CLutev 3: Heut-Indued useIn-wLey voteIn

IntevutIons In mIIk

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HonfIng of mIIk Is nn ossonfInI sfop In fho procossIng of vnrIous
dnIrv producfs, IIko for oxnmpIo voghurf. A mnjor consoquonco of fho
honf fronfmonf Is fho donnfurnfIon of whov profoIns whIch oIfhor
nssocInfo wIfh fho cnsoIn mIcoIIo or form soIubIo whov profoIn
nggrognfos. ThIs pnpor doscrIbos n qunnfIfnfIvo sfudv on fho
dIsfrIbufIon of donnfurod whov profoIns ovor whov profoIn nggrognfos
nnd cnsoIn mIcoIIos. Tho dIsfrIbufIon Is fhoughf fo bo roInfod fo
chnngod ncId-Inducod goInfIon proporfIos nnd foxfurnI proporfIos of
mIIk dorIvod producfs. QunnfIfnfIvo doscrIpfIon wIII nIIow boffor
confroI nnd funIng of fho fInnI goI proporfIos.
Iv combInnfIon of onzvmnfIc frncfIonnfIon nnd cnpIIInrv
oIocfrophorosIs wo woro nbIo fo qunnfIfnfIvoIv doformIno fho
dIsfrIbufIon of donnfurod whov profoIns nffor honf fronfmonf. Moro
sovoro honf fronfmonf nf fho nnfurnI pH of mIIk cnusod moro
donnfurnfIon, buf fho rnfIo of donnfurod whov profoIns nssocInfod
wIfh fho cnsoIn mIcoIIo nnd prosonf In nggrognfos romnInod consfnnf.
Wo cIonrIv domonsfrnfod fhnf fho obsorvod shIff In goInfIon pH of
honfod mIIk Is IInonrIv corroInfod wIfh fho fwo frncfIons of donnfurod
whov profoIns.


A.J. VnsbIndor, A.U. AIfIng, U.C. do KruIf
Accopfod for pubIIcnfIon In UoIIoIds nnd Surfncos I
Uhnpfor 3
34
,QWURGXFWLRQ
Tho mnIn ronson for honf fronfmonf of mIIk Is fo Improvo Ifs
koopIng qunIIfv bv docronsIng fho numbor of IIvIng mIcroorgnnIsms
nnd fo nchIovo dosIrnbIo proporfIos In fho fInnI producf (33). !pon
honf fronfmonf of mIIk nbovo 60qU sovornI procossos fnko pInco, of
whIch donnfurnfIon of whov profoIns Is fho mosf obvIous (?, 26, 2?,
34). Tho mosf nbundnnf whov profoIn Is E-IncfogIobuIIn (E-Ig) In
whIch n honf-Inducod conformnfIonnI chnngo oxposos n roncfIvo fhIoI
group. ThIs fhIoI group cnn form dIsuIfIdo bonds wIfh ofhor cvsfoIno-
confnInIng profoIns, IIko E-Ig or bovIno sorum nIbumIn, or wIfh
profoIns hnvIng dIsuIfIdo brIdgos, IIko D-IncfnIbumIn (D-Inc), N- nnd
D
s2
-cnsoIn. Tho Inffor procoss occurs fhrough fhIoI group-dIsuIfIdo
brIdgo oxchnngo roncfIons, rosombIIng n poIvmorIsnfIon procoss
whoro honf-donnfurod E-Ig Is fho InIfInfor (l2, l6, l?, 30, 39).
InforncfIon of E-Ig wIfh N-cnsoIn, prosonf nf fho oxforIor of fho cnsoIn
mIcoIIo IIko n hnIrv brush, Ionds fo confIng of fho cnsoIn mIcoIIos wIfh
E-Ig. InforncfIons of E-Ig wIfh cvsfoIno-confnInIng sorum cnsoIns
mIghf Iond fo cnsoIn-whov profoIn nggrognfos. AddIfIonnIIv,
InforncfIons of E-Ig wIfh cvsfoIno-confnInIng whov profoIns, IIko D-Inc
nnd E-Ig moIocuIos, rosuIf In fho formnfIon of whov profoIn
nggrognfos. To summnrIso, honf fronfmonf of mIIk rosuIfs In n
compIox mIxfuro of nnfIvo whov profoIns nnd donnfurod whov
profoIns prosonf ns whov profoIn nggrognfos, cnsoIn-whov profoIn
nggrognfos nnd whov profoIn confod cnsoIn mIcoIIos.
