**3.2 Antihypertensive activity**

Hypertension is a major risk for many disorders including coronary heart disease, stroke, heart failure, vision loss, chronic kidney disease, and dementia [28, 29]. Renin-angiotensin system (RAS) is responsible in blood pressure regulation. The liver-released angiotensin is converted to angiotensin-I (Ang-I) by renin, then the Ang-I is easily degraded to Ang-II, a potent vasoconstrictor, by angiotensin converting enzyme (ACE), leading to vasoconstriction and hypertension. Thus, hypertension can be controlled using renin and ACE inhibitors, in which the latter is more important due to its dual functions, catalyzing Ang-II (a potent vasoconstrictor) and inactivation of bradykinin (a vasodilator) [30]. However, controlling blood pressure by peptides from agricultural co-products also reported to be achieved through other mechanisms, such as nitric oxide production and blocking calcium channel and Ang-II receptor. The receptor blockers are able to hinder vasoconstriction and other functions mediated by Ang-II. Calcium channel blocker can avoid calcium availability in blood vessel cell wall and heart which make them to have a lower extent contraction, leading to relaxation and lower blood pressure [27, 31]. Arg-containing peptides have also been reported to suppress hypertension as Arg is a precursor in nitric oxide production. Nitric oxide (NO) is synthesized by the reaction of Arg and oxygen in the presence of nitric oxide synthase as a catalyst. NO is a vasodilator which act against Ang-II and a balanced level of these two compounds could lead to normal blood pressure [32]. Peptides namely, LIWKL, RPYL, RRWQWR, blocking the Ang-II receptor and HRW, a calcium channel blocker, have been reported to exert antihypertensive effects [33, 34].

The antihypertensive activity of peptides depends on several factors including amino acid composition and their position, molecular weight and charge. Normally, peptides with lower molecular weight exert higher activity due to their higher affinity to bind with ACE, in which the most potent reported peptides are di- and tri-peptide. Moreover, small peptides can stay intact through gastrointestinal digestion and epithelial transportation, reaching to blood circulation system and the target organ [31]. The structure–activity relationship of 168 dipeptides and 140 tripeptides with ACE inhibitory activity was assessed [35]. The authors reported that presence of bulky side chain and hydrophobic amino acids in dipeptides could result in higher activity, while presence of aromatic amino acids at C-terminus

**161**

*Bioactive Peptides from Agriculture and Food Industry Co-Products: Peptide Structure…*

and positively charged amino acids in the middle and hydrophobic residues at N-terminus brought them higher ACE inhibitory activity. The interaction of peptides with zinc ion in active site of ACE could effectively deactivate the enzymes. It has been reported that Leu could bind with Zn2+ by its carboxyl group and inhibit

Antihypertensive activity of hydrolysates/peptides from agricultural co-products was extensively explored by *in vitro* and *in vivo* studied, mostly based on their inhibition capacity against ACE and rat with hypertension (either SHR or hypertensive-induced rat), respectively (**Table 1**). However, clinical studies are still needed to confirm their health benefits. Clinical studies on the antihypertensive effect of IPP and VPP showed a controversial result as it reduced systolic and diastolic blood pressure in Asian case studies to a higher extent than Caucasians, while no effect was observed in Dutch and Danish cases [46, 47]. They reported that the difference could be associated to variations in genetics and dietary habit which should be taken into consideration. Kwak et al. [27] reported antihypertensive effect of black soy peptide in human trial, in which systolic blood pressure decreased in hypertensive subjects likely through reduction in ACE activity and increase in nitric oxide production. The authors concluded that the activity of black soy peptides might also be associated with higher arginine content which is a substrate for nitric oxide

**Activity Ref.**

ACE inhibitory activity, Blocking Ang-II receptor

ACE inhibitory activity ACE inhibitory activity IC502 : 41, 4.3, 0.2 and 8.5, respectively

ACE inhibitory activity IC50: 33.9

ACE inhibitory activity IC50: 0.9 and 6.9, respectively

inhibitory activity IC50: 0.7

[33]

[37]

[38]

[39]

[40]

*In vivo In vitro*

Systolic and diastolic blood pressure reduction at 600 mg/kg: −21.9

and − 15.5 mmHg, respectively, after 4 h

reduction at 10 mg/ kg in SHR: −2 mmHg

Systolic blood pressure reduction at 30 mg/kg:

−38.6 mmHg for RGL and RGM after 4 and 6 h, respectively, in

in SHR

after 6 h

−31.3 and

SHR

Flavourzyme HPY ACE

*DOI: http://dx.doi.org/10.5772/intechopen.94959*

formation, known as a strong vasodilator.

