**3.4 Antibacterial activity**

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**Peptide**

Pro-containing peptides

QPQ, QPG, QPF, LPQ, SPQ

Oat globulin Trypsin LQAFEPLR DPP-IV inhibitory

IP 13 peptides containing 6–32 amino acids

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

**Activity Ref.**

activity

activity

activity IC50: 103.5 μM

activity IC50: 0.41 mM α-amylase and β-glucosidase inhibitory activity

α-glucosidase inhibitory activity

DPP-IV inhibitory

inhibitory activity

DPP-IV inhibitory

DPP-IV inhibitory

IC50: 79.8, 70.9, 71.7, 56.7, and 78.9 μM, respectively

[57]

[58]

[59]

[60]

[61] [62]

[63]

*In vivo In vitro*

Hydrolysate α-amylase and

**Source Enzyme Hydrolysate/**

HYW20

Pepsin Trypsin Alcalsae

Alcalase

Rice bran UmamizymeG

Protease from ginger

**Plant based co-products** Wheat gluten Debitrase

*Luffa cylindrica* seed

Hemp seed meal

**Table 2.**

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

Alcalase LR, PLMLP α-glucosidase

positively charged amino acid could help to inhibit the enzyme [60].

**164**

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

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



#### **Table 3.**

*Antibacterial peptide/hydrolysate prepared from agricultural co-products.*

interactions, by which the structure of cell surface was disrupted. Subsequently, peptides could permeate to the cell and reach to cytoplasmic membrane, causing leakage of cytoplasmic fluid [78].

Antibacterial activity of hydrolysates and/or peptides has mostly evaluated by their directly exposure to pathogens and less studies have conducted to assess their application in food. A peptide, TSKYR, obtained from bovine hemoglobin [68] and hydrolysate prepared from yellowfin tuna waste [67] were added to ground beef and minced fish, respectively. The peptide, TSKYR, could reduce total viable colonies, yeasts, molds and particularly coliform bacteria within 14 days storage in a refrigerator. Moreover, the peptide (0.5% w/w) in ground beef was able to diminish lipid oxidation by 60% which was reported to be comparable to BHT. Pezeshk et al. [67] reported that hydrolysates prepared from yellowfin tuna were able to increase fish (silver carp) mince shelf-life in a refrigerator through inhibition of psychrophilic and total count bacteria as well as prevention of oxidative degradation.

#### **4. Bioavailability**

Studies revealed that peptides prepared from agricultural co-products have great potential of health promoting properties. However, bioavailability of these peptides is a challenge, in which they need to stay intact within gastrointestinal tract (GIT) and epithelial transportation to reach their target organs and exert physiological

**167**

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

PTPVP, have been reported to stay intact after in vitro GI digestion [84].

been reported to be highly susceptible to hydrolysis [86, 90, 91].

Peptides structural changes usually occur in GI tract and transepithelial transportation that would likely have effects on their physiological functions. To meet the challenge, some approaches have been applied to improve the stability of these peptides such as using permeation enhancer, enzyme inhibitor and encapsulation. Sodium caprate has been used to improve the permeability of two antihypertensive peptides, IPP and LKP, through paracellular route in Caco2 cell [98]. The authors reported that sodium caprate could intensify the peptides absorption via paracellular mechanism and inhibited PepT1 route, leading to antihypertensive effect in SHRs model. An antihypertensive peptide, RLSFNP, would degrade to RLSF, SFNP, FNP and F during the epithelial transportation. Permeation enhancers including

Epithelial permeation of bioactive peptides into blood circulation system is another challenge that affects physiological activities. Peptides may undergo some structural modification induced by brush border proteases (**Table 4**). For instance, a peptide with ACE inhibitory activity, KPLL, can be degraded to KP and LL within epithelial permeation, resulting in lower activity than the intact form [96]. The permeation could occur through four pathways, including peptide transporter 1 (PepT1), passive paracellular transportation through tight junctions, transcytosis and simple passive transcellular diffusion. Peptide properties such as size, hydrophobicity, charge and amino acid sequence are important factors affecting their absorption. Briefly, small (di- and tri-) peptides can be transported via PepT1 route, however peptide properties have effects on its efficacy. Non charged and hydrophobic peptides have higher affinity towards PepT1. Hydrophilic and negatively charged low molecular weight peptides can pass through energy-independence paracellular route. Transcytosis is an energy-dependent route, by which long chain peptides, particularly hydrophobic, can be transported. A highly hydrophobic peptide is likely transported through simple passive transcellular diffusion. To evaluate the effect of molecular weight on the permeation, Wang and Li [97] reported that hydrolysates with the molecular weight lower than 500 Da (mostly diand tri-peptides) showed higher bioavailability and were able to pass through Caco2 cell via PepT1 route, while those with the molecular weight ranging 500–1000 and 1300–1600 Da permeated through paracellular route. Besides molecular weight, peptide sequence also affects its bioavailability. A Pro-containing peptide has more stability towards brush border proteases and peptides with Leu at N-terminus have

