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Antihypertensive Peptides from

Food Proteins
Rotimi E. Aluko
Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba,
Canada R3T 2N2; email: rotimi.aluko@umanitoba.ca
Annu. Rev. Food Sci. Technol. 2015. 6:23562
Keywords
The Annual Review of Food Science and Technology is

online at food.annualreviews.org
antihypertensive peptides, food proteins, protein hydrolysates,

angiotensin-converting enzyme, renin, spontaneously hypertensive rats
This articles doi:

10.1146/annurev-food-022814-015520
c 2015 by Annual Reviews.
Copyright
All rights reserved
Abstract
Bioactive peptides are encrypted within the primary structure of food
proteins where they remain inactive until released by enzymatic hydrolysis.
Once released from the parent protein, certain peptides have the ability
to modulate the renin-angiotensin system (RAS) because they decrease
activities of renin or angiotensin-converting enzyme (ACE), the two main
enzymes that regulate mammalian blood pressure. These antihypertensive
peptides can also enhance the endothelial nitric oxide synthase (eNOS)
pathway to increase nitric oxide (NO) levels within vascular walls and
promote vasodilation. The peptides can block the interactions between
angiotensin II (vasoconstrictor) and angiotensin receptors, which can
contribute to reduced blood pressure. This review focuses on the methods
that are involved in antihypertensive peptide production from food sources,
including fractionation protocols that are used to enrich bioactive peptide
content and enhance peptide activity. It also discusses mechanisms that are
believed to be involved in the antihypertensive activity of these peptides.
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1. INTRODUCTION

Food proteins normally contain several specic peptide sequences that remain inactive as long
as they remain bonded to other amino acids within the primary structure. Treatment of proteins
with an enzyme or a combination of enzymes can lead to proteolysis that releases these peptide
sequences. The free forms of these peptides can then be used as therapeutic tools against human
chronic diseases such as hypertension. Following proteolysis, the product mixture contains a
mixture of peptides and undigested proteins. The product mixture is then centrifuged to precipitate
the heavier undigested proteins while the smaller (lighter) peptides remain in the supernatant,
which can then be freeze-dried or spray-dried into a powder. This peptide mixture is called
a protein hydrolysate and can be used as is to determine bioactive properties or can undergo
additional treatments that will separate the component peptides into different fractions. The
protein hydrolysate can be separated into various fractions on the basis of peptide size, charge, or
hydrophobicity. Active fractions can also be subjected to column chromatography-based peptide
purication to isolate homogeneous preparations that contain a single peptide (Li et al. 2014).
Protein hydrolysate, peptide fractions, and isolated homogeneous peptides can be analyzed to
determine amino acid composition or amino acid sequence (Herregods et al. 2011, Li et al. 2014),
which provides information on the structural composition of the products.
Hypertension is a chronic disease that is usually manifested as excessive high blood pressure
(systolic and diastolic values of 140 mmHg and 90 mmHg, respectively), which is characterized
by insufcient relaxation of blood vessels and reduced blood ow. If left untreated, hypertension
can lead to insufcient blood ow to vital organs, which can cause stroke and eventually death.
Human blood pressure is mainly regulated by the renin-angiotensin system (RAS), which is
maintained by two critically important proteases, renin and angiotensin-converting enzyme
(ACE). Renin (EC 3.4.23.15) is a 37-kDa enzyme that hydrolyses the Leu10-Val11 peptide
bond of angiotensinogen (a 55-kDa protein synthesized in the liver) to produce a physiologically
inactive angiotensin I, a decapeptide (Imai et al. 1983, Acharya et al. 2003). ACE (EC 3.4.15.1)
then cleaves off a dipeptide to convert angiotensin I into a vasoactive angiotensin II (an octapeptide) that binds with receptors on the vascular wall to cause blood vessel contractions. In disease
conditions, there is increased activity of renin and/or ACE, which causes abnormally high blood
levels of angiotensin II and leads to excessive blood vessel contraction but insufcient relaxation.
ACE also cleaves and inactivates bradykinin (a vasodilatory peptide), which contributes to blood
vessel relaxation insufciency. As with the RAS pathway, the production of nitric oxide (NO)
by endothelial nitric oxide synthase (eNOS) is an important mechanism for maintaining normal
blood pressure. NO is a vasodilatory agent that helps maintain regular blood vessel dilation and
normal blood pressure; therefore, when there are insufcient levels of NO (especially under
high levels of angiotensin II), there is less dilation and more contraction. On the basis of these
hypertension-inducing factors, traditional drug treatments have involved the use of compounds
that inhibit activities of renin and/or ACE as well as those that reduce/inhibit binding of
angiotensin II to its receptors or those that increase blood NO levels. However, it is well known
that antihypertensive drugs cause unpleasant negative side effects in some patients and could lead
to reduced compliance with prescribed drug treatment (Flack et al. 1997, Bremmer 2003). For
example, ACE-inhibitory drugs cause dry cough in 520% of patients in addition to other negative
side effects such as loss of taste, hyperkalemia (high levels of blood potassium), skin rashes,
hematological disorders, and angioneurotic edema (Antonios & MacGregor 1995, Semple 1995,
Gunkel et al. 1996, Tenenbaum et al. 2000). Similarly, the use of Aliskiren, a recently approved
renin-inhibitory antihypertensive drug, has been associated with gastrointestinal disorders (Abassi
et al. 2009). Therefore, there has been increased interest in developing natural compounds
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that can serve as alternatives to antihypertensive drugs. This is because the use of such natural
products is associated with reduced incidence of negative side effects. Moreover, natural peptides
have the advantages of specicity (limits negative side effects), potency (therapeutic efciency),
and low toxicity such that they can be administered at high doses (Ishida et al. 2011, Thayer
2011). In terms of bioavailability, dipeptides and tripeptides can be absorbed intact by crossing the
intestinal membrane with the help of a peptide-specic transport system, e.g., peptide transporter
1 (PepT1; a proton-dependent transporter). PepT1 uses transmembrane electrochemical proton
gradient as a broad specicity transport force to facilitate exit of small, hydrolysis-resistant
peptides from enterocytes into the surrounding bloodstream (Yang et al. 1999, Satake et al.
2002). In contrast, absorption of peptides with more than three amino acid residues by epithelial
cells occurs through receptor-mediated or nonreceptor-mediated endocytosis, referred to as
pinocytosis when the material is completely soluble (Lee 2002). A third alternative absorption
pathway is through paracellular (aqueous transport through the intercellular space between
adjacent cells) and transcellular routes, both of which allow uptake of intact peptides (Stevenson
& Keon 1998). The high hydrophilic nature of some short-chain naturally occurring peptides
enhances absorption and transport (by passive diffusion) through the paracellular route (Noach
et al. 1994). Absorption through the paracellular route has been demonstrated for an octapeptide
(Conradi et al. 1993) and a tetrapeptide (Satake et al. 2002). The transcellular route also facilitates
peptide uptake through the apical membrane brush border from where peptides are moved
through the enterocyte to the basolateral membrane (Pappenheimer & Michel 2003). Absorption
of hydrophobic peptides is facilitated through the transcellular route (Vermeirssen et al. 2004).
Food protein-derived peptides represent a suitable group of natural compounds that could
serve as alternative antihypertensive agents with less negative side effects. Research in the past
20 years has shown that peptide sequences obtained from food protein proteolysis can act similarly
to drugs as antihypertensive agents by inhibiting in vitro and in vivo renin, ACE, and angiotensin
II receptor activities in addition to enhancing blood NO levels. For example, oral administration
of sour milk to spontaneously hypertensive rats (SHRs) led to detection of active peptides in the
aorta, which was accompanied by decreased ACE activity in the aorta and lungs (Masuda et al.
1996). Matsui et al. (2004) provided evidence that an antihypertensive dipeptide (VY) absorbed
from the gastrointestinal tract (GIT) reduced SHR systolic blood pressure (SBP) by approximately
22% and can be detected in the plasma and various organs such as liver, lung, heart, and kidney.
Recent work also showed reduced renin and ACE activity levels during long-term (48 weeks) oral
administration of a hemp seed protein hydrolysate to SHRs (Girgih et al. 2014a). An egg white
peptide (RVPSL) has also been shown to reduce plasma levels of renin and angiotensin II after oral
administration to SHRs (Yu et al. 2014). Thus, there is evidence that food protein-derived peptides
can be absorbed from the GIT into the blood circulatory system. The following sections discuss
the production method, efcacy testing, and mechanism of action of antihypertensive peptides.
2. METHODS FOR PRODUCING ANTIHYPERTENSIVE PEPTIDES

