Inhibition of dipeptidyl peptidase-IV by hydrolysates of beta-lactoglobulin isolated from Gir cow milk

The current study attempts to assess the dipeptidyl peptidase – IV (DPP-IV) inhibitory potential of β-lactoglobulin (β-Lg) isolated from milk of Gir cow. Whey was separated from skim milk by precipitation of casein followed by ultrafiltration (UF) to remove lactose and salts as permeate. β-Lg was separated from the UF retentate using salting out technique. Its purity was confirmed by SDS-PAGE and RP-HPLC. Hydrolysates of β-Lg were prepared using three different enzymes viz., pepsin, trypsin and Flavourzyme at different enzyme to substrate (E:S) ratios (1:25, 1:50, 1:100) and treated for 2–12 h durations. Pepsin-treated hydrolysate showed maximum DPP-IV inhibition of 77.62 ± 0.98 % (IC₅₀ = 3.78 mg/mL). Hydrolysate was passed serially through UF filters of 10 and 3 kDa. The permeate of 3 kDa, which exhibited 52.10 ± 0.72 % DPP-IV inhibition was fractionated through RP-HPLC and time based fractions were collected. All 41 fractions were again analysed for DPP-IV inhibition activity and 3 fractions which showed maximum DPP-IV inhibition activity were chosen for LC-MS/MS analysis. One novel peptide was identified through LC-MS/MS, which showed adequate DPP-IV inhibition (IC₅₀ = 6.64 mmolL^-1).