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ScienceDirect Biopeptides from vegetable proteins: new scientific evidences Domenico Montesano1,3, Monica Gallo2,3, Francesca Blasi1 and Lina Cossignani1 In the last few decades, the search for new bioactive compounds able to fight several chronic degenerative diseases has increased. In this context, natural sources are especially appealing. Among these new products, peptides are included, for the promising bioactivities and potential applications. In fact, bioactive peptides represent specific sequences of amino acids with numerous health effects so they can be used for the formulation of new drugs, nutraceuticals and ingredients for functional foods. They can be obtained from different protein sources both of animal (milk, derivatives and eggs, meat, fish and even insects) and of vegetable origin (soy, wheat, corn, rice, barley, potatoes, algae). In particular, here it is shown an overview of recent data on some bioactive peptides of plant origin. Addresses 1 Department of Pharmaceutical Sciences, Section of Food Science and Nutrition, University of Perugia, via San Costanzo 1, 06126 Perugia, Italy 2 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, via Pansini 5, 80131 Naples, Italy Corresponding author: Gallo, Monica (
[email protected]) These authors contributed equally to this work.
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Current Opinion in Food Science 2020, 31:31–37 This review comes from a themed issue on Food bioprocessing Edited by Gabriele Rocchetti
https://doi.org/10.1016/j.cofs.2019.10.008 2214-7993/ã 2019 Elsevier Ltd. All rights reserved.
Introduction The relationship between diet and diseases is the subject of great attention by researchers, as confirmed by several studies published every year on the most authoritative scientific journals. In this context, a research field of great interest concerns food proteins and their nutritional quality [1]. Obtaining bioactive compounds, such as biopeptides, requires extraction from the matrix in which they are contained and their subsequent analysis. On the other hand, there is no valid extraction and/or analysis method for all the compounds, but the choice depends on the objectives of the study, the samples and the target compounds. Currently, traditional extraction methods such as the maceration and extraction of Soxhlet are accompanied by more modern www.sciencedirect.com
methods, such as microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), supercritical fluid extraction (SFE) and rapid solid-liquid dynamic extraction (RSLDE) [2], in which the various advances aim to increase the yield, to reduce the costs and the environmental impact. Furthermore, modifications to the extraction and analysis methods are continuously developed [3]. As is known, the biological value of proteins is defined by the type of amino acids contained in them. In this regards, foods of animal origin such as meat, fish, eggs and dairy products are considered a source of high quality proteins, even if also some plant foods stand out for the content of essential amino acids, such as amaranth, quinoa, buckwheat [4]. On the other hand, an important factor determining the nutritional quality of food proteins is the potential regulatory activity of the bioactive peptides contained in the amino acid sequence. In fact, recent studies have shown that some peptides contained within the primary sequence of proteins, released by in vitro enzymatic hydrolysis during food production processes or during in vivo digestion, can exert important biological functions [5,6], as they can bind to receptors belonging to cells implicated in specific metabolic processes. Depending on the sequence of amino acids, these peptides can exhibit immunomodulatory, antimicrobial, antioxidant, antithrombotic, hypocholesterolemic, and antihypertensive actions [7]. The numerous evidences on the beneficial effects of bioactive peptides have greatly increased the interest in these molecules in recent years; not only their bioavailability and the biological effects are studied, but their applicability and large-scale production in the food industry are also assessed [8]. In fact, the integration of these components into foods could lead to significant advances in the production of so-called ‘functional foods’, that have a beneficial influence on one or more functions of the organism [9]. As shown by recent literature, vegetable sources of bioactive peptides are increasingly considered and now represent an important starting point for obtaining these precious compounds [10,11]. Moreover, the need to implement efficient and economically viable strategies for production on an industrial scale must be taken into Current Opinion in Food Science 2020, 31:31–37
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account. In this review, alternative protein sources for the production of bioactive peptides are considered, as well as the relative biological and pharmacological effects also with a view to using these peptides as new nutraceutical products. In particular, a section is dedicated to alternative lowcost sources, for example derived from by-products and waste of agro-industrial processing related to plant foods. On the other hand, bioactive peptides were found in many plant-based foods, such as potato, soy, hempseeds, cereals, pseudocereals, algae and others, as the consequence of fermentation and enzymatic hydrolysis [11]. Following are several examples of peptides obtained from the main plant sources with their specific biological properties, as reported in recent literature and a preliminary section dedicates to current strategies for the release of peptides from protein precursors.