**Source Enzyme Hydrolysate/**

**Animal based co-product**s

Bovine whey lactoferrin

Fish (Cobia) skin

Chicken bone

Bovine bone gelatin

Bovine blood plasma

**Peptide**

hydrolysate (<3 kDa) LIWKI, RPYL and RRWQWR

WAA, AWW, IWW, WL

(Hyp)-GL and RGM-(Hyp)-GF

Pepsin YYRA Systolic blood pressure

Pepsin Partially purified

Protamex Hydrolysate

Alcalase RGL-

the enzyme activity [36].

*Bioactive Peptides from Agriculture and Food Industry Co-Products: Peptide Structure… DOI: http://dx.doi.org/10.5772/intechopen.94959*

and positively charged amino acids in the middle and hydrophobic residues at N-terminus brought them higher ACE inhibitory activity. The interaction of peptides with zinc ion in active site of ACE could effectively deactivate the enzymes. It has been reported that Leu could bind with Zn2+ by its carboxyl group and inhibit the enzyme activity [36].

Antihypertensive activity of hydrolysates/peptides from agricultural co-products was extensively explored by *in vitro* and *in vivo* studied, mostly based on their inhibition capacity against ACE and rat with hypertension (either SHR or hypertensive-induced rat), respectively (**Table 1**). However, clinical studies are still needed to confirm their health benefits. Clinical studies on the antihypertensive effect of IPP and VPP showed a controversial result as it reduced systolic and diastolic blood pressure in Asian case studies to a higher extent than Caucasians, while no effect was observed in Dutch and Danish cases [46, 47]. They reported that the difference could be associated to variations in genetics and dietary habit which should be taken into consideration. Kwak et al. [27] reported antihypertensive effect of black soy peptide in human trial, in which systolic blood pressure decreased in hypertensive subjects likely through reduction in ACE activity and increase in nitric oxide production. The authors concluded that the activity of black soy peptides might also be associated with higher arginine content which is a substrate for nitric oxide formation, known as a strong vasodilator.


*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

by giving their excess electrons.

derived peptides for 8 weeks [27].

**3.2 Antihypertensive activity**

chicken blood that showed higher antioxidant activity than those prepared from blood corpuscles with lower NCAAs [6]. Presence of NCAAs are reported to correlate with the strong antioxidant activity, because they can neutralize free radicals

Hypertension is a major risk for many disorders including coronary heart disease, stroke, heart failure, vision loss, chronic kidney disease, and dementia [28, 29]. Renin-angiotensin system (RAS) is responsible in blood pressure regulation. The liver-released angiotensin is converted to angiotensin-I (Ang-I) by renin, then the Ang-I is easily degraded to Ang-II, a potent vasoconstrictor, by angiotensin converting enzyme (ACE), leading to vasoconstriction and hypertension. Thus, hypertension can be controlled using renin and ACE inhibitors, in which the latter is more important due to its dual functions, catalyzing Ang-II (a potent vasoconstrictor) and inactivation of bradykinin (a vasodilator) [30]. However, controlling blood pressure by peptides from agricultural co-products also reported to be achieved through other mechanisms, such as nitric oxide production and blocking calcium channel and Ang-II receptor. The receptor blockers are able to hinder vasoconstriction and other functions mediated by Ang-II. Calcium channel blocker can avoid calcium availability in blood vessel cell wall and heart which make them to have a lower extent contraction, leading to relaxation and lower blood pressure [27, 31]. Arg-containing peptides have also been reported to suppress hypertension as Arg is a precursor in nitric oxide production. Nitric oxide (NO) is synthesized by the reaction of Arg and oxygen in the presence of nitric oxide synthase as a catalyst. NO is a vasodilator which act against Ang-II and a balanced level of these two compounds could lead to normal blood pressure [32]. Peptides namely, LIWKL, RPYL, RRWQWR, blocking the Ang-II receptor and HRW, a calcium channel blocker, have

