Potential Health Benefits of Biologically Active Peptides Derived from Underutilized Grains: Recent Advances in their Isolation, Identification, Bioactivity and Molecular Analysis

According to reports from the World Health Organization (WHO), nontransmissible chronic diseases, like diabetes, cardiovascular disorders, hypertension, and cancer, among others, are the main causes of death worldwide, comprising 70% of the total deaths. Therefore, there is a great interest in the search for alternative biofunctional agents that can contribute to the prevention and treatment of these types of diseases. Particularly, biologically active peptides (BAPs) represent an attractive and promising alternative due to their therapeutic potential, since they can act in similar ways to synthetic drugs. In this respect, BAPs extracted from food proteins of vegetable origin have shown antioxidant, antihypertensive, antidiabetic, anticancer, antithrombotic, anticholesterolemic, immunomodulatory, antiobesity, antiaging, and antimicrobial properties, thus showing great potential as bioactive ingredients in functional foods and pharmaceutical formulas. This chapter describes the main procedures performed for the identification and production of BAPs, as well as the health benefits of their biofunctionalities found in bioassays in vitro and in vivo, the elucidation of their mechanisms of action and the therapeutic applications of BAPs originated from underutilized vegetable sources. 

Biologically active peptides (BAPs) have gained significant research attention in the last few years due to their potential human health benefits and diverse applications. Moreover, the use of food by-products or food wastes as a protein source is highlighted as a viable technological alternative to produce BAPs, reducing food losses. However, the functionality of BAPs depends on the protein source, synthesis method, degree of hydrolysis, molecular weight, purity, and amino acid sequence and composition. Therefore, multiple efforts in the purification and molecular characterization of these compounds have been reported in recent years to know their molecular structure complexity and related bioactivity. BAPs can be obtained from different protein sources and synthesized by chemical, biological, and physical routes, alone or combined, which significantly influence their degree of hydrolysis and molecular weights. Additionally, advanced analytical techniques have been used to separate and identify BAPs. In this context, ultrafiltration membrane systems and electrodialysis systems are the most common methods used to separate BAPs with desirable molecular weights.
Likewise, multiple chromatographic techniques (HPLC, HPLC-MS/MS, UPLC/MS, RT-HPLC, and ion exchange and exclusion) have been widely used for quantitative, qualitative, and sequencing analysis of BAPs. Furthermore, emerging computational and statistical tools (in silico analysis) have been used to predict, sequence, and characterize BAPs using chemometric and chemoinformatic data. These tools facilitated the discovery and analysis of new peptides with desirable applications and functionality. Therefore, this chapter aims to discuss the current information about the molecular characterization of BAPs.

Amaranth (Amaranthus spp.) is one of the few plant species where both leaves and grains can be consumed. Among the main species used as producers of edible grains are Amaranthus hypochondriacus, A. cruentus, A. caudatus, and A. mantegazzianus. The grains of these species are characterized by their protein content (13-18%), which has high nutritional value. Additionally, the different protein fractions of the amaranth grain have been shown to contain sequences of biologically active peptides (BAPs) with multiple beneficial bio-functionalities for health. Among these, we can highlight antihypertensive, antioxidant, antitumor, antidiabetic, antithrombotic, anticholesterolemic and immunomodulatory activities. The aim of this chapter is to describe the different biological functionalities of BAPs obtained from the proteins of the amaranth grain, which can be considered a promising natural source of therapeutic agents with potential use in the prevention and treatment of various chronic diseases. 

 Dietary proteins and peptides can exert a wide variety of well-studied bioactivities, some of which are related to human well-being, health maintenance and disease prevention. These peptides can be generated by enzymatic hydrolysis, gastrointestinal simulation, or by fermentation with microorganisms. Quinoa is a pseudo cereal consumed by ancient populations for hundreds of years. It does not contain gluten, but it does contain proteins with encrypted sequences that can be released by enzymatic hydrolysis. These sequences contained in quinoa hydrolysates and peptides can exert beneficial effects on health, as they present antidiabetic, antihypertensive, antioxidant, anticancer and anti-inflammatory activities, among others. In addition, quinoa has other nutritional and bioactive compounds such as flavonoids, phenolic acids, fatty acids, vitamins and minerals. There are many studies that demonstrate the activities mentioned above, however, the exploration of in vivo models explaining the associated mechanisms are still needed. This chapter aims to understand quinoa from a functional point of view, along with presenting the biotechnological potential of grain proteins, which is currently very poorly exploited. We aim to promote quinoa cultivation, since its beneficial properties are adequate for its use in the prevention of chronic-degenerative diseases.

Millet is a small grain. Africa and Asia are the largest producers and consumers. Millet is hardier than large grain cereals and could be added to multi-season, multi-crop agriculture. It has nutritional properties such as proteins and carbohydrates, high content of fiber and unsaturated fats, essential amino acids, minerals, and vitamins, and it is considered a highly energetic cereal. The health benefits of whole millet grain consumption are risk reduction of various chronic diseases. In addition, it is characterized by having short growing seasons; it grows in poor soils and with efficient use of water. The consumption of this cereal is indicated for high-performance athletes, pregnant women, people suffering from stress or weakness, vegetarians and those seeking nutritional balance. Its largest protein fractions are albumin, prolamins and glutelins. Cereal peptides from their protein fractions have been found to possess biological activities of high interest to maintain and improve human health. Peptides from millet grains have been shown to have antioxidant, anti-inflammatory and hypoglycemic activities.

