Preparation And Separation Calcium-binding Peptide From Tilapia Protein Hydrolysate And Its Bioavailability

Tilapia as an excellent aquatic material resource rich in protein and amino acids,is not only a good source of protein in daliy diet,but also a potentially high-quality raw material for preparing a series of bioactive substances.In this study,The enzymatic hydrolysis from tilapia meat treated by different proteases.molecular weight distribution,Ca²⁺binding abilities and antioxidant abilities were investigated.The enzymatic hydrolysis with high Ca²⁺binding capacity were separated by immobilized metal chelate chromatography?IMAC?.Caco2 cells were used to evaluation peptide-calcium complex transport and uptake.It is expected that a kind of calcium supplement from tilapia,which will the enrich tilapia further processing theory in some way.The result is as follows:1.The enzymatic hydrolysis from tilapia meat treated by neutral protease,trypsin and Alcalase2.4L protease respectively.Ca²⁺binding activities were investigated.The results showed that the hydrolyzates?DH=10.5%,10.25%,7.41%?from the three kinds of protease,respectively,had the highest Ca²⁺binding activity.And the Ca²⁺binding abilitiesoftheenzymatichydrolysisisanalyzed:trypsin>Alcalase2.4L>neutral protease,the hydrolyzates?DH=7.41?had the highest Ca²⁺binding activity,and the combination rate was 78.71%.In addition,The amino acid sequence analysis showed that the hydrolyzates with higher contents of Glu,Asp,Lys and Ser had the higher Ca²⁺binding activity.2.The enzymatic hydrolysis from tilapia meat treated by neutral protease,trypsin and Alcalase2.4L protease respectively.Molecular weight distribution were investigated by the high performance size exclusion chromatography?HPSEC?.The results showed that the proportion of the component which molecular weight was not more than3kDa in enzymatic hydrolysis is analyzed:Alcalase2.4L>neutral protease>trypsin.Theinitialpeaktimeistrypsin>neutral protease>Alcalase2.4L.3.The enzymatic hydrolysis from tilapia meat treated by neutral protease,trypsin and Alcalase2.4L protease respectively.the antioxidant activities were investigated.The results showed that the hydrolyzates?DH=14.18%?treated by neutral protease show the highest antioxidantactivities,thescavengingrateof DPPH?1,1-diphenyl-2-picrylhydrazyl?radical and hydroxyl radical,as well as reducing power was93.11%,72.45%,0.88,respectively.The hydrolyzates?DH=12.84%,10.1%?treated by Alcalase2.4L and trypsin respectively show the highest antioxidant activities,The antioxidant activities of the hydrolysis first increased and then decreased with the increase of the enzymolysis time.4.The hydrolyzates with highest Ca²⁺binding activity have some antioxidant capacity.The hydrolyzates scavenging rate of DPPH radical and hydroxyl radical,as well as reducing power was81.79%,47.97%,0.45,respectively.The combinations show that some antioxidant capacity,the Ca²⁺combinations scavenging rate of DPPH radical and hydroxyl radical,as well as reducing power was77.35%,32.51%,0.29,respectively.The combination of the hydrolyzates with Ca²⁺reduced the antioxidant capacity.5.The enzymatic hydrolysis with highest Ca²⁺binding capacity were separated by IMAC.The results showed that the molecular weight of the separated component was reduced,and the small molecular protein or peptide increased.The proportion of the component which molecular weight in 5¹0 kDa reduced about 10%,the proportion of the component in1³ kDa increased 10%,and the component which molecular weight was more than 5 kDa was reach up to 70%.6.The Caco2 cell model was used to test the bioavailability of tilapia protein calcium-binding peptide?TPCBP?.The results showed that the highest calcium absorption rate was CPP-Ca group,but there was no significant difference between the TPCBP-Ca group and CPP-Ca?p>0.05?.The calcium absorption rate in the CaCl₂ group was the lowest,which was significantly different from the other two groups?p<0.05?;the absorption of inorganic calcium in the intestine was dependent on vitamin D,and TPCBP and CPP in calcium absorption were not dependent on the intake of vitamin D3;when the molar ratio of phytic acid and calcium was 0.24,the Ca²⁺absorption rate of Caco2 cells was significantly decreased?p<0.05?,The calcium absorption rate of TPCBP and TPCBP is higher than that of CaCl₂?p<0.05?.