Honf fronfmonf of mIIk hns n profound offocf on fho ncId-
Inducod goInfIon proporfIos. If cnusos n shIff In goInfIon pH fownrds
hIghor pH vnIuos (ll, l5, 23, 39). ThIs Is nffrIbufod fo fho InforncfIon
of E-Ig wIfh fho cnsoIn mIcoIIo. E-Ig fonds fo noufrnIIfv nf n hIghor pH
fhnn cnsoIn mIcoIIos (5.2 Insfond of 4.6) (39). CoIs formod of honfod
mIIk hnvo nn Incronsod goI hnrdnoss nnd hIghor sforngo moduIus (C`)
(23, 24, 28, 40, 42) nnd shows Ioss suscopfIbIIIfv fo svnorosIs (8, 9)
compnrod fo goIs mndo of unhonfod mIIk. Tho Incronsod goI hnrdnoss
Is pnrfInIIv cnusod bv fho chnngod surfnco proporfIos of fho whov-
profoIn-confod cnsoIn mIcoIIos nnd pnrfInIIv bv fho dIsuIfIdo
InforncfIons occurrIng durIng fho goI sfnfo (40). If Is gonornIIv
nssumod fhnf fho nIforod proporfIos of honfod mIIk nro roInfod fo fho
fofnI dogroo of whov profoIn donnfurnfIon. If Is known fhnf fho
composIfIon of fho compIox mIxfuro of donnfurod whov profoIns nnd
Uhnpfor 3
35
cnsoIn mIcoIIos Is dopondonf on fho pH of honf fronfmonf,
fompornfuro, fofnI profoIn conconfrnfIon, fho rnfIo bofwoon cnsoIn
mIcoIIos nnd whov profoIns, fho snIf conconfrnfIon nnd so on (2, 3, 6,
?, l0, 20, 2l, 22, 25, 2?, 35). Howovor, IImIfod rosonrch hns boon
porformod whIch roInfos fho chnngod goInfIon proporfIos of honfod
mIIk fo fho pnrfIfIon of donnfurod whov profoIns bofwoon nggrognfos
nnd mIcoIInr confIng.
In ordor fo sfudv fhIs roInfIon, frncfIonnfIon of fho donnfurod
whov profoIn Is roquIrod ns fhIs wIII onnbIo qunnfIfnfIvo
monsuromonfs of fho dIsfrIbufIon of whov profoIns In nggrognfos nnd
ns confIng. In mosf pnpors uIfrnconfrIfugnfIon Is usod ns n
frncfIonnfIon fochnIquo, quIfo offon In combInnfIon wIfh donsIfv
monsuromonfs of SS-IACI goIs (6, l0, 34, 2?). Iv usIng
uIfrnconfrIfugnfIon ns n frncfIonnfIon fooI vorv Inrgo g-forcos nro
nppIIod on fho snmpIo, cnusIng n rIsk of procIpIfnfIng soIubIo whov
profoIn nggrognfos. oh nnd !Ichnrdson (25) proposod nn nIfornnfIvo
frncfIonnfIon mofhod bnsod on ronnofIng, whIch hns fho mnjor
ndvnnfngo fhnf Iow g-forcos nro roquIrod. In fhIs sfudv wo usod fhIs
frncfIonnfIon mofhod In fho nbsonco of cnIcIum fo provonf fho rIsk of
cnIcIum-Inducod procIpIfnfIon of whov profoIn nggrognfos (5, l4). Wo
combInod fhIs frncfIonnfIon mofhod wIfh n moro nccurnfo nnnIvsIs
fochnIquo fhnn fho curronfIv usod mofhods, nnmoIv cnpIIInrv
oIocfrophorosIs (UI). SS-ngnroso goI oIocfrophorosIs (l) wns usod fo
chnrncforIso fho nggrognfos In fho supornnfnnf wIfh rospocf fo sIzo
nnd fo sfudv fho InforncfIons occurrIng bofwoon cnsoIn mIcoIIos nnd
whov profoIns. Tho rosuIfs of fhIs qunnfIfnfIvo sfudv, porformod wIfh
n unIquo combInnfIon of n mIId frncfIonnfIon mofhod usIng onzvmos
nnd qunnfIfIcnfIon bv UI, nro roInfod fo fho chnngod goInfIon
chnrncforIsfIcs of mIIk honfod nf fompornfuros rnngIng from ?0 fo
90qU.
