Dpp 4 enzyme

Whey protein hydrolysate (84 wt.% protein content), which was used as model protein in the optimization of the formulation, and the Arabic gum were kindly donated by Abbott Laboratories S.A. (Granada, Spain) and Nexira (Serqueux, France), respectively. Pullulan was supplied by Hayashibara Co., Ltd. (Okayama, Japan). Tween 20 was purchased from Sigma Aldrich (Darmstadt, Germany). Tenebrio molitor meal (68.01 wt.% protein) was kindly provided by Tebrio (Salamanca, Spain), which was ground to powder. Alcalase (subtilisin, EC 3.4.21.62) was purchased from Novozymes (Bagsvaerd, Denmark). DPP-IV enzyme (EC 3.4.14.5) and the substrate Gly-pro-p-nitroanilide were supplied by Sigma Aldrich (St. Louis, MO, USA) and stored at −20 °C until use.

DPP-IV inhibitory activity was determined according to the method of Al-Masri et al. [29 (link)]. Standard diprotin A (Sigma, St. Louis, USA) was diluted to various concentrations (0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 μg/mL) using Tris-HCl buffer (50 mM, pH 7.5) and the final volume was made to 35 μL and DPP-IV enzyme (15 μL) (Sigma, St. Louis, USA) was added to the above mixture. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the release of 1 μmol para-nitroaniline (pNA) from the substrate/min under assay conditions. After adding the enzyme, the mixture was preincubated for 10 min at 37°C to enhance the binding capacity of the inhibitor. This was followed by the addition of 50 μL of (0.2 mM) Gly-pro-p-nitroanilide (Sigma, St. Louis, USA) in Tris-HCl as a substrate. Final incubation was done at 37°C for 30 min and the reaction was terminated by addition of 25 μL of 25% glacial acetic acid. The absorbance was measured at 405 nm using microtiter plate reader (Bio-TEK, USA). Experiments were done in triplicate and the results obtained were compared with negative control.