OBTAINING PEPTIDES BY ENZYMATIC HYDROLYSIS IN DEEP EUTECTIC SOLVENTS

Bioactive peptides from dietary proteins are fragments encoded in food proteins that may be released by enzymatic hydrolysis applying proteases. The deep eutectic solvents (DES) are efficient in the complete dissolution and extraction of both polar and nonpolar compounds, presenting valuable intrinsic characteristics that enable to increase the rate of enzymatic reactions. However, the low water availability in these solvents may be a hindrance to hydrolysis reactions. The aim of this study was to evaluate the activity of different commercial proteases on soy protein isolate and bovine serum albumin in DES. The selected DES media were composed of equal molar concentrations (1:1) of choline chloride and glycerol (DES 1 - CCG) and of urea and glycerol (DES 2 - UG), due to their pH values (6,35 and 8,82, respectively), and the commercial proteases applied were Alcalase, Neutrase and Flavourzyme. The enzymatic reactions occurred at 60°C (Alcalase and Neutrase) and 50°C (Flavourzyme), for 60 min. For each assay, 1% protein was dissolved in 0.5 mL of DES containing 30% water. The reaction started with the addition of the enzyme solution (0.1 mL of 1% solution, resulting in a total addition of 50% water to the DES) and was stopped by the addition of 0.5 mL of TCA (0.44 M), which also caused precipitation of the non-hydrolyzed protein. The proteolytic activity was verified by measuring increase in the absorbance at 280 nm of the media's supernatant in comparison to a blank assay, without added enzyme. No proteolytic activity could be detected in the media and with the enzymes tested, under the assay conditions. To verify the activity of the tested enzymes, assays under identical conditions were carried out using buffer as reaction medium and soy protein isolate as substrate. Under these conditions, an activity of 1,105 U was obtained for Alcalase, 1,154 U for Neutrase and 135 U for Flavourzyme, proving the proteolytic activity of the applied enzymes. In general, DES present high viscosity, which is more pronounced in formulations containing glycerol. Both DES formulations in the present work showed considerable viscosity (84.11 mPa.s for CCG and 78.40 mPa.s for UG) and great encumbrance for homogenization, hindering the diffusion of enzymes and substrates in the reaction medium, which may explain the inability to detect the hydrolysis in the tested solvents. If this is the case, it is possible that an efficient agitation system will be able to guarantee better results under the similar conditions.