2.1. Enzymatic Protein Hydrolysis
This involves subjecting protein materials to one or more enzymes, usually at the optimum temperature and pH conditions for each protease (Pihlanto et al. 2008, Makinen et al. 2012, MemarpoorYazdi et al. 2012, Ruiz-Ruiz et al. 2013, Boschin et al. 2014, Garcia-Mora et al. 2014, Ghassem
et al. 2014, Morais et al. 2014). More than one enzyme being used to produce a single protein
hydrolysate can be added simultaneously if optimum hydrolysis conditions are similar (Wang
et al. 2011, Memarpoor-Yazdi et al. 2012, Yamada et al. 2013) or sequentially (Yang et al. 2003,
www.annualreviews.org Antihypertensive Peptides
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Vastag et al. 2011, You & Wu 2011, Rao et al. 2012a, Gu & Wu 2013, Marrufo-Estrada et al.
2013, Boschin et al. 2014, Li et al. 2014) if different. These proteases can be obtained as puried
commercially available forms (Pihlanto et al. 2008, Kazuki et al. 2009, Ruiz-Ruiz et al. 2013,
Girgih et al. 2011a, He et al. 2013a) or prepared as crude forms (Banerjee & Shanthi 2012, Nasri
et al. 2013, de Gobba et al. 2014, Garca-Tejedor et al. 2014, Ktari et al. 2014). In general, there
are no specic proteins or enzymes needed to generate antihypertensive peptides; however, an
easier-to-digest protein will generate higher peptide yield, which is compatible with potential
commercialization. Thus, various protein materials have been used to generate antihypertensive
peptides from plant, animal, and marine-based proteins. Moreover, enzymes that generate smallsize peptides (<10 amino acid residues) during protein hydrolysis seem to be more desirable
proteolysis tools because low-molecular-weight peptides may be more effective antihypertensive
agents when compared to high-molecular-weight peptides. For example, ACE-inhibitory activities (IC50 ) of <1-, 13-, 35-, 510-, and >10-kDa peptide fractions prepared from a bean protein
hydrolysate obtained by alcalase + avourzyme digestions were 0.001, 0.049, 0.107, 0.268, and
0.268 g/ml, respectively (Ruiz-Ruiz et al. 2013). Lower IC50 values indicate better inhibitory
activity when compared to higher values. The protein content of the starting raw material is irrelevant for producing desirable protein hydrolysates; however, a higher protein content of the
raw material will ensure less contamination of the hydrolysate with nonprotein materials. The
presence of nonpeptide but biologically active materials in the protein hydrolysate may confound
results obtained from the activity tests. For example, phenolic compounds are also known to have
antihypertensive properties; therefore, their removal from proteins prior to proteolysis will reduce
or eliminate this confounding effect. This aspect is illustrated by a previous study, which involved
the use of acetone to remove polyphenols from a hemp seed protein isolate prior to enzymatic
hydrolysis (Girgih et al. 2011a).
During proteolysis, it is necessary to maintain the optimum conditions required for enzyme
activity to ensure efcient release of peptides. Therefore, a constant temperature can be maintained
using a thermostated instrument, whereas a constant pH is usually maintained through the addition
of alkali or acid solution. This is because during proteolysis (peptide bond hydrolysis), protons that
are released will cause the reaction mixture pH to lower, which if not corrected by adding alkali
can lead to low pH values and enzyme inactivation. Adding alkali during proteolysis is usually one
of the reasons the salt content of the hydrolysate increases. The need to add alkali or acid solutions
may be obviated if the proteolysis is performed in a buffer solution, but this could also lead to
high salt content in the hydrolysate. A pH-Stat instrument can be used to avoid manual addition
of alkali solutions during proteolysis; the instrument constantly measures pH during hydrolysis
and automatically adds alkali when deviations to the set pH value occur. In most cases, the protein
material is simply dispersed in water to perform enzymatic hydrolysis; in other cases, the protein
material is dispersed in buffers (Zhang et al. 2013). If a single enzyme is used, the reaction is
maintained to completion at the optimum conditions and hydrolysis durations allowed, which
could range from 124 h depending on the intended product characteristics. The hydrolysis
duration has a substantial inverse relationship with peptide size, but in most cases a plateau is
reached during which further increases in hydrolysis time do not produce any effect on peptide
size or activity. For example, hydrolysis of lentil proteins with different enzymes showed that ACEinhibitory activity was time dependent and reached a maximum after which further prolongation
of proteolysis actually led to reduced peptide activity (Garcia-Mora et al. 2014). This can be
interpreted as meaning that more antihypertensive peptides can be released as protease action
progresses but a maximum number of active peptides will eventually be released. Increasing the
reaction time beyond this point could lead to a decrease in the activity of the protein hydrolysate

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because of the possibility that some of the already generated active peptides can undergo further
proteolysis to produce inactive fragments.
Garcia-Mora et al. (2014) showed that alcalase hydrolysis produced a lentil hydrolysate with a
maximum ACE-inhibitory activity of 72% at 3 h in comparison to savinase (63%, 2 h), protamex
(47%, 1 h), and corolase (51%, 2 h). Therefore, longer hydrolysis periods are more suitable than
shorter periods to generate peptides with higher levels of potential antihypertensive activities. The
enzyme type is also important; however, this is dependent on the type of protein substrate. Thus, it
is important to perform initial optimization experiments that include varying the proteolysis duration and testing hydrolysate activity to determine the digestion period associated with the highest
peptide activity. Upon completion of enzyme hydrolysis, the reaction mixture is centrifuged to
separate the materials into a supernatant (contains desired peptides), which is collected, and a
discarded precipitate (contains undigested proteins). The supernatant can be desalted, subjected
to further separations such as membrane ultraltration or column chromatography, or simply
freeze-dried. If high salt content is a concern, the protein hydrolysate can be passed through
a nanoltration membrane to obtain a retentate with minimal salt content (Picot et al. 2010).
However, because most nanoltration membranes have close to 300-Da molecular weight cut-off
(MWCO) sizes, it is likely that some small peptides, especially dipeptides, could be lost in the
permeate. Apart from customized in vitro enzymatic hydrolysis, antihypertensive peptides have
also been isolated from naturally produced muscle peptides during meat curing (Escudero et al.
2012a, 2013, 2014). This type of extraction takes advantage of the natural peptides present in
muscles or those that are released by endogenous enzymes during meat processing. For example,
acidied water was used to obtain a peptide extract from Spanish dry-cure ham; after desalting,
the peptides at 10 mg/ml concentration showed up to 60% in vitro inhibition of ACE activity
(Escudero et al. 2014).
2.2. Fermentation
This method is based on some bacteria or yeast species secreting enzymes (including proteases)
into the extracellular medium during growth. Therefore, inoculating protein materials with this
type of bacteria cell can lead to proteolysis and peptide production. In a typical experiment, an
inoculum is prepared by growing the bacterial cells on nutrient agar during incubation at 37 C
for 24 h. After harvesting the cells in sterile distilled water that contains fermentable sugars
(usually glucose), the suspension can then be used as a starter to inoculate sterile protein raw
material for fermentation. Fermentation is allowed to proceed for a period ranging from a few
hours to several days, depending on the type of fermenting microorganism and the desired
peptide product. At the end of fermentation, the product can be used as is, such as sour milk
(Nakamura et al. 1995) for blood pressure reduction, or a known quantity of the fermented
mixture can be mixed with aqueous reagents and shaken thoroughly to extract the peptides
present (Hernandez-Ledesma et al. 2005, Hayes et al. 2007, Ahn et al. 2009, Inoue et al. 2009,
Garca-Tejedor et al. 2013). The extracted solution is then centrifuged to isolate the peptides in
the supernatant, and the precipitate is discarded. The supernatant can be subjected to additional
processing such as indicated in Section 2.1. Fermented peptides can also be further treated with
in vitro enzyme digestion to enhance release of antihypertensive peptides ( Jakubczyk et al. 2013).
3. PEPTIDE FRACTIONATION AND PURIFICATION

Generally, enzymatic protein hydrolysis leads to the production of a hydrolysate that contains
peptides with a wide range of chain lengths, amino acid compositions, and antihypertensive
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efcacies. Therefore, fractionation is used to separate the hydrolysate peptides into groups based
on chain length, hydrophobicity, or net charge. Depending on the type of protein hydrolysate,
fractionation may increase (Zhang et al. 2009, Li et al. 2014) or decrease (Girgih et al. 2011b)
peptide antihypertensive potency. Peptide purication is required to isolate homogeneous
fractions, which offer better studies of structure-function properties by enhancing amino acid
sequence determination in conjunction with an antihypertensive assay. The main peptide
fractionation protocols are described below.
3.1. Membrane Ultrafiltration

