Study On Enzymolysis Technology Derived Peptides From Mactra Veneriformis And Bioactivity Of The Peptides

This study was extended from the R&D Special Fund for Public Welfare Industry from State Ocean Administration P.R. China (No:200705009) and was supported by the R&D Special Fund for Public Welfare Industry from State Ocean Administration of P.R. China(No:201305007) and A Project Funded by. the Priority Academic Program Development of Jiangsu Higher Education Institutions (No:ysxk-2010).Semi-bionic Extraction Method(SEM) was first originated by Professor Zhang Zhaowang. Mactra veneriformis was first investigated on the basis of SEM and Traditional Chinese Medicine theory.To carry out the bioactivity of Mactra veneriformis enzymatic hydrolysate,pepsin and trypsin were used to simulate enzymatic process in vivo.The obtained Mactra veneriformis enzymatic hydrolysates have a definite ACE-inhibitory and antioxidant activity.Response surface method was used to optimize the enzymatic technology.The ultratiltration was subjected to obtain different molecular mass segments of Mactra veneriformis enzymatic hydrolysate.The ACE-inhibitory activity in vitro of active site of Mactra veneriformis enzymatic hydrolysate was evaluated and the material basis on active site of Mactra veneriformis enzymatic hydrolysate was studied by UPLC-Q-TOF MS.The ACE-inhibitory peptides were obtained by Computer Assisted molecular docking screening,then furtherly.the biobioactive peptides were synthesized in vitro for validation,so as to establish an efficient,accurate research mentality and method for searching and evaluating Mactra veneriformis enzymatic biobioactive hydrolysate.This thesis was intended to provide the basis for searching and evaluating the enzymatic bioactive peptides of Jiangsu coastal shellfish and even marine bioactive peptides.Thus experimental basis and theoretical basis were provided for the further development of low-value shellfish function health products.Following parts were mainly included: 1. Literature studyResearch papers about structure-activity relationship of biobioactive peptides or amino acid sequences were referred, the progress in studies of structure-activity relationship of ACE-inhibitory peptides.antioxidant peptides and antibacterial peptides were analyzed,arranged and summarized. Research reports on molecular docking worldwide were referred, and the progress in studies on molecular docking was collected and understood.2. Preparation of biobioactive activity peptide of Mactra veneriformisTo extract biobioactive enzymatic hydrolysate from Mactra veneriformis meat residue, two gastrointestinal proteases(trypsin and pepsin) were employed to enzymatic hydrolysis at proper conditions, then the antioxidant activity and ACE inhibitory activity of the hydrolysates were investigated.Among the hydrolysates,trypsin hydrolysates had the highest antioxidant activity and ACE inhibitory activity.Different one-factor experimental design(including temperature,pH,enzyme-to-substrate ratio and reaction time) was accomplished with the enzymatic hydrolysis medium of trypsinase.According to the reponses of ACE inhibitory rate,Centural Composite model response surface design was furtherly used to optimize the enzymatic technology of Mactra veneriformis enzymatic hydrolysate.The Mactra veneriformis enzymatic hydrolysate which possessed best ACE inhibitory activity was prepared under following condition:trypsin was selected to hydrolyze at pH 8.5,48℃,2 h reaction time and enzyme-to-substrate ratio 0.6%.The ultratiltration membrane was subjected to obtain different molecular mass segments of Mactra veneriformis enzymatic hydrolysate.The cut-off molecular weight of ultratiltration membrane was 1k,3k and 5k Da,the mass segment that molecular weight less than 1k Da and 1～3k Da had higher ACE inhibitory activity,which is 83.39% and 80.20%.And the mass segment that molecular weight less than 3k Da was named as Mactra veneriformis biobioactive enzymatic hydrolysate.Investigated on the stability of temperature,pH and gastrointestinal proteases,the results showed that Mactra veneriformis biobioactive enzymatic hydrolysate had good stablility.3. Material basis of Mactra veneriformis biobioactive enzymatic hydrolysateThe molecular weight less than 1k Da had highest ACE inhibitory activity was definite and furtherly analyzed by UPLC-Q-TOF MS,which discovered 5 major peptides.By analyzing the outcome of LC-MS/MS and calculating of de novo sequencing,18 probable amino acid sequences were obtained.Owing to Discovery Studio,molecular docking of 18 peptides and ACE was studied to select and define ACE-inhibitory peptides.LL and LASPTM were screened to be the ACE-inhibitory peptides.The ACE-inhibitory peptides were prepared by synthesizing in vitro.The mass weight and amino acid sequences of synthetic peptides were confirmed by LC-MS/MS,and combining the results of ACE-inhibitory activity in vitro,the bioactivity of synthetic peptides was verified.It turned out that the amino acid sequences of synthetic peptides and Mactra veneriformis biobioactive enzymatic hydrolysate were consistent and the ACE-inhibitory activity result was consistent with the molecular docking outcome.