Purification,Identification,Promoting Calcium Uptake Activity And Mechanism Research Of Casein Phosphopeptides

Casein phosphopeptides?CPP?,having few phosphoserine groups,are bioactive polypeptides that promote calcium absorption,CPP were derived from casein hydrolysis by a single trypin or complex enzyme.CPP can be used as a calcium supplement in baby formular,health foods and special medical foods,and it has a good application prospect.In this thesis,bovine derived phosphopeptides were selected as the research material,the best source of casein protein and the best protease preparation were screened.The CPP hydrolysis process was optimized by the single factor orthogonal method.The casein phosphopeptide monomer peptides were systematically isolated and purified from casein hydrolysis and the casein phosphopeptide monomer structure was analyzed.A Caco-2 cell monolayer model was established and was used to evaluate the calcium and magnesium transport activities of casein phosphopeptide monomers.The effect of magnesium and casein phosphopeptides on calcium absorption and metabolism were investigated in rats.The experimental contents and results were as follows:?1?Process optimization for casein phosphopeptides hydrolyzed by casein.Casein and casein phosphopeptide yield were used as the experimental material and the index,respectively.Screening the origin places of casein,which included the Netherlands,France,the United States,and New Zealand.Then the protein enzymes were screened,which included trypsin,pepsin,alkaline protease and LC protease.The results showed that the casein from New Zealand and trypsin were the best.The effects of enzymolysis pH,enzymatic hydrolysis temperature,substrate concentration,enzymolysis time,and enzyme dosage on the content of casein phosphopeptides yield were investigated.Single factor orthogonal experiments were used to optimize the enzymatic hydrolysis conditions.The optimal enzymolysis conditions were as follows:substrate concentration 10%,enzymolysis pH 8.0,temperature 50�C,and hydrolysis 2 h.In order to increase the casein phosphopeptide yield in the alcohol precipitation step,the effects of pH,CaCl₂ content,ethanol concentration,temperature,and precipitation time on the yield of casein phosphopeptides were studied.The optimal alcohol precipitation conditions were:pH 5.5,CaCl₂ addition amount 0.6%,final ethanol concentration 50%,temperature 5�C,precipitation time 6 h.?2?Purification and structural identification of casein phosphopeptide.The calcium chelation ability in vitro was used as a method of screening casein phosphopeptides.The casein phosphopeptide monomers?P1,P2,P3,P4,and P5?were separated and purified from the casein hydrolysates via preparative and analytical high performance liquid chromatography.MALDI-TOF-MS,high-resolution multi-stage mass spectrometry and amino acid sequencer analysis were used to analyze the amino acid sequence.The molecular weight of P1 is 2061.69 Da,consisting of 18 amino acids,the sequence is FQpSEEQQQTEDELQDK,belongs to casein??48-63?.The molecular weight of P2 is2043.64 Da,consisting of 18 amino acids,the sequence of P2 is FQpSEEQQQTEDELQDK-NL18 and it belongs to casein??48-63?.The molecular weight of P3 is 1927.53 Da,it consists of 16 amino acids.Its sequence is DIGpSEpSTEDQAMEDIK,which belongs to casein?_s1?58-73?.The molecular weight of P4 is 1951.83 Da,it consists of 16 amino acids.The sequence is YKVPQLEIVPNpSAEER and it belongs to casein?_s1?119-134?.P5 has a molecular mass of 1890.74 Da and consists of 25 amino acids.The sequence is RELEELNVPGEIVEpSLpSpSpSEESITR and it belongs to casein??16-40?.The monomer peptides P1,P2,P3,P4 and P5 were synthetically synthesized.RP-HPLC,FT-IR,CD were used to studied the structural differences between natural and synthetic monomers.The RP-HPLC results showed that the retention times of the monomers P3 and P4 were the same,the UV absorption of the synthesized monomers was significantly higher than that of the natural monomers.FT-IR indicated that both natural and synthetic monomers P3 and P4 contained?-helix,?-turn,?-sheet and random coil.Circular dichroism analysis showed that the content of?-sheets and random coils was higher than that of?-helix ratio and?-turn ratio of natural and synthetic P3 and P4.When the concentration increased from 2 mg/mL to 4 mg/mL,the secondary structure changed from?-helix,?-turn to?