The importance of peptide size is related to absorption potential into the blood from the GIT
as well as ability to interact with target enzymes involved in blood pressure regulation. In vitro
studies have shown that smaller-size peptides may be more active than the bigger peptides (Zhang
et al. 2009, Seguro Campos et al. 2010, Ko et al. 2012a, Perez-Vega et al. 2013). However, oral
administration of low molecular weight (<3 kDa) and high molecular weight (>3 kDa) salmon
protein hydrolysate fractions to SHRs showed similar SBP-reducing effects with the latter producing a slightly better reduction (Ewart et al. 2009). Membrane ultraltration offers the possibility
of fractionating peptides into a wide range of peptide sizes using the most common membranes
with the standard MWCO sizes 1, 3, 5, and 10 kDa. However, other membrane types with 2or 6-kDa MWCO sizes have also been used for peptide separation and isolation (Lin et al. 2012,
Liu et al. 2012). Membrane fractionation can be performed to obtain a single or two products by
using a single membrane with a desired MWCO size (Fujita et al. 2001, del Castillo et al. 2007,
Hayes et al. 2007, Li et al. 2011, Liu et al. 2012, Memarpoor-Yazdi et al. 2012, Garca-Tejedor
et al. 2014) or different fractions with various membranes (Girgih et al. 2011b, Wang et al. 2011,
Ruiz-Ruiz et al. 2013). To obtain fractions that have distinct differences in peptide size, the
hydrolysate is rst passed through a membrane with the smallest MWCO size, such as the 1-kDa
membrane; the ow-through solution (permeate) is then collected as the <1-kDa peptide fraction.
The retained solution (retentate) can be passed through the 3-kDa membrane, and permeate
will contain peptides with sizes bigger than 1 kDa but less than 3 kDa (13-kDa fraction). The
retentate from the 3-kDa membrane can be passed through a 5-kDa membrane to obtain a
35-kDa permeate fraction. Finally, the 5-kDa membrane retentate is then passed through the
10-kDa membrane to obtain a permeate with 510-kDa peptides. The fractionation process can
be reversed by starting with the highest membrane MWCO size and nishing with the lowest
( Jung et al. 2006, Zhang et al. 2009). For example, the protein hydrolysate can be rst passed
through a 10-kDa membrane and the permeate collected and passed through a 5-kDa (or 6-kDa)
membrane; the retentate will be collected as the 510-kDa (or 610-kDa) fraction (Lin et al.
2012, Puchalska et al. 2014). The permeate from the 5-kDa (or 6-kDa) membrane is then passed
through a 3-kDa (or 2-kDa) membrane whose permeate is passed through a 1-kDa membrane
to obtain retentates that are 35- and 13-kDa peptide fractions. The nal permeate obtained
from the 1-kDa membrane will be taken as the <1-kDa peptide fraction. During ultraltration,
it is common to add distilled water at intervals to reduce solution viscosity and improve the rate
of peptide permeation of the membrane; this process is called dialtration and can be used to
enhance the yield and speed of the membrane ultraltration process. However, the membranes do
not have 100% efciency, and it is possible that a small amount of peptides with sizes higher than
the MWCO size could pass through into the permeate. To reduce fouling caused by peptides that
stick to the membrane during ultraltration, it is better to use membranes that have been designed
specically for protein separation. This is because membrane fouling can reduce efciency of the
ltration process and lead to low peptide yields in addition to extended ltration periods.
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3.2. Column Chromatography

Two main column chromatography methods, medium-pressure (fast protein liquid chromatography, FPLC) and high-pressure liquid chromatography (HPLC), are used for peptide separation.
FPLC separation of peptides uses mostly gel permeation or ion-exchange (cation or anion) columns
(Sheih et al. 2009, Lee et al. 2010, Wang et al. 2011, You & Wu 2011, Banerjee & Shanthi 2012,
Intarasirisawat et al. 2013, Tomatsu et al. 2013). Protein hydrolysate fractionation on an FPLC
column is designed principally for initial peptide separation followed by further purication on
an HPLC column (Murakami et al. 2004, Zhang et al. 2006, Wang et al. 2008, Balti et al. 2010,
Jang et al. 2011, Wang et al. 2011, Lee et al. 2012, He et al. 2013c, Intarasirisawat et al. 2013, Liu
et al. 2013, de Gobba et al. 2014). Therefore, fractions within each peak are pooled, freeze-dried,
and assayed for potential antihypertensive activity using ACE or renin-inhibition assay. The peak
that shows the highest in vitro enzyme inhibition activity is then subjected to HPLC separation
using a reverse-phase column (RP-HPLC) during which fractions are also collected and assayed
for activity (Wang et al. 2011; Memarpoor-Yazdi et al. 2012; He et al. 2013b,c; de Gobba et al.
2014; Girgih et al. 2014c). In most instances, a single round of RP-HPLC may not be adequate to
produce homogeneous peptide fractions; therefore, the most active fraction is usually subjected to
a second or third round of RP-HPLC separation to obtain pure peptides (Chen et al. 2013a, He
et al. 2013b). Pure peptides have also been isolated through initial ion-exchange chromatography
followed by fractionation and refractionation of the most active peaks by gel permeation chromatography (Majumder & Wu 2009, Banerjee & Shanthi 2012). Mass spectrometry can check
the purity of each peptide peak, the point at which a single specie dominates (>90%) the mass
spectrum. The mass of each peptide peak can be used to check for matching amino acid sequences
if the primary structure of the parent protein is available on public online protein databases (Hayes
et al. 2007, Gu & Wu 2013, de Gobba et al. 2014, Udenigwe et al. 2012a). Alternatively, in situ
amino acid sequencing can be performed for each peptide fraction using tandem mass spectrometry (Qian et al. 2007; Majumder & Wu 2009; Balti et al. 2010; Jang et al. 2011; Ahn et al. 2012;
Lee et al. 2012; Du et al. 2013; He et al. 2013b,c; Tanzadehpanah et al. 2013; Garca-Tejedor
et al. 2014; Girgih et al. 2014c; Norris et al. 2014; Puchalska et al. 2014) or automated Edman
degradation (Marczak et al. 2003, Jang & Lee 2005, Jung et al. 2006, Lee et al. 2006, Wang et al.
2008, Chen et al. 2013a). However, gel permeation chromatography can also be used to separate the protein hydrolysate into peptide fractions of known molecular sizes followed by assay of
each fraction for potential in vitro or in vivo antihypertensive properties (Escudero et al. 2012a,
Ruiz-Ruiz et al. 2013). Similar to ultraltration fractionation, the results from the gel permeation
chromatography separation can be used to estimate the relationship between peptide size and in
vitro or in vivo antihypertensive property.
4. ACTIVITY ASSAY
4.1. In Vitro Methods
Several methods have been devised for estimating potential antihypertensive properties of peptides;
generally, these methods involve ACE or renin activity assays. The rst ACE-inhibitory assay was
developed using hippuryl-histidyl-leucine (HHL) as a substrate; cleavage of the hippuryl-histidine
bond releases free hippuric acid, which can then be extracted into ethyl acetate. The solvent is
evaporated and the hippuric acid residue dissolved in distilled water followed by spectrophotometric measurement at 228 nm (Cushman & Cheung 1971). Alternatively, the reaction mixture can
be separated on a reverse-phase HPLC column to quantify the hippuric acid peak (Wu et al. 2002,
Wang et al. 2011). In the presence of peptide inhibitors, ACE-mediated production of hippuric
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acid is reduced, and the percentage ratio to the value in the absence of peptide can be calculated as
the percent inhibition. A major drawback of the spectrophotometric assay is that if excessive heat
is used for the ethyl acetate evaporation, some of the hippuric acid may not be dissolved in water,
which causes underestimation of enzyme activity and overestimation of peptide inhibitory capacity. The ethyl acetate may be evaporated with nitrogen gas but if solvent residues remain, there is
overestimation of enzyme activity and underestimation of peptide inhibitory activity because the
ethyl acetate also absorbs ultra-violet (UV) radiation at 228 nm, where hippuric acid is normally
measured. Moreover, the ethyl acetate extract can sometimes be contaminated with the substrate
(HHL), which also absorbs at 228 nm and can lead to overestimation of ACE activity and underestimation of peptide inhibitory activity (Wu et al. 2002). A recent report has conrmed the higher
sensitivity and precision of the HPLC method when compared to the spectrophotometric method
(Chen et al. 2013b). An alternative ACE assay involves uorimetry and uses ortho-aminobenzoic
acid-phenylalanine-arginine-lysine-dinitrophenyl-proline [abz-FRK(dnp)P-OH] as the substrate
(van Elswijk et al. 2003). ACE cleaves the arginine-lysine (RK) bond, which removes the DNP
quenching moiety and causes enhanced uorescence (excitation at 320 nm and emission at
420 nm) of the ABZ moiety. Therefore, ACE-inhibitory peptides will reduce the rate of substrate
cleavage, which is measured as less DNP release and hence reduced uorescence emission values.
Another commonly used ACE assay involves N-(3-[2-furyl]acryloyl)-phenylalanylglycylglycine
(FAPGG) as the substrate (Holmquist et al. 1979). ACE hydrolyzes FAPGG into FAP and GG;
the reaction can then be followed by continuous UV absorption measurement to determine the
decrease in absorbance at 345 nm as a result of the Phe-Gly peptide bond cleavage (Udenigwe et al.
2009). The rate of absorption decrease in the presence of peptide inhibitors will be less and can be
subtracted from the reaction rate in the absence of the inhibitor to obtain percentage inhibition.
Alternatively, the released FAP can be quantied by injecting the reaction mixture directly onto
a reverse-phase HPLC column with detection at 305 nm (Lahogue et al. 2010). In general, the
FAPGG method is simpler and faster to complete and is therefore better suited to routine analysis
of several samples when compared to the HHL assay (Shalaby et al. 2006).
The most widely used renin-inhibitory assay is based on Wang et al.s (1993) method and is
specic for human renin. This method uses an internally quenched uorogenic substrate ArgGlu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(Dabcyl)-Arg; renin hydrolyzes
the Leu-Val bond to yield a highly uorescent Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu
(called peptide-EDANS) product. The amount of peptide-EDANS released can be measured at
335345-nm excitation and 485510-nm emission wavelengths; in the presence of renin-inhibitory
peptides, the emission values are smaller and percentage inhibition can be calculated in relation
to the uninhibited reaction (Udenigwe et al. 2009, Li & Aluko 2010, Girgih et al. 2011b, He et al.
2013c, Fitzgerald et al. 2014). Another uorescence method uses substrates specic for human or
porcine renin (Takahashi et al. 2008). The human renin substrate is N-methylanthranyl (Nma)-IleHis-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys-2,4 dinitrophenyl (dnp)-D-Arg(r)-r-NH2 , and the
porcine renin substrate is Nma-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Lys(dnp)-r-r-NH2 . Hydrolysis occurs at the Leu-Val and Leu-Leu peptide bonds, respectively, for the human and porcine
substrates (Takahashi et al. 2008). The uorescent fragment (contains the Nma moiety) is then
measured at excitation and emission wavelengths of 340 and 440 nm, respectively. The high sensitivity of these uorescence methods enables identication of a wide range of renin-inhibitory
compounds because minor changes in enzyme activity can still be detected.

