**3. Bioactive peptides**

Bioactive peptides (BPs) are tiny fragments of dietary protein, consisting of 2–20 amino acids, have a molecular weight of less than three kDa, and promote health benefits. After entering the body, BPs can be absorbed in the intestine, carry out various metabolic pathways, and perform various physiological functions [4, 35, 36]. Several researchers have reported that legumes have various biological activities good for body health (**Table 3**).

The hydrolysis of legumes protein can produce these BPs. The enzymatic hydrolysis process occurs in the food processing process, for example, in the fruit ripening process, the fermentation process (producing soy sauce, tempe, natto, and other fermented products), or the germination process (producing soybean sprouts, green bean sprouts, and sprouts products others). In addition, the protein breakdown process can also be carried out by in vitro enzymatic hydrolysis, for example, using the alcalase in legume protein. Some examples of enzymatic hydrolysis are soybean hydrolysis (*Glycine max*) or mung bean hydrolysis (*Vigna radiata*), which produces BP hydrolysate as an ACE inhibitor [40, 45]. The in vitro enzymatic hydrolysis process will produce peptides with enormous structural diversity. Bioinformatics techniques using in silico studies can help select suitable peptide sources. Simulations of biological processes, such as enzyme hydrolysis, can use these in silico studies and further characterise processes and products using software/computers [61].

BPs can perform their activities and roles based on their structural properties, composition and amino acid sequence [62]. Biologically, the active peptides have similar structural properties, including the length of the amino acids, containing hydrophobic amino acids, and resistance to proteolysis [11]. For example, BPs with antioxidant activity have a length of 5–16 amino acids [63]. The structure of ACE inhibiting BPs contains arginine or lysine residues at the C-terminal will affect their activity [11]. Therefore, selecting the protease enzyme to form BPs is essential to produce biologically active peptides. For example, the Carlsberg enzyme subtilysin will hydrolyse peptide bonds with broad specificity to produce peptides with C terminal in the form of hydrophobic amino acids such as Phe, Tyr, Trp, Leu, Ile, Val and Met [64]. In addition, because of their relatively small size and high specificity, BPs can inhibit protein–protein interactions [65]. Some examples of the functional properties possessed by BPs are anti-hypertensive [66], antioxidants [67], hypocholesterolemia [68], antimicrobials [69], anti-inflammatory [70, 71], anti-cancer [59], and other functional properties. One type of BPs can have more than one functional property [9, 65]. To date, researchers are still developing comprehensive studies and reviews to confirm the therapeutic effect of BPs. This chapter will discuss the BPs of legumes and their functions.

#### **3.1 Antidiabetic**

Increased blood sugar levels are signs of diabetes caused by decreased insulin secretion, impaired insulin function, or both. In patients with T2DM, the body does not respond adequately to insulin action and the blood glucose level increases, a condition known as hyperglycemia [72]. Changing diet is one way of treating diabetes, besides losing weight, exercising, or taking drugs to increase glucose homeostasis [25]. Side effects from synthetic antidiabetic drugs are gastrointestinal disorders [73]. Other side effects are hypoglycemia and weight gain [74].

Meanwhile, some patients are intolerant of the drug [75]. Therefore, research to find BPs from food as a safe antidiabetic has recently increased to overcome these side effects [76]. Measuring the inhibitory activity of DPP-IV is one way to



#### **Table 3.**

*The amino acid sequence of several bioactive peptides from legumes.*

determine whether BPs have an antidiabetic activity or not. The role of the DPP-IV enzyme is to inactivate incretins, especially GLP-1 and GIP. GLP-1 is a glucagon-like peptide, while GIP is a glucose-dependent autotrophic insulin peptide. Incretin is a hormone that vitalising insulin secretion. So the mechanism commonly used to control T2DM is to measure how much DPP-IV inhibition is [77].