functions. Proteases and peptidases in GIT, brush border and cytoplasm are able to break down the peptide bond to a higher extent, leading to changes in structure and subsequently the activity. However, some peptides have been reported to be stable within the digestion and transportation. There are several factors affecting the peptides stability, which are associated to proteases specificities in GIT. Lower molecular weight, negatively charged, hydrophilic and acidic amino acid containing peptides are reported to be more stable against GI digestion. Negatively charged peptides from milk are more stable against GI digestion followed by positively charged and neutral peptides [79, 80]. Hydrophobic peptides reported to have less stability, which might be due to pepsin specificity towards hydrophobic amino acids [81]. Peptides containing more acidic amino acids, and also those with lower molecular weight showed more stability against GI digestion [6, 79]. Peptides with the molecular weight of larger than 3 kDa were easily digested by GI proteases, while peptides with <1 kDa mostly survived and no change in their antioxidant activity upon GI digestion [82]. Savoie et al. [83] reported that peptides from animal- (casein and cod fish) and plant- (soy and gluten) based substrates with Pro and Glu showed higher stability. Pro has a rigid ring structure bonded to β-carbon which makes it resistant against proteolytic degradation [80]. Thus, Pro containing peptides, IAGRP and

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

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

functions. Proteases and peptidases in GIT, brush border and cytoplasm are able to break down the peptide bond to a higher extent, leading to changes in structure and subsequently the activity. However, some peptides have been reported to be stable within the digestion and transportation. There are several factors affecting the peptides stability, which are associated to proteases specificities in GIT. Lower molecular weight, negatively charged, hydrophilic and acidic amino acid containing peptides are reported to be more stable against GI digestion. Negatively charged peptides from milk are more stable against GI digestion followed by positively charged and neutral peptides [79, 80]. Hydrophobic peptides reported to have less stability, which might be due to pepsin specificity towards hydrophobic amino acids [81]. Peptides containing more acidic amino acids, and also those with lower molecular weight showed more stability against GI digestion [6, 79]. Peptides with the molecular weight of larger than 3 kDa were easily digested by GI proteases, while peptides with <1 kDa mostly survived and no change in their antioxidant activity upon GI digestion [82]. Savoie et al. [83] reported that peptides from animal- (casein and cod fish) and plant- (soy and gluten) based substrates with Pro and Glu showed higher stability. Pro has a rigid ring structure bonded to β-carbon which makes it resistant against proteolytic degradation [80]. Thus, Pro containing peptides, IAGRP and PTPVP, have been reported to stay intact after in vitro GI digestion [84].

Epithelial permeation of bioactive peptides into blood circulation system is another challenge that affects physiological activities. Peptides may undergo some structural modification induced by brush border proteases (**Table 4**). For instance, a peptide with ACE inhibitory activity, KPLL, can be degraded to KP and LL within epithelial permeation, resulting in lower activity than the intact form [96]. The permeation could occur through four pathways, including peptide transporter 1 (PepT1), passive paracellular transportation through tight junctions, transcytosis and simple passive transcellular diffusion. Peptide properties such as size, hydrophobicity, charge and amino acid sequence are important factors affecting their absorption. Briefly, small (di- and tri-) peptides can be transported via PepT1 route, however peptide properties have effects on its efficacy. Non charged and hydrophobic peptides have higher affinity towards PepT1. Hydrophilic and negatively charged low molecular weight peptides can pass through energy-independence paracellular route. Transcytosis is an energy-dependent route, by which long chain peptides, particularly hydrophobic, can be transported. A highly hydrophobic peptide is likely transported through simple passive transcellular diffusion. To evaluate the effect of molecular weight on the permeation, Wang and Li [97] reported that hydrolysates with the molecular weight lower than 500 Da (mostly diand tri-peptides) showed higher bioavailability and were able to pass through Caco2 cell via PepT1 route, while those with the molecular weight ranging 500–1000 and 1300–1600 Da permeated through paracellular route. Besides molecular weight, peptide sequence also affects its bioavailability. A Pro-containing peptide has more stability towards brush border proteases and peptides with Leu at N-terminus have been reported to be highly susceptible to hydrolysis [86, 90, 91].

