F ee Radical Resea ch
ISSN: 1071-5762 (P in ) 1029-2470 (Online) Jou nal homepage: www. and online.com/jou nals/i a20
Whey p o eins: a ge s o oxida ion, o media o s o
edox p o ec ion
Linda Giblin, A. Süha Yalçın, Gökhan Biçim, Anna C. K äme , Zhi ei Chen,
Michael J. Callanan, Elena A anz & Michael J. Da ies
To ci e his a icle: Linda Giblin, A. Süha Yalçın, Gökhan Biçim, Anna C. K äme , Zhi ei
Chen, Michael J. Callanan, Elena A anz & Michael J. Da ies (2019) Whey p o eins: a ge s o
oxida ion, o media o s o edox p o ec ion, F ee Radical Resea ch, 53:sup1, 1136-1152, DOI:
10.1080/10715762.2019.1632445
To link o his a icle: h ps://doi.o g/10.1080/10715762.2019.1632445
© 2019 The Au ho (s). Published by In o ma
UK Limi ed, ading as Taylo & F ancis
G oup
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Whey p o eins: a ge s o oxida ion, o media o s o edox p o ec ion
Linda Giblin
a
,A.S
€
uha Yalc¸ın
b
,G
€
okhan Bic¸im
b
, Anna C. K €
ame
c
, Zhi ei Chen
c
,
Michael J. Callanan
d
, Elena A anz
a
and Michael J. Da ies
c
a
Teagasc Food Resea ch Cen e, Moo epa k, Fe moy, Co k, I eland;
b
Depa men o Biochemis y, School o Medicine, Ma ma a
Uni e si y,
_
Is anbul, Tu key;
c
Depa men o Biomedical Sciences, Panum Ins i u e, Uni e si y o Copenhagen, Copenhagen, Denma k;
d
Depa men o Biological Sciences, Co k Ins i u e o Technology, Co k, I eland
ABSTRACT
Bo ine whey p o eins a e highly alued dai y ing edien s. This is p ima ily due o hei amino
acid con en , diges ibili y, bioac i i ies and hei p ocessing cha ac e is ics. One o he
epo ed bioac i i ies o whey p o eins is an ioxidan ac i i y. Nume ous die a y in e en ion
ials wi h humans and animals indica e ha consump ion o whey p oduc s can modula e
edox bioma ke s o educe oxida i e s ess. This bioac i i y has in pa been assigned o
whey pep ides using a ange o biochemical o cellula assays in i o.Supe imposingwhey
pep ide sequences om gas oin es inal samples, wi h whey pep ides p o en o be an ioxi-
dan in i o, allows us o p opose pep ides om whey likely o exhibi an ioxidan ac i i y in
he die . Howe e , whey p o eins hemsel es a e a ge s o oxida ion du ing p ocessing pa -
icula ly when exposed o high he mal loads and/o ex ensi e p ocessing (e.g. in an o mula
manu ac u e). Oxida i e damage o whey p o eins can be selec i e wi h ega d o he esidues
ha a e modi ied and a e associa ed wi h he deg ee o p o ein un olding, wi h
a-Lac albumin mo e suscep ible han b-Lac oglobulin. Such oxida i e damage may ha e
ad e se e ec s on human heal h. This e iew summa ises how whey p o eins can modula e
cellula edox pa hways and con e sely how whey p o eins can be oxidised du ing p ocessing.
Gi en he ex ensi e p ocessing s eps ha whey p o eins a e o en subjec ed o, we conclude
ha oxida ion du ing p ocessing is likely o comp omise he posi i e heal h a ibu es associ-
a ed wi h whey p o eins.
ARTICLE HISTORY
Recei ed 4 Ma ch 2019
Re ised 5 June 2019
Accep ed 6 June 2019
KEYWORDS
An ioxidan ; bioa ailable;
gas oin es inal diges ion;
glyca ion; in an o mula;
oxida i e damage;
p ocessing; acemised
amino acids; whey p o eins
In oduc ion
Milk p o eins (whey p o eins (20%) and caseins
(80%)) a e high-quali y sou ces o amino acids (AA) in
he human die . Bo ine milk p o eins and pep ides
play impo an oles in human heal h no jus in
e ms o nu i ion bu also in e ms o hei no able
bioac i i ies. The majo bo ine whey p o eins a e
a-Lac albumin (a-Lac) and b-Lac oglobulin (b-Lg) wi h
immunoglobulins, bo ine se um albumin (BSA) and
lac o e in as mino p o eins (Table 1). Whey p o eins
p o ide a comple e p o ein sou ce and a e ich in
bo h sulphu -con aining and b anched-chain AAs.
Whey p o eins do no coagula e unde he acidic con-
di ions p esen in he s omach, and a e conside ed
o be “ as p o eins”since hey each he jejunum
sho ly a e en e ing he gas oin es inal ac and
ha e a diges ible indispensable AA sco e o 1.09 [1].
Whey p o eins exhibi a wide ange o bioac i i ies
including an ioxidan , an ibac e ial, an i ungal,
an i i al, an ihype ensi e, an i h ombo ic, opioid and
immunomodula o y p ope ies [2]. Consequen ly,
whey p oduc s a e ecognised as alue-added ing e-
dien s and a e commonly used in he spo s nu i ion
ma ke , nu i ional be e ages o he elde ly and in an
o mula (IF) [3]. These p oduc s a e also p ized in ood
o mula ion as hey imp o e p oduc quali y by wa e -
binding, s abilising ae a ed ood p oduc s and ac ing
as emulsi ying agen s [4]. Whey p oduc s can be sup-
plied as ing edien s o ood o mula ions as whey p o-
ein concen a e (WPC), whey p o ein isola e (WPI) o
as whey hyd olysa es (WH) all o which di e in he
deg ee o p ocessing and p o ein con en (Table 2).