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4.2. In Vivo Methods

Historically, the SHR has been the most used genetically hypertensive rat model for testing
the in vivo antihypertensive effects of food protein-derived protein hydrolysates and peptides.
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This is because the SHR is the closest model of human arterial hypertension and has been used
extensively for antihypertensive research in safety and efcacy testing (Feld et al. 1981, Tschudi
et al. 1994, Curtis et al. 1995, Zheng et al. 1997, Kvam et al. 1998, Sipola et al. 2001, Sato
et al. 2002, Suetsuna et al. 2004, Wood et al. 2005, Katayama et al. 2007, Hiwatashi et al. 2010,
Sanchez et al. 2010, Herregods et al. 2011, Lu et al. 2011). Due to growth differences, male SHRs
are more commonly used than females to test efcacy of antihypertensive compounds. This is
because male SHRs can grow up to 300 g within a 15-week period in comparison to a maximum
weight of approximately 200 g for females (http://www.criver.com/products-services/basicresearch/find-a-model/spontaneously-hypertensive-%28shr%29-rat). The males can develop systolic and diastolic blood pressure values of up to 203 and 176 mmHg, respectively;
similar values for females are 191 and 154 mmHg, respectively (http://www.criver.com/
products-services/basic-research/find-a-model/spontaneously-hypertensive-%28shr%29rat). SHRs can survive up to 66 weeks independent of whether they receive antihypertensive
treatments (Feld et al. 1981) and can therefore serve as useful models to determine long-term
effects of antihypertensive peptides. A recent work has shown that the plasma renin activity in
SHRs is almost twice that of the normotensive equivalent (Girgih et al. 2014a); therefore, the
SHR provides an excellent model to test renin-inhibitory peptides. Moreover, the SHRs have
been shown to respond positively, which was measured as decreased blood pressure when treated
with antihypertensive agents that inhibit renin (Wood et al. 2005; Hiwatashi et al. 2010; Chou
et al. 2013; Girgih et al. 2014a,c) and ACE (Curtis et al. 1995; Miguel et al. 2010; Sanchez et al.
2010; Fernandez-Musoles et al. 2013; Girgih et al. 2014a,c) activities. Previous works have also
shown that the SHR is a good model for testing ability of antihypertensive peptides to cross the
gastrointestinal barrier and be present within the blood circulatory system (Masuda et al. 1996,
Matsui et al. 2004). Recent works have shown that ACE-inhibitory peptides (VPP, IPP, AHIII)
not only reduced the blood pressure of SHRs but induced endothelium relaxation through
increased NO production (Hirota et al. 2011, Ko et al. 2012b). Therefore, the SHR also serves
as a suitable animal model to determine the NO-enhancing and vasorelaxation properties of
antihypertensive peptides. Most tests involve oral administration of the peptides but intravenous
injection has also been reported (Lee et al. 2012). However, a polycystic kidney disease rat model
was also recently used to show effective blood pressure reduction by a pea protein hydrolysate
during an 8-week oral feeding trial (Li et al. 2011).
5. MECHANISM OF ACTION
In most cases, the exact cause of human systemic hypertension is unknown but the disease is
characterized by excessive RAS enzyme activities, which lead to abnormally high levels of plasma
angiotensin II. The high plasma angiotensin II level causes excessive blood vessel constriction with
reduced vasorelaxation and enhanced superoxide radical production, which lead to increased NO
degradation. The net effect is high blood pressure and the pathological condition of hypertension.
Bioactive peptides have been shown to produce antihypertensive effects mainly as a result of
actions that involve inhibition of RAS enzyme activities (ACE and renin); however, enhanced
production of NO has also been demonstrated. For example, in long-term experiments, it has
been shown that plasma, lung, kidney, and aorta renin and/or ACE activities as well as angiotensin
II levels can be downregulated by antihypertensive protein hydrolysates (Wang et al. 2012,
Fernandez-Musoles et al. 2013, Girgih et al. 2014a). However, long-term oral administration of
antihypertensive corn peptides reduced ACE activity in lungs but not in the plasma (Huang et al.
2011). The antihypertensive effect of an egg protein-derived tripeptide (IRW) was associated with
upregulation of the NO synthesis pathway in addition to ACE inhibition (Majumder et al. 2013).
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5.1. ACE Inhibition

Protein hydrolysates have been shown to inhibit in vitro and in vivo activities of ACE, but activity
is highly dependent on the type of protease(s) used or the proteolysis conditions, in addition to
the nature of the raw material (Ewart et al. 2009, Ko et al. 2012a, Marrufo-Estrada et al. 2013, He
et al. 2013a, Alashi et al. 2014). For example, hydrolysis of whey protein concentrate (WPC) with
alcalase was found to produce protein hydrolysates with higher levels of ACE-inhibitory activities
than similar hydrolysates produced from avourzyme and pancreatin (Morais et al. 2014). Li
et al. (2006) also showed that the antihypertensive activity of an alcalase mung bean hydrolysate
was greater than that of the neutrase hydrolysate. The differences in product activity are due
to peptide bond hydrolysis specicity differing among enzymes and, as such, each hydrolysate
containing peptide mixtures that differ in amino acid composition and chain length. Even for
the same enzyme and starting protein material, varying the enzyme-to-substrate ratio (E:S) can
produce hydrolysates with different ACE-inhibitory properties, further illustrating the importance
of peptide characteristics. For hydrolysis of WPC, there was increased ACE-inhibitory activity of
the hydrolysates at high E:S values, which indicates that the presence of more enzyme molecules
could enhance production of active peptides. However, the relationship between the enzyme
concentration and ACE-inhibitory activity of the WPC protein hydrolysates was not exactly linear.
The exact reason for this nonlinearity is unknown but may be related to inactivation (degradation)
of active peptides at some enzyme levels. In general, E:S values used for food protein hydrolysate
production can range from 0.1 to 10%, but hydrolysis should be optimized so as to not use excessive
enzyme levels that will not translate to better activity of the products.
Modes of action of certain protein hydrolysates and peptides have shown that ACE inhibition
takes place through competitive, noncompetitive, uncompetitive, or even mixed-type peptideenzyme interactions. For example, isolated peptides from collagen hydrolysates were shown to
inhibit ACE activity in a competitive manner, which indicates interaction with the active site
and competition with the substrate to block proteolysis (Banerjee & Shanthi 2012). In contrast,
Memarpoor-Yazdi et al. (2012) showed that egg white lysozyme-derived peptides inhibited ACE
activity in a noncompetitive manner, which suggests interaction with enzyme proteins at locations
other than the active site. Memarpoor-Yazdi et al. (2012) also showed that interaction of the
lysozyme peptides with ACE led to a reduction in -helix, whereas unordered conformation
increased. The increased level of disordered fraction could have led to the reduced catalytic ability
of the ACE molecule. An almond protein hydrolysate containing <1-kDa peptides was also found
to inhibit ACE activity in a noncompetitive manner (Wang et al. 2011).
Several studies have examined the role of peptide chain amino acid position on the potential for higher ACE inhibition. Even though most of these studies involve statistical modeling
coupled with in vitro experiments, they are relevant to our ability to design peptides with high
antihypertensive potency. This is because knowledge of critical amino acid residues required on
a peptide chain can allow selection of specic enzymes that will liberate peptides with desirable
amino acid contents or sequences. For example, if arginine and/or lysine are important for ACE
inhibition, then trypsin could be an effective proteolysis tool. Similarly, thermolysin has a high
propensity to hydrolyze peptide bonds that involve branched-chain amino acids (BCAAs), and its
hydrolysate will contain higher levels of these amino acids. BCAAs, especially valine and isoleucine,
are components of the famous milk tripeptides (VPP and IPP) that were identied as potent ACE
inhibitors and antihypertensive agents very early in bioactive peptide research (Nakamura et al.
1995). Proline, especially when present at the C-terminal, has also been shown to potentiate
high ACE inhibition; therefore, prolyl endopeptidases and other proteases that generate prolinecontaining peptides may be useful protein hydrolysis tools for producing strong antihypertensive
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peptides. Aromatic (or bulky) amino acids (AAAs) can also potentiate high ACE-inhibitory activity
of peptides and enhance antihypertensive effects. Thus, enzymes such as pepsin, chymotrypsin,
and alcalase provide important protein cutting tools for producing AAA-enriched peptides with
potentially strong ACE-inhibitory and antihypertensive properties. In terms of specic amino acid
positions and enhanced ACE-inhibitory activity of peptides, it has been shown that for dipeptides,
the presence of AAAs or hydrophobic side chains is important (Wu et al. 2006b). For tripeptides, the presence of an AAA at the C-terminal coupled with a positively charged residue in the
middle position and a hydrophobic residue at the N-terminal was shown to be important for
strong ACE inhibition (Wu et al. 2006b). Wu et al. (2006b) then used their statistical model to
predict various peptide sequences, which were then validated through in vitro ACE-inhibition
assay; LRW, IKP, and FW were shown to have experimental IC50 values of 0.23, 2.75, and
5.89 M, respectively. For longer peptides, it was shown that the ACE-inhibitory activity was
highly dependent on the amino acid sequence C-terminal tetra residue section (Wu et al. 2006a).
Thus, for a strong ACE-inhibitory tetrapeptide, the following amino acids are critical: C1 (tyrosine, proline, phenylalanine), C2 (phenylalanine), C3 (arginine, histidine, tryptophan, phenylalanine), and C4 (valine, isoleucine, methionine). To enhance ACE-inhibitory potency of pentapeptides and longer chains, these amino acid residues are critical at the four C-terminal positions:
C1 (tyrosine, cysteine), C2 (histidine, tryptophan, methionine), C3 (valine, leucine, isoleucine,
methionine), and C4 (tryptophan). However, a recent work also determined the favorable amino
acid residues for the C-terminal pentapeptide residues of strong ACE-inhibitory peptides, which
were reported as follows (Sagardia et al. 2013): C1 (glycine, leucine, alanine, valine, isoleucine),
C2 (arginine, valine, threonine), C3 (aspartic acid, asparagine, lysine), C4 (tryptophan, tyrosine,
cysteine), and C5 (valine, isoleucine). All the reports agree that the C-terminal sequence of a peptide contains the critical amino acid residues necessary for strong ACE inhibition. There is also
some agreement with regard to specic amino acid requirements, as all the models reported the
importance of a positively charged amino acid at the C2 and tryptophan at the C4 of long-chain
(>4 residues) peptides. However, there are still major differences in the reported required amino
acids for other C-terminal positions, which may be due to variations in modeling tools and peptide
databases. A major agreement between these peptide structure-activity modeling studies is that
proline did not appear important for C-terminal activity, which is contrary to previous reports for
milk peptides. The reason for this discrepancy is unknown but it could be that the physicochemical
properties of individual amino acids on which the statistical modeling is based are not enough to
provide accurate predictions for all possible potent ACE-inhibitory amino acid combinations.
5.2. Renin Inhibition

