Food Protein-Derived Bioactive Peptides Production Processing and Potential Health Benefits

R:Co

ncise

Revie

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Food

Scien

ce

Food Protein-Derived BioactivePeptides: Production, Processing,and Potential Health BenefitsChibuike C. Udenigwe and Rotimi E. Aluko

Abstract: Bioactive peptides (BAPs), derived through enzymatic hydrolysis of food proteins, have demonstrated potentialfor application as health-promoting agents against numerous human health and disease conditions, including cardiovasculardisease, inflammation, and cancer. The feasibility of pharmacological application of these peptides depends on absorptionand bioavailability in intact forms in target tissues, which in turn depends on structure of the peptides. Therefore,production and processing of peptides based on important structure-function parameters can lead to the production ofpotent peptides. This article reviews the literature on BAPs with emphasis on strategic production and processing methodsas well as antihypertensive, anticancer, anticalmodulin, hypocholesterolemic, and multifunctional properties of the foodprotein-derived peptides. It is recommended that future research efforts on BAP should be directed toward elucidationof their in vivo molecular mechanisms of action, safety at various doses, and pharmacological activity in maintaininghomeostasis during aberrant health conditions in human subjects.

Keywords: bioactive peptides, functional food, human health, hypertension, multifunctional

IntroductionThe human body is constantly subjected to physiological im-

balances and exposure to extrinsic toxic substances that perturbnormal functions leading to various health conditions. These aber-rations can be controlled by physiological homeostasis, or throughthe use of health-promoting agents especially in acute and chronicconditions (Ames and others 1993). It is generally established thatthe nutritive and non-nutritive constituents of food can be usedto modify the risk of developing or aggravating human diseaseconditions. In this regard, functional foods and nutraceuticals haveemerged as adjuvant or alternative to chemotherapy especially inprevention and management of human diseases, and for main-taining optimum health state (Kris-Etherton and others 2002).This area of research has increasingly become the subject of vari-ous research programs as the health and well-being of consumersgradually became the primary focus of the food industry. Thereis a growing trend and interest in the use of food protein-derivedpeptides as intervention agents against chronic human diseasesand for maintenance of general well-being. These peptides areproduced by enzymatic hydrolysis of food proteins to release thepeptide sequences, followed by posthydrolysis processing to iso-late bioactive peptides (BAPs) from a complex mixture of otherinactive molecules (Wang and Gonzalez De Mejia 2005; Korho-nen and Pihlanto 2006; Hartmann and Meisel 2007; Aluko 2008a).These peptides are different from naturally occurring BAPs, such asendorphins, because they are generated by proteolysis of native

MS 20110594 Submitted 5/10/2011, Accepted 9/12/2011. Authors are withDept. of Human Nutritional Sciences and author Aluko is also with the RichardsonCentre for Functional Foods and Nutraceuticals, Univ. of Manitoba, 196 InnovationDrive, Winnipeg, MB R3T 2N2, Canada. Author Udenigwe is also with Dept. ofFood Science, Univ. of Guelph, Guelph, ON N1G 2W1, Canada. Direct inquiriesto author Aluko (E-mail: [email protected]).

food proteins. By definition, BAPs discussed in this review arefood protein-derived peptides that possess beneficial pharmacolog-ical properties beyond normal and adequate nutrition (Hartmannand Meisel 2007). The food processing steps lead to concentrationof the active peptides with the enhancement of the physiologicalactivity of the products, which could also be nutritionally benefi-cial as a source of essential amino acids. This approach can providethe opportunity for diversification of the use of agricultural cropsand animal products beyond basic nutritional purposes, especiallyas a source of active ingredients for formulation of food productswith health benefits.

Production and Processing of Food Protein-DerivedBAPs

Typical food sourcesNumerous animal and plant food proteins have been exploited

as sources of BAPs. Several studies on BAPs were conducted usinganimal proteins mostly milk proteins, casein and whey, egg andmeat muscle proteins have also yielded BAPs. In addition, severalBAPs have been produced from marine protein sources, includingfish, salmon, oyster, macroalgae, squid, sea urchin, shrimp, snowcrab, and seahorse (Table 1). Typical plant food proteins used forthe production of BAPs include soy, pulses (lentil, chickpea, pea,and beans), oat, wheat, hemp seed, canola, and flaxseed. Basedon the current literature, food proteins are selected as sources ofBAPs based on 2 major criteria (1) a pursuit of value-added useof abundant underutilized proteins or protein-rich food indus-try by-products, and (2) utilization of proteins containing specificpeptide sequences or amino acid residues of particular pharmaco-logical interest. While these criteria are individually important, acombination of the 2 approaches can lead to the strategic selectionof proteins that can produce high yields of defined potent peptidesequences. Recently, a QSAR-based in silico method was recentlyproposed for the prediction of food protein sources that can yield

C© 2011 Institute of Food Technologists R©doi: 10.1111/j.1750-3841.2011.02455.x Vol. 71, Nr. 1, 2012 � Journal of Food Science R11Further reproduction without permission is prohibited

R:ConciseReviewsinFoodScience

Food protein-derived bioactive peptides . . .

Tab

le1–

Sourc

esan

dbio

acti

vepro

per

ties

of

mar

ine

pro

tein

-der

ived

hyd

roly

sate

san

dpep

tides

.

Tre

atm

ent/

pep

tide

Mar

ine

pro

tein

Pro

teas

epro

per

ty/s

equen

ceO

utc

om

eR

efer

ence

Ani

mal

stud

ies

Chu

msa

lmon

Com

plex

prot

ease

Pept

ides

(mos

tly30

0to

860

Da)

prod

uced

Imm

une

stim

ulan

tY

ang

and

othe

rs(2

009)

(Onc

orhy

nchu

ske

ta)

colla

gen

afte

rna

nofil

trat

ion,

desa

linat

ion,

and

cryo

conc

entr

atio

n;fe

d0

to1.

35g/

kgbo

dyw

eigh

tto

ICR

mic

efo

r4

wk

Pept

ide

trea

tmen

ten

hanc

edm

itoge

n-in

duce

dly

mph

ocyt

epr

olife

ratio

n,na

tura

lkill

er(N

K)

cell

activ

ity,s

plee

nC

D4+

The

lper

cells

,and

secr

etio

nof

cyto

kine

s(I

L-2,

IL-5

,IL-

6,IF

N-γ

);no

effe

ctob

serv

edon

mac

roph

age

activ

ity;

pept

ides

have

pote

ntia

lfor

use

asim

mun

est

imul

ant

for

dise

ase

prev

entio

nSa

me

asab

ove

but

with

irra

diat

ion-

indu

ced

Mul

tifun

ctio

nalp

rope

rty

Yan

gan

dot

hers

(201

0)im

mun

esu

ppre

ssio

nin

the

ICR

mic

ePr

otec

ted

agai

nst

gam

ma

radi

atio

nin

duce

dim

mun

osup

pres

sion

byau

gmen

ting

CD

4+T

help

erce

lls,

enha

ncin

gsp

leen

IL-1

2,re

duci

ngto

talN

F-κ

Bth

roug

hI-

κB

indu

ctio

n,an

din

hibi

tion

ofsp

leno

cyte

apop

tosis

via

incr

ease

inan

tiapo

ptot

icB

cl-2

and

decr

ease

inpr

oapo

ptot

icB

axPe

ptid

esw

ere

fed

at0%

to9%

to4-

wk-

old

Ant

ioxi

dant

Lian

gan

dot

hers

(201

0)Sp

ragu

e–D

awle

yra

tsun

tilna

tura

ldea

thD

ose-

depe

nden

tin

hibi

tion

ofag

e-re

late

dde

crea

sein

antio

xida

nten

zym

esan

dlip

idpe

roxi

datio

n;de

crea

sed

spon

tane

ous

tum

orin

cide

nce

inra

tsPa

cific

oyst

ers

(Cra

ssos

trea

Cru

depr

otea

seso

lutio

nfr

omPe

ptid

es(<

3kD

a)pr

oduc

edaf

ter

Mul

tifun

ctio

nalp

rope

rty

Wan

gan

dot

hers

(201

0)gi

gas)

Bac

illus

sp.S

M98

011

mem

bran

eul

trafi

ltrat

ion

atla

b,pi

lot

and

plan

tsc

ale;

fed

0to

1m

gsa

mpl

e/d

for

14d

toB

ALB

/cm

ice

with

tran

spla

nted

mur

ine

S180

sarc

oma

Dos

e-de

pend

enti

nhib

ition

(up

to48

%)

ofgr

owth

ofsa

rcom

ain

mic

e;en

hanc

edN

Kce

llsac

tivity

,spl

een

lym

phoc

yte

prol

ifera

tion,

and

mac

roph

age

phag

ocyt

icra

te;t

heob

serv

edim

mun

ostim

ulat

ion

coul

dbe

resp

onsib

lefo

rth

ean

titum

orac

tivity

Jelly

fish

colla

gen

Prot

amex

Adm

inist

ered

at0,

5,or

10m

L/kg

/dfo

rA

ntio

xida

ntD

ing

and

othe

rs(2

011)