Howe e , nume ous p ocessing s eps om milk o
whey powde o ood o mula ion inhe en ly expose
whey p o eins hemsel es o edox modi ica ions. This
e iew summa ises how whey p o eins can modula e
cellula edox pa hways and con e sely how whey p o-
eins can be oxidised du ing p ocessing.
CONTACT Linda Giblin [email p o ec ed] Teagasc Food Resea ch Cen e, Moo epa k, Fe moy, Co k, I eland
ß2019 The Au ho (s). Published by In o ma UK Limi ed, ading as Taylo & F ancis G oup
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/Licenses/by/4.0/), which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
FREE RADICAL RESEARCH
2019, VOL. 53, NO. S1, 1136–1152
h ps://doi.o g/10.1080/10715762.2019.1632445
Whey p o eins and hei abili y o modi y
edox pa hways –s udies in animal models
and humans
The an ioxidan and de oxi ying bioac i i ies o whey
p o eins a e mos likely linked o hei con ibu ion o
glu a hione (GSH) syn hesis, ecen ly e iewed in
Co ochano e al. [5]. Whey p o eins a e ich in Cys wi h
b-Lg con aining 5 Cys esidues, a-Lac has 8 Cys, BSA
has 35 Cys and lac o e in con ains 34 Cys, al hough in
each case he majo i y o Cys is p esen as disulphide
bonds. The hiol (R-SH) g oup o Cys eac s apidly wi h
many oxidan s. This AA when p esen wi h Gly and Gln
in he ipep ide, GSH, is an impo an co ac o and
an ioxidan in mammalian cells and issues. Reduced
GSH, is eadily oxidised o he disulphide species, oxi-
dised glu a hione (GSSG), wi h he la e hen eadily
ecycled by he enzyme GSH educ ase, a he expense
o nico inamide adenine dinucleo ide phospha e
(NADPH), back o i s educed o m. O he oxidised spe-
cies can howe e also be o med om GSH including
GSH sulphonamide and oxy acids, wi h he la e spe-
cies being i e e sible p oduc s. GSH de oxi ies a num-
be o endogenous and exogenous oxins including
oxic me als, pe oleum dis illa es, lipid pe oxides, qui-
nones, bili ubin and p os aglandins h ough di ec con-
juga ion. Cell lines (e.g. C2C12, MRC-5, PC12, Caco
2
,
HUVEC, 9HTEo, HepG2, and REPE-1) exposed o a ious
whey p oduc s (WPC and WPI) ha e documen ed
inc eases in GSH le els wi h some excep ions [5]. Whey
p oduc s ha e also been epo ed o inc ease ac i i ies
o he p o ec i e enzymes supe oxide dismu ase (which
emo es supe oxide adicals) and ca alase (which
educes H
2
O
2
o wa e ) and dec ease le els o eac i e
oxygen species, lipid pe oxida ion and DNA damage in
cellula assays [5]. Howe e using cell lines wi h whole
p o eins has i s limi a ions, no leas o which is he
al e ed edox homeos asis o immo alised cell lines [6]
and he nonphysiological exposu e o cells o in ac
die a y p o eins.
Human o animal in e en ion ials wi h die s ha
include whey p oduc s a e he bes assessmen o
impac on cellula edox pa hways al hough eliable
and consis en da a wi h well-de ined bioma ke s is
a he limi ed. In a signi ican numbe o cases gene ic
and non-speci ic assays o an ioxidan ac i i y ha e
been used, which is no ideal. Table 3 lis s a and
mouse s udies wi h a ocus on edox pa ame e s, whe e
animals consumed di e en whey p oduc s (WPC and
WPI) a a dosage o 0.02–1 g/kg body mass o e a
pe iod o 7–84 d. Redox bioma ke s we e measu ed in
li e , b ain, e y h ocy e, muscle, se um, kidney, colon,
sali a y gland o pa a hy oid gland in he p esence o
absence o a ious s esso s (exe cise, diabe es, hea ,
b ain inju y, hepa o oxici y, dyslipidaemia, schizoph e-
nia o a la oxin). Al hough con lic ing da a ha e been
epo ed [7], he majo i y o ials ha e epo ed
inc eases in GSH and o he an ioxidan ma ke s.
Howe e exac ly which species, and wha concen a-
ions, a e esponsible o hese changes is unclea in
mos cases. Human die a y whey in e en ion ials
wi h a ocus on edox eadou s ha e been e iewed
p e iously by Co ochano e al. [5], wi h addi ional ials
de ailed in Table 4. These in e en ion ials gene ally
ec ui pa icipan s o , on a e age, a 3-week s udy wi h
o wi hou an exe cise ou ine, measu ing plasma GSH
as an indica o o whole body edox s a us. Many s ud-
ies epo inc eases in plasma GSH om ime ze o wi h
whey in e en ion bu o he s obse e no e ec on
plasma GSH le els (Table 4)[8,9]. Whe he o no a sin-
gle edox analy e de ec ed a e y low le els in he
plasma is a good bioma ke o edox s a e a a global
o local issue le el is deba able [10,11].
The e is also some e idence om in e en ion
ials ha whey p o eins may dec ease plasma GSH le -
els, cause hea damage and li e inju y [12,13]. O al
Table 1. Bo ine whey p o eins.