Unlike ACE inhibition, there have been only very few reports on the renin-inhibitory properties of food protein-derived peptides. The reason for this areas slow development could be due
to renin being a more difcult enzyme to inhibit and the costs associated with the assay being
signicantly higher than those associated with ACE. One of the earliest works identied three
dipeptides from an alcalase digest of yellow pea seed proteins; IR, KF, and EF had 9.2, 17.8, and
22.7-mM IC50 values, respectively (Li & Aluko 2010). On the basis of these three peptides, it
was suggested that a hydrophobic residue at the N-terminus combined with a bulky amino acid
residue at the C-terminus could enhance renin inhibition by dipeptides. Udenigwe et al. (2009)
showed that a axseed protein hydrolysate fraction with a more cationic character inhibited renin
activity more than the fraction with less cationic properties. Moreover, kinetic studies showed the
cationic fraction inhibited renin through an uncompetitive mode of action, which suggests lack
of afnity for the enzyme active site. More recent works have shown the in vitro renin-inhibitory
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capacities for various food protein hydrolysates such as hemp seed (Girgih et al. 2011b, 2014a),
rapeseed (He et al. 2013a,b,c), canola (Alashi et al. 2014), macroalga (Fitzgerald et al. 2012), kidney
bean (Mundi & Aluko 2014), African yam seed (Ajibola et al. 2013), and chicken skin (Onuh et al.
2013). A quantitative structure-activity relationship (QSAR) study of dipeptides showed that the
presence of a bulky amino acid at the C-terminal with a hydrophobic amino acid at the N-terminal
favored higher levels of renin-inhibitory activity than peptides without C-terminal hydrophobic
residues (Udenigwe et al. 2012b). Using the QSAR model, four tryptophan-containing antihypertensive dipeptides (IW, LW, VW, and AW) were predicted to have the highest potential as renin
inhibitors. However, in vitro validation of their renin-inhibitory activity showed IW to be most
active (IC50 = 2.3 mM), followed by LW (IC50 = 3.2 mM); VW and AW were inactive. Thus,
the structural similarity between isoleucine and leucine may be responsible for this distinguishing inhibitory capacity when compared to other tryptophan-containing dipeptides. Using pure
peptide sequences found in rapeseed protein hydrolysates, it has been shown that higher renin
inhibition is dependent on the ability of the peptide to reduce the -helix and -sheet fractions of
the enzyme in addition to forming several hydrogen bond interactions with the active site residues
(He et al. 2014). Recent work showed that hemp seed peptides (WYT, SVYT, WVYY, IPAGV)
also interacted with the active site of renin; specically, WYT formed electrostatic bonds with the
aspartic acid residues that participate in enzyme catalysis (Girgih et al. 2014b). Therefore, these
works have shown that certain food protein-derived peptides can bind to active or nonactive sites
of renin to modulate catalytic activity. In vivo conrmation of activity was also obtained for the
hemp seed protein hydrolysate, which was shown to lower plasma renin activity in SHRs during
long-term oral feeding (Girgih et al. 2014a). However, it should be stressed that previous works
have not been able to establish a correlation between the renin-inhibitory activities and ACEinhibitory activities of food protein-derived peptides. Therefore, it seems the mode of action of
these peptides against ACE is different from that against renin.

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5.3. Nitric Oxide Production

By inhibiting excessive accumulation of angiotensin II in the blood, antihypertensive peptides
will reduce superoxide free radical (an NO degradation agent) levels and contribute to increased
plasma NO levels. Moreover, as part of their multifunctional properties, food protein-derived
peptides have been shown to enhance the eNOS pathway, which is responsible for producing
NO. For example, the antihypertensive peptide IRW that was isolated from egg ovotransferrin
was shown to restore eNOS expression in the SHR mesenteric artery and aorta (Majumder et al.
2013). Using cultured vascular endothelial cells, it was demonstrated that addition of the milk
protein-derived ACE-inhibitory tripeptides (VPP and IPP) led to increased NO production and
better endothelium-dependent relaxation of aortic rings (Hirota et al. 2011). Increased production
of eNOS leads to higher levels of NO in the vascular system, which can enhance blood vessel
relaxation and lead to blood pressure reduction. The effects of VPP and IPP were inhibited when
coincubated with NOS inhibitors, which conrmed the production of NO as contributing to the
vasorelaxative action of these antihypertensive peptides. Similarly, oral administration of a silk
broin hydrolysate to SHRs led to 8, 13, and 22% increases in plasma NO content at daily doses
of 100, 600, and 1,200 mg/kg body weight (bw), respectively, which was accompanied by reduced
levels of vasoconstrictive endothelin (Yuan et al. 2012).
5.4. Angiotensin II Receptor Blockage

There is scant information on the angiotensin II receptor-blocking effects of food protein-derived
peptides. AT1 is the main angiotensin receptor in vascular walls, and binding of angiotensin II
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leads to blood vessel contractions. The antihypertensive drug valsartan prevents interaction of
angiotensin II with AT1 , which contributes to vascular relaxation. Similarly, RPYL, a lactoferrinderived peptide, has been shown to have concentration-dependent inhibition (up to 62% at
300 M) of interaction between angiotensin II and human angiotensin AT1 receptors in an in vitro
model (Fernandez-Musoles et al. 2013). RPYL also inhibited angiotensin I-dependent vascular
contraction, which suggests ACE inhibition as an additional mechanism of its antihypertensive
activity.

6. BLOOD PRESSUREREDUCING EFFECTS

The ability of protein hydrolysates to reduce blood pressure is highly dependent on several factors,
which include resistance to structural degradation and functional inactivation by gastrointestinal
enzymes. Because the antihypertensive activity of peptides is dependent on amino acid composition
and sequence, the loss of amino acid residues as a result of proteolysis within the GIT can lead to
reduced or increased potency or complete loss of activity. Peptides must also be absorbed from
the GIT and transported through the blood circulatory system to elicit antihypertensive effects.
To conrm GIT stability, the protein hydrolysates or peptides can be treated in vitro with pepsin
followed by pancreatin (or trypsin + chymotrypsin) at 37 C and under appropriate pH conditions;
this is referred to as simulated GIT digestion (Yang et al. 2003, Jang et al. 2011, Escudero et al.
2014, Garcia-Mora et al. 2014, Puchalska et al. 2014). A comparison of the ACE or renin-inhibitory
activity before and after treatment can then be made. A reduction or loss of in vitro inhibitory
activity after the simulated GIT digestion (Banerjee & Shanthi 2012, Puchalska et al. 2014) signies
potential inactivity (no reduction in blood pressure) if the protein hydrolysate is provided orally.
However, if the in vitro activity is unchanged (Katayama et al. 2007, Wang et al. 2008, Jimsheena
& Gowda 2010, Lee et al. 2010, Vercruysse et al. 2010, Ko et al. 2012a, Escudero et al. 2014)
or increases (Ewart et al. 2009, Jang et al. 2011, Aleman et al. 2013, Garcia-Mora et al. 2014)
after the simulated GIT digestion, the protein hydrolysate has potential in vivo activity and can be
tested in a suitable animal hypertension model. For peptides, the original sequence could undergo
fragmentation into shorter peptides that are less or more active. In addition, within the blood
vessels the peptides must not become substrates for renin and ACE to avoid structural degradation
and loss of activity. Similar to the simulated GIT treatment, the protein hydrolysate or peptide can
also be incubated with ACE (Yang et al. 2003, Rao et al. 2012b) or renin followed by determination
of in vitro activity (ACE or renin inhibition). The results obtained will be interpreted similarly to
those explained above for simulated GIT treatment. For example, incubation of spinach Rubisco
peptides with ACE showed that peptide MRWRD was fragmented into a more potent MRW
and a less potent RW, whereas peptide MRW was unaffected (Yang et al. 2003). Lee et al. (2012)
also showed that the ACE-inhibitory activity of peptides VAW and MKR was not affected by
preincubation with ACE. To the contrary, incubation of peptide LRIPVA with ACE led to LR
and IP fragments with 120 times less activity. Peptide IAYKPAG was also fragmented by ACE
into IAY and KP with reduced ACE-inhibitory activity (Yang et al. 2003). Similarly, the ACEinhibitory activity of peptide RGY was decreased after incubation with ACE (Rao et al. 2012b).
On the basis of these principles, Fujita et al. (2000) classied protein hydrolysates and peptides
into the following three main categories. In category one, true inhibitors are the peptides that
maintain their ACE-inhibitory values. Category two comprises peptides that exhibit reduced ACE
inhibition after incubation with ACE and are called substrates. Prodrug peptides, to the contrary,
are converted into true inhibitors after incubation with ACE or GIT proteases and belong to
category three. Therefore, only true inhibitors and prodrug peptides will likely exert in vivo blood
pressurelowering effects.
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6.1. Protein Hydrolysates

