Transfer And Absorption Mechanism Of Casein-dervied Angiotensin-I-converting Enzyme Inhibitory Peptides Based On Caco-2 Cell Model

Food proteins can be hydrolyzed to produce active peptide that have become a research focus in the functional food.Bovine milk containing the abundant proteins,can produce different active peptides in vitro digestion.it confirmed that these active peptides have physiological functions in animal experiments.Most of these active peptides are composed by more than three amino acids residues,but less study on absorbiton mechanism of more than three amino acids of peptide in intestinal tract has been reported,Clarifying active peptide absorbed mechanism in intestinal tract is particularly important for active peptide mechanism of action and functional evaluation.This research uses milk casein as raw material,and it was digested in the vitro environment,discussing ACE(Angiotensin converting enzyme)inhibitory peptides in the gastrointestinal digestion,analyzing change of hydrolysate by RP-HPLC,and identifying the sequence of peptides by LC-MS/MS;Fluorescent tags casein was digested in vivo,using the laser confocal microscope,RP-HPLC,and full wavelength function readout instrument to detect FITC-casein hydrolysate mucosa absorption in rat intestinal;Finally,establishing the Caco-2monolayer cell model,six kinds of ACE inhibitory peptides in the intestinal absorption and absorption mechanism was studied by adding different inhibitors.The main results were as follows:Firstly,Bioactivity of milk casein derived peptides in vitro gastrointestinal digestion was investigated.Hydrolysis of the gastrointestinal digestion reached maximum for 25.51 ± 0.41% among the mimic gastric,intestinal and gastrointestinal digestion;ACE activity in vitro and DPPH free radicals detection method was used to detected the ACE inhibitory activity and antioxidant activity of hydrolysate respectively.digesting within 1h,DPPH free radicals clearance increased rapidly to 18.25 ± 0.72%,25.82 ± 1.15% and 41.78 ± 0.71%,respectively;2 h gastrointestinal hydrolysate reached the maximum among thethree digestive way and DPPH free radical clearance rate was 44.27±0.93%.ACE inhibition rate of gastrointestinal digestive juices was significantly greater than the two separate digestion ways,and ACE inhibitory activity of 2 h hydrolysate was maximum to 68.03±1.14%.The secondary mass spectrometry of LC-MS/MS identified 21 pieces of ACE activity peptides and these peptides still need further verification.Secondly,Absorption of FITC-casein in rats was studied.FITC-casein used SDS-PAGE electrophoresis to evaluate the stability of FITC-labeled casein with labeling rate 13.91%,in the gastrointestinal digestive juices.The results showed that when the artificial gastric juice and intestinal juice digest for 5 h,molecular weight of digest was concentrated on about 11 kDa with significant fluorescent strength;Tricine-SDS-PAGE electrophoresis was used to detect gastrointestinal 2h-digestive juice,and protein bands were mainly concentrated under the 5 kDa with significant fluorescent strength.So FITC-casein can be used to track the gastrointestinal absorption.Rats lavage experiment elucidated that the fluorescence intensity of intestinal epithelial cells reached 1047.12 ± 157.82 RFU in the 1 h-lavage,and significantly incresed to 2037.44±119.31 RFU in the 3 h-lavage.Laser confocal microscopy showed that bovine milk casein in rats could be rapidly absorbed.Establishing intestinal capsule not flip model confirms that FITC-casein hydrolysate under 5 k Da could be absorbed by intestine,the degree of absorption rate is duodenum>jejunum>ileum absorb>colon.The duodenum is the main absorbtion position.Finally.ACE inhibitory peptide absorption mechanism was researched based on Caco-2 cells the model.The evaluation indexes result of Caco-2 single cell membrane model was as following: the 16-day cultivation showed a good cell morphology,uniform growth,and forming a single-layer film;Papp of fluorescent yellow was 6.06×10-7;AP/BL side enzyme activity ratio of 7.60 in the 15 days,in addtion,alkaline phosphatase was mainly distributed in the AP side withobvious polarization phenomenon;Monolayer film resistor of 417?·cm2,these index were in accord with entired Caco-2 monolayer film.The indexes mentioned above showed the Caco-2 monolayer cell model is successful established.Selecting the six kinds of ACE inhibitory peptides produced in gastrointestinal digestion conditions to investigate the transport mechanism.Thesix kinds of ACE inhibitory peptides IPP,RYLGY,LHLPLP,AYFYPEL,RPKHPIKHQ and WQVLPNAVPAK were syntheted and discussed in this paper.Taking RYLGY as a model,The main factors influencing the RYLGY across membrane and transfer capacity are optimized,and ensure the best conditions was the peptide concentration of 5mmol/L,the transfer time of 90 min,at pH7.4and the transport direction was AP-BL.Using transmembrane transport inhibitor,for example,deoxidation cholic acid sodium,Gly-pro,verapamil,sodium azide and MK-571 sodium to research six kinds of ACE inhibitory peptide absorption mechanism.Six kinds of ACE inhibitory peptides absorbed and efflux mechanism elucidated as below.Six kinds of ACE inhibitory peptides were paracellular route transfering through the intestinal epithelial cells;IPP transfering exists an effect of efflux needing ATP;Efflux mechanism of RYLGY,LHLPLP and RPKHPIKHQ is taking P-glycoprotein as vector needing ATP to provide energy;The AYFYPEL takes the form of P-glycoprotein as vector which execute effect of efflux;However,the WQVLPNAVPAK takes MRP2 as vector and needs ATP.The RP-HPLC showed that six kinds of peptides could transfer through Caco-2 monolayer cell membranes with entire amino acid residue form.Finally,it found that three long peptides in intestinal epithelial cells can be partially hydrolyzed,only part of peptides through Caco-2 monolayer cell membranes with the complete form with detecting of the three long peptide of BL side by LC-MS/MS.In together,Casein can produce the bioactive peptides in vitro gastrointestinal digestion.The polypeptide of FITC-casein hydrolysate may be absorbed by intestinal tract in vivo digestion.And absorption mechanism of Caco-2 cell model showed that the identifying six kinds of ACE inhibitory peptides can be absorbed in entire amino acid residues by intestinal epithelial cells with the long peptides partially hydrolyzed.The main transfer mechinism was paracellular route.