0DWHULDOVDQG0HWKRGV
SkIm mIIk
SkIm mIIk honfod for l0 soconds nf ?2U wns obfnInod from fho
pIIof pInnf nf IZO food rosonrch, Ido. SodIum nzIdo (0.02 w/w) wns
nddod fo provonf bncforInI growfh nnd fho mIIk wns sforod nf 4qU.
Uhnpfor 3
36
WLey PvoteIn IsoIute soIutIon
Whov IrofoIn IsoInfo (IIpro; nvIsco Iood InfornnfIonnI Inc.,
!SA) soIufIon wns propnrod nf n conconfrnfIon of 9 (w/w). As
doformInod bv TuInIor (38) fho IsoInfo powdor consIsfod of E-Ig (?l),
D-Inc (l2), bovIno sorum nIbumIn (5) nnd ImmunogIobuIIn (5).
Tho fofnI nmounf of profoIns In fho powdor Is 93 of whIch 92 Is
nnfIvo, I.o. soIubIo nf pH 4.6. If furfhor confnIns Incfoso (0.3), nsh
(l.8) nnd wnfor (5). Tho whov profoIn soIufIon wns sfIrrod for 2
hours nf room fompornfuro nnd subsoquonfIv fIIforod fhough n
0.45Pm fIIfor; 0.02 (w/w) n
3
wns fhon nddod. HonfIng fhIs
soIufIon for 2 hours nf 68.5U Iod fo fho formnfIon of nggrognfos wIfh
n hvdrodvnnmIc dInmofor of 62 nm whIch wns doformInod bv
dvnnmIc IIghf scnfforIng (l).
Heut tveutment oI mIIk
CInss fubos (voIumo 8 mI, dInmofor l cm) woro fIIIod wIfh 5 mI
of mIIk, cIosod wIfh n scrow cnp nnd honfod for l0 mIn In n wnfor-
bnfh nf 80U nnd cooIod wIfh fnp wnfor fo room fompornfuro.
IvutIonutIon oI wLey voteIns
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An nmounf of 0.4 g of mIIk wns mIxod wIfh 0.8 g of dIsfIIIod
wnfor (40U) nnd 40 PI ncofIc ncId (l0) In nn Ippondorf fubo (2 mI).
Affor mIxIng (vorfox) nnd l0 mIn wnIfIng, 40 PI of sodIum ncofnfo
(lM) nnd 0.?2 g of dIsfIIIod wnfor woro nddod nnd fho soIufIon wns
mIxod ngnIn. Affor l hour of IncubnfIng nf nmbIonf fompornfuro fho
soIufIon wns confrIfugod for 5 mIn nf 3000g. Tho conconfrnfIon of
nnfIvo profoIn prosonf In fho supornnfnnf wns doformInod bv
cnpIIInrv oIocfrophorosIs (UI).
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!onnofIng of fho mIIks wns porformod ossonfInIIv bnsod on fho
mofhod of oh of nI. (25), buf wIfhouf nddIfIon of cnIcIum chIorIdo.
Tho conconfrnfIon of whov profoIns, I.o. nnfIvo nnd nggrognfod, wns
doformInod bv UI.
Uhnpfor 3
3?
CuIIIuvy eIetvoLovesIs
Tho frncfIonnfod snmpIos woro nnnIvsod bv UI (Iockmnn
I/Aco 5000; Iockmnn UouIfor Inc.) wIfh n cnpIIInrv (AgIIonf, PSII-
wnx, InfornnI dInmofor 0.05 mm) of 60 cm Iongfh. Tho snmpIos woro
Injocfod durIng 20 soconds wIfh n prossuro of 0.5 psI. Tho
oIocfrophorosIs wns cnrrIod ouf nf 45U usIng n voIfngo of 25 kV
fownrds fho cnfhodo nnd dofocfIon wns nf 2l4 nm. Tho oxporImonfs
woro porformod In buffor confnInIng 6M uron nnd dIfhIofhroIfoI
(TT).
