PRETREATMENT OF QUINOA PROTEIN BY HIGH-PRESSURE HOMOGENIZATION: EFFECT ON ENZYMATIC HYDROLYSIS AND ANTIHYPERTENSIVE PROPERTIES OF RELEASED PEPTIDES

High-pressure homogenization (HPH) has been proposed as a novel strategy to enhance the degree of proteolysis (DH) in the production of bioactive peptides. However, it has received little attention for plant proteins so far. In this study, HPH was used as a pretreatment before enzymatic hydrolysis of quinoa protein isolate (QPI). The effect of HPH on protein hydrolysis (monitored by the analysis of DH and SDS-PAGE), the release of peptides (total peptide concentration-PC), as well as on angiotensin I converting enzyme (ACE) inhibitory activity was investigated. QPI dispersion (5%, w/v based on protein content) was processed using a high-pressure homogenizer at different pressure levels (50, 100 or 180 MPa) for a single cycle, and an inlet temperature of 25 °C. Subsequent proteolysis was performed by incubating HPH-pretreated QPI (pH 8.0) with alcalase (50 μL enzyme/g protein) at 50 °C for 4 h. Untreated QPI hydrolyzed at atmospheric pressure (0.1 MPa) was used as control. Freeze-dried protein hydrolysates were analyzed. Results showed that pretreatment with HPH at 50 MPa significantly reduced DH of QPI from 15.98% to 13.53% as compared to the control, whereas higher-pressure levels did not show a significant effect. SDS-PAGE of non-hydrolyzed QPI showed multiple intense bands corresponding to polypeptides with apparent molecular weights ranging from ~16 to ~190 kDa. Bands above 100 kDa disappeared after hydrolysis, with or without HPH pretreatment, revealing that high molecular weight QPI subunits were susceptible to enzymatic hydrolysis by alcalase. Also, HPH pretreatment and increasing pressure improved the hydrolysis of some low molecular weight components, as evidenced by a slight decrease in band intensities around 70 and 30 kDa. In both cases, the respective protein subunits were cleaved into lower molecular weight fragments (<15kDa), most likely composed of short peptides and free amino acids. HPH at 50 MPa induced minor structural changes in QPI, resulting in reduced alcalase activity but without compromising peptide production. PC remained unchanged. Furthermore, it is assumed that excessive hydrolysis occurred under all experimental conditions which likely led to higher concentrations of free amino acids that might have interfered with PC results. Hydrolysates obtained from HPH-pretreated QPI inhibited ACE (IC50=0.04 mg/mL) more effectively than the untreated sample, regardless of pressure level. The antihypertensive potential of quinoa protein hydrolysate was closely related to the release of low-molecular-weight peptides, which were favored by HPH. Finally, increasing pressure above 50 MPa resulted in no further improvement in the DH or ACE-inhibitory activity, indicating that 50 MPa was the optimal operating pressure and, also, with lower energy costs. However, additional studies on shorter hydrolysis times must be conducted to better elucidate the effects of pressure level on QPI proteolysis. The findings of this study demonstrate the potential of HPH pretreatment in combination with enzymatic hydrolysis for the development of new functional ingredients from quinoa proteins. It is noteworthy that this novel technology has certain inherent advantages that could be attractive to the food industry.