**3.7 Anti-cancer**

Many researchers are experimenting with cancer therapy using BPs from various legumes. The results are more promising, cheaper, and safer than cancer treatment using surgery, chemotherapy, or radiotherapy, which have adverse side effects due to the emergence of drug resistance and radio-resistance [113]. Extensive exploration has shown that a high intake of legumes can significantly reduce the risk of colorectal adenoma [114, 115] BPs with anti-cancer activity have a relatively low molecular weight (**Table 3**), as isolated from black soybean by-products have the sequence Leu/Ile-Val-Pro-Lys [116]. In comparison, lunasin from soybeans contains 43 amino acids [58]. The hallmark of lunasin is the Arg-Gly-Asp sequence which

#### *Bioactive Peptides from Legumes and Their Bioavailability DOI: http://dx.doi.org/10.5772/intechopen.99979*

functions for adhesion to the extracellular matrix, and the 8 Asp sequence to bind chromatin [117]. Kim and co-workers [118] said that hydrophobic BPs isolated from soybeans could act as anti-cancer. Some legumes also have higher hydrophobic amino acids that are similar in levels to soybeans, such as mung bean, chickpea, and velvet bean (**Table 1**), thus potentially producing anti-cancer peptides.

The mechanism of inhibition of tumour growth by BPs varies depending on the variety of legume sources, namely by induction of extrinsic apoptosis [119], induction of chromatin condensation [59] or inhibition of inflammatory processes [120]. BP isolated from chickpea (*Cicer arietinum* L.) inhibits the proliferation of breast cancer cells effectively [60]; this BP has the sequence ARQSHFANAQP. Meanwhile, BP from *Phaseolus vulgaris* (cultivar extra-long autumn purple) can also inhibit the proliferation of human tumour cells by inducing apoptotic bodies and nitric oxide [59]. Apoptosis (programmed cell death) is a complex process coordinated by specific target proteins and, in many cases, possibly responsible for the potential anticancer effects [121]. Lunasin can reduce the incidence of skin tumours by 70% [58]. Also, it inhibits gastrointestinal cancer cells [122] and cardiovascular and immunological disorders [123]. The researcher reported that consumption of legumes could reduce the risk of 10 kinds of chronic diseases, including breast cancer, lung cancer and colon cancer [124]. Consumption of legumes in higher amounts will lead to a lower risk of death from cancer [125].

## **4. Bioacessability, stability, and bioavailability of bioactive peptides**

Bioaccessibility, stability, and bioavailability are the main concerns in utilising bioactive peptides (BPs) from food ingredients to remain active in maintaining a healthy body. Bioaccessibility is the first step in the digestive system so that nutrients/BPs out of the food tissue and transported across the intestinal epithelial barrier into the blood circulation system. BP transport processes may involve passive transport (paracellular or passive diffusion) or active routes [126]. During the nutrient transport process, the stability of the material must be kept high, so the bioavailability of nutrients is maintained to be utilised by target cells or tissues. In the digestive tract, nutrients are released from the food matrix and converted into chemical forms that can bind to and enter intestinal cells or pass between them. Dietary factors can also affect the bioavailability of the BPs contained. Interactions between peptides and components of the food matrix can modulate their digestibility and alter the absorption route of the peptide [10]. The release of nutrients in the small intestine starts from chewing, which involves digestive enzymes in the mouth and then in the stomach mixed with acids and enzymes in gastric juices. This whole process is a process for making nutrients biologically accessible [127].

Although the number of active components in the food consumed is abundant, it cannot necessarily prevent disease because it depends on the amount available to function in target organs or tissues [128]. Bioavailability is the number of bioactive compounds that organisms can use effectively [129]. For example, when food contact with the mouth or gastrointestinal tract, various interactions can affect the bioavailability of food nutrients (e.g., the presence of fat can increase the bioavailability of quercetin in food) [130]. In studying the role of bioactive compounds in human health, several factors can inhibit the bioavailability of the active components for use in target organs or tissues [131]. For example, fruit antioxidants mixed with macromolecules form a food matrix such as carbohydrates, fats, and proteins [132].

From a nutritional point of view, bioavailability refers to several nutrient fractions or bioactive compounds that are ingested and can reach the systemic circulation and can finally be utilised [133]. Besides that, bioavailability is the

fraction of a nutrient stored or available for a particular physiological function [134]. Another definition, bioavailability, is the amount of active metabolite from the oral dose fraction reaching systemic circulation [135]. The bioavailability of oral BPs is limited because their release from the plant matrix is affected by: solubility in GI fluids, permeability in intestinal epithelial cells, enzymatic and chemical reactions in the GI tract [136]. Four essential steps are required to absorb bioactive compounds effectively: (a) release from the food matrix; (b) incorporation into bile salt micelles; (c) absorption by epithelial cells; and finally; (d) incorporation into the cyclomicron secretion into the lymphatic system.