Bo ine whey p o ein Concen a ion (g/L) in bo ine milk Molecula mass (kDa) AAs
To al 7
b-Lac oglobulin 3.5 18.3 162
a-Lac albumin 1.2 14.2 123
Immunoglobulins 0.7 150–900 Va iable
Bo ine se um albumin 0.4 66.4 583
Lac o e in 0.02–0.35 80 700
Table 2. Composi ion o di e en bo ine whey p oduc s.
Whey powde ype P o ein (%) Fa (%) Lac ose (%)
Whey p o ein concen a e (WPC) 34–80 1–74–52
Whey p o ein isola e (WPI) 90–95 0.5–1 0.5–1
Hyd olysed whey p o ein (WH) 80–90 0.5–8 0.5–10
FREE RADICAL RESEARCH 1137
Table 3. Animal s udies wi h di e en whey p oduc s and he an ioxidan esponses epo ed.
Bo ine whey p oduc , sou ce
a
Dose and ime Species/ issue/o gan/disease Resul s
b
Re e ence
WPC, Dai y
Coope a i e Poland
0.3 g/kg; 7–14 d Ra ; sali a y gland,
pa o id gland
"GSH; "GSH-Px; "SOD; WPC
imp o ed edox
homeos asis in
sali a y glands
[131]
WPC, Dai y
Coope a i e Poland
0.3–0.5 g/kg; 7–21 d Ra ; li e "hepa ic GSH; "hepa ic MDA [132]
WPC, Da isco 0.02–0.04–0.06–0.08–0.1 g/kg
bw (30 d)
Ra ; CCl
4
hepa o oxici y; li e #MDA; WPC p e en ed li e
damage induced by CCl
4
[133]
Un-dena u ed WP,
P o he
V
R
, I aly
18%; 3 weeks Ra ; CCl
4
hepa o oxici y; li e "GSH [134]
WP, G een Land o Food
Indus ies, Egyp
10%; 28 ds Ra ; CCl
4
hepa o oxici y; li e , #MDA; "GSH [135]
WPC, Da isco 0.1 g/mL; 30 d Ra ; CCl
4
hepa o oxici y; li e , #MDA; "CAT; "GST [136]
WPI, Da isco 0.1 g–0.2 g/kg; Ra ; lipolysaccha ides/
li e , se um
"SOD; #DNA damage;
#Caspase-3 (apop osis)
[137]
WPC, Da isco 0.3 g/kg; 30 d Ra ; s la oxin-con amina ed
die ; li e , es es
"GSH, #LP, #DNA damage;
WPC p e en s geno oxici y
o a la oxins
[138]
WPC, Da isco 0.3 g/kg; 30 d Ra ; a la oxin-con amina ed
die ; li e , es es
"GSH, #LP, #DNA damage;
WPC educed oxida i e
s ess induced by a la oxins
[139]
WPC, P obio ica 0.15 g/kg; 8 weeks Ra ; exe cise model;
muscle, li e
WP inc eased hepa ic GSH
and p o ec ed agains
exe cise induced muscle
p o ein oxida ion
[140]
WPI, Diamond Whey, I aly 20% Mouse; exe cise model;
muscle, li e
#LP; #GSH/GSSG [141]
WPC, Camillo ek, India 0.3 g/kg; 28 d Ra ; e y h ocy e om young
and aged animals
#T-SH; #sialic acid; "ipid
hyd ope oxides; "p o ein
ca bonyls; WPC es o es
edox s a us in e y h ocy es
[142]
WPI, Da isco 0.15 g/kg; 28 d Ra ; non-alcoholic a y li e "hepa ic GSH, #hepa ic MDA [143]
WP, Immunocal 3.3% wice daily; 28 d Mouse; auma ic b ain inju y "GSH/GSSG; p e en ed
educ ion o GSH/GSSG
in b ain
[144]
WPI, Bioplex Nu i ion, USA 100 g; 12 weeks Mouse; b ain mi ochond ia #MDA; #4-hyd oxyalkenals;
inc eased b ain
mi ochond ia ac i i y
[145]
WP, Immunocal 0.66 g/kg; 60 d Mouse; ALS model T ea men p e en ed disease-
associa ed educ ions in
whole blood and spinal
co d issue GSH
[146]
WPC 0.3 g/kg; 28 d Ra ; neu odegene a ion; b ain "Beclin-1; "A g-3 (Au ophagy);
"FRAP; #PC; "T-SH; #ROS;
#NO; "ace ylcholines e ase
[142]
WPI, Immunocal 0.33 g/mL; 6 weeks Mouse; schizoph enia
model; b ain
"GSH/GSSG a ios; "GSH [147]
WP, Tu key WP supplemen ed die ; 21 d Ra ; bu n inju y model;
li e , kidney
Whey p o ein supp essed
bu n-induced changes in
hepa ic and enal issue
[148]
WP, Tu key WP supplemen ed die ; 21 d Ra ; expe imen al lapa o omy
and colonic anas omosis;
li e , abdominal wall, colon
Whey p o ein
supplemen a ion inc eased
GSH and supp essed MDA
in di e en issues
[149]
WPI, Balance Muscle
Technologies, New Zealand
150—250 g/kg; 4 weeks Ra ; HLAP; colon No change DNA damage [150]
WP, mozza ella cheese 10 g/100 g; 6 weeks Ra ; i on o e load; oxici y;
plasma, e y h ocy e
"SOD, "GSH, no change GSH-
Px in e y h ocy es; no
change plasma Vi E; WP
had an ioxida i e and
an igeno oxic e ec s
[7]
WP, Immunocal 0.1 g; 4 weeks Mu ine; i on o e load/ oxici y #MDA; #Hexanal; "GSH-
Px; "GSH
[151]
a
WPI: whey p o ein isola e; WP: whey p o ein, ype no speci ied; WPC: whey p o ein concen a e.
b
GSH: glu a hione; GSH –Px: glu a hione pe oxidase; CAT: ca alase; GPT: glu amic-py u ic acid ansaminase; ALP: alkaline phospha ase; SOD: supe oxide
dismu ase; GST: glu a hione S- ans e ase; GSSG: oxidised glu a hione; LP: plasma lac a e; T-SH: o al hiol; PC: p o ein ca bonyl; ROS: eac i e oxygen
species; NO: ni ic oxide; MDA: malondialdehyde, CCl
4
: ca bon e achlo ide.