6w

kto

D-g

alac

tose

-ind

uced

agin

gIC

Rm

ice

Pept

ides

decr

ease

dse

rum

and

hepa

ticm

alon

dial

dehy

de;

incr

ease

dgl

utat

hion

epe

roxi

dase

(GSH

-Px)

and

supe

roxi

dedi

smut

ase

(SO

D)

Invi

trost

udie

sSe

ahor

seA

mix

ture

of6

prot

ease

sSH

P-1

(a15

amin

oac

ids-

cont

aini

ngA

nti-

infla

mm

ator

yR

yuan

dot

hers

(201

0)pe

ptid

e)iso

late

daf

ter

ion-

exch

ange

chro

mat

ogra

phy

and

RP-

HPL

C;h

uman

chod

rocy

tic(S

W-1

353)

and

oste

obla

stic

(MG

-63)

cells

trea

ted

with

10to

100

μg/

mL

ofSH

P-1

SHP-

1do

wn-

regu

late

dT

PA-i

nduc

edco

llage

nre

leas

ein

cells

via

decr

esas

ein

colla

gena

ses

1an

d3

expr

essio

n;as

soci

ated

with

bloc

king

phos

phor

ylat

ion

ofN

F-κ

Ban

dp3

8ki

nase

casc

ade

lead

ing

tode

crea

sed

NO

prod

uctio

n,iN

OS,

and

CO

X-2

;ben

efici

alef

fect

sfo

rtr

eatm

ent

ofar

thri

tisG

iant

squi

d(D

osid

icus

giga

s)Se

ven

prot

ease

sin

clud

ing

Cru

depr

otei

nhy

drol

ysat

esco

ntai

ning

aM

ultif

unct

iona

lpro

pert

yA

lem

anan

dot

her

(201

1)ge

latin

Alc

alas

e(A

lc),

Esp

eras

e(E

sp),

and

Neu

tras

e(N

eu)

rang

eof

low

-an

dhi

ghm

olec

ular

size

pept

ides

Alc

-an

dN

eu-p

repa

red

sam

ple

show

edbe

tter

AC

Ein

hibi

tion;

Esp

-pre

pare

dsa

mpl

eex

hibi

ted

mos

tpo

tent

cyto

toxi

cef

fect

agai

nst

MC

F-7

(hum

anbr

east

)an

dU

87(g

liom

a)ca

ncer

cells

(IC

50of

0.13

and

0.10

mg/

mL,

resp

ectiv

ely)

;all

sam

ples

also

show

edan

tioxi

dant

prop

ertie

s(F

RA

Pan

dm

etal

chel

atio

n)

(Con

tinue

d)

R12 Journal of Food Science � Vol. 71, Nr. 1, 2012

R:Co

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Revie

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Food

Scien

ceFood protein-derived bioactive peptides . . .

Tab

le1–

Continued

Tre

atm

ent/

pep

tide

Mar

ine

pro

tein

Pro

teas

epro

per

ty/s

equen

ceO

utc

om

eR

efer

ence

Roc

kfish

(Seb

aste

shu

bbsi)

Alc

alas

e,Fl

avou

rzym

ePe

ptid

esri

chin

Gly

,Pro

,Ala

,Glu

Mul

tifun

ctio

nalp

rope

rty

Kim

and

othe

rs(2

011)

gela

tinM

oder

ate

free

radi

cal(

DPP

H,s

uper

oxid

e,hy

drox

yl,a

lkyl

)sc

aven

ging

prop

erty

;AC

Ein

hibi

tion

with

IC50

of0.

82m

g/m

LA

tlant

icsa

lmon

(Sal

mo

Alc

alas

e,pa

pain

Dip

eptid

esA

la-P

roan

dVa

l-A

rgiso

late

dA

CE

inhi

bitio

nG

uan

dot

hers

(201

1)sa

lar

L.)

skin

colla

gen

afte

rR

P-H

PLC

Ala

-Pro

and

Val-

Arg

inhi

bite

dA

CE

activ

ityw

ithIC

50of

0.06

and

0.33

mg/

mL,

resp

ectiv

ely

20-

and

4-fo

lds

mor

epo

tent

than

the

crud

ehy

drol

ysat

esTu

nada

rkm

uscl

ePa

pain

(PA

)or

Prot

ease

Isol

ated

ado

deca

pept

ide

Leu-

Pro-

His-

Val-

Ant

ican

cer

Hsu

and

othe

rs(2

011)

by-p

rodu

ctX

XII

I(P

R)

Leu-

Thr

-Pro

-Glu

-Ala

-Gly

-Ala

-Thr

(1)

and

ahe

ndec

apep

tide

Pro-

Thr

-Ala

-Glu

-G

ly-G

ly-V

al-T

yr-M

et-V

al-T

hr(2

)af

ter

gelfi

ltrat

ion

and

RP-

HPL

C

Pept

ides

1an

d2,

from

PAan

dPR

,res

pect

ivel

y,do

se-d

epen

dent

lyin

hibi

ted

brea

stca

ncer

(MC

F-7)

cells

prol

ifera

tion

with

IC50

of8.

1an

d8.

8μ

M,r

espe

ctiv

ely

Snow

crab

(Chi

onoe

cete

sPr

otam

exLo

wm

olec

ular

size

net-

char

ged

pept

ide

Ant

ican

cer

Doy

enan

dot

hers

(201

1b)

opili

o)by

-pro

duct

frac

tions

(cat

ioni

c=

KC

l2,a

nion

ic=

KC

l1)

wer

ege

nera

ted

afte

rfr

actio

natio

nof

the

crud

ehy

drol

ysat

esby

elec

trod

ialy

sis-u

ltrafi

ltrat

ion

atpH

3,6,

and

9

Cat

ioni

cfr

actio

n(K

Cl2

,pH

6)sh

owed

mos

tpo

tent

inhi

bito

ryac

tivity

agai

nst

the

viab

ility

oflu

ng(A

549)

,br

east

(BT

549)

,col

on(H

CT

15),

and

pros

tate

(PC

3)ca

ncer

cells

at1:

10an

d1:

100

dilu

tions

Shri

mp

shel

lby-

prod

uct

Cry

otin

enzy

me

Hig

hm

olec

ular

size

(<10

,10

to30

,and

Ant

ican

cer

Kan

nan

and

othe

rs(2

011)

>30

kDa)

gast

roin

test

inal

resis

tant

olig

opep

tide

frac

tions

All

sam

ples

show

edtim

e-de

pend

ent

inhi

bitio

nof

prol

ifera

tion

ofC

aco-

2(c

olon

)an

dH

epG

2(li

ver)

canc

erce

lls(u

pto

60%

inhi

bitio

nby

frac

tions

<10

and

10to

30kD

a)Pu

rple

sea

urch

inN

eutr

ase,

papa

in,p

epsin

,or

Pept

ides

wer

efr

actio

nate

d(<

1,1

to3,

3to

Ant

ioxi

dant

Qin

and

othe

rs(2

011)

(Stro

ngyl

ocen

trotu

snu

dus)

gona

dtr

ypsin

5,5

to10

kDa)

bym

embr

ane

ultr

afiltr

atio

nA

llsa

mpl

esex

hibi

ted

antio

xida

ntpr

oper

ties

(DPP

Hsc

aven

ging

and

FRA

P)bu

tth

e<

1kD

afr

actio

nssh

owed

the

best

activ

ities

Abb

revi

atio

ns:N

F-κ

B=

nucl

ear

fact

or-κ

B;I

κ-B

=in

hibi

tor

ofN

F-κ

B;D

PPH

=2,

2-di

phen

yl-1

-pic

rylh

ydra

zylr

adic

al;F

RA

P=

ferr

ic-r

educ

ing

antio

xida

ntpo

wer

;AC

E=

angi

oten

sinI-

conv

ertin

gen

zym

e;IL

=in

terl

euki

n;IF

N=

inte

rfer

on;

TPA

=12

-O-t

etra

deca

noyl

phor

bol-

13-a

ceta

te;d

etai

led

revi

ewab

out

mar

ine-

deri

ved

bioa

ctiv

epe

ptid

esca

nbe

foun

din

Kim

and

Wije

seka

ra(2

010)

,Har

nedy

and

Fitz

gera

ld(2

011)

,Wils

onan

dot

hers

(201

1),a

ndFi

tzge

rald

and

othe

rs(2

011)

.