Buckwheat (Fagopyrum esculentum Moench) is a pseudocereal with a high content of amino acids, such as lysine; its lack of gluten makes it an attractive product with high nutritional potential that could be included in the human diet. It contains several compounds such as phenolics, carotenoids, organic acids, vitamin E and Bcomplex vitamins, and macro and microelements. Its high protein quality and peptide content are relevant in F. esculentum. Additionally, biologically active peptides (BAPs) have been associated with the prevention of some chronic and neurodegenerative diseases. Consequently, the agro-industrial, cosmetic, food and pharmaceutical sectors have shown a strong interest in this pseudocereal due to its wide range of compounds. This chapter is a compilation of scientific contributions related to buckwheat properties, including generalities, nutritional value, bioactive compounds, BAPs, and finally, perspectives and conclusions. 

Sorghum (Sorghum bicolor L. Moench) is an important staple food crop grown worldwide, easy to grow and drought tolerant. In recent years, sorghum grain has been increasingly regarded as a promising feedstock for the production of bioactive compounds. In particular, its main protein (kafirins fraction) has been used for the generation of biologically active peptides due to its high hydrophobic amino acid content and its hypoallergenicity. Several studies have shown that sorghum-derived peptides have antioxidant, anticancer, antimicrobial and anti-inflammatory activities, among others. These beneficial health effects could be attributed to the fact that in general, peptides derived from kafirins present glutamic acid and non-polar amino acids: proline, leucine and alanine. It has been reported that the presence of these hydrophobic amino acids in peptides facilitates their interaction with free radicals by neutralizing them, in addition to their low molecular weight and the position of the amino acids in the peptide sequence. Therefore, sorghum is a cereal with high potential for the production of biologically active peptides with benefits for human health.

There is a great diversity of lupin grains; however, their varieties share a high protein content, thus making them an essential ingredient for substituting or complementing other foods, as well as a source of interest for obtaining hydrolysates and biologically active peptides. At present, it should be noted that numerous studies have shown positive effects of these hydrolysates and peptides in the prevention and/or treatment of diseases, due to their anti-inflammatory, hypocholesterolemic, antidiabetic and hypoglycemic effects; however, it is still necessary to carry out bioavailability and biodigestibility studies to develop forms of inclusion in foods that allow their greater absorption, distribution and metabolism.

Mung bean (Vigna radiata L.) is a protein-rich pulse mainly cultivated in Asia, where its consumption has been associated with positive health outcomes. Mung bean protein is especially rich in leucine, lysine, phenylalanine, and tyrosine amino acids and it contains the 8S α-globulin as the major seed storage protein. Proteinderived products from pulses, such as protein concentrates, hydrolysates, and purified peptide fractions are becoming popular functional foods. Mung bean peptides are enzymatically generated using gastrointestinal and non-gastrointestinal proteases. Protein hydrolysates generated by one or a combination of enzymes have been demonstrated to exert different biological potentials, including antioxidant, antihypertensive, anticancer, and hypocholesterolemic effects. These properties are attributed to the amino acid sequences, the type of enzyme used for hydrolysis, and the purification method. More robust experimental designs must be performed to understand the role and mechanisms of these bioactive peptides with in vivo studies and clinical trials. Furthermore, there is a lack of information related to the incorporation of bioactive peptides into a food matrix while preserving their bioactivity. This chapter provides an overview of the central aspects of mung bean physical structure and chemical composition, protein characteristics, enzymatic production, and the biological potential of mung bean protein hydrolysates and peptides.

At present, it is relevant to investigate new sources of nutrients with beneficial activity for humans, so the scientific community has proposed to investigate different legumes such as beans, soybeans, and chickpeas. Chickpea is a grain with high nutrient (lipids, minerals, protein, and carbohydrates) content and is commonly used in Mediterranean cuisine. However, chickpea is a grain with high protein content, which has attracted the attention of researchers, as it is a new source for the obtainment of peptides with biological activity; in that sense, peptides with antioxidant, antihypertensive, hypocholesterolemic, anticancer, and antidiabetic activity have already been reported. This chapter summarizes the most recent information about the biological activity of peptides from chickpea (Cicer arietinum L.) grain. 

