Preparation And Product Development Of Antioxidant Peptides From Rana Chensinensis Bone

Rana chensinensis bones are by-products of Rana chensinensis,which are rich in bone protein.In this study,the technology of ultrasonic assisted extraction(UAE)was used to optimize the degreasing process before enzymatic hydrolysis of Rana chensinensis bone,and the bones were compared before and after pretreatment.Then we should select two kinds of proteases to hydrolyze the rana chensinensis bones in two steps to prepare the antioxidant peptides;We determined the antioxidant activity of the different molecular weights of the bone peptides;And the most active peptides were used as raw materials to prepare the Rana chensinensis bone peptide chewable tablets.The main results are as follows:(1)Pretreatment process of Rana chensinensis bones:The pretreatment of Rana chensinensis bones was carried out by ultrasonic assisted degreasing.The protein content and fat residual were used as indicators to evaluate the ratio of liquid to material,ultrasonic temperature and ultrasonic time.Then we carry out the response surface optimization test.The optimal conditions were as follows::the ratio of liquid to material was 6m L/g,the ultrasonic temperature was 38?,and the ultrasonic time was 21 min.Under this condition,the protein content of the bones was 48.53%,which was 21.90% and 11.64% higher than that of the non-pretreated and not ultrasound pretreated,respectively.And the residual fat was 0.98%,which was 86.85% and 81.92% higher than that of the non-pretreated and not ultrasound pretreated,respectively.The degree of hydrolysis was 75.83% and 32.78 % higher than that of the other two groups,respectively;Structural analysis showed that pretreatment had no significant effect on the secondary structure of the Rana chensinensis bones protein,but it changed the microstructure of the bone meal surface,so that the surface structure of the bone particles was loos,which wasbeneficial to the subsequent enzymatic hydrolysis and the improvement of hydrolysis degree.(2)Preparation of antioxidant peptides by enzymatic hydrolysis of Rana chensinensis bone by two-step enzyme hydrolysis:We selected neutral protease and alkaline protease by single enzyme hydrolysis to hydrolys bones of stepwise.The neutral protease was chosen as the first enzyme,and the degree of hydrolysis was used as an index to evaluate the ratio of material to liquid,the amount of enzyme,the p H and the hydrolysis time,and the orthogonal experiment was carried out.The optimum conditions were as follows:the ratio of material to liquid was 1:30,the amount of enzyme was 7%,the p H was 6.5,and the hydrolysis time was 100 min.Under this condition,the degree of hydrolysis of bones was 26.81 %.We used the alkaline protease next to hydrolyze the bone,the degree of hydrolysis and the DPPH free radical scavenging rate were used as the index to evaluate the amount of enzyme,the p H and the hydrolysis time,and we carried out the response surface optimization test.The optimal conditions were as follows:The enzyme content was 5.57%,the p H was 8.94,and the enzymolysis time was 97 min,the DPPH free radical scavenging rate was 41.89% and 90.43%,respectively.(3)Isolation and determination of antioxidant activity of the peptide of Rana chensinensis bone:The hydrolysates were separated by ultrafiltration into 6 different molecular weight of >30k Da,10 k Da~30k Da,3k Da~10k Da,1k Da~3k Da,<1k Da and the not ultrafiltrations',and we evaluated their antioxidant activity by 5methods of the DPPH free radical scavenging rate,reducing power,metal ion chelating activity,hydroxyl radical(·OH)scavenging activity and the total antioxidant activity.The results showed that DPPH free radicals and reduction power had the relatively high scavenging rate of 1~3k Da,3~10k Da,<1k Da,the relatively high chelating activity of metal ions were <1k Da and 1~3k Da,the relatively high elimination rate of·OH was <1 k Da,and the antioxidant activity of the relatively high was 1~3k Da and 3~10k Da.There were no significant differences in the results of different indicators,and the highest antioxidant activity of the peptide are allconcentrated in the range of <10k Da,and the low molecular weights' of peptides were higher than the high molecular weights'.Therefore,we selected the <10k Da peptide for subsequent product research.(4)Preparation of Rana chensinensis bone peptide chewable tablets :We selected the wet granulation process,were used particle yield as the index to evaluate the drying temperature,drying time,and PVP ethanol concentration of the mixture,and the orthogonal experiment was carried out.The optimal conditions were as follows:the drying temperature was 60?,the drying time was 90 min,and the10% PVP concentration of ethanol containing was about 50,and the particle yield was 92.81%.We choosed the formula of Rana chensinensis bone peptide chewable tablets,first,we choosed the optimal of peptide milk powder and the type of flavoring agent and sour agent,then we conducted a single factor optimization test,and the orthogonal experiment was carried out.The optimal conditions were as follows:20%of the peptide milk powder,40% of the sorbitol,3% of the malic acid,25% of the starch and 12% of the mannitol.The sensory score was 92.4,and the product was round,smooth surface,milky whit,uniform colo,milk suitable,no smell,and tasted sweet and sour,creamy texture,moderate hardness,and no particles feel.The quality differences and microbiological indicators are both in line with national standards,the hardness and other structural indicators have no significant difference to the sale of milk tablets,and the chewable tablets are suitable for drying or sealed environment at room temperature.