Vol. 71, Nr. 1, 2012 � Journal of Food Science R13



BAPs (Gu and others 2011). This approach could lead to the se-lection of excellent protein sources of BAPs only when details ofthe structure-function properties of active sequences are known.Moreover, detailed experimental work is needed to confirm actualproduction of the peptides, reproducibility, and substantiation ofthe feasibility of use of the in silico prediction method.

Production and processing methodsBAPs are encrypted in the primary structure of plant and an-

imal proteins as inactive amino acid sequences but they can bereleased by fermentation, food processing, and enzyme-catalyzedproteolysis in vitro or in the digestive tract after human consump-tion (Hartmann and Meisel 2007; Aluko 2008b). In most cases,these protein hydrolysates and peptides have demonstrated bet-ter bioactivity compared to their parent proteins, and this showsthat hydrolysis of peptide bonds is important in liberating thepotent peptides. Several factors affect the bioactive properties ofthe peptides including the enzymes used for hydrolysis, processingconditions, and the size of the resulting peptides, which greatlyaffects their absorption across the enterocytes and bioavailabilityin target tissues. Most reported BAPs are produced by in vitro en-zymatic hydrolysis or fermentation. After selecting an appropriatefood protein, enzymatic hydrolysis is performed using single ormultiple specific or nonspecific proteases to release peptides of in-terest. Simulated gastrointestinal enzymatic process has also beenused to mimic normal human digestion of proteins to evaluatethe possibility of releasing potent BAPs after normal consumptionof food proteins. The latter strategy could be cost-effective sinceextensive processing of the peptide product will not be needed.Some factors to consider in producing BAPs include hydrolysistime, degree of hydrolysis of the proteins, enzyme–substrate ratios,and pretreatment of the protein prior to hydrolysis. For example,thermal treatment of proteins can enhance enzymatic hydrolysis(Inouye and others 2009) possibly by increasing enzyme–proteininteractions due to thermal-induced unfolding of the proteins. Inaddition, sonication and hydrostatic pressure treatments of foodproteins have separately resulted in enhanced hydrolysis and re-lease of potent BAPs (Quiros and others 2007; Wu and Majumder2009). Furthermore, it is feasible to scale-up production of pep-tides from laboratory scale to pilot and industrial plant scales withconserved peptide profiles and bioactivity of the resulting products(Wang and others 2010).

A challenge often faced in food protein-derived peptide researchis to obtain high-yield peptide products with potent bioactivity.This limitation results in carrying out further processing of theenzymatic food protein hydrolysates. Therefore, after protein hy-drolysis, the resulting peptide product is further processed based onphysicochemical and structural properties of the constituent pep-tides in a bid to enhance bioactivity. The peptide properties thatare often focused on include size, net charge, and hydrophobicity,depending on the targeted pharmacological uses. Membrane ultra-filtration and size-exclusion chromatography can be used to con-centrate peptides of defined molecular weight ranges, especiallyfor obtaining fractions containing low molecular weight peptidesthat can withstand further in vivo proteolytic digestion. In addi-tion, reverse-phase HPLC on a hydrophobic column matrix can beused to fractionate peptides based on their hydrophobic properties(Pownall and others 2010), especially when studying the structure-function properties of peptides. Peptide fractions of particularnet charges can be obtained by chromatography using selectiveion-exchange columns (Li and Aluko 2005; Pownall and others2011). This processing approach is very useful especially when

the molecular disease targets are inactivated by molecules withstrong net positive or negative charges. In addition, a novel mem-brane technology known as electrodialysis-ultrafiltration (EDUF)can be used to separate cationic, anionic, and neutral peptides ofdefined molecular sizes (Firdaous and others 2009). This methodhas demonstrated high efficiency in selectively separating and con-centrating low molecular size BAPs with net charges. Recently,the EDUF process was used to successfully fractionate net posi-tively and negatively charged BAPs of low molecular sizes (300to 700 Da) from snow crab by-product hydrolysates (Doyen andothers 2011a, 2011b). Moreover, particular amino acids can beenriched in food peptide mixtures using adsorbent materials. Forexample, a peptide fraction rich in branched chain amino acidsand low in aromatic amino acids can be obtained after proteinhydrolysis by passing of the hydrolysates through a column packedwith activated carbon or simply by mixing with activated car-bon (Adachi and others 1993; Udenigwe and Aluko 2010). Thesefractionation processes often result in appreciable peptide yield de-pending on prevalence of the amino acid residues or peptides ofinterest within the hydrolysate product. Furthermore, extensivebioassay-guided purification steps can be carried out in order toproduce pure peptides for further analysis especially for structure-function studies. The limitation of the latter process is the low yieldof the resulting peptides, which may have to be synthesized forfurther studies. The low peptide yield decreases the feasibility ofusing food proteins as sources of BAPs. Therefore, for commercialproduction of functional food products containing BAPs, it will beworthwhile to develop applicable food-grade processing methodsthat will yield high amounts of highly active peptide mixtures.This approach requires an understanding of the structural require-ments of the peptides for bioactivity, and exploiting the uniquestructural features in concentrating the particular peptides of in-terest during processing. In summary, the processes commonlyused for the production and processing of BAPs are shown inFigure 1.

Food Protein-Derived BAPs and Human HealthAs shown in Figure 2, food protein hydrolysates have ex-

hibited potent biological activities such as antihypertensive,antioxidant, immunomodulatory, anticancer, antimicrobial, andlipid-lowering activities (Meisel 2004; Wang and Gonzalez DeMejia 2005; Korhonen and Pihlanto 2006; Pihlanto 2006; Aluko2008a, 2008b), which are largely due to their constituent peptides.The specific bioactivity of food peptides against various molec-ular disease targets depends primarily on their structural prop-erties such as chain length and physicochemical characteristicsof the amino acid residues, for example, hydrophobicity, molec-ular charge, and side-chain bulkiness (Pripp and others 2005).Generally, the activity of these peptides against molecular diseasetargets are regarded as lower than synthetic peptidomimetics anddrugs, but the use of dietary BAPs in intervention against humandiseases offers many advantages, including safety of the naturalproduct, low health cost, and the additional nutritional bene-fits of the peptides as source of beneficial and essential aminoacids. The current literature contains a vast amount of infor-mation on food protein-derived BAPs with physiologically rel-evant bioactive properties. These peptides range in sizes fromdi-, tri-, and oligopeptides to high molecular weight polypep-tides (Erdmann and others 2008; Hernandez-Ledesma and others2009a). Based on the bioactivities, a number of peptide-basedfood products have been developed and commercialized for


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use as human health-promoting agents; see review articles byKorhonen and Pihlanto (2006), Hartmann and Meisel (2007),and Fitzgerald and others (2011) for comprehensive lists of thesefood products and their health claims.

Antihypertensive peptidesPhysiological regulation of blood pressure (BP). BP is

physiologically controlled by the renin-angiotensin system (RAS)and the kinin-nitric oxide (NO) system (Figure 3). The RAS in-volves activation of angiotensinogen by the proteolytic activity ofrenin which converts it to angiotensin (AT)-I. This reaction isthe first and rate-limiting step of the RAS pathway. AT-I is thencleaved at the histidyl residue from the C-terminus by the activ-ity of angiotensin I-converting enzyme (ACE) to produce AT-II.AT-II is a powerful vasoconstrictor that functions by binding toreceptors, located in tissues all over the body, to elicit physiolog-ical reaction cascades that lead to blood vessel contractions thatmaintain normal BP. However, in pathological conditions, thereis excessive level of AT-II, which causes severe blood vessel con-tractions and limited relaxation to produce high BP. Moreover,the kinin-NO system is involved in the production of bradykinin,which exerts its antihypertensive effects by eliciting reactions thatincrease intracellular Ca2+ concentration leading to activation ofnitric oxide synthases (NOS) that produce NO, a powerful va-sodilator. ACE degrades bradykinin, and increased concentrationof ACE leads to dual effects such as the prevention of vasodilationand the activation of vasoconstriction. Based on the roles of ACEin the RAS pathway, inhibitors of this enzyme have been used as

antihypertensive agents (Ibrahim 2006). Moreover, direct inhibi-tion of renin can potentially provide better control of elevated BPthan ACE inhibition since it prevents the synthesis of AT-I, whichcan be converted to AT-II in some tissues via an ACE-independentalternative chymase-catalyzed pathway (Segall and others 2007).

ACE-inhibiting food protein-derived peptides. RAS-modulating food-derived peptides are primarily targeted againstACE activity. The pioneering work on naturally occurringACE-inhibiting antihypertensive peptides from snake (Bothropsjararaca) venom (Ferreira and others 1970; Ondetti and oth-ers 1971) sprouted several investigations on the use of food

Food Protein-Derived Peptides

Antioxidant Activities

AnticancerProperties

Anti-inflammatory Properties

ImmunomodulatoryProperties

AntimicrobialActivity

Lipid-loweringProperties

AntihypertensiveActivity

MultifunctionalProperties

Liver Disease Treatment (High Fischer ratio

peptides)

Figure 2–Bioactive properties of food protein-derived peptides relevant tothe promotion of human health and disease prevention.