FRAP: e ic- educing an ioxidan powe .
1138 L. GIBLIN ET AL.
Table 4. Human in e en ion ials wi h whey p oduc s and physiological esponse.
Bo ine whey p oduc
a
Desc ip ion o he s udy Resul s
b
Re e ence
WPI undena u ed Open-labeled clinical ial, 38 pa ien s wi h
non-alcoholic s ea ohepa i is we e gi en 20 g
whey p o ein isola e o 12 weeks.
Imp o emen s in li e biochemis ies, inc eased
plasma GSH, o al an ioxidan capaci y, and
educed hepa ic mac o esicula s ea osis in
pa ien s compa ed o ime ze o (s udy s a ).
[152]
WPI 23 cance pa ien s ecei ed 4 0g WPI plus
2.64 mg zinc and 0.76 mg selenium o al
snack o 12 weeks compa ed o a con ol
g oup (n¼19) who ecei ed 40 g
mal odex in.
Baseline assessmen s a 6 and 12 weeks.
Con ols showed a signi ican ly lowe %
change in plasma GSH le els whe eas he e
was a signi ican ime-dependen inc ease in
he in e en ion g oup om ime ze o o 12
weeks and be ween whey and mal odex in
in e en ion a bo h 6 weeks and 12 weeks.
[153]
WP A pilo open-label s udy o die a y
supplemen a ion wi h p essu ised whey in
27 cys ic ib osis pa ien s. Whey dosage:
20 g/d in pa ien s <18 yea s and 40 g/d in
olde pa ien s o 1 mon h.
Whole blood glu a hione le els did no change
om ime ze o o 1 mon h. O al
supplemen a ion wi h p essu ised whey
imp o es nu i ional s a us and can ha e
addi ional bene icial e ec s on in lamma ion
in pa ien s wi h cys ic ib osis.
[8]
WPC immunocal 10 cys ic ib osis pa ien s ecei ed whey
p o ein isola e (20 g/d) o 3 mon hs
compa ed o a cys ic ib osis con ol g oup
(n¼11) who ecei ed a casein placebo.
46.6% inc ease om ime ze o was obse ed in
he lymphocy e GSH le els wi h whey.
[154]
WPI 5 d o ene gy balance, ene gy de ici , and
esis ance exe cise a e ene gy de ici . 8
males and 7 emales heal hy esis ance-
ained subjec s comple ed esis ance
exe cise and consumed ei he placebo o
30 g whey p o ein immedia ely pos -exe cise.
Muscle biopsies we e ob ained a 1 and 4 h
in o eco e y in each ial. Res ing p o ein
le els o au ophagy- ela ed gene p o ein 5
dec eased a e ene gy de ici compa ed
wi h placebo.
[155]
WPI Acu e s udy 4 h: Heal hy, 9 seden a y male
subjec s ed wo doses (0.8 o 1.6 g/kg
body mass).
No e ec o he supplemen a ion a ei he
dose o e he 4-h sampling pe iod on blood
glu a hione concen a ion.
[9]
Ch onic s udy: 18 males subjec ed o 6-week
ae obic (bike) aining pe iod and whey
supplemen a ion
The ae obic aining pe iod esul ed in
signi ican ly lowe glu a hione
concen a ions in whole blood, an e ec
ha was mi iga ed by WPI supplemen a ion.
WPI WPI (40 g/d) we e supplemen ed o 31
o e weigh people wi h impai ed as ing
glucose/DM2 o 12 weeks.
An inc ease in glu a hione pe oxidase, a
dec ease in u ic acid and no change in
glu a hione educ ase, o al an ioxidan
s a us, oxida i e damage, in lamma ion and
glucose ma ke s we e obse ed a 12 weeks
compa ed o ime ze o.
[156]
Signi ican imp o emen s in an h opome ic
pa ame e s and a mass we e also de ec ed.
WPI Whey p o ein supplemen a ion (30 g, h ee
imes pe day) and esis ance aining in 10
o e weigh young men compa ed o a
con ol g oup and esis ance aining
only g oup.
Inc eased le els o o al an ioxidan capaci y
and GSH was obse ed a e whey
supplemen a ion compa ed o o he
ea men g oups and compa ed o p e- es .
Plasma i amin C le els we e signi ican ly
inc eased wi h whey supplemen a ion
compa ed o p e- es . Al hough exe cise can
lead o an ioxidan sys em imp o emen and
educe some ca dio ascula isk ac o s
among o e weigh subjec s, he combina ion
o esis ance aining and whey consump ion
was mo e e ec i e.
[157]
WPI immunocal The e ec s o WPI supplemen a ion (20 g/d) o
6 mon hs on 15 Pa kinson’s disease pa ien
was compa ed o 16 pa ien s on soy p o ein
supplemen a ion.
Signi ican inc eases in plasma concen a ion o
educed GSH and he a io o educed o
oxidised glu a hione we e ound in he 15
WPI-supplemen ed pa ien s compa ed o
baseline. This was associa ed wi h a
signi ican dec ease o plasma le els o
homocys eine.
