Study On Preparation Of Magnetic Restricted Access Affinity Mediaand Purification Of Angiotensin-I-Converting Enzyme(ACE) Inhibitory Peptides

With the improvement of people's safety awareness of food and medicine and self-care awareness,the concept of using non-drug therapy to achieve disease treatment and prevention has been accepted by more people.The isolation of angiotensin-I-converting enzyme?ACE?inhibitory peptide from natural products and the development of functional food with blood pressure regulation to control,alleviate and adjuvant treat hypertension have become research hot-spots in recent years.At present,the separation of ACE inhibitory peptides is mainly a combination of several different separation methods,and each step of separation needs to be carried out multiple times,which makes the separation process less efficient and costly.In order to meet the needs of the functional food and medical fields,it is not only necessary to further develop new natural ACE inhibitory peptides strcture,but also to study how to rapidly and efficiently isolate ACE inhibitory peptides from natural products.Based on the background of rapid separation of ACE inhibitory peptides from natural products,in this study,we prepared a magnetic restricted access affinity media?RAAM?which combines the advantages of magnetic separation,immobilized metal affinity separation and restricted access media and applied it for rapid and efficient separation of ACE inhibitory peptides from enzymatic hydrolysate of casein and pinctada martensi meat.The prepared RAAM in this study can efficiently block the interference of macromolecular proteins while enrich the ACE inhibitory peptide in one step separation,which improving the separation efficiency and reducing the purification cost.The main research contents are as follows:?1?Magnetic silica microspheres?mSiO₂?were successfully prepared by reverse microemulsion method.The prepared mSiO₂ microspheres possess good sphericity,and super paramagnetic properties.The particle size distribution of prepared mSiO₂ microspheres is about 200-250 nm.The prepared mSiO₂ microspheres can be rapidly separate from liquids.The mSiO₂ microspheres were subjected to amination modification and the maximum amino content was 0.5042 mmol·g^-11 under the optimum reaction condition.The mSiO₂ microspheres were activated by epichlorohydrin?ECH?,the epoxy density can reach up to 155.8?mol·g^-1under the optimum condition.Immobilized metal affinity media?IMAM?was prepared via immobilizing Cu²⁺on the surface of mSiO₂ microspheres by iminodiacetic acid?IDA?,the Cu²⁺chelation amount could reach 49.36?mol·g^-1.The effects of adsorption condition of IMAM for BSA were investigated.The experimental results showed that the maximum adsorption capacity of BSA was 76.34 mg·g^-1.?2?Restricted access affinity media?RAAM?was prepared by modifying IMAM with mPEG and its adsorption performance was investigated.RAAM was successfully prepared by grafting aldehyde modified mPEG onto the surface of IMAM via Schiff base formation.The preparation conditions of RAAM grafted with different molecular weight?MW?mPEG were optimized.Under the optimal condition,the maximum grafting rates of RAAM grafted with mPEG?MW1000,1900,5000?were 10.33%,12.42,14.17%,respectively.The adsorption performance of RAAM grafted with different molecular weight mPEG in single system and binary system which set BSA as a macromolecular protein model and FFVAP as a small molecule peptide model were investigated.The results show that RAAM due to the grafted mPEG could effectively block the adsorption of BSA and the higher molecular weight of grafted mPEG,the better resistance to BSA and the higher adsorption capacity of FFVAP.The NH₄Cl+NaCl system was selected as the eluent,the maximum recovery rate of FFVAP can reach 75.02%.The recycle performance of the media was examined and the results showed that the prepared RAAM had good reusability,which greatly reduced the purification cost.The interaction mechanism between RAAM and BSA was determined by zeta potential,fluorescence spectroscopy and microcalorimetry.The results of zeta potential indicated that electrostatic attraction existed in the binding process of RAAM and BSA.Meanwhile,RAAM showed a repulsive force against BSA due to the grafted mPEG.The adsorption of BSA on the surface of media depends on the competing results of the two forces.The results of fluorescence spectroscopy showed that the adsorption media can quench the fluorescence of BSA.The quenching mechanism is that the adsorption media formed a complex with BSA which belonged to the static quenching mechanism.The results of microcalorimetry showed that the higher the molecular weight of mPEG grafted on RAAM surface,the greater heat generated.This results indicated that the grafted mPEG on the surface of RAAM provided a certain energy barrier for effectively blocking BSA.?3?Casein was synergistically digested with trypsin and pepsin to obtain hydrolysate with ACE inhibitory activity and the casein hydrolysate was purified with RAAM.The results of gel permeation chromatography?GPC?showed that RAAM could block the adsorption of macromolecular proteins and effectively enriched ACE inhibitory peptide in casein hydrolysate.Compared with the enzymatic hydrolysate,the ACE inhibition rate was improved 55.93%after purification by RAAM.The components purified by RAAM were further separated by reversed phase high performance liquid chromatography?RP-HPLC?and the eluted fraction with the highest inhibitory activity was selected and then identified by mass spectrometry.A novel ACE inhibitory peptide was identified as LLYQEPVLGPVR with an IC₅₀0 value of 273.5±5.4?mol·L^-1.According to the Lineweaver-Buck double reciprocal method,the inhibition mode of purified ACE inhibitory peptide LLYQEPVLGPVR was determined to be non-competitive inhibition.Molecular docking results showed that the ACE inhibitory peptide LLYQEPVLGPVR bind with ACE through hydrogen bond and the binding site was located at the inactive site of ACE,the non-competitive inhibition mode of LLYQEPVLGPVR was further demonstrated from the molecular structure.?4?Pinctada martensi meat was digested with alkaline protease to obtain hydrolysate with ACE inhibitory activity.The pearl oyster?pinctada martensi?meat protein hydrolysate?POMPH?was purified with RAAM and the purified components were further separated by RP-HPLC.Two ACE inhibitory peptides with high inhibitory activity were identified as HLHT and GWA,and their IC₅₀0 values were 458.1±3.2?mol·L^-11 and 109.3±1.5?mol·L^-1,respectively.According to the Lineweaver-Buck double reciprocal method,the inhibition mode of purified ACE inhibitory peptide HLHT was determined to be non-competitive inhibition while GWA was determined to be competitive inhibition.Molecular docking results showed that the ACE inhibitor peptide HLHT binds to the inactive site of ACE while GWA binds to the active site,further demonstrating their inhibition modes from the molecular structure.The effective antihypertensive effect in vivo was confirmed by intravenous injection of POMPH in SD rats.