Selection of food protein source

Enzymatic hydrolysis

Protein isolation

Inactivation of enzyme(s)

Post-hydrolysis processing Ultrafiltration Electrodialysis-

ultrafiltration

Chromatography Activated carbon column

Ion-exchangeRP-HPLC

Size-exclusion

Anion-exchange

Cation-exchange

Net anionic peptide fractions

Peptidefractions of

different sizes

Peptide fractions of different

hydrophobicity

Net cationic peptide fractions

Cationic, anionic and neutral

peptide fractions

HPLC purification

Pure peptides

BCAA-rich peptide fractions

Figure 1–Schematic diagram showing stepstoward the production and processing of foodprotein-derived bioactive peptides.




protein-derived peptides as antihypertensive agents. Till date, sev-eral ACE-inhibiting peptides have been reported from an enor-mous list of plant and animal food proteins most especially milk,fish, egg, and soy proteins (see review articles by FitzGerald andothers 2004; Hartman and Meisel 2007; Aluko 2008a, 2008b; Erd-mann and others 2008). The technological aspect of productionof antihypertensive food-derived peptides has also been reviewed(Aluko 2007). The amino acid sequences of a number of thesepeptides have been identified and related to their biological ac-tivities. The peptide inhibitory concentration that reduced ACEactivity by 50% (IC50) were reported to be as low as 2, 5, and 9 μMfor Val-Ala-Pro (αs1-casein f25–27), β-casein-derived Ile-Pro-Pro(f74–76), and Val-Pro-Pro (f84–86), respectively (Nakamura andothers 1995; FitzGerald and others 2004).

The mechanism of ACE inhibition by food protein-derivedpeptides has been studied and found to be via competitive inhi-bition (Sato and others 2002). This mode of enzyme inhibition ischaracterized by competition of the peptides with ACE substratefor the enzyme catalytic sites. Moreover, some peptides have alsoexhibited noncompetitive (for example, Leu-Trp and Ile-Tyr)and uncompetitive (for example, Ile-Trp and Phe-Tyr) modes ofinhibition (Sato and others 2002) where the peptides bind othersites on the enzyme leading to changes in enzyme conformationand decreased activity. The above example shows that a singleamino acid substitution, even with isomers, can greatly influencethe nature of interactions between peptides and ACE. Thus, anunderstanding of the structural basis for potency has resulted inthe discovery of more potent peptides. The molecular mechanismof ACE inhibition by peptides has been reviewed (Li and others2004; Phelan and Kerins 2011) and hydrophobic amino acidresidues of peptides are important structural feature for potency.QSAR studies by partial least squares projection of latent structure(PLS) indicated that C-terminal bulky hydrophobic amino acids(for example, Pro, Trp, Phe, and Tyr) and N-terminal aliphaticamino acids (for example, Leu, Ile, and Val) are necessary structuralfeatures of dipeptides and tripeptides for ACE inhibition (Wuand others 2006a) and that the last 4 C-terminal predominantly

Angiotensinogen

Angiotensin-I

Angiotensin-II

AT receptor-mediated vasoconstriction

Elevated blood pressure

Bradykinin

Inactive peptide fragments

Angiotensin I-converting enzyme

(ACE)

Renin

Nitric oxide-mediated vasodilation Bioactive

peptides

Chymase

Figure 3–The blood pressure regulating renin-angiotensin system (RAS)pathway showing potential molecular targets (renin and angiotensin-converting enzyme, ACE) for bioactive peptides. Inhibition of renin re-duces the possibility of producing angiotensin-II via an ACE-independentchymase-catalyzed reaction.

hydrophobic amino acid residues in 4 to 10 amino acid-containingpeptides are important determinants for ACE inhibition (Wu andothers 2006b). Moreover, amino acids with positive charge onthe ε-amino group (for example, Arg and Lys) also contributesubstantially to ACE inhibition if present at the C-terminal ofpeptides, possibly by interacting with anionic allosteric bindingsites different from the active site of ACE (Vermeirssen and others2004).

Renin-inhibiting food protein-derived peptides. In ad-dition to ACE inhibition, recent studies have demonstrated thatfood-derived peptides can also inhibit the activity of renin. Thisnew approach to antihypertensive therapy by food-derived pep-tides can potentially provide better BP-lowering properties thaninhibiting only ACE activity. The initial work reported that hy-drolysis of flaxseed protein with different proteases followed byultrafiltration yielded low molecular size (<1 kDa) fractions thatexhibited moderate renin-inhibitory activities with IC50 of 1.22to 2.81 mg/mL (Udenigwe and others 2009a). These peptidefractions inhibited renin activity in vitro through a mixed-type in-hibition mode and also potently exhibited ACE inhibition at lowconcentrations. Thus, their dual roles as ACE and renin inhibitorscan potentially enhance their antihypertensive effects. Similarly,other studies reported the presence of renin inhibitors in enzy-matic hydrolysates of pea and hemp seed protein isolates. Girgihand others (2011) demonstrated that a simulated gastrointestinaldigested hemp seed protein hydrolysate inhibited renin (IC50 of0.81 mg/mL) and ACE (IC50 of 0.67 mg/mL) activities in vitro.The crude protein hydrolysate was found to be more potent thanunhydrolyzed hemp seed protein and fractionated peptides of var-ious molecular sizes as renin inhibitor. Moreover, Li and Aluko(2010) isolated 3 dipeptides (Ile-Arg, Lys-Phe, and Glu-Phe) fromalcalase-prepared pea protein hydrolysates with the ability to mod-erately or weakly inhibit renin activity with IC50 of 9.2, 17.8,and 22.6 mM, respectively. The fact that these dipeptides ex-hibited lower activity than the crude peptide fraction suggests apossible synergistic activity of the peptides. Table 2 shows the se-quence, renin- and ACE-inhibitory activities of the pea dipeptides.Based on these data, the structural requirements of dipeptides forrenin inhibition was recently elucidated by PLS-based chemomet-rics and supported by experimental studies. Udenigwe and others(2011) recently reported that the presence of an N-terminal hy-drophobic low molecular weight amino acid (for example, Ile, Leu,Ala, and Val) and a C-terminal bulky amino acid (for example, Trp,Phe, and Tyr) is required for potency against human renin. Thesefeatures are similar to dipeptide structural requirements for ACEinhibition although there was no correlation between ACE- andrenin-inhibitory activities of the dipeptides (Table 2). Based onthese PLS models, previously reported antihypertensive dipeptide(Ile-Trp) was discovered as the most potent renin-inhibiting dipep-tide and an effective ACE inhibitor (Table 2). These dual-potentactivities in modulating RAS enzymes may have contributed tothe pronounced BP-lowering activity of Ile-Trp compared to theother dipeptides (Sato and others 2002). These studies can lead tothe discovery of safe natural highly potent antihypertensive agentsand can also contribute to the elucidation of alternative mecha-nisms of action of previously reported food-derived BP-loweringpeptides.

In vivo studies of food protein-derived antihypertensivepeptides. There has been some correlation between in vitro RASenzyme inhibition and hypotensive activity of BAPs and viceversa. Recently, ACE- and renin-inhibitory hempseed proteinhydrolysates induced a pronounced decrease in systolic BP (SBP)


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(−30 mmHg) in spontaneously hypertensive rats (SHR) after 8 hof oral gavage of 200 mg of sample/kg body weight (Girgih andothers 2011). The popular milk-derived tripeptides, Ile-Pro-Pro

Table 2–Predicted and observed renin-inhibitory activities(RI,%) of dipeptides and the corresponding ACE-inhibitory ac-tivities (ACEI); peptides with potency in inhibiting both RASenzymes can be used as effective blood pressure-lowering agentduring hypertension depending on bioavailability.