[158]
WPI immunocal o i o ec amin O al supplemen a ion wi h wo di e en Cys-
ich whey p o ein o mulas o 30 HIV-
in ec ed pa ien s. Pa ien s we e andomised
o a supplemen al die wi h a daily dose o
45 g o whey p o eins.
O al supplemen a ion wi h P o ec amin
signi ican ly inc eased plasma GSH le els in
pa ien s wi h ad anced HIV-in ec ion
compa ed o baseline.
[159]
WPC A double-blind clinical ial o 4 mon hs wi h 9
HIV-in ec ed child en (6 yea s) who ecei ed
whey p o ein (mon h 1 ¼20% RDA p o ein,
mon h 2 ¼30% RDA, mon h 3 ¼40% RDA,
WPC supplemen a ion signi ican ly inc eased
e y h ocy e GSH le els and signi ican ly
dec eased CD8þcells compa ed o baseline.
[160]
(con inued)
FREE RADICAL RESEARCH 1139

ga age o adul male Sp ague Dawley a s (n¼6) wi h
he en i onmen al pollu an ac olein (0.005 g/kg
body weigh /d) o 30 d esul ed in a signi ican
dec ease in GSH le els (8.38 ± 1.17 nmol/mg p o ein) in
ed blood cells compa ed o he con ol g oup
(11.31 ± 1.63 nmol/mg p o ein) (p<.05) [12]. Addi ion
o whey p o ein a a dosage o 0.2 g/kg body weigh /d
did no hal he loss o GSH. In addi ion, his co- ea -
men wi h whey p o ein exace ba ed an obse ed
inc ease in plasma homocys eine le els and c ea ine
kinase le els induced by ac olein, such ha he le els
o hese ma ke s in he co- ea men g oup we e signi i-
can ly highe han in he con ols. This led he au ho s
o hypo hesise ha in ake o ac olein oge he wi h
whey p o eins may cause hea damage in a s [12].
Li e may also be ad e sely a ec ed by whey ea -
men . G€
u gen e al. [13] in es iga ed li e heal h in
Wis a albino male a s (n¼10) a e a whey p o ein
die o 5 d (sho - e m) o 4 weeks (long- e m). Hepa ic
inju y was obse ed by abno mal hepa ocy e his ology
and signi ican ly inc eased le els o se um aspa a e
amino ans e ase and hepa ic in e luekin-1bin a s
ha consumed whey compa ed o he con ol g oup,
wi h he ma ke s o li e inju y wo sening wi h inc eas-
ing ime o exposu e o whey p o ein die (p<.05).
Bioa ailable an ioxidan whey pep ides
In addi ion o p o iding essen ial AAs and educed
hiols (Cys esidues), whey p o eins also con ibu e pep-
ides wi h po en ial an ioxidan ac i i y [5,14,15].
Howe e o be bioac i e beyond he gu , whey pep i-
des mus su i e gu ansi and be bioa ailable o hei
a ge [16]. The ha sh condi ions o he uppe gas o-
in es inal ac unc ion o hyd olyse p o eins in o indi-
idual AAs o anspo ac oss he in es inal ba ie .
Se e al ecen s udies ha e acked he a e o whey
p o eins du ing uppe gas oin es inal diges ion using
in i o diges ion models [14,17–20] o gas ic [18,21]
and jejunal e luen s [22,23] om pigs [18,21,24] and
humans [19,22,23] pos consump ion o a ious dai y
oods (IF, WPI, skim milk powde , unpas eu ised milk,
whey powde , and lac o e in). Based on hese s udies,
Table 5 lis s loca ions wi hin indi idual whey p o eins
om which pep ides ha e been iden i ied in he in es-
inal phase. b-Lg has ou gu - esis an “ho spo s;”con-
sis ing o agmen s ( ) wi h he ollowing amino acid
esidues om he p ima y sequence (41–58), (92–100),
(126–138) and (149–154). a-Lac also has 4 ho spo s;
(17–27), (63–68), (80–90) and (97–102). BSA has 5;
(11–18), (107–114), (219–224), (489–495) and
(514–518). Lac o e in has 7; (67–77), (140–145),
(216–228), (289–295), (309–318), (332–337) and
(592–594). This sugges s ha hese egions o he p i-
ma y sequence a e somewha esis an o gas oin es-
inal diges ion and pep ides om hese ho spo s may
su i e he gu long enough o be anspo ed ac oss
he in es inal ba ie . Ce ainly, he p esence o p oline
and/o aspa ic acid, o glu amic acid esidues wi hin a
pep ide appea s o con e a esis ance o gas oin es-
inal diges ion [23]. I should also be no ed ha pep ide
p o iles di e acco ding o deg ee o p ocessing [24].
Table 5 also de ails whey pep ides ha ha e been
epo ed o ha e po en ial edox ac i i y enc yp ed
wi hin hese ho spo s [5,25–35]. These whey pep ides
ha e been epo ed o show edox ac i i y in he e ic-
educing an ioxidan powe assay (FRAP), 2,20-azino-
bis(3-e hylbenzo hiazoline-6-sul onic acid) (ABTS) assay,
oxygen adical abso bance capaci y assay (ORAC) o he
1,1-diphenyl-2-pic ylhyd azyl (DPPH) adical assay. I
should howe e be no ed ha all o hese assays a e
gene ic in i o assays conduc ed in he absence o
al e na i e a ge s, and hence he da a canno be ead-
ily ansla ed o mo e complex sys ems. In some cases,
howe e , hey ha e also been shown o boos cellula
an ioxidan s a us, albei in i o [5,15]. C oss ma ching
gas oin es inal esis an ho spo s o an ioxidan bio-
ac i i y allows us o p opose a lis o po en ial an ioxi-
dan whey pep ides likely o a i e in he bloods eam,
pos whey consump ion. Li le is known abou pep ides
de i ed om whey ci cula ing in he bloods eam.