Peptides structural changes usually occur in GI tract and transepithelial transportation that would likely have effects on their physiological functions. To meet the challenge, some approaches have been applied to improve the stability of these peptides such as using permeation enhancer, enzyme inhibitor and encapsulation. Sodium caprate has been used to improve the permeability of two antihypertensive peptides, IPP and LKP, through paracellular route in Caco2 cell [98]. The authors reported that sodium caprate could intensify the peptides absorption via paracellular mechanism and inhibited PepT1 route, leading to antihypertensive effect in SHRs model. An antihypertensive peptide, RLSFNP, would degrade to RLSF, SFNP, FNP and F during the epithelial transportation. Permeation enhancers including

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

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

*Lisinibacillus sphaericus Bacillus thuringiensis B. cereus Clostridium perfringens B. subtilis*

*E. coli Shigella dysenteriae P. aeruginosa S. aureus B. subtilis S. pneumoniae*

*Listeria monocytogenes L. monocytogenes* biofilm

*Porphyromonas gingivalis* (PG)*, Candida albicans* (CA)

MIC (μg/ml) 150

MIC (μg/ml) 29

MIC (μg/ml) 0.25

IC50: (PG, CA; μM)

> > 8

289, ND4 ND, 75.6 ND, 78.5 [72]

[73]

[74]

[75]

interactions, by which the structure of cell surface was disrupted. Subsequently, peptides could permeate to the cell and reach to cytoplasmic membrane, causing

Antibacterial activity of hydrolysates and/or peptides has mostly evaluated by their directly exposure to pathogens and less studies have conducted to assess their application in food. A peptide, TSKYR, obtained from bovine hemoglobin [68] and hydrolysate prepared from yellowfin tuna waste [67] were added to ground beef and minced fish, respectively. The peptide, TSKYR, could reduce total viable colonies, yeasts, molds and particularly coliform bacteria within 14 days storage in a refrigerator. Moreover, the peptide (0.5% w/w) in ground beef was able to diminish lipid oxidation by 60% which was reported to be comparable to BHT. Pezeshk et al. [67] reported that hydrolysates prepared from yellowfin tuna were able to increase fish (silver carp) mince shelf-life in a refrigerator through inhibition of psychrophilic and total count bacteria as well as prevention of oxidative degradation.

Studies revealed that peptides prepared from agricultural co-products have great potential of health promoting properties. However, bioavailability of these peptides is a challenge, in which they need to stay intact within gastrointestinal tract (GIT) and epithelial transportation to reach their target organs and exert physiological

leakage of cytoplasmic fluid [78].

Rice bran KVDHFPL

*Minimum inhibitory concentration.*

**Plant-based co-products**

Hydrolysate prepared by Alcalase

CAILTHKR obtained by Protamexhydrolyzed meal

obtained by bromelainhydrolyzed bran

LRRHASEGGHGPHW EKLLGKQDKGVIIRA SSFSKGVQRAAF obtained by pepsinhydrolyzed bran

*Antibacterial peptide/hydrolysate prepared from agricultural co-products.*

Palm kernel cake

*Jatropha curcas* meal

*1*

*2*

*3*

*4*

**Table 3.**

*Inhibition zone.*

*No inhibition.*

*Not detected.*

**166**

**4. Bioavailability**

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**Table 4.**

*Peptide modification within epithelial permeation and their transportation route across Caco-2 cell.*

sodium glycocholate hydrate, sodium deoxycholate and Na2EDTA as well as enzyme inhibitors, bacitracin and leupeptin, have been applied to improve the intact peptide bioavailability [99]. Na2EDTA was the most effective to enhance RLSFNP absorption through enlarging intracellular junctions. They also reported that bacitracin could exert permeation enhancer activity beyond its protease inhibitory effect. Permeation enhancer is believed to cause damages in cell membrane in case of long-term usage, leading to inflammation. However, major destructive effects were not observed by using bacitracin in rat intestine [100]. Besides, encapsulation of RLSFNP by liposome could also facilitate the intact peptide transportation through transcytosis in Caco2 cell [101]. In addition, Li et al. [102] used nano-encapsulation of antidiabetic peptides made by chitosan coated liposome to maintain the stability of peptides.
