 amino acides.Among the examed six kinds of proteases, Alcalase 2.4L showed the highest actctiviyt on catalyzeding the hydrolysis of ox bone collagen, followed by Trypsin, Promamex and Flavourzyme. The effect of several key factors, including enzyme dosage, substrate concentration,reaction temperature, pH value and reaction time, on the hydrolysis of ox collagen by Alcalase 2.4L were investigated. Results showed that the optimal conditions were enzyme dosage of 40μL/g, substrate concentration of 80g/L, reaction temperature of 60℃, pH value of 8.5, and reaction time of 3h, under which DH value reached 13.5%. Kinetic study showed that the maximum velocity and Km value were of 0.3444 mol/L·h and 1.6362 mol/L, respectively.Hydrolysis degree of the reaction showed obviously effect on O2-·scavenging activity of the collagen peptide which reached 63.86%-65.81% when the hydrolysis degree of the reaction was within 6.8%-7.9%. The product showed high·OH scavenging activity, which was increased with increasing of hydrolysis degree and the highest valued was achieved as 99.84% (DH 7.9%). Similar influence curve of hydrolysis degree on the Fe3+ reducibility of the product was found and the highest value of Fe3+ reducibility was obtained by controlling the DH value of 7.9%. Collagen peptide also showed high anti-lipid peroxidation activity. When the reaction DH value was within 7.9%-13.4%, the anti-lipid peroxidation activity of the peptide was found of 74.78%-81.35%. Disappointedly, the effect of the peptide on DPPH·scavenging was lower than the original materials. Considering the total antioxidation of hydrolysis product, the best collagen peptide was prepared by controlling the reaction DH of about 7.9% (reaction time 30min)GPC analysis showed that, within the examined range of DH values, a linear dependence of the antioxidant activity of the peptides on the DH of reaction was found with the linear equation was y=-153.27x+3328.4 (R2=0.9923). Collagen peptide with highest total antioxidation contains 14-15 amino acids with its average molar weight of 2025Da. collagen peptide with highest total anti-radicals activity conclude 78.3% functional components and the rate of functional peptide extraction reached 7.31%.This study not only enriches our knowledge of enzymatic modification of natural compounds, but also find novel route to the enzymatic preparation of natural antioxidants. Besides, this research also forms a theoretical and experimental foundation for the further development of collagen with various sources...