The biological effects of a BP depend on its capacity to survive until it reaches the target organ. Thus, the main requirement of a BP is its stability or resistance to gastrointestinal enzyme hydrolysis, brush border and serum peptidase. Experimental evidence shows that the length of the peptide chain determines the ability of BPs to pass through the intestinal epithelium in humans by different mechanisms. For small peptides, it is possible to transport through active basolateral, while for large peptides through a transport mechanism mediated by exocytotic-vesicles [137].

However, many peptides are biologically active but are unlikely to be absorbed in the gastrointestinal tract via local effects or receptors that release hormones and cell signalling in the gut. Such BPs affect gastric emptying, gastrointestinal transport, nutrient absorption (amino acids, glucose, lipids) and composition of the colon microflora. They may also regulate food intake [138].

In addition to the presence of specific residues, charge, and molecular weight, hydrogen bonding potential and amino acid hydrophobic tend to affect the bioavailability resistance of BPs to proteases and enzyme hydrolysing peptides [11, 139, 140]. Lunasin, a BP isolated from soybeans and cereal (wheat, barley and rice), has 43 amino acids (MW 5.4 kDa), displays a helical structure and contains nine aspartic acid residues in the C-terminal region. Lunasin is highly bioavailable, heat-stable (100°C, 10 min), and anti-cancer against carcinogenic chemicals. In vivo digestibility of lunasin-fortified soy protein was studied in mice fed for four weeks [141].

During transit in the central digestive tract, the structural properties of the peptide will influence the stability of BPs, including molecular weight, charge, amino acid sequence, and hydrophobicity [126]. Tests using Sprague Dawley rats showed that the highest absorption of ACE inhibitor BPs was in the jejunum [7]. The results showed that BPs with 2–6 amino acids were easy to absorb than proteins and free amino acids [142]. Small (di- and tripeptide) and large (10–51 amino acids) peptides can pass through the intestinal barrier and exhibit their biological function at the target tissue level. However, as the molecular weight of BPs increases, their chances of passing through the intestinal barrier decrease further [143]. The presence of proline and proline hydroxyl will result in resistance of BPs to digestive enzymes, especially a tripeptide with Pro-Pro at the C-terminal [144]. In another study, the number of peptides in human plasma increased depending on the dose of the BP administered. Thus, it concluded that the saturation of BP transporters could affect the number of peptides that can enter the peripheral blood [145].

Encryption of BPs in their natural protein structure may protect these BPs from gastric digestion. Another way to protect BPs is to modify structural proteins such as phosphorylation of serine, threonine, or tyrosine can prevent hydrolysis by digestive proteases. As a result, protein or peptides have a greater chance of being absorbed in target organs or tissues [146]. Stability also depends on the degree of hydrophobicity/hydrophilicity. The more hydrophobic the structure, the more difficult it is to attack by proteases [147].

Therefore, it explained that the difference in bioavailability of BPs between in vitro and in vivo tests (after oral consumption), which may be smaller or larger,

*Bioactive Peptides from Legumes and Their Bioavailability DOI: http://dx.doi.org/10.5772/intechopen.99979*

occurs due to an increase or decrease in BPs after being catalysed by gastrointestinal proteases. A simulation test of the gastrointestinal digestion process of several tempe legumes (*Phaseolus lunatus* L, *Canavalia ensiformis* L, *Mucuna pruriens*) showed that the proteolysis process by the digestive enzyme pepsin-pancreatin increased ACE inhibitory activity [7, 31, 34]. Another example is that BPs' antioxidant activity in vivo is more significant than in vitro [148]. The shape of the molecular structure also influences the stability of BPs. For example, a small BP (YPI) isolated from ovalbumin has additional stability when tested in GI digestive system simulation. BPs (YPI) and peptides containing P at the C end (RADHP and ADHP) are stable. If their structure is slightly modified by adding one or two amino acids to the C end (e.g. RADHPF, RADHPFL, FRADHPFL), they become unstable in simulated GI hydrolysis [149].

Finally, the use of BPs in nutraceutical and pharmacology for human health is still limited. For that, it is necessary to evaluate: (1) degradation of BPs by proteases in the digestive tract, which can affect bioaccessibility, stability, and bioavailability; (2) the existence of technology that allows modification of the structure of BPs such as (a) phosphorylation of amino acids in BPs to make them more resistant to hydrolysis by digestive enzymes; or (b) increase the amino acid hydrophobic at the N-terminal or C-terminal [150].

#### **5. Technology for bioactive peptides**

In general, protein-rich foods that undergo processing involving protease enzymes will produce peptides. However, not all peptides resulting from protein hydrolysis of foodstuffs will become bioactive peptides (BPs) beneficial to body health. The structural properties of BPs (composition, amino acid sequence, hydrophobic amino acid content, and resistance to digestive enzymes) will determine their beneficial functional properties [126], such as, example anti-diabetic, antihypertensive, cholesterol-lowering, antioxidant, and other functional properties.

