This study was carried out for separation and characterization of calcium binding peptides derived from enzymatic hydrolysates of cheese whey protein(CWP), and to provide the basic information for the development of therapeutic formula, which improve the bioavailability of calcium.
CWPs that were heated at 100 ℃ for 10 min. were hydrolyzed by various proteolytic enzymes (trypsin, papain W-40, protease S, neutrase 1.5 and pepsin), and then properties of hydrolysate, separation and identification of calcium binding protein, analysis of calcium binding ability and characterization of calcium binding peptides were investigated.
Experiment 1. Separation, hydrolysis and hydrolysates properties of CWP.
1. The degree of hydrolysis(DH) of heat-denatured CWP were higher than those of native CWP by various commercial enzymes. The DH of tryptic hydrolysates was shown the highest(37.72%) in heat-denatured CWP, and lowered in order of pepsin, protease S, papain W-40 and neutrase 1.5.
2. The non protein nitrogen(NPN) and disulfide bond(-SS- bond) of heat-denatured CWP were the highest in tryptic hydrolysates, but the lowest in neutrase 1.5 hydrolysates. The free sulfhydryl group(free SH group) was shown the highest in peptic hydrolysates.
3. BSA, β-LG and α-LA in tryptic hydrolysates by SDS-PAGE were almost eliminated, and molecular weight of peptides derived from tryptic hydrolysates were smaller than 7 KDa. Also, optimum reaction time of tryptic hydrolysate was three hours.
4. Solubility of hydrolysates that hydrolyzed by commercial enzymes were observed the lowest in the range of pH from 3.5 to 5.5, which are the isoelectric regions of β-LG and α-LA, while hydrolysates were solubilized well in the range of pH below pH 3 and above pH 6. Solubility of tryptic hydrolysates added with CaCb was lower than those of other commercial enzymatic hydrolysates, in all range of pH.
5. It has been confirmed that α-LA was mostly eliminated, but β-LG was not affected by heat treatment in reverse phase HPLC analysis. The hydrolysis patterns of all enzymatic hydrolysates were similar to those of SDS-PAGE.
Experiment 2. Separation and identification of calcium binding protein.
1. By ion exchange chromatography, tryptic hydrolysates were shown a peak from F-2(0.25M NaCl) and F-3(0.5M NaCl), respectively, and F-2(peak) was shown the calcium binding ability.
2. Protein recovery rate was observed the highest(96%) in tryptic hydrolysates, and protein recovery rate in other proteolytic hydrolysates were higher than 89%. Calcium recovery rate was the highest(43.6%) in tryptic hydrolysates, but other proteolytic hydrolysates were observed low recovery level(1.4 to 7.8%), in F-2 and F-3.
3. Peaks with calcium binding ability were eluted by preparative HPLC of tryptic F-2(peak). By SDS-PAGE, fractions of peak 3, 4 and 5(below 5 KDa) were identified as low molecular weight peptides(between 1.4 and 3.4 KDa). It was confirmed as calcium binding peptides.
Experiment 3. Characterization of calcium binding peptides.
1. From the molecular weight distribution analysis of heated-CWP hydrolysates, content of peptides that are smaller than 10 KDa have been shown the highest (73.44%) in tryptic hydrolysates, but the lowest content(33.51%) of the peptides in proteolytic hydrolysate by neutrase 1.5. Moreover 50% of F-2(peak) was shown to have low molecular weight peptides, which is smaller than 5 KDa.
2. The most abundant amino acid was Glu(30.08%), but Thr, Ser and Met were not detected in calcium binding peptides by amino acid analysis. Asp, Lys, Glu and Leu, which are known as the calcium binding site, were detected abundantly in calcium binding peptides.
3. Peptide sequence of calcium binding peptides were observed the Val-Tyr-Val -Glu-Glu-Leu-Lys(41-47) and Ile-Pro-Ala-Val-Phe-Lys(78-83) in β-LG sequence, Phe-Leu-Asp-Asp-Asp-Leu-Thr-Asp(80-87) and He-Leu~Asp-Lys(95-98) in aα-LA sequence.
4. Regarding the degree of in vitro solubilization of calcium ion under duodenal condition, calcium binding peptides from CWP was 97.7%, but 95.3% in CPP from CN.
Therefore, calcium binding peptide from cheese whey protein hydrolysate will be a suitable food supplement, which can be applied for therapeutic formula related to osteoporosis, to enhance the bioavailability of calcium ion by inhibition of calcium precipitation in upper small intestine.