Initial testing of potential antihypertensive activity of protein hydrolysates involves in vitro inhibition of renin and/or ACE activities. Hydrolysates that show highest levels of in vitro activities
are then usually orally administered to SHRs to determine blood pressurereducing ability (Fujita
et al. 2001, Herregods et al. 2011); however, in some instances all the hydrolysates are orally
administered (Li et al. 2006, Girgih et al. 2011b, He et al. 2013a, Alashi et al. 2014). To determine
the effect of peptide size, membrane ultraltration fractions with dened peptide size ranges are
also orally administered for comparison with the whole (unfractionated) hydrolysate (Girgih et al.
2011b). SHR tests are usually carried out either on a short-term (024 h) basis with blood pressure measurements at 12 h intervals or on a long-term basis consisting of 28 weeks of feeding
experiments. The short-term trials provide useful information on daily efciency of the protein
hydrolysate (Li et al. 2006, Nakano et al. 2006, Wang et al. 2008, Girgih et al. 2011b, Wang et al.
2011, Ko et al. 2012a, Alashi et al. 2014), whereas long-term trials are used to determine either
preventive or treatment effects (Yang et al. 2004, Nakano et al. 2006, Wang et al. 2008, Huang
et al. 2011, Wang et al. 2011, Lin et al. 2012, Liu et al. 2012, Fernandez-Musoles et al. 2013,
Yamada et al. 2013, Girgih et al. 2014a). To determine preventive effects, the protein hydrolysate
is given to young (growing) SHRs. Blood pressure is measured weekly and compared with that of
SHRs that consumed the placebo diet (Yang et al. 2004, Nakano et al. 2006, Girgih et al. 2014a).
Treatment potential can be determined by feeding the adult rats (with full-blown hypertension)
the protein hydrolysate coupled with weekly blood pressure measurements (Girgih et al. 2014a).
Direct use of protein hydrolysates as antihypertensive agents has the benet of reduced costs
given the absence of the need for peptide separation and purication. Moreover, the synergistic
effects of different peptides present in protein hydrolysates could produce better blood pressure
reducing effects than when the peptides have been separated into less heterogeneous mixtures.
For example, separation of a hemp seed protein hydrolysate into <1-kDa or 13-kDa peptide
fractions resulted in reduced efcacy as a blood pressurereducing agent when compared to the
unfractionated hydrolysate (Girgih et al. 2011b). The peptide fractions showed a maximum of
18 mmHg reduction after 6 h of oral administration to SHRs when compared to 30 mmHg
after 8 h for the unfractionated hydrolysate. More importantly, the unfractionated hydrolysate
was still active (19 mmHg), whereas the peptide fractions were inactive after 24 h of oral administration. The demonstrated greater antihypertensive effectiveness of unfractionated hydrolysate
is consistent with synergistic effects of varying peptide sizes. Table 1 shows the blood pressure
reducing effects of different food protein hydrolysates after oral administration to SHRs. The data
show the different protein sources, peptide production tools, and protein hydrolysate doses. More
importantly, Table 1 shows that the antihypertensive effect can be directly dose dependent, like
the silk broin, or not, like the soybean and almond. Table 1 also shows different rates of blood
pressurelowering activity; the mushroom peptide extract produces a maximum decrease within
30 min ( Jang et al. 2011), whereas protein hydrolysates such as whey (Wang et al. 2012), sesame
(Nakano et al. 2006), and rapeseed (He et al. 2013a) take 824 h. These results demonstrate
that potency of antihypertensive protein hydrolysates is highly dependent not only on starting
raw material but also the type of enzyme used for proteolysis. Commercial products showed that
gelatins had no effect on the blood pressure of SHRs after oral administration (Herregods et al.
2011), which suggests lack of absorption, minimal presence of bioactive peptides, or structural
degradation of active peptides in the GIT. However, subsequent hydrolysis of the gelatins with
thermolysin produced a protein hydrolysate that reduced SHR SBP by almost 17 mmHg after
6 h; thus, the gelatin was broken down by thermolysin into antihypertensive peptides (Herregods
et al. 2011). The thermolysin-induced antihypertensive gelatin peptides acted through inhibition
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Table 1 Effects of oral administration of food protein-derived protein hydrolysates on short-term (24 h) changes in the
systolic blood pressure (SBP) of spontaneously hypertensive rats
SBP reduction

Protein source
Protease(s)
Dose (mg/kg bw)
(mmHg)a
References
300
17 after 6 h
Herregods et al. 2011
Pepsin
1,000
10 after 6 h
Li et al. 2014
Trypsin
1,000
22 after 6 h
Li et al. 2014
Pistachio
Pepsin + Trypsin
1,000
22 after 6 h
Li et al. 2014
Fish
Thermolysin
500
15 after 4 h
Fujita et al. 2001
Sesame seed
Thermolysin
30 after 8 h
Nakano et al. 2006
Canola meal
Pancreatin
200
15 after 6 h
Alashi et al. 2014
Canola meal
Pepsin
200
24 after 4 h
Alashi et al. 2014
Canola meal
Trypsin
200
5 after 8 h
Alashi et al. 2014
Canola meal
Alcalase
200
34 after 4 h
Alashi et al. 2014
Canola meal
Chymotrypsin
200
15 after 6 h
Alashi et al. 2014
Rapeseed meal
Proteinase K
100
5 after 8 h
He et al. 2013a
Rapeseed meal
Thermolysin K
100
9 after 8 h
He et al. 2013a
Rapeseed meal
Alcalase
100
24 after 8 h
He et al. 2013a
Rapeseed meal
Flavourzyme
100
17 after 6 h
He et al. 2013a
Rapeseed meal
Pepsin + Pancreatin
100
20 after 24 h
He et al. 2013a
Almond
Neutrase + N120P
400
17 after 2 h
Wang et al. 2011
Almond
Neutrase + N120P
800
21 after 2 h
Wang et al. 2011
Bamboo shoot
None (aqueous extract)
20
11 after 4 h
Liu et al. 2013
Bamboo shoot
50
18 after 4 h
Liu et al. 2013
Bamboo shoot
100
27 after 5 h
Liu et al. 2013
Mung bean
Alcalase
600
30 after 6 h
Li et al. 2006
Spanish ham
4.56
38 after 6 h
Escudero et al. 2012a
Spanish ham
1.48
28 after 6 h
Spanish ham
8.7
24 after 6 h
Dairy whey
Crude enzyme extract
400
22 after 4 h
Tavares et al. 2012
Oyster
Pepsin
20
16 after 4 h
Wang et al. 2008
Gelatin
Thermolysin
Pistachio
Pistachio
Mushroom
600
48 after 0.5 h
Jang et al. 2011
Rapeseed
Pepsin
500
7 after 4 h
Marczak et al. 2003
Rapeseed
Alcalase
500
16 after 4 h
Marczak et al. 2003
Soybean
Protease D3
50
25 after 2 h
Kodera & Nio 2006
Soybean
Protease D3
500
40 after 2 h
Kodera & Nio 2006
Soybean
Protease D3
1,000
50 after 2 h
Kodera & Nio 2006
Jellysh
Pepsin + Papain
200
23 after 2 h
Liu et al. 2012
Jellysh
Pepsin + Papain
400
28 after 4 h
Liu et al. 2012
Jellysh
Pepsin + Papain
800
31 after 2 h
Liu et al. 2012
Shrimp
Pepsin
300
17 after 4 h
Zhang et al. 2009
Shrimp
Pepsin
600
27 after 6 h
Zhang et al. 2009
Shrimp
Pepsin
900
35 after 6 h
Zhang et al. 2009
Bovine casein
A combination of 3 enzymes
20 after 2 h
Yamada et al. 2013
Bovine casein
10
28 after 2 h
Yamada et al. 2013

(Continued )
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Table 1 (Continued )