Several low molecular weight BPs can induce insulin stimulation in blood intake, for example, the peptides present in fermented soybeans [78] or fermented kidney beans [9]. Some BPs that are isolated from black bean (*Phaseolus vulgaris* L) protein hydrolyzate effectively inhibits glucose transporter 2 (GLUT2) and glucose transporter, which depends on sodium 1 (SGLT1), which functions to lower blood glucose levels [39]. The BPs found in legumes (such as kidney beans, *Phaseolus vulgaris* L with ten amino acids) have antidiabetic properties [9] (**Table 3**). The table states that the process can produce antidiabetic peptides, including fermentation, germination, or enzymatic hydrolysis.

#### **3.2 Anti-hypertensives**

Controlling hypertension is essential to reduce the risk of cardiovascular complications such as coronary heart disease (which causes heart disease) and stroke, congestive heart failure, irregular heart rhythm, and renal failure [3, 79]. A healthy diet is a way to control hypertension. Eating foods high in BPs is very healthy. Several studies have shown that food ingredients derived from legumes have an anti-hypertensive function. The preparation of BPs uses three ways: fermentation of materials into fermented products, germination, and enzymatic hydrolysis. **Table 3** shows some of the research results.

The anti-hypertensive activity was measured by measuring the inhibitory activity of the ACE (Angiotensin I-converting Enzyme). The ACE will cut angiotensin I to produce angiotensin II (vasoactive peptide). This angiotensin II compound will bind to receptors on the walls of blood vessels causing contraction of blood vessels so that blood pressure rises [80]. The presence of BPs will bind to the ACE enzyme, thus inhibiting the action of ACE, and as a result, blood pressure can drop. Some legumes that are recognised to contain BPs that lower blood pressure include garden beans (*Pisum sativum*) [48], green beans [20, 40], soy (*Glycine max*) glycinin [41], kidney bean [9], and pigeon pea [49]. Fermented products also have anti-hypertensive activity, such as douchi, a traditional Chinese food fermented soybean [8]. Other fermented products are Korean soybean paste fermented with mixed cultures of bacteria [81, 82], and many other products from legumes.

Research on anti-hypertensive BPs from food is still being studied [65]. Antihypertensive BPs (isolated from food) have a higher tissue binding affinity than synthetic drugs, resulting in slower tissue loss [83]. For vigorous anti-hypertensive activity, the position of specific amino acid residues is critical. For example, valine and isoleucine are essential for ACE inhibition [84]. Increased ACE inhibitory activity occurs when the C-terminal is Proline [84]. Therefore, the strategy to produce peptides with high anti-hypertensive activity is to hydrolyse protein to produce proline containing peptides.

#### **3.3 Hypo-cholesterolemic**

Many researchers have studied and reviewed the ability of BPs as cholesterollowering agents [65]. The human body needs healthy cholesterol levels to produce vitamin D and steroid hormones, and bile acids. However, arteriosclerosis can occur when cholesterol in the blood forms plaque in the arteries. As a result, it can reduce oxygen supply to the heart, which leads to cardiovascular disease. While chemicals that lower blood cholesterol can cause liver damage or failure, myopathy [85] and diabetes [86, 87], or some people are sensitive to statins (cholesterol-lowering drugs) [88]. Therefore, the research for BPs that can lower cholesterol has increased over the years [65]. **Table 3** shows BPs in legumes (such as red beans and soybeans with 4–16 amino acids) with hypocholesterolemic activity.