The antihypertensive activity of peptides depends on several factors including amino acid composition and their position, molecular weight and charge. Normally, peptides with lower molecular weight exert higher activity due to their higher affinity to bind with ACE, in which the most potent reported peptides are di- and tri-peptide. Moreover, small peptides can stay intact through gastrointestinal digestion and epithelial transportation, reaching to blood circulation system and the target organ [31]. The structure–activity relationship of 168 dipeptides and 140 tripeptides with ACE inhibitory activity was assessed [35]. The authors reported that presence of bulky side chain and hydrophobic amino acids in dipeptides could result in higher activity, while presence of aromatic amino acids at C-terminus

been reported to exert antihypertensive effects [33, 34].

Although antioxidant activity of agricultural co-product has been widely evaluated, few studies were conducted to assess the activity *in vivo* and there is still a gap for clinical trial. The chemical *in vitro* studies are not able to reflect the activity in biological systems due to their complicated physiological conditions. In addition, cellular evaluation might be able to provide a comparable environment to biological systems. Antioxidant properties of peptides from fish sauce increased with the increasing of peptides concentration based on chemical assays, while these peptides could act as pro-oxidants in higher concentration (>50 μg L-leucine equivalent/ml) in cellular experiments [24]. Hydrolysates prepared from corn gluten meal and ham seed meal as well as ACFL, a peptide from horse mackerel viscera, increased the level of antioxidant enzymes based on *in vivo* models upon exposure to oxidative stress [10, 25, 26]. In a human trial study, a reduction in plasma malondialdehyde and an increase in SOD level was observed after daily ingestion of 4.5 g black soy

**160**


#### **Table 1.**

*Peptides/hydrolysates derived from agricultural co-products involved in antihypertension activity.*

#### **3.3 Antidiabetic activity**

The diabetes is a chronic health problem which involved 463 million adults in 2019 and it is estimated to reach 700 million by 2045. In the health disorder, the elevated blood glucose cannot be treated properly due to either pancreas failure in insulin production (type-I) or insulin resistance in the body (type-II), in which the latter comprised the majority of about 90–95% [48].

The diabetes type-I treatment is associated to insulin injection, while the diabetes type-II can be prevented by controlling the pathways, by which the blood glucose elevates. The enzymes, α-amylase and α-glucosidase, play roles in carbohydrate digestion through breaking them down to oligosaccharides and subsequently

**163**

*Bioactive Peptides from Agriculture and Food Industry Co-Products: Peptide Structure…*

derived from agricultural co-products are summarized in **Table 2**.

Chicken feet Neutrase Hydrolysate Reduction

Collagenase GA-Hyp

**Peptide**

GPA GP-Hyp

TQMVDEEIM-EKFR

Trypsin VAGTWY Reduction

VAAA KAAVT, YGAE, ANVST and TKAVEH

**Activity Ref.**

activity

activity IC50: >20, 5.03 and 2.51 mM, respectively

activity IC50: 86.3 and 69.84 μM, respectively

activity IC50: 210 μM

activity IC50: 44.7 μM

activity IC50: 49.6 and 41.9 μM, respectively

activity

STC-1

cholecystokinin (CCK) and GLP-1 release in enteroendocrine STC-1 cells, DPP-IV inhibitory

DPP-IV inhibitory activity, IC50: 141 μM Stimulation of GLP-1 secretion in

DPP-IV inhibitory

DPP-IV inhibitory

DPP-IV inhibitory

DPP-IV inhibitory

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

*In vivo In vitro*

of glycemia in glucoseintolerant rats at 300 mg/kg

of glucose level in mice at 300 mg/kg

Hydrolysate Stimulation

Trypsin IPAVF DPP-IV inhibitory

Flavourzyme GPAE, GPGA DPP-IV inhibitory

to glucose, which is easily absorbed from intestine to blood, leading to hyperglycemia. In addition, dipeptidyl peptidase IV (DPP-IV), a protease located on endothelial, epithelial and some other cells, could easily degrade hormones stimulating insulin secretion during food ingestion, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), and cause dysregulation of blood glucose. Therefore, many studies have attempted to find bioactive peptides to inhibit these enzymes, so that the diabetes type-II would be cured or prevented. Bioactive peptide with *in vitro* enzyme inhibitory and *in vivo* antidiabetic activities