Jakobsson e al. [36] quan i ied a-Lac (140–250 mg/L
se um/L human milk/kg body weigh ) by adioimmuno-
assay in blood plasma om 1 mon h old b eas ed
Table 4. Con inued.
Bo ine whey p oduc
a
Desc ip ion o he s udy Resul s
b
Re e ence
mon h 4: 50% RDA) compa ed o
con ol g oup.
WPI, p essu ised 2-week pe iod, 18 heal hy males and 18
heal hy emales we e andomised in o h ee
di e en g oups. Each g oup inges ed 15,
30, o 45 g/d p essu ised whey p o ein in
he mo ning in ba o ma o 14 d.
Inc eases in lymphocy e GSH le els om p e o
pos supplemen a ion was a ec ed by he
amoun o whey p o ein inges ed, wi h
45 g/d o whey supplemen a ion o e
2 weeks inc easing lymphocy e GSH by 24%
om baseline.
[161]
a
WPI: whey p o ein isola e; WP: whey p o ein, ype no speci ied; WPC: whey p o ein concen a e.
b
GSH: glu a hione.
1140 L. GIBLIN ET AL.
in an s (n¼3), 30–60min a e eeding. Kui unen e al.
[37] also epo ed ha plasma om 20 ull e m in an s
was posi i e o a-Lac and b-Lg, le els o which
declined o e ime (almos 60% educ ion by 8 mon hs
o age) as he in an gu ba ie ma u ed. No in o ma-
ion on he sequences o he bioa ailable pep ides was
p o ided. Howe e , we ha e ecen ly iden i ied a num-
be o an ioxidan whey pep ides pos in i o gas o-
in es inal diges ion capable o a e sing he igh
junc ions o Caco
2
-HT29 monolaye s (a widely accep ed
model o he in es inal ba ie ) and a i ing in he
basola e al compa men [15].
Redox modi ica ion o whey du ing p ocessing
As whey p oduc s ypically unde go se e al p ocessing
s eps om milk o inal ood ma ix, he e is subs an ial
e idence ha p ocessing can induce o exace ba e
edox eac ions ha modi y whey p o eins. A he ou -
se , milk p oduced o consume consump ion is hea -
ea ed o kill bac e ia and inc ease shel li e. Mul iple
me hods a e used including low (e.g. 15 s a 74 C),
high (e.g. 15 s a 90C), and ul ahigh (e.g. 145 C o a
ew seconds) empe a u e ea men s. Mos comme cial
milk is also homogenised by high p essu e ea men o
educe a globule size. Bo ine liquid whey is hen p o-
duced by ei he he enzyma ic ea men o milk (swee
whey) o by he addi ion o acids o mine als (acid
whey) bo h o which esul in he p ecipi a ion, and
he e o e emo al, o caseins. Signi ican modi ica ions
on whey p o eins occu wi h p ocessing, due o hea
ea men (pas eu isa ion and sp ay d ying), exposu es
o high p essu es (e.g. du ing homogenisa ion), ligh
exposu e, he use o s e ilisa ion/disin ec ion agen s
such as H
2
O
2
(pe mi ed a concen a ions 16 mM in
he USA [38]), high pH alues (employed o gi e hyd o-
lysed p o ein samples e.g. o IF) and long- e m expos-
u e o educing suga s (e.g. lac ose, glucose, galac ose.
and p oduc s om hese).
Oxida ion, glyca ion, and acemisa ion o
whey p o eins
The mal ea men o milk and milk p oduc s can esul
in a signi ican inc ease in he le el o oxida i e modi i-
ca ion and o ma ion o p o ein ca bonyls (wi h he
Table 5. Whey pep ide sequences esis an o uppe gas oin es inal diges ion and an ioxidan pep ides enc yp ed wi hin
hese sequences.
Whey p o ein
a,b
Ho spo s: loca ion o pep ide sequences esis an
o gas oin es inal diges ion
c
An ioxidan whey pep ides wi hin hese ho spo s
b,c,d
b-Lg
[14,20,22,23,162]
(41–58) (40–48), (42–46), (42–47), (43–49), (43–51), (45–57),
(50–56), (52–61), (55–61), (56–62), (58–61)
[26–28,32,33]
(92–100) (89–96), (92–100), (94–100), (95–101), (95–110), (96–105),
(96–100)
[25,27–29]
(126–138) (122–131), (122–134), (123–131), (123–134), (123–135),
(124–131), (125–135)
[25,30]
(149–154) (145–149), (149–156), (150–160), (151–162)
[26,27,31]
a-Lac
[14,20,22,23]
(17–27) (13–19), (15–22), (16–23), (19–20), (25–26)
[27,33]
(63–68)
(80–90) (82–88)
[27]
(97–102) (99–102), (101–104)
[15,34]
BSA
[14,22]
(11–18)
(107–114)
(219–224)
(489–495)
(514–518)
Lac o e in
[14,19]
(67–77)
(140–145)
(216–228) (228–229)
[35]
(289–295)
(309–318)
(332–337)
(592–594)
a
Whey p o eins: b-Lac oglobulin (b-Lg), a-Lac albumin (a-Lac), and bo ine se um albumin (BSA).
b
Re e ences a e in squa e b acke s.
c
Pep ide loca ion ( agmen ) on p ima y sequence o whey p o ein is s a ed as name o whey p o ein, agmen (posi ion om amino acid o amino acid)
o example b-Lg (41–58).
d
Amino acid in i alic indica es he s a o end o he an ioxidan pep ide sequence alls ou side he gu esis an ho spo s.