The broad bean progenitor was a local wild vegetation, which was discovered in a prehistoric Natufian culture site. It is in symbiosis with Rhizobium leguminosarum for nitrogen fixation. It has a large genetic diversity and belongs to the third largest family of angiosperms, with over 16,000-19,000 species. Commonly, the genotypes are V. faba var. major, minor, equina and paucijuga. Some of its applications, for example, are as pills for Parkinson´s disease or hypertensive patients due to its L-Dopa, and high potassium and low sodium contents, respectively. Likewise, bread, biscuits, pasta, emulsions and beverages can be fortified with broadbean flour, improving the protein content. The majority of proteins contained in the broad bean are globulins (80%), followed by albumins (20%), and, in a lesser amount, glutelins (15%) and prolamins (6%). Globulins are composed of legumin and vicilin/convicilin. Broad bean is a cheap and healthy source of protein. Therefore, it can produce biologically active peptides; for example, NPN-1 can decrease muscle wasting; protein hydrolysates are hypocholesterolemic; VFTI-G1 is anticarcinogenic (IC50=30μM); moreover, a protease inhibitor isolated from a broad bean, is useful in the treatment of fungal disease in HIV-infected patients (51.2% inhibition at 32 μM). In addition, fraction F1 has antityrosinase activity IC50=0.140; and fabatins have moderate activity against E. coli, E. Hirae and P. aeruginosa. The future of product developments in food and pharmacology lies in a combination of breakthroughs in genetics, physiology of the gut, hydrolysis, extrusion and purification of BAPs.

Recently, proteins and peptides have become important ingredients in functional foods due to the emergence of knowledge about their properties and biological activities. Many biologically active peptides (BAPs) have been studied from different animal and vegetal origins, principally legumes, such as soybeans and beans. BAPs have been shown to have anticancer, antitumoral, antioxidant and antimutagenic activities. Cacao, an ancient crop that originated in the Mesoamerican region, contains several bioactive compounds, e.g., polyphenols, alkaloids, flavanols, procyanidins and peptides. Cocoa beans contain 50% lipids and 14% protein, with albumins and globulins prevailing. The principal amino acids from cocoa protein are lysine, arginine, serine, proline, alanine, leucine, valine and methionine, and they allow the elucidation of the peptide’s bioactivity. Cocoa BAPs show antitumoral, antioxidant, antihypertensive and antidiabetic activities and obesity prevention. This work demonstrates the potential of all its BAPs.

Chia seed (Salvia hispanica L.) is rich in nutraceutical compounds with multiple benefits for human health and with great potential for its use in food. Interest in the study of this seed has been increasing year by year. Currently, studies have been based on the beneficial potential of chia proteins as a low-cost source of vegetable protein. Meanwhile, other studies have been based on the residual use of chia cake, a residue from the extraction of the chia oil industry. Bioactive peptides from chia have been shown to possess the inhibitory potential of molecular targets of hypertension, diabetes, adipogenesis, microbial, cancer, and aging. This chapter aims to provide an overview of chia bioactive peptides and their importance as a seed with high value for its protein content. 

Consumer demand for products with health benefits beyond simple nutrition is the market driver for nutraceuticals and functional foods. The development of these products has been on the rise since the last decade as consumers became more aware of the consequences of lifestyle diseases. This scenario has also benefited from the growing economy, changing lifestyles and consumption patterns. Researchers and the food industry are exploring novel sources of bioactive components and attention has been given to underutilized grain proteins. This chapter aims to review the incorporation of underutilized grains into nutraceuticals and functional foods. The recent advances and challenges in food processing techniques to develop these products are presented. Moreover, comprehensive details on the improvement of product properties with the incorporation of underutilized grains, by means of nutritional, techno-functional and bioactivity, as well as sensorial analysis are given. Finally, the health promoting effects of peptides encrypted in the protein fraction of these grains will be discussed. 

In recent years, the use of cosmetics and personal care products has increased worldwide, due to their corrective, decorative and hygienic functions. The development of new cosmetic formulations based on the use of bioactive compounds has expanded rapidly due to consumer demand for products with protective and therapeutic functions derived from natural sources. Skin aging is a complex biological process influenced by a combination of endogenous or intrinsic and exogenous or extrinsic factors. Since skin health and beauty are considered to be one of the main factors representing overall “well-being” and the perception of “health” in humans, several antiaging strategies have been developed in recent years. There are two main groups of agents that can be used as antiaging components, antioxidants and cellular regulators. Antioxidants, such as vitamins, polyphenols and flavonoids, reduce collagen degradation by reducing the concentration of free radicals in tissues. Cell regulators, such as retinols, peptides and growth factors, have direct effects on collagen metabolism and influence collagen synthesis.

Adequate nutrition is essential for good health. The characterization of the molecules present in grains - used in the human diet for thousands of years - and their hydrolysates are currently a growing trend, due to their potential benefits on health, low cost and minimal adverse effects. Grains contain many bioactive compounds, such as carbohydrates, lipids and proteins; the latter can be cleaved by chemical hydrolysis or enzymatic processes and generate smaller fragments named peptides. The continuous attack of pathogenic microorganisms and viruses on humans makes necessary a continuous search for new antimicrobial and antiviral molecules; in this respect, several studies have found antimicrobial and antiviral properties in some peptides, with some of these deriving from underutilized grains. In respect to this, an exhaustive bibliographic search of these findings was performed by the use of the online search engines NCBI, Google Scholar and Google. Therefore, this chapter aims to describe the state-of-the-art scientific findings about the effects of peptides proceeding from underutilized grains against microorganisms, particularly focusing on the antiviral potential effects.