*DOI: http://dx.doi.org/10.5772/intechopen.94959*

**Source Enzyme Hydrolysate/**

Mare whey Papain NLEIILR

Crude protease extracts from smooth hound and cuttlefish hepatopancreas

*in vitro* GI digestion by pepsin and pancreatin

**Animal based co-products**

Collagen from pig and cattle skin, fish scale and chicken feet

β-Lactoglobulin from bovine whey

Atlantic salmon

skin

Cuttlefish viscera

Bovine haemoglobin

### *Bioactive Peptides from Agriculture and Food Industry Co-Products: Peptide Structure… DOI: http://dx.doi.org/10.5772/intechopen.94959*

to glucose, which is easily absorbed from intestine to blood, leading to hyperglycemia. In addition, dipeptidyl peptidase IV (DPP-IV), a protease located on endothelial, epithelial and some other cells, could easily degrade hormones stimulating insulin secretion during food ingestion, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), and cause dysregulation of blood glucose. Therefore, many studies have attempted to find bioactive peptides to inhibit these enzymes, so that the diabetes type-II would be cured or prevented. Bioactive peptide with *in vitro* enzyme inhibitory and *in vivo* antidiabetic activities derived from agricultural co-products are summarized in **Table 2**.


*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

**Activity Ref.**

ACE inhibitory activity IC50: 8.9, 8.9 and 4.9, respectively

Renin and ACE inhibition activity

ACE inhibitory activity IC50: 3.6

inhibitory activity IC50: 46.7

Renin and ACE inhibitory activity

inhibitory activity

[29]

[41]

[42]

[28]

[43]

[44]

[45]

*In vivo In vitro*

in human plasma by 32% at 50 mg administration

Systolic blood pressure reduction at 100 mg/kg

reduction at 50 mg/kg

−9.5 mmHg after 2 h

Systolic blood pressure reduction at 200 mg/kg

−37 mmHg after 8 h

in SHR1 : −20 and − 35 mmHg after 6 h for hydrolysates and the peptides <1 kDa, respectively

in SHR:

in SHR:

Hydrolysate ACE

**Peptide**

and RVWCP

Whey IW ACE activity reduction

peptides <1 kDa including NL, QL, FL, HAL, AAVL, AKTVF, TPLTR

Trypsin AY Systolic blood pressure

Cottonseed Papain FPAIGMK ACE

Autolysis ARIYH, LRKGNLE

Alcalase Hydrolysate,

Thermoase Partially purified

Trypsin and Alcalase

hydrolysate (3–5 kDa)

**Source Enzyme Hydrolysate/**

Poultry viscera

Wheat bran

Corn gluten meal

Flaxseed protein isolate

Red seaweed (*Porphyra columbina*) by-product

*1*

*2*

**Table 1.**

**Plant based co-products**

The diabetes is a chronic health problem which involved 463 million adults in 2019 and it is estimated to reach 700 million by 2045. In the health disorder, the elevated blood glucose cannot be treated properly due to either pancreas failure in insulin production (type-I) or insulin resistance in the body (type-II), in which the

*Peptides/hydrolysates derived from agricultural co-products involved in antihypertension activity.*

The diabetes type-I treatment is associated to insulin injection, while the diabetes type-II can be prevented by controlling the pathways, by which the blood glucose elevates. The enzymes, α-amylase and α-glucosidase, play roles in carbohydrate digestion through breaking them down to oligosaccharides and subsequently

**162**

**3.3 Antidiabetic activity**

*Spontaneously hypertensive rat.*

*IC50 based on μg/ml.*

latter comprised the majority of about 90–95% [48].