Protein source
SBP reduction
(mmHg)a
Protease(s)
Dose (mg/kg bw)
Bovine casein
100
Bovine casein
Yeast (Debaryomyces hansenii )
200
20 after 2 h
Garca-Tejedor et al. 2013
Bovine casein
Yeast (Kluyveromyces lactis)
200
12 after 1 h
Bovine casein
Yeast (Kluyveromyces
marxianus)
200
20 after 2 h
Bovine
lactoferrin
Yeast (Debaryomyces hansenii )
200
18 after 1 h
Bovine
lactoferrin
Yeast (Kluyveromyces lactis)
200
10 after 1 h
Bovine
lactoferrin
Yeast (Kluyveromyces
marxianus)
200
23 after 1 h
Dairy whey
Alcalase
240
32 after 8 h
Wang et al. 2012
Silk broin
Alcalase
100
11 after 4 h
Zhou et al. 2010
Silk broin
Alcalase
600
20 after 4 h
Zhou et al. 2010
Silk broin
Alcalase
1,200
42 after 4 h
Zhou et al. 2010
Hemp seed
Pepsin + Pancreatin
200
30 after 8 h
Girgih et al. 2011b
Flaxseed
Thermoase
200
29 after 4 h
Nwachukwu et al. 2014
42 after 2 h
References
Yamada et al. 2013
Yellow eld pea
Thermolysin
200
19 after 4 h
Li et al. 2011
Flaxseed
Trypsin + Pronase
200
18 after 2 h
Udenigwe et al. 2012a
Palmaria palmata
Papain
50
34 after 2 h
Fitzgerald et al. 2014
Some values are estimates obtained from graphs in the respective publications.
of ACE activity, as shown by the inhibition of angiotensin I-evoked contraction of isolated rat
aortic rings. The lack of contraction in the presence of the thermolysin gelatin peptides indicates
that ACE molecules were blocked from converting angiotensin I into angiotensin II, the latter
being the contractile agent.
Peptide structural characteristics such as type and position of specic amino acids on the peptide chain have been shown to be important for ACE or renin inhibition and ultimately blood
pressurereducing effects. Pistachio protein hydrolysates produced through pepsin, trypsin, or
pepsin + trypsin hydrolyses were shown to reduce blood pressure in SHRs after oral administration; however, the effect was stronger for the trypsin and pepsin + trypsin hydrolysates with a
22-mmHg decrease in SBP when compared to the maximum decrease of 10 mmHg for the pepsin
hydrolysate (Li et al. 2014). The pepsin + trypsin hydrolysate was shown to contain ACKEP as
the most active peptide. ACKEP shares some structural similarity with antihypertensive drugs
such as Lisinopril and Enalapril, as they each contain proline at the C-terminal. Thus, the blood
pressurereducing ability of pepsin + trypsin hydrolysates may be attributed to the presence of
proline, which enhanced binding of the peptides to the ACE active site and resulted in substrate
binding blocking.
6.2. Purified Peptides

Using consecutive HPLC separation techniques, it is possible to obtain homogeneous peptide
preparations that can be subjected to amino acid sequence determination. These peptides can be
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either synthesized or isolated during the HPLC separations and then used to determine blood
pressurelowering effects in SHRs. Table 2 shows some of the food protein-derived peptides that
have been identied and tested for in vivo efcacy. With a few exceptions, the results conrm that
shorter-chain peptides could provide better antihypertensive effects than the longer-chain peptides; this may be due to faster rates of absorption and resistance to physiological clearance (longer
persistence in the body). As shown for IRW, the antihypertensive effect may also be dose dependent. One of the early reports was the blood pressurereducing effect of milk tripeptides (VPP
and IPP), which were shown to be active components of sour milk (Table 2). Since then, various
peptide doses have been reported to be effective blood pressurereducing agents. For example,
Kontani et al. (2014) showed that the peptide IHRF reduced SHR blood pressure up to a maximum
of approximately 18 mmHg at 5 mg/kg bw 7 h after oral administration, whereas a 15-mg/kg bw
dose led to a 39-mmHg decrease (Table 2). A recent report showed 3, 7, and 10 mg/kg bw peptides produced dose-dependent decreases in SBP for two lactoferrin-derived peptides (DPYKLRP
and LRP) after oral administration to SHRs (Garca-Tejedor et al. 2014). The mechanism of
action was conrmed to be through in vivo inhibition of plasma ACE activity, which decreased
in SHRs that were given the two peptides. Various casein-derived peptides at 410 mg/kg bw
were also shown to be effective antihypertensive peptides when tested in SHRs; results suggested
that shorter peptides were more effective than longer peptides, as shown in Table 2 (Miguel
et al. 2010). This may be due to the possibility that longer peptides suffer structural degradation
in the GIT, leading to absorption of fragments with lower antihypertensive potency. For some
peptides, there seems to be a direct relationship between the measured in vitro activity and blood
pressurereducing effects in SHRs, as shown in Table 2 for the spinach Rubisco peptides. Peptides
VNP and VWP isolated from rice protein showed, however, very similar in vitro ACE-inhibitory
activities, but VWP produced a greater decrease in SBP (38 mmHg) when compared to VNP
(29 mmHg), also shown in Table 2. Thus, it is possible that ACE inhibition alone may not be
responsible for the blood pressurelowering effects of peptides; other in vivo effects such as renin
inhibition and enhanced NO production could be contributing to the observed antihypertensive
results.
Although short-chain peptides have been proposed as being more effective blood pressure
reducing agents, a tuna dark musclederived peptide containing 13 amino acids (WPEAAELMMEVDP) was shown to reduce SHR SBP by 17 mmHg (Qian et al. 2007). A 21-amino acid
residue peptide (GDLGKTTTVSNWSPPKWKDTP) was isolated from tuna frame protein and
also shown to reduce SBP in SHRs (Lee et al. 2010). In vitro analysis showed both peptides
inhibited ACE activity in a noncompetitive manner, which suggests binding to nonactive sites.
A recent report also showed that a tridecapeptide (IRLIIVLMPILMA) obtained from a papain
digest of the red seaweed (Palmaria palmata) protein had SBP-reducing effects (up to 34 mmHg)
in SHR (Fitzgerald et al. 2014). However, due to the long chain length it is possible that the active forms of these peptides are shorter sequences produced through proteolytic cleavage during
passage through the GIT or during in vivo interactions with ACE or renin. For example, in vitro
simulated gastrointestinal digestion of IRLIIVLMPILMA resulted in two peptide products, IR
and LIIVLMPILMA (Fitzgerald et al. 2014). Similarly, the concentration of peptide VKKVLGNP (isolated from porcine skeletal muscle peptic hydrolysate) decreased with time during in
vitro incubation with ACE (Katayama et al. 2007). However, the ACE-inhibitory activity did not
change during the incubation period, which suggests that VKKVLGNP was converted into active
peptide fragments. In general, the currently available data do not provide denitive information
on type or sequence of amino acids required for effective peptide antihypertensive activity when
provided orally.
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Table 2 Effects of oral administration of food protein-derived peptides on systolic blood pressure (SBP) of spontaneously
hypertensive rats

Max. SBP
Protease(s)
Dose
(mg/kg bw)
reduction
(mmHg)a
Peptide sequence
Protein source
IHRF
Rice glutelin
Chymotrypsin
18
Kontani et al. 2014
References
IHRF
Rice glutelin
Chymotrypsin
15
39
Kontani et al. 2014
DPYKLRP
Lactoferrin
Yeast extract
10
27
PYKLRP
Lactoferrin
Yeast extract
10
21
YKLRP
Lactoferrin
Yeast extract
10
20
KLRP
Lactoferrin
Yeast extract
10
12
LRP
Lactoferrin
Yeast extract
10
27
GILRP
Lactoferrin
Yeast extract
10
20
IAK
Casein
Synthesized
21
Miguel et al. 2010
HLPLP
Casein
Synthesized
24
Miguel et al. 2010
YAKPVA
Casein
Synthesized
23
Miguel et al. 2010
HPHPHLSF
Casein
Synthesized
10
16
Miguel et al. 2010
KKYNVPQL
Casein
Synthesized
10
12
Miguel et al. 2010
LVYPFTGPIPN
Casein
Synthesized
10
28
Miguel et al. 2010
WQVLPNAVPAK
Casein
Synthesized
18
Miguel et al. 2010
VPP
Sour milk
Fermentation
20
Nakamura et al. 1995
IPP
Sour milk
Fermentation
18
Nakamura et al. 1995
MRW
Spinach Rubisco
Pepsin + Pancreatin
30
20
Yang et al. 2003
MRWRD
Spinach Rubisco
Pepsin + Pancreatin
30
14
Yang et al. 2003
IAYKPAG
Spinach Rubisco
Pepsin + Pancreatin
100
15
Yang et al. 2003
DY
Bamboo shoot
10
18
Liu et al. 2013
AAATP
Spanish ham
26
Escudero et al. 2013
VNP
Rice
Alcalase + Trypsin
29
Chen et al. 2013a
VWP
Rice
Alcalase + Trypsin
38
Chen et al. 2013a
IRW
Egg
ovotransferrin
Thermolysin + Pepsin
10
Majumder et al. 2013
IRW
Egg
ovotransferrin
Thermolysin + Pepsin
15
22
Majumder et al. 2013
AVF
Insect
Pepsin + Trypsin +
Chymotrypsin
13
Vercruysse et al. 2010
VF
Insect
Pepsin + Trypsin +
Chymotrypsin
19
Vercruysse et al. 2010
GQP
Mushroom
27
Lee et al. 2004
DKVGINYW
Dairy whey
Synthesized
15
Tavares et al. 2012
DAQSAPLRVY
Dairy whey
Synthesized
10
Tavares et al. 2012
KGYGGVSLPEW
Dairy whey
Synthesized
20
Tavares et al. 2012
YFP
Yellown sole
Chymotrypsin
10
22
Jung et al. 2006
IY
Rapeseed
Alcalase
7.5
10
Marczak et al. 2003
VW
Rapeseed
Alcalase
7.5
11
Marczak et al. 2003
RIY
Rapeseed
Alcalase
7.5
11
Marczak et al. 2003
VWIS
Rapeseed
Alcalase
12.5
13
Marczak et al. 2003