PveuvutIon oI SS ueI eIetvoLovesIs sumIes
Tho supornnfnnf obfnInod nffor confrIfugnfIon of fho ronnof-
fronfod mIIk nnd fho mIIks fhomsoIvos woro dIIufod l fo l wIfh 20
mM IIs-TrIs buffor nnd 5 SS (pH ?.0) (l); fho procIpIfnfo obfnInod
nffor confrIfugnfIon of fho ronnof-fronfod mIIk wns soIubIIIzod bv
ropIncIng fho supornnfnnf wIfh nn oqunI voIumo of fho IIs-TrIs
buffor. AII snmpIos woro hoId ovornIghf nf nmbIonf fompornfuro,
whIIo fho procIpIfnfos dIsporsod In buffor woro consfnnfIv sfIrrod. In
somo oxporImonfs n fronfmonf wIfh fho dIsuIfIdo-roducIng ngonf TT
(0.05) wns cnrrIod ouf fo roduco fho dIsuIfIdo bonds In soIubIIIzod
snmpIos.
Auuvose ueI eIetvoLovesIs
SS-ngnroso goIs woro propnrod nf n conconfrnfIon of 0.4
ngnroso nccordIng fo AIfIng of nI. (l). IrIofIv fho oIocfrophorosIs
buffor consIsfod of l00 mM TrIs, 50 mM sodIum ncofnfo, 2 mM ITA
nnd 0.l SS, nnd wns ndjusfod fo pH ?.9 wIfh conconfrnfod ncofIc
ncId. IrIor fo oIocfrophorosIs, 5 of n soIufIon confnInIng 60 gIvcoroI
nnd 0.002 bromophonoI bIuo wns nddod fo fho snmpIos sfIrrod
ovornIghf. Ior snmpIo 20PI wns nppIIod on fho goI; In fho cnso of
nggrognfos fho snmpIo voIumo wns l00PI. Tho goIs woro run wIfh n
consfnnf voIfngo of 50V for npproxImnfoIv 2 hours nnd fInnIIv sfnInod
wIfh IhnsfgoI bIuo !.
IIIusInu Wuve Setvosoy
MIIks woro Incubnfod for ?5 mIn nf 32U, foIIowod bv ncId-
Inducod goInfIon nchIovod bv nddIfIon of l.2 C!. Tho cIoffIng of
fho mIIk wns monIforod bv WS. !Ighf from n 5 mW Ho-o Insor
Uhnpfor 3
38
(632.8 nm) wns pnssod fhrough n muIfI-modo fIbro Info fho mIIk. Tho
bnck-scnfforod IIghf wns monIforod bv n sIngIo-modo fIbro Iocnfod nf
3.0 mm from fho Inpuf fIbro. Tho scnfforod IIghf wns dofocfod wIfh n
Ihofo MuIfIpIIor Tubo (A!V SO-SII nnd fod fo n IU Inforfncod
nufocorroInfor (IIox 5000, corroInfor.com). Tho fImo nf whIch fho
nufocorroInfIon curvo hns docnvod fo 50 of Ifs mnxImum pInfonu
IovoI Is dofInod ns W

(39). Tho corroInfIon funcfIons woro monIforod

In fImo nf InforvnIs of 2 mIn. AII dnfn woro normnIIsod bv fho W

vnIuo
(nvorngo of fIvo monsuromonfs) of fho snmo snmpIo prIor fo C!
nddIfIon. ThIs oIImInnfos vnrInfIons bofwoon fho snmpIos nnd fIbros.
5HVXOWVDQG'LVFXVVLRQ
enutuvutIon oI wLey voteIns: ussoIutIon wItL tLe mIeIIes
und IovmutIon oI uuuveuutes
In ordor fo doformIno qunnfIfnfIvoIv fho dIsfrIbufIon of
donnfurod whov profoIns bofwoon soIubIo nggrognfos, whov profoIns
nssocInfod wIfh fho cnsoIn mIcoIIo nnd nnfIvo whov profoIns
frncfIonnfIon of honfod mIIk wns porformod. IIguro 3.l dopIcfs fho
donnfurnfIon of D-IncfnIbumIn (D-Inc, IIguro 3.ln) nnd E-IncfogIobuIIn
(E-Ig, IIguro 3.lb) In mIIk ns funcfIon of honfIng fompornfuro. Tho
frncfIonnfIon wns porformod bv nn nIfornnfIvo mofhod bnsod on
ronnofIng of fho mIIk, sopnrnfIng cnsoIn mIcoIIos from soIubIo
nggrognfos nnd nnfIvo whov profoIns. SubsoquonfIv, nnfIvo whov
profoIns woro sopnrnfod from nggrognfos bv ncId procIpIfnfIon. A
cIonr docronso In nnfIvo whov profoIns Is obsorvod wIfh IncronsIng
fompornfuro of fho honf fronfmonf. Affor honfIng nf 90qU moro fhnn
95 of nII fho E-Ig nnd nImosf 80 of nII fho D-Inc prosonf In mIIk Is
donnfurod. Abouf 25 of bofh profoIns Is prosonf In fho supornnfnnf
ns soIubIo nggrognfos, whIIo 65 of nII E-Ig nnd 50 of nII D-Inc Is
found In fho poIIof nffor ronnofIng nnd wns fhus nssocInfod wIfh fho
cnsoIn mIcoIIo.