-sheet-antiparallel.?3?The purity and chemical composition of CPP1?CPP mixture from optimal enzymolysis conditions?and CPP2?commercially available CPP mixture?were determined by national standards,the results showed that the purity of commercially available CPP2was significantly higher than that of CPP1.RP-HPLC chromatogram analysis showed that the components of CPP1 and CPP2 were complex,and the content of the high-polarity peptides of CPP2 was relatively less than that of CPP1.The results of bulk density,tap density,Hausner ratio,Carr's index,and angle of repose show that the powder flowability of CPP2 was better than that of CPP1.The solubility index showed that the solubility of CPP1 in 30%ethanol solvent was significantly higher than that of CPP2.?4?The calcium and magnesium transport activity of casein phosphopeptides in Caco-2 cell model.Caco-2 cell model was established to determine the transport of calcium and magnesium ions in the basolateral side at different time points.Effects of casein phosphopeptide monomer on calcium transport:casein phosphopeptide mixture and all the monomers?P1,P2,P3,P4 and P5?both significantly increased the calcium transport compared to the control group.The calcium transport amount in monomer peptides groups was significantly higher than that of casein phosphopeptides mixture group?p<0.05?.Monomer peptide P5 had the best calcium transport capacity among all the monomer peptides.Effect of casein phosphopeptide monomer on magnesium transport:The magnesium transport results have the same trend as the calcium transport results.Casein phosphopeptide mixture and all the monomers?P1,P2,P3,P4 and P5?both significantly increased the magnesium transport amount compared with the control group.The magnesium transport amount in monomer peptides groups was significantly higher than that of casein phosphopeptides mixture group?p<0.05?.The magnesium transport amount in monomer peptide P5 was the highest among all the monomer peptides.Effects of casein phosphopeptide monomer peptide on calcium transport in the presence of magnesium under the conditions:casein phosphopeptide mixture and monomers?P1,P2,P3,P4 and P5?significantly increased magnesium transport in the presence of calcium.The most obvious effect in monomer P5 group was found.Interestingly,monomer peptides?P1,P2,P3,P4 and P5?did not increase the calcium transport amount in the presence of magnesium.Furthermore,the calcium transport amount in monomer peptides?P1,P2,P3,P4 and P5?was even lower than that of the blank control group.?5?The effect of casein phosphopeptides on calcium absorption metabolism in rats in the presence/absence of magnesium.The blank control group,calcium carbonate group and P5 monomer peptide group were set up to study the effects of P5 on calcium absorption and metabolism in animals.In P5 group,the serum calcium,femur length and femur calcium content were significantly increased compared with the control group,the serum alkaline phosphatase and urinary pyridine were significantly reduced compared to the control group.The urinary pyridine and calcium content in the femur in P5 group had obvious effect than that of calcium carbonate group.The final body weight,weight gain,and organ index of the rats did not change significantly,no any adverse physiological phenomena was found,it revealed that casein phosphopeptide monomer peptide P5 did not affect the normal growth of the rats and had no side effects.In order to investigate the effect of casein phosphopeptides on calcium absorption metabolism in rats in the presence of magnesium,the blank control group,magnesium deficiency group,magnesium supplement group,high magnesium supplement group,supplement CPP group,magnesium supplement CPP group,high magnesium supplement CPP group were designed.The body weight,femoral physicochemical characteristics,serum biochemistry,urine biochemistry indicators were analyzed.The effects of CPP on calcium absorption and metabolism in animals were studied in the presence or absence of magnesium.The lack of magnesium leaded to a decrease in the rate of bone formation and an increase in bone resorption.Magnesium or CPP supplementation in diet can increase bone formation and prevent bone resorption compared to the magnesium deficiency group.CPP could significantly increase femur bone mineral density and serum osteocalcin content in the existence of magnesium.There was a synergistic effect in inhibiting serum parathyroid hormone content,decreasing urinary deoxypyridinoline content,and increasing femur length.The results showed that the CPP was benefit for bone growth and inhibits bone resorption while magnesium supplymentation in diet.?6?Mechanism research of casein phosphopeptids enhance calcium uptake.It was reported that there were two main mechanisms for intestinal Ca²⁺absorption,including a passive non-saturable paracellular pathway and an active transcellular pathway.Western blotting was used to detect the amount of calcium channel protein TRPV6 which was associated with the active absorption of transcellular pathways.Transmembrane electric resistance was monitored which was associated with the passive absorption of paracellular pathway.The TRPV6 amount and TEER were studied in the effects of P5 on promoting calcium absorption.During the calcium transport in the Caco-2 monolayer model,there was no significant difference in the TEER between the control group and the monomer P5 group.P5 did not promote calcium absorption through stimulating the paracellular pathway.The expression of calcium channel protein TRPV6 in Caco-2 after treatment with P5 was significantly up-regulated,indicating that P5 promotes calcium absorption through stimulating the active transcellular pathway.