Current strategies for the release of peptides from protein precursors Various methods for obtaining bioactive peptides have been considered: some techniques involve the use of hydrolytic enzymes, such as trypsin, pepsin, alkalase, others use microbial fermentation that allows the proteolytic enzymes of lactic flora and finally through synthesis techniques [12]. The main techniques used for the synthesis of bioactive peptides are: chemical synthesis, synthesis by recombinant DNA technology and enzymatic synthesis. Currently, enzymatic hydrolysis represents the main process for the recovery of these molecules from both animal and plant sources. Most commercial enzymatic preparations frequently used are from animal and microbial sources. Although the use of plant proteases is still relatively limited to papain and bromelain from papaya and pineapple, respectively, the application of new plant proteases is increasing. A recent review by Mazorra-Manzano et al. [13] shows the latest knowledge in the use and diversity of plant proteases for bioactive peptides release from food proteins including both available commercial plant proteases as well as new potential plant sources. In addition, this work described the properties of peptides released by plant proteases and health benefits associated in the control of disorders such as hypertension, diabetes, obesity, and cancer. In alternative to enzymatic methods, in several studies the potential effects of bacterial strains in the production of bioactive peptides were evaluated. Fermentation techniques, depending on the type of fermenting microorganism and the desired peptide product, can last from a few hours to several days [14]. However, nowadays the use of enzymes to produce bioactive peptides is the most widespread method, in fact this method is faster and the reactions are more easily controlled than those using microorganisms. To this aspect contributes the remarkable specificity of substrate that allows the development of protein hydrolysates with both well-defined chemical and nutritional characteristics. Moreover, even for the aspect of food safety, enzymatic reactions are more Current Opinion in Food Science 2020, 31:31–37
suitable as they do not leave residues of organic solvents or other potentially toxic products for human health [15].
Bioactive peptides from cereals and legumes Cereals and legumes are the main sources of the vegetable-derived peptides, being both rich sources of proteins with a complementary spectrum of amino acids. Antioxidant properties and potential mechanisms of hydrolyzed proteins and peptides from cereals have been recently reviewed [16]. Karami et al. reported the antioxidant, anticancer and angiotensin converting enzyme (ACE)inhibitory activities of bioactive peptides from wheat germ protein hydrolysates, identified using nano-LC– MS/MS analysis [17]. Peptides with high antioxidant activity were obtained after the hydrolysis of rice bran protein under in vitro gastrointestinal digestion and the successive fractionation [18]. Also bioactive peptides from legume proteins have interesting biological activities and potential nutraceutical applications. It was reported that some lupine biopeptides, with molecular profile determined by molecular exclusion chromatography, exert anti-inflammatory effects and improve the immune and antioxidant status in human peripheral lymphocytes [19]. It was recently reported the antioxidant and ACE-inhibitory activities of protein hydrolysates from Mung bean, characterized by UV, circular dichroism (CD) spectroscopy and Fourier transform infrared (FTIR) spectroscopy [20]. A review article by Gonza´lez-Montoya et al. [21] is focused on the antiproliferative properties of the bioactive peptides from the main legumes: soybean, peas, chickpeas, common beans, fava beans, lentils, lupins.
Dietary peptides from soy Soy protein is the major plant source that produces peptides with biological/pharmacological properties, that exhibit antihypertensive, anticholesterol, and antioxidant activities, and seem to prevent cancer [22]. The processing of soy protein into peptides in the gastrointestinal tract greatly increases their healthful effects by exposing active groups within the amino acid chain. Some soy peptides like lunasin and soymorphins possess several properties and play a role in the prevention of multiple chronic diseases [23]. A work by Wang et al. [24] reports the preparation of bioactive peptides from soy proteins with antidiabetic, antihypertensive, antioxidant and identification of peptides inhibiting a-glucosidase. The results obtained allow to consider the hydrolysed peptides from soy proteins as promising natural ingredients for nutraceutical and/or functional food formulation. Cannabis sativa as new source of biopeptides
Hemp (Cannabis sativa L.) is a source of nutritious seeds that have been used as human food for thousands of years [25]. In particular, hemp seed cake, a by-product of cold oil processing, represents a food waste material of high www.sciencedirect.com
New scientific advances on vegetable biopeptides Montesano et al. 33
nutritional value. In a recent research by Hadnapev et al., it was used as starting raw material for bioactive peptides production [26]. Alkali extraction followed by isoelectric precipitation was employed for hemp protein isolation. Subsequently, the influence of different enzymes (alcalase and pancreatin), as well as the degree of hydrolysis on the kinetics of different molecular weights peptides production and their antioxidant potential was investigated. The obtained results showed that the peptides characterized by the highest degree of hydrolysis exhibited the strongest antioxidant activity. Moreover, the properties of the obtained hydrolysates were dependent on the type and specificity of the employed protease, as well as the hydrolysis time. Another study by Nongonierma and FitzGerald [27] demonstrated the potential benefit of hydrolysing plant protein substrates before oral ingestion with the view of releasing dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. In this research, four plant protein isolates from hemp, pea, rice and soy were hydrolysed with three enzyme preparations. The results obtained showed that food protein hydrolysates contained peptide sequences with DPP-IV inhibitory properties which may find use to improve serum glucose regulation in type 2 diabetics.
