Simultaneous release of peptides and phenolics with antioxidant, ACE-inhibitory and anti-inflammatory activities from pinto bean (Phaseolus vulgaris L. var. pinto) proteins by subtilisins
Introduction
Cardiovascular diseases (CVD) are the biggest cause of global mortality (World Health Organization, 2014). Raised blood pressure (hypertension) is a major cardiovascular risk factor that has been considered as a key target for controlling CVD-related mortality and improving global health. Healthy diets consisting of low sodium and unsaturated fat intake as well as increased consumption of fruits, vegetables and legumes have shown a high impact for reducing the prevalence of hypertension (World Health Organization, 2015).
Nutritionally, legumes are recognized as a good source of proteins, starch, dietary fibre, minerals and vitamins (Vaz Patto et al., 2015). Legume consumption has been associated to the prevention of CVD (Arnoldi et al, 2015, Rebello et al, 2014). Positive health effects of legumes on CVD prevention have been ascribed to the presence of a variety of naturally occurring bioactive compounds. Legume proteins are sources of bioactive peptides exhibiting blood pressure-lowering (angiotensin I converting enzyme (ACE)-inhibitory), anti-inflammatory and antioxidant activities once released upon gastrointestinal digestion or enzymatic treatments that may occur during food processing (López-Barrios, Gutiérrez-Uribe, & Serna-Saldívar, 2014). Legumes are also rich in phenolic compounds ranging from 1.1 to 68 mg/g of dry weight (Vaz Patto et al., 2015). Phenolic acids and flavonoids are the most abundant phenolics in legumes and they generally occur as free and bound forms, the latter representing up to 50% of the total phenolic content (Gutiérrez-Uribe et al, 2011, Yeo, Shahidi, 2015). Scientific data evidence the potential of polyphenols to improve cardiovascular health through an array of actions including antioxidant, anti-inflammatory, antihypertensive and anti-atherosclerotic activities (Del Rio et al., 2013). Bioactivities and bioavailability of plant polyphenols may be affected by interactions between these compounds and food matrix components (Wang, He, & Chen, 2014). Some processing technologies such as germination, fermentation and enzymatic treatments (Acosta-Estrada, Gutiérrez-Uribe, & Serna-Saldívar, 2014) have been evaluated to avoid these disadvantages increasing soluble to bound phenolics ratio.
The common bean (Phaseolus vulgaris L.) is the most important legume specie besides soybean in terms of economic value and represents one third of the total world production of pulses (22.5 Mt/year) (FAOSTAT, 2013). Common beans have received increased attention because of their beneficial physiological effects in reducing cardiometabolic risk factors and a broad range of chronic diseases (Hayat, Ahmad, Masud, Ahmed, & Bashir, 2014). Dry beans are the staple food of many countries in America and Asia; however, despite their nutritional value and beneficial effects common beans are largely underexploited and their worldwide consumption is declining (Kohajdová, Karovičová, & Magala, 2013). In this context, one valuable approach to enhance common bean intake and uses is their application in the design of novel and healthy foods.
Aimed at a better exploitation of legume food products, production of functional hydrolysates containing bioactive peptides from legume seed storage proteins is a topic of great scientific interest (Garcia-Mora et al, 2015, Luna Vital et al, 2014, Torruco-Uco et al, 2009, Valdez-Ortiz et al, 2012). However, the effect of proteolysis on the release of bound phenolics and how phenolic compounds contribute to the bioactivity of legume hydrolysates have been unexplored. In this study, we examine the effectiveness of two proteases from Bacillus spp. (also known as subtilisins) to release bioactive peptides and phenolic compounds with antioxidant, ACE inhibitory, and anti-inflammatory activities from pinto bean (P. vulgaris var. pinto) proteins.
Section snippets
Chemicals and materials
DC (detergent compatible) protein assay reagents were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Tripeptide Abz-Gly-Phe(NO2)-Pro was purchased from Cymit-Quimica (Barcelona, Spain). Dimethyl sulphoxide and 2,2′-azinobis 3-ethylbenzothiazoline 6-sulphonate (ABTS) were purchased from Merck Millipore (Darmstadt, Germany). Dulbecco's phosphate buffer saline 10× and reagents for gel electrophoresis were from Life Technologies (Madrid, Spain). All other reagents were purchased from
Proteolytic pattern of hydrolysis
Fig. 1 shows the electrophoretic protein profile under denaturing and non-reducing conditions of the pinto bean protein concentrate (control) and their respective hydrolysates obtained at different times (30–120 min). Protein concentrate (lane C(-)) showed intense bands with molecular weights between 97 and 10 kDa. Bands corresponding to phaseolin (25, 45 and 50 kDa) and phytohaemagglutinins, α-amylase inhibitor and α-amylase β subunit (32 and 18, 15 kDa, respectively) were readily digested to
Discussion
Health organizations recommend pulse consumption as part of a healthy diet due to vast evidence of their protective and therapeutic effects on chronic diseases (Rebello et al., 2014). As a consequence, initiatives have been developed to improve their production, intake and use. In this context, the use of legumes as functional ingredients for the development of healthy foods is being explored (López-Barrios et al., 2014). Different legume proteins are being studied for their potential to
Conclusions
This study demonstrates that there is a benefit in the application of subtilisins to release bioactive peptides and phenolic compounds from pinto bean proteins with potential antioxidant, ACE inhibitory and anti-inflammatory activities. Enzyme type and hydrolysis time were important factors affecting the bioactive composition of hydrolysates. Pinto bean hydrolysates obtained by Alcalase after 120 min were characterized by a higher concentration of small peptides <3 kDa. Interestingly, this is
Acknowledgements
This research was cofunded by the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Union through the projects AGL2010-16310 and AGL2013-43247-R and FEDER programme, respectively. P. Garcia-Mora thanks MINECO for providing a PhD fellowship. E. Peñas thanks the Spanish Ministry of Economy and Competitiveness, CSIC and European Social Fund for “Ramón y Cajal” contract.
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