#### **Table 2.**

*Examples of agricultural by-product peptides and hydrolysates exhibiting antidiabetic activity.*

In a clinical study, Goudarzi and Madadlou [64] indicated that hydrolysate prepared from whey proteins stimulated insulin production, so that plasma glucose got back to normal level in postprandial hyperglycaemia cases, while the hydrolysate had no effect in prehypertensive cases. Although studies indicated that hydrolysates/peptides might stimulate secretions of hormones involving in insulin production [55, 56, 64], most studies have focused on major enzymes involving in carbohydrate digestion and DPP-IV. The structure–activity relation of peptides possessing the diabetes-involving-enzyme inhibition has not been completely understood yet. Nongonierma et al. [57] identified di- and tri-peptides inhibiting DPP-IV in wheat gluten hydrolysate. These peptides had some main characteristics including the presence of Pro at carboxyl terminus or penultimate position and Phe or Leu at amino terminus. Li-Chan et al. [54] described that peptides with DPP-IV inhibitory activity required presence of hydrophobic amino acids, particularly Pro, as Pro placed at 1–4 (preferably at second) positions from N-terminal end and bounded with Leu, Val, Phe, Ala and Gly. Dipeptides as X-Pro with X as a small size hydrophobic amino acid would likely be an effective inhibitor. Presence of hydrophobic and aromatic amino acids at N-terminal end of peptides with DPP-IV inhibitory activity was also reported by Lima et al. [65]. Ren et al. [63] evaluated the α-glucosidase inhibitory capacity of peptides from hemp seed and indicated that hydrophobicity of peptides was a prime factor affecting inhibitory activity and molecular weight as a second priority. The authors have also reported that larger molecular weight peptides could also enhance α-glucosidase activity. α-Amylase is another enzyme involving in carbohydrate digestion and it has been reported that presence of branched and aromatic amino acids such as Lys, Phe, Tyr and Trp and positively charged amino acid could help to inhibit the enzyme [60].

**165**

*Bioactive Peptides from Agriculture and Food Industry Co-Products: Peptide Structure…*

The antibacterial activity of hydrolysates/peptides has been studied to a lesser extent when compared to other aforementioned properties. Hydrolysates/peptides with antibacterial activity have been obtained from co-products of milk, seafood, meat and others which are summarized in **Table 3**. Conventional antibiotics and preservatives are extensively applied to control pathogens, which lead to antibioticresistant strains. Therefore, an alternative antimicrobial agent has been sought. Peptides with antibacterial properties could be one of alternative agents as they are non-toxic and could act against both Gram-negative and Gram-positive as well as antibiotic-resistant bacteria [76]. Typically, chemical antibiotics have specific targets and bacteria can develop various defense strategies towards antibiotics. In contrast, antimicrobial peptides target cell membrane and can cause serious dam-

The antibacterial activity of these peptides is associated to their molecular weight, charge and hydrophobicity [67]. Peptides are attached by negativelycharged residues of cell membrane, like lipopolysaccharides and lipoteichonic acid on Gram-negative and Gram-positive bacteria, respectively, through electrostatic

**Source Peptide/Hydrolysate Test bacteria Activity Ref.**

*Flavobacterium psychrophilum, Renibacterium salmoninarum*

*Listeria. monocytogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa*

Total viable colonies Coliform bacteria Yeasts Molds

> *E. coli S. aureus Salmonella typhimurium Streptococcus mutans*

*E. coli S. aureus S. typhimurium S. mutans*

> *E. coli L. innocua*

*Bacillus cereus* IZ2

MIC1

(mg/ml) 2

MIC (mg/ml) 0.5

Reduce microbial counts in ground beef stored in refrigerator

> (mm): 2.30–2.55

MIC (mg/ml) 130/65

MIC (mg/ml) 260/130

IZ (mm) 9

5

0.5 0.5 0.5

260/130 NI3 /260 NI/260

260/130 NI/260 NI/260

7.5

[66]

[67]

[68]

[69]

[70]

[71]

*DOI: http://dx.doi.org/10.5772/intechopen.94959*

age which make it difficult to develop resistance [77].

Hydrolysate prepared by pepsin

Partially purified (<3 kDa) hydrolysate prepared by Protamex

TSKYR obtained by pepsin-hydrolyzed hemoglobin

Hydrolysate prepared by Alcalase, Flavourzyme, Protamex, trypsin and papain

Crude hydrolysate prepared by trypsin/ partially purified (<3 kDa)

prepared by trypsin/ partially purified (<3 kDa)

Hydrolysate prepared by Protamex

Cow whey Crude hydrolysate

**3.4 Antibacterial activity**

**Animal based by-products**

*Rainbow trout* Viscera

Yellowfin tuna viscera

Bovine hemoglobin

Porcine blood proteins: albumin globulin

Camel whey

Snow crab co-products