Predicted Observed ACEIDipeptide RI (%)a RI (%)a (IC50 μM)b

Ile-Arg 34.7 49.1 691.8Leu-Arg 36.2 33.9 n.d.Asn-Arg 30.2 25.3 n.d.Lys-Phe 15.1 28.7 28.1Glu-Phe 18.3 22.3 2980.0Gln-Phe 19.8 12.0 n.d.Arg-Phe 11.4 6.3 93.3Ser-Phe 21.9 15.9 130.2Tyr-Ala n.d. 15.1 460.0Phe-Lys 7.7 8.9 n.d.Phe-Glu 3.2 1.8 n.d.Phe-Gln 4.2 8.6 51.2Phe-Thr n.d. 20.4 n.d.Ala-Trp 81.2 n.a. 34.8Val-Trp 71.1 n.a. 7.1Leu-Trp 69.0 37.1 38.9Ile-Trp 66.2 69.1 4.7

n.d. = no data; n.a. = no activity.apredicted and observed RI (%) for the dipeptides were analyzed at concentration of 3.2mM, data were derived from Udenigwe and others (2011);bACEI data were derived from Wu and others (2006a), Li and Aluko (2010),Udenigwe and others (2011), and BIOPEP database (http://www.uwm.edu.pl/biochemia/index.php/en/biopep).

and Val-Pro-Pro, are the active ingredients of hypotensive productsCalpis AMEEL S and Evolus, and these tripeptides were reportedto reduce SBP by −28.3 and −32.1 mmHg, respectively, in SHR(Nakamura and others 1995). In mildly hypertensive human sub-jects, Calpis reduced SBP and diastolic BP (DBP) by −14.1 and−6.9 mmHg, after consumption of 95 mL/d of the sour milkproduct for 8 wk (Hata and others 1996). Moreover, a random-ized placebo controlled trial using 70 Caucasian subjects with stage1 hypertension demonstrated that consumption of 7.5 mg of Ile-Pro-Pro per day for 4 wk reduced SBP and DBP by 3.8 and 2.3mmHg, respectively (Boelsma and Kloek 2010). Therefore, theextent of activity of these peptides may be dependent on the na-ture of delivery system, dose, study duration, genetics, and stagesof hypertension. In addition, a dairy whey protein-derived peptideproduct (BioZate) exhibited hypotensive activity in hypertensivehuman volunteers by decreasing SBP by −11 mmHg and DBPby −7 mmHg after 6 wk of consuming 20 g of the productper day (Pins and Keenan 2002). Contrary to these studies, a re-cent studies have demonstrated the lack of significant BP-loweringactivity by putatively antihypertensive lactotripeptide-containingproducts in prehypertensive and hypertensive subjects following24-h ambulatory BP monitoring (ABPM) (Van Mierlo and others2009; Usinger and others 2010). These observations indicate thatBP measurements such as ABPM can provide more precise dataas opposed to single-point clinical/office measurements. Table 3shows summaries of recent human clinical trials using lactotripep-tides and the contradictory outcomes.

The inhibition of the physiological activities of ACE or reninby food protein-derived peptides ultimately lead to reduction inthe amount of circulating AT-II, elevated level of bradykinin,

Table 3–Human clinical studies with lactotripeptide (LTP)-based products on different stages of hypertension; contrasting resultscould be due to notable differences in study design, blood pressure measurement tool, study population, dose, and peptide deliveryvehicle.

Peptide or proteinhydrolysate Treatment Outcome Reference

Dairy drink containing LTP(Ile-Pro-Pro andVal-Pro-Pro)

Multicentre crossover studywith untreated hypertensivewhite subjects: study 1, 69subjects received 200 g/d ofdairy drink withlactotripeptides; study 2, 93subjects received 100 g/d ofsame with 350 mgpotassium for 4 wk; ABPMand OBPM

Contrary to previous reports, peptide products didnot have any significant effect on mean 24-hambulatory SBP and DBP compared to placeboin the 2 studies; office BP decreased but nodifference was observed in treatments comparedto placebo

Van Mierlo and others(2009)

LTP (Ile-Pro-Pro)-enrichedmilk protein hydrolysates(MPH)

Seventy prehypertensive andstage 1 hypertensivesubjects consumed 15 mgof encapsulated MPH orplacebo daily for 4 wk;OBPM

MPH decreased SBP and DBP by −3.8 and −2.3mmHg, respectively; no difference was found inplasma renin activity, AT-I or AT-II; nosignificant change in BP observed inprehypertensive subjects compared to placebo;MPH was well tolerated and safe to the subjects

Boelsma and Kloek (2010)

Peptides from Lactobacillushelveticus fermented milk(FM) containing LTP(Ile-Pro-Pro andVal-Pro-Pro)

Ninety-four prehypertensiveand borderline hypertensivesubjects consumed 150-mLor 300-mL FM or placebodaily for 8 wk; ABPM andOBPM

FM showed no significant effect on SBP and DBPcompared to placebo using both ABPM andOBPM; no effects on plasma lipids; observedeffect was not superior to effects of lifestyleintervention for lowering BP

Usinger and others (2010)

LTP product (AmealPeptide) Ninety-one previously treatedand treatment-naive (newlydiagnosed) stage 1 and stage2 hypertensive subjectsreceived a twice-daily75-mg dose of peptideproduct for 6 wk; 24-hABPM and OBPM

ABPM showed peptide-induced decreased daytimeSBP (−3.6 mmHg) and mean 24-h SBP (−2mmHg); OBPM was not reliable for BPmeasurement due to detected “placebo effect”which was minimal using ABPM; effect ondaytime SBP was more pronounced intreatment-naive subjects compared to placebo

Germino and others(2010)

Abbreviations: ABPM = 24-h ambulatory BP; OBPM = office BP measurements; AT = angiotensin; SBP = systolic blood pressure; DBP = diastolic blood pressure.




decrease in ACE-induced contraction with concomitant decreasein elevated BP (Sipola and others 2001; Ruiz-Gimenez and others2010). For example, oral intake of a 100-mL drink containing 3 mgof Val-Tyr twice a day for 4 wk resulted in decreased plasma AT-IIand aldosterone and increased AT-I with associated reduction inSBP by −9.3 mmHg in human subjects with mild hypertension(Kawasaki and others 2000). The observed pattern in the vasoactivepeptides (AT) indicates in vivo ACE inhibition by Val-Tyr treat-ment. However, BP decrease was also observed in prehypertensivehuman subjects after administration of milk-derived Ile-Pro-Prowithout significant change in plasma AT-I, AT-II, and renin ac-tivity (Boelsma and Kloek 2010). This suggests the possibility ofexistence of alternative routes for the activity of peptides. For ex-ample, a recent study showed that ACE-inhibiting trypsin-digestedamaranth glutelins activated NO production through induction ofendothelial NOS in cultured endothelial cells with concomitantinduction of smooth muscle relaxation in isolated rat aortic seg-ments (Barba de la Rosa and others 2010). The observed activitywas attributed to the ability of the peptides to induce phosphoryla-tion of endothelial NOS at Ser117 residue. Therefore, antihyper-tensive effects of food-derived peptides can be mediated throughvarious other pathways other than modulation of RAS enzymeactivity, though the observed effects in the study above could beas a result of increased bradykinin arising from ACE inhibition.

Meta-analyses. A number of meta-analyses have been con-ducted on the hypotensive effects of food protein hydrolysatesand peptides. Pripp (2008) reported a −5.3 and −2.4 mmHgdecrease in SBP and DBP, respectively, in a meta-analysis of 17clinical trials using peptides and protein hydrolysates from dif-ferent sources (milk and fish). The heterogeneity of the samplesconstitutes a limitation for this study since it would be difficult tocompare these data with studies that used homogeneous peptidesamples. In another meta-analysis of 12 randomized controlledtrials, Xu and others (2008) observed a similar effects on BP(−4.8 mmHg SBP, −2.2 mmHg DBP) with lactotripeptides (Val-Pro-Pro and Ile-Pro-Pro) in 623 prehypertensive and hypertensivesubjects; more pronounced effect was observed with only hyper-tensive subjects. The homogeneity of the population studied mayseem to have contributed to the uniformity of the results. A re-cent meta-analysis using 18 trials showed that the lactotripeptidesdecreased both SBP and DBP by slightly lower magnitude (−3.73and −1.97 mmHg, respectively) and the effects were dependenton ethnic factors (Cicero and others 2011). Thus, the authors ob-served that the tripeptides-induced lowering of elevated BP wasmore pronounced in Asian subjects compared to Caucasian sub-jects, and was independent of age of subjects, length of study,dose of the lactotripeptides, and baseline BP. With the moderatepeptide-induced decreases in BP, the consumption of hypotensivefood-derived peptides can be combined with lifestyle changes inorder to achieve substantial BP-lowering effects in severe hyper-tension.

Food protein-derived antioxidant peptidesDietary consumption of antioxidants can supplement the en-

dogenous enzymatic and nonenzymatic antioxidant systems againstoxidative stress (Fang and others 2002). Although synthetic foodantioxidants have been widely applied in the food industry forfood preservation, the use of food-derived peptides has gener-ated interest as both food preservative and health products. Thereis abundant literature information on several food protein hy-drolysates and peptides with antioxidant properties in variousoxidative reaction systems. Plant and animal food protein sources

of antioxidant peptides include pea, soy, fish, quinoa, flaxseed,milk casein, whey, and egg (Aluko and Monu 2003; Pih-lanto 2006; Humiski and Aluko 2007; Erdmann and others2008; Udenigwe and others 2009b). The antioxidant proper-ties of these peptides include scavenging or quenching of reac-tive oxygen species (ROS)/free radicals and inhibition of ROS-induced oxidation of biological macromolecules such as lipids,proteins, and DNA. The radical-quenching activities of foodantioxidants are due to the ability of the antioxidants to par-ticipate in single electron transfer reaction (Huang and others2005); thus, the abundance of peptidic amino acid residues that cantransfer electrons to the free radicals at physiological pH can con-tribute to enhanced antioxidative property. Other mechanisms ofantioxidant activity of peptides include transition metal chelatingactivity and ferric reducing power.