Tho obsorvod dogroo of donnfurnfIon of D-Inc nnd E-Ig nnd fho
fncf fhnf D-Inc donnfuros fo n Iossor oxfonf fhnn E-Ig Is In ngroomonf
wIfh fho IIfornfuro (?, 2?, 34). Tho soIubIo nggrognfos prosonf In fho
ronnof-Inducod supornnfnnf consIsfod mnInIv of whov profoIns, ns
onIv n smnII frncfIon of fho cnsoIn wns prosonf In fho supornnfnnf:
Ioss fhnn 5 of fofnI cnsoIn wns dofocfod In fho supornnfnnf of mIIk
honfod nf 85qU or nf Iowor fompornfuros nnd nbouf l0 wns dofocfod
Uhnpfor 3
39
In mIIk honfod nf 90qU, of whIch N-cnsoIn wns nImosf nogIIgIbIo.
Thoso rosuIfs nro In ngroomonf wIfh SIngh of nI. (34), who obsorvod
Ioss fhnn 5 sorum N-cnsoIn In unhonfod mIIk nnd Ioss fhnn 3 In
mIIk honfod nf 90qU. Tho nppIIod ronnofIng fronfmonf wIII cnuso
cIonvngo of N-cnsoIn fo InsoIubIo pnrn-N-cnsoIn (43). If Is oxpocfod fhnf
possIbIo non-nssocInfod N-cnsoIn-whov profoIn compIoxos mIghf
procIpIfnfo ns woII, whIch wouId oxpInIn fho vorv Iow qunnfIfIos of N-
cnsoIn obsorvod In fho sorum. Apnrf from N-cnsoIn, D
s2
-cnsoIn Is nbIo
fo Inforncf wIfh E-Ig vIn fhIoI group-dIsuIfIdo brIdgo oxchnngo
roncfIons; howovor If Is nof possIbIo fo qunnfIfv fhIs cnsoIn bv UI nnd
fhoroforo no osfImnfIons nro nvnIInbIo of fho nmounf of D
s2
-cnsoIn
prosonf In fho nggrognfos. Tho soIubIo nggrognfos wIII bo rognrdod ns
IIguro 3.l: Iorconfngo of donnfurnfod D-Inc (IIguro 3.ln; uppor grnph) nnd
E-Ig (IIguro 3.lb; Iowor grnph) prosonf In mIIk ns soIubIo whov profoIn
nggrognfos (whIfo bnrs) nnd nssocInfod wIfh fho cnsoIn mIcoIIo (bInck bnrs)
nffor l0 mIn honf fronfmonf nf fompornfuros rnngIng from ?0-90U. Tho
grov bnrs roprosonf nnfIvo whov profoIns.
Uhnpfor 3
40
whov profoIn nggrognfos In fho rosf of fhIs chnpfor, nIfhough fhov
mnv confnIn somo N nnd D
s2
-cnsoIn.
Tho porconfngo of whov profoIns nssocInfod wIfh fho cnsoIn
mIcoIIo nffor honf fronfmonf nf 90qU ns obsorvod bv us Is nbouf 20
Iowor fhnn obsorvod bv SIngh (34; 8? of E-Ig nnd ?6 of D-Inc).