FREE RADICAL RESEARCH 1141
si es o some o hese cha ac e ised [39]) and c osslinks.
The le els o hese ma e ials ha e been p oposed as a
ma ke o milk powde quali y [40]. The highes le els
o p o ein oxida ion p oduc s ha e been epo ed o be
p esen in powde ed IF [39,41–44]. a-Lac has been
epo ed o show enhanced oxida ion compa ed o
b-Lg [45]. A ecen s udy has epo ed ha he mal
ea men can induce educible (disulphide) c osslinks
in isola ed b-Lg (100 mM), bu no isola ed a-Lac
(150 mM) in bo h he absence and p esence o H
2
O
2
(500 mM), which appea s o be associa ed wi h he p o-
ein un olding and he accessibili y o he ee Cys-121
esidue on b-Lg [46,47]. Blocking his hiol p e en ed
c oss-link o ma ion, unde lining he impo ance o his
esidue. Mixed c oss-links be ween b-Lg and a-Lac we e
de ec ed when bo h p o eins we e co- ea ed [47].
Disulphide c oss-links can also be gene a ed ia hiol-
disulphide exchange (i.e. nonoxida i e) eac ions, wi h
hese occu ing ia a ack o a ee hiol (and usually he
mo e eac i e anion o m) on a disulphide bond, wi h
his esul ing in an exchange o pa ne s, and hence
c oss-link o ma ion [48,49].Thisp ocessoccu smo e ap-
idly a highe empe a u es and on p o ein un olding, as
his inc eases he accessibili y o eac ion si es [48]. These
eac ions can be enhanced by he addi ion o ee hiols
[50] and highe Ca
2þ
concen a ions [51].
Exposu e o bo h hea and H
2
O
2
appea s o ha e a
g ea e e ec han ei he agen alone, wi h his
asc ibed o he occu ence o a mechanism in ol ing
he o ma ion o a sul enic acid (RSOH species) a
Cys121 in b-Lg media ed by H
2
O
2
, once his esidue is
made accessible by he mal un olding [46,47]. Limi ed
loss o Me and T p (a e y high H
2
O
2
concen a ion)
has also been de ec ed wi h bo h b-Lg and a-Lac on
ea men wi h hea and H
2
O
2
[46,47]. Inc eased expos-
u e o hea (longe imes and highe empe a u es) and
highe concen a ions o H
2
O
2
ha e been shown o
enhance he ex en o modi ica ion, as do combina ions
o hese wo ac o s [46,47]. No p o ec ion agains dam-
age was de ec ed when adical apping agen s we e
included, indica ing ha hese a e molecula ( wo elec-
on) and no adical (one elec on) eac ions [46].
These da a indica e ha oxida i e damage can be
selec i e wi h ega d o he esidues ha a e modi ied,
and ha un olding o he p o eins is a c i ical ac o
wi h ega d o he ex en o damage. Dec easing hea
loads and oxidan exposu e would he e o e be
expec ed o minimise whey p o ein modi ica ion.
Pho o-oxida ion a ising om ligh exposu e o milk
p epa a ions con aining ibo la in ( i amin B
2
,an
endogenous componen o milk) can induce changes in
milk p o ein s uc u e (e.g. polyme isa ion, seconda y
and e ia y s uc u e o speci ic p o eins), as well as
inducing he o ma ion o p o ein ca bonyls, he Ty
oxida ion p oduc s, di- y osine (di-Ty ) and 3,4-dihy-
d oxyphenylalanine (DOPA), and he T p-de i ed spe-
cies, N- o mylkynu enine (NFK) and kynu enine (Kyn)
[52,53]. Di e en oxidan sys ems can he e o e gene -
a e di e en pa e ns and ex en s o damage, and
in ol e al e na i e mechanisms, hough he o e all
e ec s (e.g. agg ega ion) may be simila . Ligh exposu e
has been used as a non he mal echnology o con ol
pa hogens and ex end p oduc shel -li e bu his may
esul in inc eased o ma ion o ca bonyls and hence
p o ein damage [54].
P esence o oxida ion, glyca ion, and
acemised whey p o eins in IF
Whey p o ein modi ica ion is a po en ial heal h isk
o in an s ed wi h IF, and he e is con incing da a
ha indica es ha b eas milk has conside able heal h
bene i s ( e iewed [55]). Table 6 summa ises he la ge
numbe o di e en ypes and le els o p o ein modi-
ica ions de ec ed in IF samples. A a mac omolecula
le el, hese include bo h educible (p esumed o be
disulphide-linked species, due o hei loss on dio hio-
h ei ol ea men ) and (mul iple ypes o ) non educi-
ble p o ein agg ega es [56]. E idence has been
p esen ed o he c oss-linked species di-Ty (a species
a ising om adical eac ions), as well as lysinoalanine
(LAL), lan hionine (LAN), and he p ecu so species o
he la e wo p oduc s, dehyd oalanine (DHA) (Table
6)[56,57]. Whe he he DHA a ises om base-ca a-
lysed o adical-media ed eac ions a e unknown.
Signi ican le els o p o ein ca bonyls a e also p esen
in he samples, as de ec ed by assaying o al ca bonyl
con en and also by using immunoblo ing on p o-
eins sepa a ed by SDS-PAGE, wi h he la e expe i-
men s indica ing ha a signi ican quan i y o he
ca bonylsisp esen onhigh-molecula -massagg e-
ga es [56]. The exac na u e o he p o eins in ol ed
and he si es wi hin hese p o eins emain o be
de e mined, hough i is clea ha he e a e signi i-
can s uc u al changes o he IF p o eins induced by
p ocessing [58].
