Introduction: Hypertension is the result of angiotensin converting enzyme (ACE) activity in the vessel wall membrane. This enzyme converts angiotensin I to angiotensin II which results in vessel wall stiffness and an increase in blood pressure. Inhibition of ACE activity is a therapy for hypertension. In addition to synthetic inhibitors, some bioactive peptides (which are the products of protein proteolysis) have been identified as ACE inhibitors. Bread is a widely consumed bakery product all over the world. During dough fermentation, yeast proteases hydrolyze wheat flour proteins to prepare amino acid for cell growth. Natural cereal proteases are considered to be the other sources of protease. Proteolysis produces peptides in dough, which are bake-stable and have physiological effects on human body. Soy protein is a valuable plant protein, reported to be a source of peptides with ACE inhibitory activity and can be used to induce diversity in peptide species during dough fermentation. In this study, a completely randomized factorial design was created to evaluate the effect of the type of soy protein derivative, wheat flour substitution level and fermentation time on the ACE inhibitory activity of dough bioactive peptides.
Materials and Methods: Wheat flour was substituted with 3 soy protein derivatives, including soy protein isolate, extruded soy protein and soy protein hydrolysate at 5 and 10%. Moreover, fermentation time was adjusted at 30, 60 and 90 min. Dough aqueous extract was evaluated in terms of molecular weight distribution using SDS-PAGE technique. The extract was then filtered through 3KDa membrane to separate short-chain peptides (theoretically <30 amino acids). Peptide concentration was determined using UV absorbance difference. The peptide solution was tested for the degree of hydrolysis based on OPA complexation reaction and ACE inhibition activity using FAPGG as the reaction substrate at two peptide concentrations. The experiments were triplicated and data were analyzed by ANOVA and Fisher`s mean comparison test using MINITAB software.
Results and Discussion: Based on the SDS-PAGE pattern, it was observed that samples had a high level of low molecular weight peptides fraction were those enhanced with extruded soy proteins and soy protein hydrolysate. This results indicated that the addition of soy protein derivatives led to a higher content of short-chain peptides compared with wheat dough. The results also showed that all the examined variables, i.e. the type of protein, substitution degree and fermentation time, significantly affected the degree of hydrolysis and ACE inhibition activity of the separated peptides. The maximum degree of hydrolysis was observed in samples with soy protein hydrolysate- which was expected to have greater peptides diversity. This might be the reason for the higher ACE inhibition activity observed for these samples. Addition of Soy protein extrudate resulted in a higher degree of hydrolysis compared with soy protein isolate revealing that the extrusion technique caused to increase the protein susceptibility to proteolysis during fermentation along with the higher content of broken amino acid chains. The higher wheat flour substitution level resulted in a higher degree of hydrolysis, while in the case of ACE inhibitory activity, it was not significant. Overall, longer fermentation time increase the degree of hydrolysis, but led to lower ACE inhibition activity, probably due to active peptides hydrolysis. Wheat flour itself had a high level of ACE inhibition activity at the shortest fermentation time, compared with composite flours, while this activity was reduced at extended fermentation time. IC50 was the highest for the samples containing soy protein hydrolysate, surely a benefit from the initial proteolysis. In conclusion, the wheat flour substitution with 5% soy protein hydrolysate substitution,would lead to reasonable ACE inhibition activity and is suggested for bread formulation with hypertension lowering effect. It also needs more research to be done in order to evaluate substitution degrees lower than 5%, because it was observed that peptides diversity was more important than high hydrolysis degree. Overall, soy protein extrusion enhanced proteolysis and short-chain peptides production during fermentation which is a better option compared with isolated soy protein.