The Preparation Of Casein Peptide And Study Of Sequence Characteristics Of Casein-derived Peptides That Can Be Absorbed In The Intestine

Casein is an important source of bioactive peptides,but bioactive peptides from casein only paly its biological function in the body as a complete form,which could transmit through the intestinal epithelial cells into the circulation.The small intestine is a major site of material absorption.and the biggest barrier of the absorption of peptides.Whether polypeptides can be successfully transported to the blood by the small intestine is related to the sequences of peptides.So,studying the sequence characteristics of casein peptides which can be absorbed by the small intestinal can provide a theoretical basis for the utilization and industrialization for bioactive peptides derived from casein.In this research,casein peptides were hydrolysez by enzyme from casein protein.Caco-2 cell model was established for transport experiment of casein peptides,and then casein peptides sequences were identified and analysed by UPLC-MS.1.Casein peptides were hydrolysed by trpsin and trypsin protease.The hydrolysis conditions were optimized by single factor experiments and response surface experiments.Then peptides were isolated by Sephadex G-10,and the separation conditions were optimized.The amount of trypsin protease at E/S 5.45%,pH 7.13,temperature 51.99?,concentration of substrate 5%,hydrolysis time 3h were found as optimum conditions.Under this condition,peptide concentration was 0.157mg/mL and the degree of hydrolysis was 16.02%.The optimized separation conditions by Sephadex G-10 was defined that the volume of sample was 80 mg/mL,sample volume was 3 mLand the flow rate was 1.5 mL/min.The amount of alkaline protease at 5.22%,pH at 8.04,temperature at 54.44?,concentration of substrate at 5%,hydrolysis time at 3h were found as the optimal conditions of alkaline protease group.Under this condition,the polypeptide concentration was 0.252 mg/mL,and the degree of hydrolysis was 23.82%.The optimized separation conditions by Sephadex G-10 was defined that the volume of sample was 80mg/mL,sample volume was 2mL and the flow rate was 2 mL/min.2.Caco-2 cell model was established and validated throgh determination of the membrane resistance,alkaline phosphatase and fluorescein sodium permeation rate.The model could be uesd to simulated intestinal absorption.Then casein peptide was transported with the model.After that,the sequences characteristics of casein peptides such as molecular size,charge,hydrophobic,and amino acids were researched.The results showed that dipeptides were the biggest part of transported peptides and pentapeptides were the least part.Partial peptide may be hydrolysed by enzyme on the brush border membrane.The net charge of most of etrapeptides and pentapeptides were not zero.For most part of peptides,C-or N-of amino acids were Val and Leu which had larger side chain volume.What'more,parts of tetrapeptide and pentapeptides contained Pro.Amino acids connected with Pro have large sied chain volume.These sequence features may related to the peptide transporters.