Structure Characterization Of Calcium-bound Sunflower Seed And Peanut Proteolytic Peptides And Their Effects On Calcium Transport Of Caco-2 Cells

In this study,calcium-coupled sunflower seeds and peanut proteolytic peptides were used as the research object,and were separated and purified by ultrafiltration,calcium ion chelate affinity chromatography,reversed-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry.The Caco-2monolayer model was used to study the effect of calcium-binding sunflower seeds and peanut proteolytic peptides on calcium transport in intestinal cells.The main findings are as follows:1.Calcium-bound sunflower seeds and peanut proteolytic peptides were separated by ultrafiltration to obtain two high-calcium-binding fractions,namely sunflower proteolytic peptide K₄ with calcium binding capacity of 172.50 mg/g?1³kDa?;peanut glycolysis peptide H₁??10 kDa?with a calcium binding of 220.74 mg/g.Further,calcium ion chelate affinity chromatography was used to separate and purify the K₄ component and H1 component.KF₂ and HF₂ with higher calcium binding were separated and their calcium binding levels were 332.19 mg/g and 354.54 mg/g,respectively.Liquid chromatography-mass spectrometry was used to identify the primary structure of KF₂ and HF₂ components of sunflower seeds with high binding capacity.Seven calcium-binding peptides were identified from the KF₂ fraction,and theiraminoacidsequenceswereasfollows:1.FVTPEEEQQQMHQR;2.LAGGISSEHIQGQQQFQGQQGR;3.LDTQNHENOLDENQRR;4.FLAGNPQAQ AQSQQQQQR;5.GGIGIGGYGTTGHHGL;6.NDRNQEVVAIIVDDVNNPANQLDF QAK;7.QYGGQQTQTVER.Its characteristics include"EEEQQQ,EQ-QQQ-QQ,Q-Q-QQQQQ,DD,NN,RR,HH"linked characteristic sequences.Eight calcium binding peptides were identified from the HF₂ fraction,and their amino acid sequences are as follows:1.LLGFGINAENNHR;2.VIQQGQATVTVANGNNR;3.NESEEEGAIVTVRGGLR;4.IVQNLRGENESEEQGAIVTVK;5.TNNNDNQLDQ FPRR;6.LEENAGGEQEERGQRRR;7.FQGQDQSQQQQDSHQKVHR;8.FEGEDQS QQQQQDSHQKVR.Features include:"NNHR,QQ-Q,NNR,E-EEE,EE-EEQ,NNNDN,Q-QDQ-QQQ,EE-EQ-EE-Q,QQ-QQQQ,Q-QQQQQ-Q,RR,RRR"And other connected feature sequences.From the aforementioned calcium-binding peptides,sunflower seed proteinase peptide FVTPEEEQQQMHQR and peanut phytopeptide IVQNLRGENESEEQGAIVTVK with high affinity to the protein library were selected for synthesis by chemical solid phase synthesis and the calcium binding of the two peptide chains was determined.The calcium binding amount of the sunflower proteinase peptide FVTPEEEQQQMHQR was 220.11 mg/g,and the calciumbindingcapacityofthepeanutproteolyticpeptide IVQNLRGENESEEQGAIVTVK was 179.70 mg/g,indicating that the synthetic peptide has a higher calcium binding amount.2.Through the Caco-2 monolayer cell model,CaCl₂ and CPP-Ca were used as control groups to study the effect of sunflower seeds and peanut proteolytic peptide-calcium complexes on intestinal calcium transport.The results showed that the calcium-transporting amount of peptide-calcium complex of sunflower seed was20.72?g/well;the calcium transport of peanut peptide-calcium complex was24.63?g/well;the calcium-promoting translocation was significantly higher than that of CaCl₂ group and CPP group.No significant difference.The results showed that sunflower seeds and peanut proteolytic peptides all had a good ability to promote intestinal calcium absorption,which was equivalent to that of CPP.