Some factors that may affect the antioxidant activity of foodprotein hydrolysates include specificity of proteases used for hy-drolysis, degree of hydrolysis, and the structural properties of theresulting peptides, including molecular size, hydrophobicity, andamino acid composition (Pihlanto 2006). The amount of histidine,cysteine, proline, methionine, and aromatic amino acids have beenreported to contribute to the antioxidant activity of food pep-tides. Structure-function studies using a number of synthetic pep-tides revealed that histidine residue of peptides can chelate metalion, quench active oxygen, and scavenge .OH (Chen and others1996; Chen and others 1998) and these properties were attributedto its imidazole group, which can participate in hydrogen atomtransfer and single electron transfer reactions (Chan and Decker1994). Similar potent antioxidant activity has also been reportedfor a histidine-containing dipeptide, carnosine (β-Ala-His), de-rived from muscle cells (Chan and others 1994). Moreover, theaddition of hydrophobic amino acids, proline and leucine, to theN-terminus of a dipeptide His-His resulted in enhanced antioxida-tive property of the peptides, and these new peptides also displayedsynergistic effects when combined with nonpeptide antioxidants(Chen and others 1996). Hydrophobic amino acids are importantfor enhancement of the antioxidant properties of peptides sincethey can increase the accessibility of the antioxidant peptides to hy-drophobic cellular targets such as the polyunsaturated chain of fattyacids of biological membranes (Chen and others 1998). Moreover,the electron-dense aromatic rings of phenylalanine, tyrosine, andtryptophan residues of peptides can contribute to the chelating ofpro-oxidant metal ions whereas phenylalanine can also scavenge˙OH radicals to form more stable para-, meta-, or ortho-substitutedhydroxylated derivatives (Sun and others 1993). Therefore, thespecific contribution of individual amino acid residues to the an-tioxidant activity of a peptide depends largely on the nature ofthe ROS/free radical and the reaction medium. However, it is notclear how these “antioxidant” amino acid residues contribute tothe antioxidant activity of a peptide mixture typical of food proteinhydrolysates, or the possible positive or negative contributions ofother amino acid residues present in the hydrolysates. It is impor-tant to delineate these possible amino acid contributions in orderto strategically process the hydrolysates to yield peptide mixturescontaining amino acid residues of interest.

In addition to the direct antioxidant activity due to the sulfhydrylfunctional group, cysteine residues of peptides can also serveas precursor for the synthesis of glutathione (γ -L-glutamyl-L-cysteinylglycine), a ubiquitous cellular antioxidant tripeptide,thereby contributing toward regeneration of the physiological an-tioxidant defense system (Meisel 2005). Moreover, food-derivedpeptides can also display antioxidant property by induction of


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gene expression of proteins that protect cellular components fromoxidative stress-induced deterioration. In endothelial cells, adipeptide Met-Tyr derived from sardine muscle protein stimu-lated the expression of heme oxygenase-1 and ferritin leading toa sustained cellular protection from oxidative stress (Erdmann andothers 2006). In addition, a recent study observed that casein hy-drolysates generated with different proteases exhibited varying an-tioxidant activities, independent of degree of hydrolysis, in humanJurkat T cells by increasing cellular catalase activity and amount ofreduced glutathione but without any effect on superoxide dismu-tase (SOD) activity (Phelan and others 2009a; Lahart and other2011). Although the casein hydrolysates showed dose-dependentdecrease in viability and growth of the human Jurkat T cells, lowerdoses retained the beneficial antioxidant properties without any ef-fect on membrane integrity. In D-galactose-induced aging ICRmice, oral intake of jellyfish collagen hydrolysates prepared withProtamex induced increase in SOD and glutathione peroxidasewith concomitant decrease in serum and hepatic malondialde-hyde, an oxidative stress marker (Ding and others 2011). Althoughthese effects are promising, it will be worthwhile for researchersto focus more on the effects of these peptides in human subjects inorder to evaluate mechanisms of action and possible application ofthese peptides in formulating health-promoting food products.

Food protein-derived calmodulin (CaM)-binding peptidesCaM is a ubiquitous negatively charged 148-amino acid

(16.6 kDa) Ca2+-binding protein that is involved in the activa-tion of several important proteins in response to increased in-tracellular Ca2+ concentration (Ikura and others 1992; Hooksand Means 2001). Some clinically important enzymes that re-quire Ca2+/CaM activation include endothelial and neuronalNOS, cyclic nucleotide phosphodiesterase 1 (CaMPDE), adeno-sine triphosphatase, phospholipase A2, adenylate cyclase, andprotein kinase II (Itano and others 1980). Thus, CaM playsimportant roles in several cellular processes including cell growth,cell proliferation, neurotransmission, vasodilation, and smoothmuscle contraction (Cho and others 1998). Therefore, CaM-binding natural compounds can be used for the prevention andamelioration of diseases induced or exacerbated by increased activ-ity of CaM-dependent enzyme (Martınez-Luis and others 2007).

Considering the roles of CaM in human health conditions,CaM-binding agents can be used as multifunctional agents forameliorating disease conditions. The amino acid sequences ofmany natural CaM-binding proteins and peptides revealed thepresence of repeated positively charged (cationic) and hydropho-bic amino acid residues at the CaM-binding sites (O’Neil andDeGrado 1990). These structural features are thought to be moreimportant than the specific amino acid sequence in determin-ing affinity of peptides for CaM (Kizawa and others 1995). Theaffinity of the cationic residues for the net negatively chargedCaM led to a rationale to use cationic peptides as CaM-bindingagents (Itano and others 1980; Barnett and others 1983). A num-ber of food protein-derived cationic peptides have been reportedto bind CaM leading to the inhibition of CaM-dependent en-zymes. Earlier works by Kizawa and others (1995) and Kizawa(1997) reported the isolation of CaM-binding peptides from ca-sein, specifically αs2-casein (f164–179, f183–206, f183–207, andf90–109), which inhibited CaMPDE activation with IC50 val-ues of 38, 6.9, 1.1 and 1.0 μM, respectively, without any effectson the basal PDE activity. These activities are lower than theinhibition of CaM-induced PDE activity by an anti-CaM drug(calmidazolium) with IC50 of 0.12 μM and a microbial metabolite

(KS-505a) with IC50 of 0.065 μM (Martınez-Luis and others2007) although αs2-casein f183–207 and f90–109 show potentialfor further consideration. Based on the work with αs2-casein pep-tides, our laboratory has explored other cationic amino acid-richfood protein sources for the production of CaM-binding peptides.Li and Aluko (2005) reported that pea protein-derived cationicpeptide fraction inhibited CaM-dependent protein kinase II activ-ity via the competitive mode of inhibition. In other similar studies,2 cationic peptide mixtures fractionated from Alcalase-preparedflaxseed protein hydrolysates bound CaM with concomitant inhi-bition of the activities of endothelial and neuronal NOS (Omoniand Aluko 2006a, 2006b) via the mixed-type and noncompeti-tive modes of inhibition, respectively. The authors reported thatthese activities were due to decreased α-helix/unfolding of CaMand increased rigidity of the Ca2+/CaM complex due to bind-ing of the cationic peptides. Moreover, a recent study have alsoshown that cationic peptide fractions from egg white lysozymecan simultaneously inhibit CaMPDE and also act as antioxidants(You and others 2010), which makes these peptides good candi-dates for use against multiple disease conditions. These cationicpeptides can be easily purified from inactive enzymatic food pro-tein hydrolysates using ion-exchange columns or electrodialysis,due to their unique physicochemical characteristics (Kizawa andothers 1995). Although these peptides have shown unique inter-action with CaM leading to potent inhibition of CaM-dependentenzymes, there is currently a dearth of information on absorp-tion, bioavailability, and pharmacological activity of food protein-derived CaM-binding peptides in ameliorating specific humanhealth and disease conditions.