Biopeptides from marine organisms Marine organisms are rich sources of structurally diverse bioactive compounds with various biological activities. Therefore, seafood has an essential role in the human diet and is not only a reliable source of protein, but it also has a nutritional impact due to its lipids, vitamin and mineral constituents. A book by Abbas et al. [28] reported the various functional compounds of seafood focusing on their potential use and health benefits. Among the various types of seafood containing functional compounds with beneficial health effects, spirulina (Arthrospira platensis), an unicellular blue algae, known for its high protein content and therapeutic properties, can be mentioned. Several studies in vivo and in vitro show its effectiveness in treatments of anemia, hepatotoxicity, cardiovascular diseases, hyperglycemia, hyperlipidemia, immunodeficiency, inflammatory processes, and enhancement of immune resistance in several types of cancer, reduction of cholesterol, HIV, and other viral diseases. Consequently, it has been considered as a generally recognized as safe (GRAS) ingredient since 2003 and defined as food of the future. These attributes, combined with its disseminated cultivation techniques, sustainable production, and commercial popularity, make Spirulina an attractive source for exploration and production of bioactive peptides [29]. In another paper, Alzahrani et al. [30] reported interesting data on Nitzschia laevis, a common pennate marine diatom, as compared to spirulina and chlorella, another well-known microalga, concluding that the hydrolysates obtained from their protein fraction showed strong in vitro antioxidant activities. www.sciencedirect.com
Bioactive peptides from other vegetable sources Garlic (Allium sativum L.)
This plant has been known since ancient times and historically represents a significant antioxidant potential also used in folk medicine and for the treatment of ageing-related disorders. In the literature there are many studies on the antiglicative properties of bioactive peptides extracted from aged garlic compared to the fresh one. This is because aged garlic has a greater concentration of organosulfur compounds, which are potent antioxidants and free radical scavengers. A study by Shi et al. [31] has investigated the antiglycative effect of active peptides in fresh garlic extract by electron spin resonance (ESR) spectroscopy. The results of this study showed that the water-soluble active peptides with small side chains of fresh garlic had a high inhibitory effect on glycation in aqueous systems, when compared with hydrophobic ones with long side chains.
Quinoa (Chenopodium quinoa L.)
The cereals and flour of the quinoa pseudocereal represent a food with an high nutritional value, in fact they contain an elevated quantity of proteins, minerals and vitamins. In particular, quinoa proteins are rich in amino acids such as lysine, threonine and methionine, commonly lacking in cereals, and are close to the ideal protein balance recommended by FAO [32,33]. The authors evaluated the antioxidant potential of quinoa flour, after fermentation with autochthonous and selected lactic bacteria. Specifically, biopeptides were identified, characterized and evaluated for antioxidant properties in vitro, also using human keratinocytes NCTC 2544. The autochthonous lactic acid bacteria, therefore, were shown to promote the release of antioxidant peptides by means of proteolysis of native proteins. It is possible to state that the fermentation process applied to quinoa flour with a selected starter can be used to produce a functional food ingredient, a food supplement or pharmaceutical preparations.
Miscellaneous
In addition to these reviewed products, there are many other plant-derived products that are able to provide bioactive peptides by different hydrolysis methods. In the Table 1 are summarized the main sources of bioactive peptides with the relative biological properties.
Recovery of bioactive peptides from byproduct and processing waste Waste generated by agro-industries [43] can be a rich source of valuable compounds, among which proteins, and therefore could become a sustainable alternative to reduce malnutrition and hunger in developing countries [44]. Current Opinion in Food Science 2020, 31:31–37
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Table 1 Sources of bioactive peptides with the relative biological properties Source
Biological properties
Reference
Potato Rapeseed Lupine Cowpea
Antioxidant ACE inhibitory and antihypertensive Anti-inflammatory Anti-inflammatory, benefits against cancer, diabetes, and cardiovascular disease Antioxidant and ACE-inhibitory Anti-inflammatory Prevention of chronic diseases ACE-inhibitory Antioxidant and ACE-inhibitory
[34] [35] [36] [37]
Wheat Pyropia yezoensis Amaranth Walnut Cocoa
Currently, a large amount of by-products and various waste is produced annually from the processing of microalgae, soybean meal, olive, cherry, rapeseed flour, and so on. Generally these by-products contain significant amounts of proteins, peptides and amino acids. Bioactive peptides production from waste and by-products does not significantly differ from the production from food vegetables, and enzymes are commonly used to hydrolyse proteins [45]. A review by Lemes et al. [46] describes recent advances in bioactive peptide technology, such as: new strategies for transforming bioactive peptides from residual waste into added-value products; nanotechnology for the encapsulation, protection and release of controlled peptides, use of techniques of large-scale recovery and purification of peptides aiming at future applications in pharmaceutical and food industries. Table 2 reports the summary of some bioactive peptides obtained from byproducts and agro-industrial waste mainly by enzymatic hydrolysis. Recent examples in this field include the production of bioactive peptides from date seeds, that constitute a waste or can be used as animal feed. Different enzymes were used for the hydrolysis of date seed proteins by individual or sequential treatment and several bioactivities were investigated with positive results [47]. Garcia et al. [48] reported that waste material derived from the processing of cherry represented a starting point to obtain peptides showing antioxidant and antihypertensive properties. Peach seeds contain more than 40% of