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

Cholesterol reduction by peptides can occur due to inhibition of cholesterol micelle formation, inhibition of lipase activity and strong bile acid-binding [89]. Peptides from fermented soy milk show the ability to bind bile acids [90]. The solubility of cholesterol in lipid micelles will be reduced due to BPs [91], resulting in inhibition of cholesterol absorption in Caco-2 cells with one layer. For example, peptides from cowpeas can inhibit HMGCoA reductase and reduce the dissolution of cholesterol micelles in vitro [92]. A 36% reduction in plasma cholesterol levels could occur in the livers of rats consuming the a'-subunit. The tight binding of BPs with taurocholate, deoxytaurocholate, and glycodeoxycholate can also lead to decreased cholesterol absorption in the intestine [93]. Soybean peptides (LPYP, IAVPGEVA and IAVPTGVA) can activate the LDLR-SREBP 2 pathway to increase LDL uptake effectively. For moderate hypercholesterolemia, 30 g/ml lupine protein consumption effectively reduced the Proprotein Convertase Subtilisin/Kexin type 9 enzyme (PCSK9). Inhibiting HMGCoA reductase activity on HepG2 cells may explain the hypocholesterolemic effect of lupine protein hydrolysate [94]. In addition, peptides cause the regulation of lipoprotein b-VLDL cholesterol receptors to increase in rat liver [95].

#### **3.4 Antioxidants**

The antioxidant properties of peptides have more to do with their composition, structure, and hydrophobicity [62]. The amino acid sequence of these peptides can determine different biological activities. Amino acids Tyr, Trp, Met, Lys, Cys, and His are examples of amino acids that cause antioxidant activity [96]. BPs from some legumes have antioxidant properties, for example, soy peptides with 4–16 amino acids [42, 43] (**Table 3**). This table also shows that BP of Leu-Leu-Pro-His-His from soybean β-conglycinin hydrolysate has antioxidant properties. The amino acid leucine or proline at the N-end can increase its antioxidative activity [35]. Amino acids with aromatic residues can donate protons to electron-deficient radicals. This property enhances the radical scavenging character of amino acid residues. Amino acids in the C-terminal region can increase the antioxidant activity higher than in the N-terminal region. This increase in antioxidative activity relates to the nature of the electronic, hydrophobic, steric, and hydrogen bonding amino acids in the area [39]. Soy milk has significant antimutagenic and antioxidant activity. So fermented soy milk probably can prevent mutagenic and oxidative damage [97]. Consumption of douchi (fermented soy food) extract will increase the activity of SOD (Superoxide dismutase) in the liver and kidneys of mice. This consumption also reduces the serum TBARS (Thio Barbituric Acid Reactive Substance), which will increase catalase activity (CAT). These results may indicate the involvement of BPs and free amino acid components from douchi extract as antioxidants [98].

#### **3.5 Antimicrobial**

The ability of BPs as antimicrobial peptides (AMP) has also been widely researched and studied [65]. For example, Pina-Pérez and Ferrús-Pérez [55] studied AMP from several legumes against bacterial pathogens that cause foodborne diseases. AMP is generally active against a broad spectrum of microorganisms, including bacteria (Gram+ and Gram-), fungi, and viruses [99]. Some AMPs also show additional activity, such as antioxidant activity [100], immunomodulation [101] and wound healing activity [102]. Therefore, this AMP may be a better choice of antibiotics for pathogenic bacteria resistant to conventional antibiotics.

AMP has various characteristics, including amino acid length (between 12 and 50), amino acid composition, charge and position of disulfide bonds [103]. AMP isolated from soybeans showed that long-chain peptides had higher AMP activity

than short peptides [55]. AMP interacts with microbes due to positive charges or hydrophilic and hydrophobic (amphipathic) terminal amino acids, recognised as a prominent structural motif. The charge, hydrophobicity and length of cationic AMP are directly related to their potential as antimicrobials [103]. AMP will cause changes in permeability and osmotic disturbances in bacterial cell membranes [104]. AMP can directly kill bacteria by creating pores through the bacterial cell membrane [101] or interacting with macromolecules in microbial cells [105]. The structure and sequence of peptide amino acids are the main factors for whether or not it is effective as an antimicrobial [104]. Some AMPs are rich in positively charged amino acids (arginine and lysine). Such AMP can enter microbial cells by inducing energy-dependent endocytic pathways such as micropinocytosis [106]. **Table 3** shows some of the AMP amino acid sequences from soybeans.