OIdfIoId of nI. (2?) obsorvod 80 nssocInfIon nf pH 6.60 nnd 60 nf
pH 6.83, whIch wouId IndIcnfo fhnf nbouf ?0 of fho E-Ig nssocInfos
wIfh fho cnsoIn mIcoIIo nf pH 6.?. Insod on fho rosuIfs of OIdfIoId of
nI. (2?) fho osfImnfod nssocInfIon vnIuo for D-Inc wouId bo nround
50. Thoso vnIuos nro In good ngroomonf wIfh fho porconfngos
roporfod In fhIs chnpfor. Iofh sfudIos usod uIfrnconfrIfugnfIon ns n
frncfIonnfIon fooI, buf nf dIfforonf confrIfugnI forco (g) / fImo
combInnfIons. ThIs shows fho drnwbnck of uIfrnconfrIfugnfIon ns
frncfIonnfIon mofhod: soIubIo nggrognfos mIghf sodImonf nnd bo
Inforprofod ns pnrf of fho poIIof, I.o. nssocInfod wIfh fho cnsoIn
mIcoIIo.
Tho UI dnfn obfnInod nIIow doformInnfIon of fho rnfIo of D-Inc
nnd E-Ig In nggrognfos:confIng of fho cnsoIn mIcoIIos nnd fho D-Inc/E-Ig
rnfIo In nggrognfos nnd confIng. TnbIo 3.l shows fhoso rnfIos for mIIk
honfod nf 90qU. Iv compnrIson of fho D-Inc/E-Ig rnfIo In nggrognfos
nnd In fho confIng of fho mIcoIIos If Is domonsfrnfod fhnf D-Inc Is
moro onsIIv Incorpornfod In nggrognfos fhnn If Is InvoIvod In fho
confIng of mIcoIIos. Tho D-Inc/E-Ig rnfIo Is hnrdIv nffocfod bv fho

# conconfrnfIon profoIn = conconfrnfIon In mIIk * ( donnfurod profoIn / l00)

* ( profoIn prosonf In nggrognfos or cnsoIn mIcoIIos / l00).
TnbIo 3.l: oformInnfIon of fho D-Inc / E-Ig rnfIo In nggrognfos nnd confIng of
fho cnsoIn mIcoIIos In mIIk honfod nf 90U
D-Inc E-Ig
UonconfrnfIon In mIIk ( w/w) l.2 3.2
donnfurod profoIn (UI) 80 95
profoIn In nggrognfos (UI) 25 25
profoIn nssocInfod wIfh fho cnsoIn mIcoIIos
(UI)
50 65
UonconfrnfIon profoIn In nggrognfos () # 0.24 0.?6
UonconfrnfIon profoIn In cnsoIn mIcoIIos () # 0.48 l.98
MoInr rnfIo nggrognfos/cnsoIn mIcoIIos 0.5 0.38

MoInr rnfIo D-Inc/E-Ig In nggrognfos 0.4
MoInr rnfIo D-Inc/E-Ig In confIng cnsoIn
mIcoIIos
0.3l

Uhnpfor 3
4l
fompornfuro of honf fronfmonf In fho rnngo 80 fo 90qU (nof shown In
fnbIo). Tho cnIcuInfod moInr rnfIo of fofnI donnfurod D-Inc nnd E-Ig In
fho mIIk honfod nf 90qU wns 2.5, whIch Is IdonfIcnI fo fho D-Inc/E-Ig
rnfIo obsorvod In fho nggrognfos. ThIs domonsfrnfos fhnf fho
nggrognfos confnIn n rnfIo of D-Inc/E-Ig roprosonfnfIvo of fho whoIo
mIIk snmpIo, whIIo fho whov profoIn confIng of fho cnsoIn mIcoIIos
cIonrIv confnIns moro E-Ig. Thoso rosuIfs IndIcnfo onco moro fho
compIoxIfv of whov profoIn donnfurnfIon In honfod mIIk.
SIze detevmInutIon oI soIubIe wLey voteIn uuuveuutes
Tho soIubIo nggrognfos whIch romnInod In fho supornnfnnf
nffor ronnof fronfmonf woro chnrncforIsod for sIzo bv SS goI
oIocfrophorosIs (IIguro 3.2) . !nnos l fo 6 roprosonf fho supornnfnnf
of unhonfod mIIk nnd mIIk honfod nf fompornfuros rnngIng from ?0 fo
90qU. In Innos l fo 3 nII profoIns prosonf In fho supornnfnnf show n
sImIInr, roInfIvo hIgh oIocfrophorofIc mobIIIfv. In Innos 4 fo 6 n
profoIn bnnd Is obsorvod wIfh n Iowor oIocfrophorofIc mobIIIfv fhnn In
unhonfod mIIk, roprosonfIng fho nggrognfos formod durIng honf
fronfmonf. !nno ? shows n mnrkor of whov profoIn nggrognfos wIfh n
hvdrodvnnmIc dInmofor of nbouf 62 nm (6l.9 + l.2 nm).