Hypolipidemic and hypocholesterolemic peptidesProtease-aided hydrolysis of food proteins can also release pep-

tide sequences that possess cholesterol and lipid-lowering activi-ties. Food protein sources of hypocholesterolemic and hypolipi-demic peptides include soy protein (Nagaoka and others 1999;Aoyama and others 2000; Cho and others 2007), milk protein(Kirana and others 2005), buckwheat protein (Kayashita and others1997), egg white protein (Manso and others 2008), and fish pro-tein (Wergedahl and others 2004). However, enzymatic hydrolysiscan also lead to reduced lipid-lowering activity of food proteins(Kayashita and others 1997). Most literatures on lipid-loweringpeptides were focused on soy protein hydrolysates and peptides.The hypocholesterolemic and hypolipidemic properties of soy pro-tein hydrolysates reported in animals (Aoyama and others 2000)and in humans (Hori and others 2001) have been partly attributedto the soy 7S globulin (β-conglycinin). The α+α′ subunit ofthis protein strongly upregulated the expression of low-densitylipoprotein (LDL) receptor in cultured hepatocytes leading to anincrease in LDL uptake and degradation (Lovati and others 1998).The peptide region responsible for the activity has been identifiedfrom the α′ subunit and sequenced (Lovati and others 2000). This24-amino acid peptide that corresponds to position 127 to 150of the α′ subunit displayed potential in modulating cholesterolhomeostasis by increasing LDL receptor-mediated LDL uptake inHep G2 cells (Lovati and others 2000). Moreover, Cho and others(2008) also identified an octapeptide (FVVNATSN) from the en-zymatic digest of soy protein as the most active stimulator of LDLreceptor transcription in Hep T9A4 human hepatic cells. Thus,proteolytic digestion of the soy protein was important for releasingmore active small peptides with improved cardioprotective prop-erty. This has also been demonstrated in a study by Mochizuki andothers (2009) that produced BAPs from purified isoflavone-free




soy 7S β-conglycinin using bacterial proteases. The resulting7S-peptides showed hypotriglyceridemic properties by alteringgene expressions related to triacylglycerol synthesis and also de-creased Apo B-100 accumulation in Hep G2 cells partly due to in-crease in LDL receptor mRNA expression (Mochizuki and others2009). Apo B-100 is a functional component of very low-densitylipoprotein (VLDL) and its degradation reduces VLDL synthesis.These observations supported a previous study that showed that soyβ-conglycinin possesses beneficial effects on plasma triacylglycerolin humans (Kohno and others 2006).

In addition to alterations of gene expressions, soy protein hy-drolysates and constituent peptides also exhibited hypocholes-terolemic activity by binding bile acids and neutral sterolsin the intestine leading to increased fecal removal (Cho andothers 2007; Yang and others 2007). The ability of the soy proteinhydrolysates to bind bile acids may depend in part on their insol-uble high molecular weight peptide fraction rich in hydrophobicamino acids (Higaki and others 2006), as earlier observed for highmolecular weight fraction of the tryptic digest of buckwheat pro-tein (Kayashita and others 1997). This shows that even thoughlarge BAPs may not be able to cross the intestinal epithelium intoblood circulation to exert their beneficial lipid-lowering effects inthe hepatocytes and other cellular locations, they might be use-ful in cholesterol homeostasis by enhancing fecal removal of bileacids and exogenous cholesterol from the intestine depending ontheir hydrophobic properties. Two soybeans-derived peptide prod-ucts based on lunasin (Lunasin XP

R©and LunaSoyTM) have been

commercialized as cholesterol-lowering food ingredients (SoyLabs2011). Lunasin is a 43 amino acid-containing polypeptide (molec-ular weight 5.4 kDa) found in soybeans, barley, rye, and wheat(Wang and others 2008; Hernandez-Ledesma and others 2009a).Lunasin exerts its hypocholesterolemic activity by blocking acety-lation of histone H3 Lys14 residue thereby reducing the pro-duction of HMG-CoA reductase with concomitant decrease incholesterol biosynthesis; lunasin also increases cellular productionof LDL receptors leading to removal of plasma LDL cholesterol(SoyLabs 2011). Structure-function studies are needed to under-stand the structural requirements for the lipid-lowering propertiesof peptides. Further larger human studies are required to confirmthe beneficial effects of these products in different hypercholes-terolemic populations, and to evaluate the overall contributiontoward management of cardiovascular disease.

Anticancer peptidesPeptides with anticancer properties have also been reported from

foods. A number of studies on anticancer peptides have been fo-cused on lunasin (Wang and others 2008; Hernandez-Ledesmaand others 2009a). The anticancer property of lunasin is predom-inantly against chemical and viral oncogene-induced cancers, andbased on the modulation of histone (H) acetylation and deacety-lation pathways specifically by inhibiting histone acetyl transferase(HAT). This leads to inhibition of acetylation of H3 and H4,repression of cell cycle progression (arrest at G1/S phase), andapoptosis in cancer cells (Hernandez-Ledesma and others 2009a).Although lunasin showed excellent potential as anticancer agentin cell cultures, its large molecular size raises questions as to its ab-sorption and use as an orally bioavailable health-promoting agent.Dia and others (2009a) reported that 4.5% of lunasin was ab-sorbed in human subjects that consumed lunasin-containing soyprotein. They also observed other lunasin-derived peptide se-quences in the plasma, which could be attributed to degradation bygastrointestinal proteases and plasma peptidases. Another study re-

ported efficient absorption of lunasin from rye consumption intothe liver, kidney, and blood, and the tissue-derived extracts retainedthe anticancer HAT-inhibitory property of the parent molecule(Jeong and others 2009). It has been suggested that the activityof protease inhibitors present in lunasin-containing whole foodcontributed to the resistance of lunasin against gastrointestinal di-gestion as opposed to its synthetic form (Hernandez-Ledesma andothers 2009a). Additional studies are needed to understand themechanism of lunasin activity, absorption kinetics into the bloodcirculation and cancer cell targets, and application as effective food-derived anticancer nutraceutical.

In addition to lunasin, other soy protein-derived peptides havealso shown promising activities for anticancer therapy. Wang andothers (2008) reported that enzymatic hydrolysates from dif-ferent soy varieties inhibited the viability of cultured leukemiacells (L1210) with IC50 values of 3.5 to 6.2 mg/mL, whichwere significantly lower than the activity of lunasin (IC50 of0.078 mg/mL). Moreover, a lunasin-containing glutelin fraction ofAmaranthus hypochondriacus, when digested with trypsin, inducedprogrammed cell death (apoptosis) in cervical cancer (HeLa) cellsby 30% and 38% at 1 and 5 μg/mL, respectively (Silva-Sanchezand others 2008). It was not reported whether the anticancerpeptides were derived from lunasin primary sequence or fromother protein precursors present within the fraction. A similarstudy also demonstrated that a soy protein-derived hydrophobicpeptide fraction exhibited cytotoxicity with IC50 of 0.16 mg/mLagainst macrophage-like murine tumor cell line (P388D1) by ar-resting cell cycle progression at the G2/M phases (Kim and others2000). Recently, Hsu and others (2011) isolated 2 large pep-tides (Leu-Pro-His-Val-Leu-Thr-Pro-Glu-Ala-Gly-Ala-Thr andPro-Thr-Ala-Glu-Gly-Gly-Val-Tyr-Met-Val-Thr) from tuna darkmuscle by-product hydrolyzed with papain and protease XXII.These peptides exhibited dose-dependent antiproliferative activ-ities against cultured breast cancer (MCF-7) cells with IC50 of8.1 and 8.8 μM, respectively. Thus, enzymatic hydrolysis of foodproteins can release BAPs with anticancer properties. This wasclearly demonstrated in a study where the antiproliferative activityof A. mantegazzianus protein hydrolysates was twice the activity ofthe parent protein (Barrio and Anon 2010). Recently, low molec-ular size peptides from Pacific oyster hydrolysates were reportedto induce dose-dependent inhibition of growth of transplantedmurine sarcoma in BALB/c mice possibly via increased immunos-timulation (Wang and others 2010). Other marine food-derivedanticancer peptides are presented in Table 1. Based on these stud-ies, detailed animal studies and clinical human trials are highlyneeded to evaluate the physiological anticancer activities of thesepeptides.

Immunomodulatory and anti-inflammatory peptidesImmunomodulation involves suppression or stimulation of hu-

man immune functions. Immunomodulatory food peptides act byenhancing the functions of immune system including regulationof cytokine expression, antibody production, and ROS-inducedimmune functions (Hartmann and Meisel 2007; Yang and others2009). For example, a tryptic digest of rice protein improved im-mune function by promoting phagocytosis and increasing super-oxide anion production in human polymorphonuclear leukocytes(Takahashi and others 1994). In addition, egg-derived peptidesalso showed immunostimulating activities and were used to in-crease immune functions during cancer immunotherapy (Mineand Kovacs-Nolan 2006). Moreover, a recent work showed thatoral administration of a pea protein hydrolysate to mice led to


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reduced NO production by activated macrophages as well as re-duced secretion of the proinflammatory cytokines, tumor necrosisfactor (TNF)-α and interleukin (IL)-6, by up to 35% and 80%,respectively (Ndiaye and others 2011). In human volunteers, con-sumption of 3 g/d of wheat protein hydrolysate for 6 d increasedactivity of natural killer cells (Horiguchi and others 2005). Fur-thermore, another study demonstrated that whey protein-derivedpeptides can activate cellular immune functions (Gauthier andothers 2006).