Modi ica ions ha e also been de ec ed on IF p o eins
a he AA le el, wi h e idence epo ed o he o ma-
ion o T p oxida ion p oduc s (N- o mylkynu enine,
NFK; kynu enine, Kyn; and 3-hyd oxykynu enine,
3OHKyn), di-Ty , Phe-de i ed ma e ials (3-hyd oxyPhe,
m-Ty ) and he Me oxida ion p oduc , me hionine sul -
oxide [56,59]. Some o hese species a e consis en wi h
he species de ec ed on isola ed whey p o eins
1142 L. GIBLIN ET AL.
exposed o speci ic oxidan sys ems (e.g. hea /H
2
O
2
and
also ligh in he p esence o ibo la in) [46,47,52,60,61].
A numbe o he species de ec ed a e consis en wi h
he occu ence o adical eac ions (e.g. di-Ty , which
appea s o be only o med by adical eac ions), bu
o he s (e.g. me hionine sul oxide) can be o med by
bo h adical and molecula ( wo elec on) p ocesses
(e.g. di ec oxida ion by H
2
O
2
)[62]. A numbe o hese
ma e ials may unde go addi ional eac ions (e.g. edox
cycling) ha may exace ba e damage [63].
Fu he mo e, some o hese p oduc s (e.g. hose om
T p) may ha e mul iple biological ac i i ies and con ib-
u e o disease, i aken up [64].
IF also con ain signi ican le els o ad anced glyca ion
endp oduc s (AGE) ma e ials including he ea ly s age
p oduc u osine, and well-cha ac e ised AGEs including
N
E
-(ca boxyme hyl)lysine (CML), N
E
-(ca boxye hyl)lysine
(CEL), py aline, and p o ein c osslinks, such as pen osi-
dine ( om Lys and A g esidue linked by a pen ose),
glyoxal lysine dime (GOLD), me hylglyoxal lysine dime
(MOULD), and A g-de i ed p oduc s such as a gpy imidine
and hyd oimidazolone isome s [56,65–72]. The le els o
hese ma e ials ha e been examined using a wide a ie y
o bo h di ec and indi ec me hodologies [66,73,74]. AGE
is a chemically he e ogeneous g oup o compounds. AGE
o ma ion in dai y p oduc s in ol e Mailla d eac ions, bu
Table 6. Selec ed alues o concen a ions (nmol/mg p o ein excep whe e o he wise indica ed) o p o ein oxida ion p oduc s,
ad anced glyca ion endp oduc s (AGEs) and acemised AAs p esen in powde ed in an o mula samples.
Concen a ions o oxida ion p oduc s in in an o mula samples
P oduc s In ac p o ein IF Hyd olysed p o ein IF
P o ein ca bonyls 2.88
a
8.6–60.9 [41]
19.4 [42]
6.5 [40]
0.85
b
18.3 [42]
Dehyd oalanine (DHA) 0.47
a
0.58
b
Lan hionine (LAN) 0.01
a
0.09
b
Lysinoalanine (LAL) 0.03
a
Below limi o de ec ion [57]
18.2 ng mg
1
p o ein [42]
150–920 ng mg
1
p o ein [163]
Highe alues de ec ed in liquid IF
samples [164,165]
0.29
b
63.3 ng mg
1
p o ein [42]
Me a- y osine 0.97
a
1.83
b
Kynu enine 1.58
a
4.54
b
3-hyd oxykynu enine 2.82
a
7.40
b
Me hionine sul oxide 64.97
a
24.82
b
Di y osine 3.12
a
2.95
a
1.3 [42]
6.46
b
1
b
6.8 [42]
Ca boxyme hyllysine (CML) 36.3
a
ng mg
1
p o ein
60.1 ng mg
1
p o ein [42]
12 and 145 ng mg
1
o low- empe a u e
ea ed, and high- empe a u e hea ed IF
espec i ely [125]
1.2 ng mg
1
p o ein [66]
81.0
b
ng mg
1
p o ein
212.4 ng mg
1
p o ein [42]
Fu osine 2.3
a
mgmg
1
p o ein
5.6 mgmg
1
p o ein [42]
2.7 mgmg
1
p o ein [66]
8.0–19.4 mgmg
1
p o ein [74]
13.215.5 mgmg
1
p o ein [166]
0.21–0.81 mgmg
1
p o ein [167]
0.251.05 mgmg
1
p o ein [168]
7.0 mgmg
1
p o ein [169]
3.5
b
(mgmg
1
p o ein)
2.4 mgmg
1
p o ein [42]
2.6 mgmg
1
p o ein [74]
0.52–0.81 mgmg
1
p o ein [167]
0.60.9 mgmg
1
p o ein [168]
D-His 2.7
a,c
2.5
b,c
D-Lys 1.6
a,c
4.3
b,c
D-Phe 1.4
a,c
3.4
b,c
D-Ty 1.1
a,c d
D-A g 5.5
a,c
32.0
b,c
D-Ala 1.3
a,c d
D-Ile 0.3
a,c d
D-Leu 0.7
a,c
1.4
b,c
D-Me 7.2
a,c d
D-Se 0.5
a,c d
D-Val 4.3
a,c
0.3
b,c
Da a om [113] excep whe e o he wise indica ed.
a
Mean da a om h ee comme cial b ands.
b
Da a om h ee eplica e measu emen s om a single comme cial b and.
c
Da a exp essed as % o D-isome ela i e o o al (D þL) isome s.
d
No de ec ed.
FREE RADICAL RESEARCH 1143
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