Food processing processes related to conventional BPs production include cooking, ripening, fermentation and germination. In principle, the processing involves protease enzymes, e.g., chymotrypsin, trypsin, papain, thermolysin, and others) [151], either in the form of free or immobilised enzymes. For food processing by fermentation, protease enzymes derived from microbes are used in the process, while for germination, the enzymes are from growing seeds. Production of BPs increased by regulating the types of enzymes, microbes used, and germination time. Combining these processes (enzymatic process followed by fermentation, or vice versa) will increase the production of BPs so that it is more optimal [152]. The conventional production of the BPs product was a low amount and purity, making it less effective for the industrial scale [153]. So this conventional method for producing BPs does not necessarily involve a separation and purification process, but the production of functional foods containing healthy BPs in the form of fermented food products [153].

The process technology used to produce functional or nutraceutical food will affect the functional, nutritional and biological properties of the protein in the food. Therefore, several things to pay attention to, namely: (1) the effect of using a thermal (or non-thermal) process on the components of the food produced, including its effect on its functional properties and preservation capabilities; (2) available extraction processes and formulations and their optimization; (3) innovative and sustainable applications that can be developed [127]. In addition, consideration of the choice of processing technology must also be based on the desired nutritional function and appearance and sensory properties (such as colour, texture, and taste in the mouth) to be attractive to consumers [154]. Thermal processes can encourage non-enzymatic Maillard reactions between amino groups and reducing sugars [155]. This process will produce colour, sensory properties that affect consumer acceptance and reduce the activity of BPs [155, 156]. The use of thermal processes (e.g. boiling, cooking, blanching, frying, and sterilising) for softening cell walls and inactivate microorganisms and enzymes to make the shelf life longer [127]. The development of non-thermal processes has several weaknesses; for example, the use of nanofiltration membranes requires energy [157]. Freeze-drying, encapsulation, and solvent extraction techniques are costly. To overcome this limitation, food technology experts must develop new alternative technologies (technology that can maintain bio-accessibility, stability, bio-availability and bio-activity of active components). Including BPs, processed food ingredients and the form of pure isolates (capsules or nanocapsules).

The production of BPs has become more accessible, faster, and more effective with the development of science and technology. Production of BPs on an industrial scale usually uses an enzyme hydrolysis process. So the BPs production process uses computer equipment and database search algorithms to predict target peptides and their properties. By selecting the correct protease enzyme through the database, it is possible to select the protein-enzyme combination, in-silico hydrolysis, and the nature of the peptide to be produced [152, 153, 158]. This in-silico hydrolysis method is a functional and widely practised approach for producing legume BPs (**Table 3**).

The legume or various food peptides resulting from enzymatic hydrolysis was then fractionated and purified using a combination of various chromatographic techniques [158–160]. Isoelectric focusing and ultrafiltration are separate macromolecular compounds (such as protein and pectin). Meanwhile, extraction techniques use solvents or supercritical solutions to isolate small molecule bioactive compounds such as antioxidants [157, 161, 162]. This extraction technique, combined with thermal technology (e.g. pasteurisation or spray drying), has been applied to functional foods. This conventional food processing technology is well documented and well established, but its application for the isolation of BPs still needs development and improvement.

The weakness of current technology is that there is still a need for studies on product safety for health. For example, advanced technologies such as cold plasma, nanotechnology, ultrasound, and others, are thought to affect advanced lipid oxidation processes and cause cell tissue damage. For this reason, the effect of this advanced technology on the safety and health of the food components produced needs to be studied to obtain a complete understanding. In this case, it is necessary to adapt the product and technology to the desired functional properties of the active ingredient. For example, modification or interaction with other macronutrients (e.g. dietary fibre) can increase the bio-availability of bioactive compounds [163].

On the other hand, encapsulation technology using legume protein ingredients as a material is also a technique for providing chemical compounds found naturally in plants and other nutraceutical compounds (such as vitamins, minerals, BPs, or others). Thus this encapsulation allowing these compounds (including BPs) to enter the body and undergo release and degradation by enzymes digestion [164]. Other technologies used to protect the active ingredients or nutraceuticals (such as BPs and others) are encapsulation, edible films and coatings, and vacuum impregnation. One may be promising is nutrigenomics, where the active ingredients are given to individuals on a Taylor-made basis according to the genetic characteristics of each individual [165].

Although several researchers have evaluated and characterised BPs that BPs isolated from food have potential bioactive activity and therapeutic functions, and have high bio-availability (bio-accessibility) (due to the support of excellent and modern processing technology), however, all of them can only have a positive impact on human health when combined with healthy living habits [4].
