Preparation Of Peptides With Antioxidative Activity From Sinonovacula Constricta

Bio-active peptides which were proved to play an important role in human health,is becoming one of research hot spots in the field of food science and nutrition.Marine shellfishes are a kind of important raw material for the preparation of bio-active peptides due to its rich protein.Sinonovacula constricta which belongs to Solen,Veneridae,Lamellibranchia,is a kind of aquatic animal with rich protein,and thus could be used as a good material to produce bio-active peptides.This is not only helpful to make full use of sinonovacula constricta protein,but also helpful for the development of deep processed products and broaden the usage of sinonovacula constricta resources.In this study,sinonovacula constricta was used to prepare antioxidant peptides by enzymolysis method.Purified peptides were obtained from the hydrolysate through a series of separating and purifying procedures.And MALDI-TOF/TOF and LC-MS/MS were adopted to analyze its amino acid sequences.The main research contents and conclusions are listed as follows:1.Direct drying method,Kieldahl method and Soxhlet extraction method were adopted separately to measure the moisture,protein and fat content of the fresh sinonovacula constricta.The results showed that:the moisture content in fresh sinonovacula constricta was 80.74%;protein and fat content were 15.51%and 2.35%.It suggested that sinonovacula constricta is a kind of aquatic animal with high protein but low fat.It is suitable for the preparation of bio-active peptides.2.Alkaline protease,neutral protease,TPCK-tiypsin and chymotrypsin were chosen to hydrolyze sinonovacula constricta protein separately.The yield of poly-peptides was taken as evaluation index,and orthogonal experiment,base on single factor experiment,was adopted to optimize enzymolysis process of the four kinds of protease.Experimental results were showed as follows:The optimal enzymolysis parameters for alkaline protease were:pH=9.5,the amount of enzyme was 3045U/g,the solid-liquid ratio was 1:3.0(m:V),the enzymolysis temperature was 45? and the enzymolysis time was 2.5hr.Under the optimal hydrolytic conditions,the yield of poly-peptides was up to(81.3 ± 0.4)mg/g.The optimal enzymolysis parameters for neutral protease were:pH=7.0,the amount of enzyme was 1200U/g,the solid-liquid ratio was 1:2.5(m:V),the enzymolysis temperature was 40 ? and the enzymolysis time was 2.0hr.Under the optimal hydrolytic conditions,the yield of poly-peptides was up to(79.2 ± 0.7)mg/g.The optimal enzymolysis parameters for TPCK-trypsin were:pH=7.8,the amount of enzyme was 18000U/g,the solid-liquid ratio was 1:3.5(m:V),the enzymolysis temperature was 37 ? and the enzymolysis time was 2.0hr.Under the optimal hydrolytic conditions,the yield of poly-peptides was up to(71.6 ± 0.4)mg/g.The optimal enzymolysis parameters for chymotrypsin were:pH=8.5,the amount of enzyme was 1904.4U/g,the solid-liquid ratio was 1:2.0(m:V),the enzymolysis temperature was 55 ? and the enzymolysis time was 2.5hr.Under the optimal hydrolytic conditions,the yield of poly-peptides was up to(73.8 ± 0.7)mg/g.3.Polyethersulfone ultrafiltration membrane,with the molecular weight cut-off of 10000Da and 5000Da,were adopted to separate products of enzyme hydrolysis produced under the optimal enzymolysis condition.Products of enzyme hydrolysis with the molecular weight larger than 10000Da,between 5000 and 10000Da,and less than 5000Da were obtained.The antioxidant capacity of products of enzyme hydrolysis,and its separated parts with different molecular weight distribution was evaluated separately by scavenging capability of DPPH·,·OH and·O2-.By comparing the antioxidant activity of the products of enzyme hydrolysis,and it's separated portions with different molecular weight distribution.It was showed that seperated portion from the hydrolysate produced by neutral protease with the peptides molecular weightless than 5000Da was proved to be with the highest antioxidant capacity.Therefore,neutral protease was chosen as the most suitable enzyme to produce antioxidant peptides.4.Neutral protease was chosen to hydrolyze sinonovacula constricta protein,and the ultrafiltered portion(named SCNH)was further fractionated by chromatographic methods using SephadexG 15 gel filtration and get five components named SCNH1,SCNH2,SCNH3,SCNH4 and SCNH5.The antioxidant activity of these five components was evaluated by scavenging capability of DPPH·,·OH and ·O2-.The results showed that SCNH2 component had the highest antioxidant activity.Semi preparative high performance liquid chromatography was adopted for further separation and purification to get four components named SCNH2-1,SCNH2-2,SCNH2-3 and SCNH2-4.The antioxidant activity of these four components was evaluated by scavenging capability of DPPH·,·OH and·O2-.The results showed that SCNH2-3 component had the highest antioxidant activity.MALDI-TOF/TOF and LC-MS/MS were used to identify the peptide in SCNH2-3.The results of amino acid sequencing suggested the peptide in the fraction of SCNH2-3 was Phe-Gln-Lys-Asn-Arg-Leu-Phe-Phe-Tyr(FQKNRLFFY 1261.681Da).