(Continued )
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Table 2 (Continued )
Protease(s)
Dose
Max. SBP
reduction
(mg/kg bw)
(mmHg)a
Peptide sequence
Protein source
VKKVLGNP
Porcine muscle
Pepsin
10
24
Katayama et al. 2007
References
RPR
Pork meat
Synthesized
33
Escudero et al. 2012b
PTPVP
Pork meat
Synthesized
26
KAPVA
Pork meat
Synthesized
34
KRVIQY
Porcine myosin
Pepsin
10
23
Muguruma et al. 2009
VKAGF
Porcine myosin
Pepsin
10
17
Muguruma et al. 2009
MKP
Bovine casein
Alcalase + Trypsin +
Protease N
0.1
35
Yamada et al. 2013
VEGY
Microalgae
Alcalase
10
23
Ko et al. 2012a
YH
Edible seaweed
Synthesized
50
50
Suetsuna et al. 2004
KY
Edible seaweed
Synthesized
50
45
WY
Edible seaweed
Synthesized
50
46
IY
Edible seaweed
Synthesized
50
33
FQ
Buckwheat
sprout
Synthesized
0.1
42
Koyama et al. 2013
VAE
Buckwheat
sprout
Synthesized
0.1
38
Koyama et al. 2013
VVG
Buckwheat
sprout
Synthesized
0.1
38
Koyama et al. 2013
DVWY
Buckwheat
sprout
Synthesized
0.1
59
Koyama et al. 2013
FDART
Buckwheat
sprout
Synthesized
0.1
32
Koyama et al. 2013
WTFR
Buckwheat
sprout
Synthesized
0.1
36
Koyama et al. 2013
MAW
Cuttlesh
Synthesized
10
13
Balti et al. 2012
AHSY
Cuttlesh
Synthesized
10
14
Balti et al. 2012
VYAP
Cuttlesh
Synthesized
10
22
Balti et al. 2012
VIIF
Cuttlesh
Synthesized
10
19
Balti et al. 2012
WYT
Hemp seed
Pepsin + Pancreatin
30
13
Girgih et al. 2014c
WVYY
Hemp seed
Pepsin + Pancreatin
30
34
Girgih et al. 2014c
SVYT
Hemp seed
Pepsin + Pancreatin
30
24
Girgih et al. 2014c
PSLPA
Hemp seed
Pepsin + Pancreatin
30
40
Girgih et al. 2014c
IPAGV
Hemp seed
Pepsin + Pancreatin
30
36
Girgih et al. 2014c
LY
Rapeseed
Alcalase
30
26
He et al. 2013b
TF
Rapeseed
Alcalase
30
12
He et al. 2013b
RALP
Rapeseed
Alcalase
30
16
He et al. 2013b
GHS
Rapeseed
Pepsin + Pancreatin
30
17
He et al. 2013c
WMP
Yellow eld pea
Thermolysin
30
39
Aluko et al. 2014
ADMFPF
Yellow eld pea
Thermolysin
30
25
Aluko et al. 2014
IRLIIVLMPILMA
Palmaria palmata
Papain
50
33
Fitzgerald et al. 2014
Some values are estimates obtained from graphs in the respective publications.
253
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7. FUTURE PERSPECTIVES

Research into production, characterization, and efcacy testing of antihypertensive peptides continues to evolve rapidly. Current efforts include the use of in silico methods to perform virtual
enzyme hydrolysis of proteins for which the released peptide sequences can be synthesized and
tested for activity. If in vitro activity (usually against renin and ACE) is conrmed, actual enzyme
hydrolysis can be performed to obtain peptides that can then undergo in vitro and in vivo testing.
This in silico approach could obviate the current time-consuming (and expensive) hit-and-miss
approach whereby various enzymes are thrown at proteins followed by testing of each hydrolysate
to identify active products. However, availability of the primary structure of several more proteins
is required to expand the in silico method beyond the currently limited structurally characterized food proteins. Additional information on structure-function properties of antihypertensive
peptides is also needed to develop better enzyme digestion tools that will release peptides with
desirable amino acid sequences. Finally, more human intervention trials are necessary to provide efcacy data that meet current regulatory requirements in order to allow a health claim of
antihypertensive activity on peptide-formulated food products or nutraceuticals.
DISCLOSURE STATEMENT
The author is not aware of any afliations, memberships, funding, or nancial holdings that might
be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
The research program of Dr. Aluko is funded by the Natural Sciences and Engineering Research
Council of Canada (NSERC) and the Manitoba Agri-Food Research & Development Initiative
(ARDI).
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Volume 6, 2015
An Amazing Journey
Larry McKay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Physical Modication of Food Starch Functionalities
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Nanostructured Fat Crystal Systems
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Antimicrobial Food Equipment Coatings: Applications and Challenges
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Non-Nutritive Sweeteners and Obesity
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Genetic Mechanisms of Prebiotic Oligosaccharide Metabolism in
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Electrostatic Coating Technologies for Food Processing
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The Role of Oxygen in Lipid Oxidation Reactions: A Review
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Colloids in Food: Ingredients, Structure, and Stability
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Antihypertensive Peptides from Food Proteins
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Pickering Emulsions for Food Applications: Background, Trends, and Challenges
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The Nutraceutical Bioavailability Classication Scheme: Classifying Nutraceuticals
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Comparative Analysis of Intestinal Tract Models

C.F. Williams, G.E. Walton, L. Jiang, S. Plummer, I. Garaiova,
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Bacillus and Other Spore-Forming Genera: Variations in Responses
and Mechanisms for Survival
Aleksandra Checinska, Andrzej Paszczynski, and Malcolm Burbank p p p p p p p p p p p p p p p p p p p p 351
Protein-Polysaccharide Interactions to Alter Texture
Fred van de Velde, Els H.A. de Hoog, Alexander Oosterveld, and R. Hans Tromp p p p p p 371
High Hydrostatic Pressure Processing: A Promising Nonthermal

Technology to Inactivate Viruses in High-Risk Foods
Fangfei Lou, Hudaa Neetoo, Haiqiang Chen, and Jianrong Li p p p p p p p p p p p p p p p p p p p p p p p p p p 389
Human Norovirus as a Foodborne Pathogen: Challenges
and Developments
Matthew D. Moore, Rebecca M. Goulter, and Lee-Ann Jaykus p p p p p p p p p p p p p p p p p p p p p p p p p p 411
Principles and Application of High PressureBased Technologies in
the Food Industry
V.M. (Bala) Balasubramaniam, Sergio I. Martnez-Monteagudo,
and Rockendra Gupta p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 435
Challenges and Solutions to Incorporation of Nutraceuticals in Foods
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Statistical Aspects of Food Safety Sampling
I. Jongenburger, H.M.W. den Besten, and M.H. Zwietering p p p p p p p p p p p p p p p p p p p p p p p p p p p p 479
Diet-Based Strategies for Cancer Chemoprevention: The Role of
Combination Regimens Using Dietary Bioactive Components
Christina DiMarco-Crook and Hang Xiao p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 505
Collagen and Gelatin
Dasong Liu, Mehdi Nikoo, Gokhan Boran, Peng Zhou, and Joe M. Regenstein p p p p p p p p 527
Indexes
Cumulative Index of Contributing Authors, Volumes 16 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 559
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Errata
An online log of corrections to Annual Review of Food Science and Technology articles may
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FO06CH11-Aluko

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

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Antihypertensive Peptides from

Annu. Rev. Food Sci. Technol. 2015. 6:23562

The Annual Review of Food Science and Technology is

antihypertensive peptides, food proteins, protein hydrolysates,

This articles doi:

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

2. METHODS FOR PRODUCING ANTIHYPERTENSIVE PEPTIDES

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

3. PEPTIDE FRACTIONATION AND PURIFICATION

www.annualreviews.org Antihypertensive Peptides

3.1. Membrane Ultrafiltration

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

3.2. Column Chromatography

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

4.2. In Vivo Methods

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

5.1. ACE Inhibition

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

5.2. Renin Inhibition

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

5.3. Nitric Oxide Production

5.4. Angiotensin II Receptor Blockage

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

6. BLOOD PRESSUREREDUCING EFFECTS

6.1. Protein Hydrolysates

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

Annu. Rev. Food Sci. Technol. 2015.6:235-262. Downloaded from www.annualreviews.org

Dose (mg/kg bw)

Herregods et al. 2011

Fujita et al. 2001

Nakano et al. 2006

Alashi et al. 2014

Alashi et al. 2014

Alashi et al. 2014

Alashi et al. 2014

Alashi et al. 2014

Wang et al. 2011

Wang et al. 2011

None (aqueous extract)

Liu et al. 2013

None (aqueous extract)

Liu et al. 2013

None (aqueous extract)

Liu et al. 2013

None (aqueous extract)

Escudero et al. 2012a

None (aqueous extract)

Escudero et al. 2012a

None (aqueous extract)

Escudero et al. 2012a

Crude enzyme extract

Tavares et al. 2012

Wang et al. 2008

None (aqueous extract)

Jang et al. 2011

Marczak et al. 2003