The anti-inflammatory properties of food-derived peptideshave been reported mostly in modulating endotoxin-inducedproduction of proinflammatory responses in macrophages.For example, soy lunasin and lunasin-like peptides exhibitedanti-inflammatory properties by decreasing ROS production,TNF-α, IL-6, IL-1β, nuclear factor-κB (NF-κB) levels, anddown-regulation NO/PGE2 synthesis and inducible NOS/COX-2 expressions in activated macrophages (Dia and others 2009b;Gonzalez de Mejia and Dia 2009; Hernandez-Ledesma and others2009b). The activity of lunasin was due to the suppression of nu-clear translocation of p65/p50 subunits of NF-κB in RAW264.7macrophage, which reduces binding of NF-κB to target genes withconcomitant inhibition of proinflammatory markers gene activa-tion and the gene products, for example, IL-6, inducible NOS,COX-2 (Gonzalez de Mejia and Dia 2009). In addition, caseinhydrolysates were reported to increase concanavalin A (ConA)-stimulated T helper (Th)-1 produced IL-2 level but not Th-2 pro-duced IL-10 in human Jurkat T cells; this shows that the peptidesacted via T cell-mediated immune response and not humoral orantibody-mediated immune response (Phelan and others 2009a).Furthermore, food-derived peptides have also protected againstradiation-induced immunosuppression. A recent study reportedthat a peptide fraction from Chum salmon collagen hydrolysatesprotected against gamma radiation-induced immunosuppressionin mice by augmenting CD4+ Th cells, enhancing spleen pro-duction of IL-12, inducing I-κB thereby reducing NF-κB, andinhibiting splenocyte apoptosis (Yang and others 2010). As withanticancer and CaM-binding peptides, future clinical interventionstudies using these peptides are needed to evaluate their efficacy,pharmacokinetics, and possible use for the formulation of func-tional food products.

Multifunctional peptidesMultifunctional peptides have been discovered from some

food proteins and have been reported to possess more thanone significant physiologically relevant bioactive property. Studieson milk proteins demonstrated that a hexapeptide (TTMPLW)derived from αS1-casien (f194–199) by trypsin-catalyzed di-gestion exhibited both ACE-inhibitory and immunomodula-tory activities (Meisel 2004) while a β-lactoglobulin-derivedβ-lactorphin (YLLF) inhibited ACE activity and also possessedopioid-like activity (Antila and others 1991; Mullally and others1997). In addition, several other milk-derived peptides suchas α-lactorphin (YGLF), α-immunocasokinin (TTMPLW), β-casomorphin-7 (YPFPGPI), and β-casokinin (AVPYPQR) areregarded as multifunctional, some possessing in vivo bioactive prop-erties (Meisel 2004). Moreover, crude chymotryptic α-casein hy-drolysates displayed several in vitro bioactivities such as ACE andpropyl endopeptidase inhibition, antioxidant, Zn2+-binding, andantibacterial activities (Srinivas and Prakash 2010). Four peptides(GFHI, DFHING, FHG, and GLSDGEWQ) present in beef sar-coplasmic protein hydrolysate were reported to possess anticancer,antimicrobial, and ACE-inhibitory properties (Jang and others

2008). Other partially purified food peptides from quinoa/peaproteins and hen egg white lysozyme have also displayed mul-tifunctionality as ACE-inhibiting antioxidants (Aluko and Monu2003; Humiski and Aluko 2007) and CaMPDE-inhibiting antiox-idants (You and others 2010), respectively. The multifunctionalbioactive properties of protein hydrolysates and peptides derivedfrom marine foods are shown in Table 1. The multiple bioactivitiesdisplayed by these peptides can increase their impact toward theamelioration of more than one disease target or multiple symptomsof a disease, such as cardiovascular disease, since many human dis-eases are interrelated in terms of etiology and progression. There-fore, the conditions for generating and processing bioactive foodprotein hydrolyastes can be carefully designed to yield multifunc-tional peptides with diverse applications in maintaining optimumhuman health.

Delivery and Bioavailability of BAPsBAPs can be administered using different vehicles such as bev-

erages and bakery products (reviewed by Fitzgerald and others2011; Hernandez-Ledesma and others 2011). In vitro bioactivityof peptides does not generally translate into in vivo pharmaco-logical effects due to concerns about absorption, bioavailability,and susceptibility of the peptides to degradation into inactive frag-ments by physiological enzymes (Vermeirssen and others 2004;Hernandez-Ledesma and others 2011). For example, peptides de-rived from milk proteins MAP1 and MAP2 showed in vitro ACE-inhibitory activity (MPH2 was more potent than MPH1) but onlyMAP1 reduced BP of hypertensive subjects (Boelsma and Klooek2010). Thus, in order to use food-derived peptides as enterallypotent health-promoting agents, they must show stability againstgastrointestinal proteases and be absorbed through the enterocytesto the serum without degradation by brush border and serumpeptidases (FitzGerald and others 2004). This ensures that theoriginal peptide sequences that displayed in vitro bioactivity areconserved and delivered to the cellular sites of action. Microen-capsulation has been explored for delivery of BAPs to enhancetheir stability and absorption (Rocha and others 2009; Hwangand others 2010). Moreover, bioavailability of BAPs depends onphysicochemical properties of the peptides such as charge, molec-ular size, lipophilicity, and solubility; smaller peptides are trans-ported across the enterocytes through intestinal-expressed peptidetransporters whereas oligopeptides may be absorbed by passivetransport through hydrophobic regions of membrane epithelia ortight junctions (Darewicz and others 2011). In this regard, it wouldbe desirable to investigate the bioactivity of small peptides (dipep-tides, tri-peptides, and small oligopeptides); peptides of small sizeshave demonstrated in vivo bioactivity, resistance to peptidolysis andcan be absorbed intact into blood circulation (Matsui and oth-ers 2002; Foltz and others 2007). Lactotripeptides (Ile-Pro-Pro,Val-Pro-Pro) were detected in nanomolar amounts when givenwith yogurt as delivery medium in fasted and fed states innormotensive subjects (Foltz and others 2007). Furthermore,peptides that act in the gastrointestinal tract (for example,cholesterol-binding and anoretic peptides) do not have to be ab-sorbed to exert their biological properties (Wang and GonzalezDe Mejia 2005).

Safety of BAPsTill date, there has been little concern about safety of food

protein-derived BAPs since the body would normally hydrolyzefood proteins into peptides (Wang and Gonzalez De Mejia 2005)and food-grade enzymes and processes are utilized for industrial




production of peptides. Numerous studies have shown lack oftoxicity of these peptides in cell cultures. The safety aspects ofmilk-derived peptides have been reviewed (Phelan and others2009b). Moreover, a recent study reported that both single dose(2000 mg/kg) and repeated daily dose (1000 mg/kg for 4 wk)of casein hydrolysates containing antihypertensive peptides (αs1-casein f90–94 and f143–149) resulted in no adverse effects onclinical (blood biochemical, hematology, organ weight ratios,histopathological) parameters or mortality in rats (Anadon andothers 2010). The sample doses represent peptide amounts thatare well above those needed to observe pharmacological activities.Furthermore, in prehypertensive and hypertensive human subjects,lactotripeptide (Ile-Pro-Pro)-rich milk protein hydrolysates werefound to be well tolerated and without any significant adverseeffects on serum chemistry and urine parameters when comparedwith subjects that received placebo treatment (Boelsma and Klooek2010; Germino and others 2010). Therefore, food protein-derivedBAPs are generally safe but care should be taken to avoid process-ing techniques that would negatively affect peptide quality andsafety.

ConclusionsThe current literature has shown that peptides derived from en-

zymatic food protein hydrolysates possess remarkable multifunc-tional activities relevant to the sustenance of human health. Thisresearch area is continuously growing with the discovery of newmolecular disease targets. While a lot of information exists onthe various bioactivities of food protein-derived peptides, futureresearch efforts should be directed toward evaluation of in vivohealth-promoting effects, bioavailability, and pharmacokinetics inhuman subjects, elucidation of the molecular mechanisms of actionand overall possible use as health-promoting agents in food sys-tems. Moreover, the safety of these peptide-based products shouldalso be evaluated prior to commercialization especially after ex-tensive food processing that may affect the natural integrity andquality of the constituent peptides.

AcknowledgmentsThe research program of REA is supported by the Natural Sci-

ences and Engineering Research Council of Canada (NSERC).CCU acknowledges the support from NSERC through anAlexander Graham Bell Canada Graduate Scholarship for his doc-toral studies.

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Food Protein-Derived Bioactive Peptides Production Processing and Potential Health Benefits

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