[38] [39] [40] [41] [42]
proteins and can constitute a cheap source of bioactive peptides. They were investigated for the production of antioxidant peptides using different enzymes, with the best bioactivity produced by hydrolysis with thermolysin [49]. Tomato seed contains proteins of high nutritional value and nutraceutical properties, so they can be recovered for application as food additives. Mechmeche et al. [50,51], in some studies have developed a simple and inexpensive method to produce bioactive peptides from tomato seed meal isolate using Lactobacillus plantarum and their antioxidant activity was also evaluated. In another paper, Esteve et al. [52] proposed a new strategy for the revalorization of olive waste material using proteases to obtain bioactive peptides. A flour with about 22% protein, hydrolysed with specific enzymes, provided hydrolysates with antioxidant and antihypertensive capacity. Brewers’ spent grain is the most abundant by-product generated in the beer-brewing process and, due to its high content of protein, it represents an attractive compound in human nutrition. A proteinenriched isolate from brewers’ spent grain was hydrolysed using some enzymes and six ACE inhibitory peptides were identified [53]. The proteins from the potato starch industry byproduct is a promising source, as several health benefits may be associated with their hydrolysates. Waglay et al. [54] investigated the efficiency of some selected proteases and characterized the
Table 2 Peptides and their biological properties from agro-industrial plant-based waste or by-products Source
By-product/waste
Biological properties
Reference
Date Cherry Peach Tomato Olive Barley Potato
Seeds Seeds Seeds Seeds Flour Brewers’ spent grain Potato starch industry by-product
[47] [48] [49] [50,51] [52] [53] [54]
Cauliflower Palmaria palmata
Cauliflower by-products Red alga
Antioxidant and ACE inhibitory Antioxidant and antihypertensive Antioxidant Antioxidant Antioxidant and antihypertensive ACE inhibitory ACE inhibitory, antioxidant, lipolysis stimulating, anti-cholesterol ACE inhibitory ACE inhibitory
Current Opinion in Food Science 2020, 31:31–37
[55] [56]
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New scientific advances on vegetable biopeptides Montesano et al. 35
hydrolysate properties. Cauliflower by-products are a potential source of value-added compounds. Chiozzi et al. [55] developed an analytical strategy for the production of purified bioactive peptides from cauliflower waste proteins, by testing two different extraction protocols and screening different enzymes for protein hydrolysis, in this way, they identified three novel ACE-inhibitory peptides. Palmaria palmate is a red alga which is rarely eaten and is usually discarded in the production of kombu (Saccharina japonica), although it is becoming popular as a foodstuff. Some authors reported that thermolysin digestion produces two peptides with ACE inhibitor activity, that can be considered as one more example of revalorization of waste [56].
Final considerations and recommendations The interest for health-promoting functional foods, dietary supplements and pharmaceutical preparations containing bioactive peptides is markedly increasing. A focal point is represented by the fact that the sequences that show the same bioactivity can be released from both animal and plant native proteins. More recently, the scientific community investigated the possibility to obtain bioactive peptides from plants. Several researches showed that plant-derived peptides display a myriad of activities ranging from antimicrobial, anticancer, anticholesterol to beneficial effects for cardiovascular health. In addition, studies in humans have established the effects of identified plant peptides on cancer, cell proliferation, and their use as preventative control agents for diseases such as diabetes and high blood pressure. The peptides reviewed generally have shown excellent health-promoting properties and potentially prevent many diseases by making them extremely suitable for nutraceutical applications. However, more studies are required to further identify their target organs, and elucidate their biological mechanisms of action in order to be potentially used as functional foods or even therapeutics for the prevention or treatment of chronic diseases. In particular, further clinical studies are needed to better understand the gastrointestinal stability, bioavailability and safety of these peptides for their use as drugs, nutraceuticals or functional foods. Moreover, industrial scale-up technologies are required to ensure cost of production of these bioactives remains feasible. Finally, from the last paragraph it seems relevant the effort of the scientific community to derive high valueadded products from waste from agro-industrial processing, in accordance with the concepts of circular economy and sustainability, fundamental to guarantee respect for the ecosystem.
Conflict of interest statement Nothing declared. www.sciencedirect.com
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors.
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Current Opinion in Food Science 2020, 31:31–37