Compared with un-separated -hydrolysates whose DPP-IV inhibition rate was 60.90 0.54%), F1 and F2 displayed weaker DPP-IV inhibitory activity (with DPP-IV inhibition rate of 27.53 1.77% and 56.92 0.54%, respectively). potential source of natural DPP-IV inhibitor. CPI-203 0.05). The DPP-IV inhibition rate of hydrolysates enhanced as hydrolysis time improved from 0 h to 4 h, and reached the maximum value at 4 h (82.30 1.39%). However, with the elongation of hydrolysis time increased to 5 h, CPI-203 the DPP-IV inhibition rate of the acquired hydrolysates was 76.47 0.95%, which was not significantly higher than that of 4 h hydrolysates ( 0.05). Therefore, the 4 h hydrolysates were selected for further analysis. Open in a separate window Number 1 Degree of hydrolysis (DH) (A) and Dipeptidyl peptidase-IV (DPP-IV) inhibitory activity (B) of bovine -lactalbumin hydrolysates. Bovine -lactalbumin was hydrolyzed with alcalase (enzyme/substrate percentage = 5%, 0C5 h). The DPP-IV CPI-203 inhibition rate was measured using 1.0 mg/mL of hydrolysate (final assay concentration, protein basis). Ideals are indicated as means SD of 3 self-employed determinations. Different characters indicate significant variations ( 0.05). 2.2. DPP-IV Inhibitory Activity of Bovine -Lactalbumin Hydrolysates Fractionated by Sephadex G-25 Gel Filtration Chromatography The acquired bovine -lactalbumin hydrolysates with the highest DPP-IV inhibitory activity were separated by Sephadex G-25 gel filtration chromatography, and three individual fractions (F1, F2, F3) were acquired, as demonstrated in Number 2A. DPP-IV inhibitory activity of bovine -lactalbumin hydrolysates and each portion was identified at a concentration of 0.5 mg/mL. Compared with un-separated -hydrolysates whose DPP-IV inhibition rate was 60.90 0.54%), F1 and F2 displayed weaker DPP-IV inhibitory activity (with DPP-IV inhibition rate of 27.53 1.77% and 56.92 0.54%, respectively). However, the F3 portion possessed more potent DPP-IV inhibitory CPI-203 activity than un-separated -hydrolysates (with DPP-IV inhibition rate of 62.88 0.61%, 0.05) (Figure 2B). Therefore, the F3 portion was performed for further purification by RP-HPLC. Open in a separate window Number 2 Elution profile (A) and dipeptidyl peptidase-IV (DPP-IV) inhibitory activity (B) of bovine -lactalbumin hydrolysate fractions acquired by Sephadex G-25 gel filtration chromatography. The DPP-IV inhibition rate was identified using samples at concentration of 0.5 mg/mL (final assay concentration). Ideals are indicated as means SD of 3 self-employed determinations. Different characters indicate significant variations ( 0.05). 2.3. DPP-IV Inhibitory Activity of F3 Fractionated by RP-HPLC To further separate the highest DPP-IV inhibitory portion, RP-HPLC was used to purify F3 portion acquired by Sephadex G-25 gel filtration chromatography. As demonstrated in Number 3A, after 30 min elution, 13 major peaks were detected, based on which CPI-203 the fractions (numbered from F3-1 to F3-13) were collected. All the fractions were subjected to DPP-IV inhibitory activity dedication at a concentration of 0.024 mg/mL (final assay concentration). Results showed that F3-8 and F3-11 fractions exhibited the highest DPP-IV inhibitory activity with ideals of 28.10 0.41% and 28.40 0.45%, respectively (Figure 3B). Open in a separate window Number 3 High-performance liquid chromatography profile of F3 portion from Sephadex G-25 gel filtration chromatography (A) and dipeptidyl peptidase-IV (DPP-IV) inhibitory activity of fractions (B). The DPP-IV inhibition rate was identified using sample of 0.024 mg/mL (final assay concentration). Ideals are indicated as means SD of 3 self-employed determinations. Different characters indicate significant variations ( 0.05). 2.4. Recognition of DPP-IV Inhibitory Peptides F3-8 and F3-11 fractions acquired by RP-HPLC were subjected to LC-MS/MS analysis for peptide sequence identification. As outlined in Table 1, the peptide sequences of the F3-8 and F3-11 were Glu-Leu-Lys-Asp-Leu-Lys-Gly-Tyr (ELKDLKGY) and Ile-Leu-Asp-Lys-Val-Gly-Ile-Asn-Tyr (ILDKVGINY), respectively. Number 4A showed the LC-MS/MS spectrum of single-charged ion with 483.26996, which matched to sequence ELKDLKGY corresponding to bovine -lactalbumin f (30C37). Number 4B showed the LC-MS/MS spectrum of single-charged ion with 517.79901, which matched to sequence ILDKVGINY corresponding to bovine -lactalbumin f (114C122). Mature amino acid sequences of bovine -lactalbumin are demonstrated in Number 5, and peptide sequences recognized in F3-8 and F3-11 fractions are underlined. Open in a separate window Number 4 Recognition of DPP-IV inhibitory peptides. (A) LC-MS/MS spectrum of single-charged ion with 483.26996. Following sequence interpretation and database searching, the MS/MS spectrum of ion with 483.26996 was matched to peptide ELKDLKGY. LAG3 (B) LC-MS/MS spectrum of single-charged ion with 517.79901. Following sequence interpretation and database searching, the MS/MS spectrum of ion with 517.79901 was matched to peptide ILDKVGINY. Peptide sequences are displayed with ion fragments observed in the spectrum. Open in a separate window Number 5 Mature amino acid sequences of bovine -lactalbumin. Peptide sequences recognized in the bovine -lactalbumin fractions are underlined. Table 1 Liquid chromatography-electrospray ionization tandem mass spectrometry (MS) recognition.