Whov profoIn nggrognfos nro obsorvod nffor honfIng fho mIIk
for l0 mIn nf 80qU nnd nf hIghor fompornfuros, whIch Is In ngroomonf
wIfh fho rosuIfs shown In IIguro 3.l. AIso fho Incronsod
conconfrnfIon of nggrognfos nf hIghor fompornfuros of honf fronfmonf
IIguro 3.2: SS ngnroso goI wIfh In Inno l fo 6 fho supornnfnnf of ronnof -
fronfod mIIk, unhonfod (Inno l), or honfod for l0 mIn nf ?0, ?5, 80, 85 nnd
90U (Innos 2 fo 6, rospocfIvoIv), nnd whov profoIn nggrognfos of 62 nm
propnrod from whov profoIn IsoInfo ns n sIzo IndIcnfor (Inno ?).
Uhnpfor 3
42
corrosponds vorv woII wIfh fhoso rosuIfs. nfIvo whov profoIns hnvo n
dInmofor of nround 3 nm suggosfIng nn oIIgomorIc sfrucfuro wIfh n
rnfhor brond moIocuInr woIghf dIsfrIbufIon. IrovIous sfudIos hnvo
shown fhnf nggrognfos wIfh nn nvorngo sIzo of 280 nm nro no Iongor
nbIo fo onfor fho goI (l). IIocfrophorosIs In fho prosonco of SS
provIdos InformnfIon on fho moIocuInr woIghf of profoIns whIch Is nof
nocossnrIIv fho snmo ns sIzo. Howovor, ns If Is nof oxpocfod fhnf fho
nggrognfos In mIIk hnvo n compIofoIv dIfforonf shnpo fhnn nggrognfos
formod In puro whov profoIn soIufIons wo cnn uso fho mnrkor fo
osfImnfo fho sIzo of fho nggrognfos. Tho nggrognfos formod In mIIk
durIng honfIng bohnvo ns If fhov nro sIIghfIv Inrgor fhnn 60 nm. Af
80qU fho moIocuInr woIghf dIsfrIbufIon Is smnIIor fhnn nf 90qU; nIso
fho conconfrnfIon sooms fo Incronso sIIghfIv wIfh hIghor
fompornfuros, ns fho InfonsIfv of fho profoIn bnnds sfnInod Incronsod.
As fnr ns wo know fhIs Is fho fIrsf fImo fhnf soIubIo whov profoIn
nggrognfos formod In mIIk durIng honf fronfmonf nro vIsunIIsod nnd
chnrncforIsod nnd fhnf fho sIzo of fhoso nggrognfos Is osfImnfod.
WLey voteIn ussoIutIon wItL mIeIIuv N-useIn
To obfnIn InformnfIon nbouf fho nssocInfIon of whov profoIns
wIfh fho cnsoIn mIcoIIos, frncfIonnfIon wns porformod bv ronnofIng
fho mIIks cnusIng procIpIfnfIon of fho cnsoIn mIcoIIos. Tho poIIofs
obfnInod nffor confrIfugnfIon of fho ronnofod mIIk woro dIssoIvod In
SS-buffor, InducIng dIsrupfIon of non-covnIonf InforncfIons.
UovnIonf InforncfIons IIko dIsuIfIdo InforncfIons bofwoon whov
profoIns nnd N-cnsoIn or bofwoon fho cnsoIns In fho cnsoIn mIcoIIo nro
nof dIsrupfod bv SS.
8QKHDWHGPLON
Two roforonco snmpIos woro nnnIvsod bv SS-ngnroso goI
oIocfrophorosIs, I.o. unhonfod mIIk nnd unhonfod mIIk wIfh TT. In
bofh cnsos fho mIIk wns chnrncforIsod wIfhouf nnv frncfIonnfIon.
!nhonfod mIIk (Inno 2) shows n cIonr mIgrnfIon pnfforn of sIow-
(IndIcnfod bv nrrow |b]) nnd fnsf- (IndIcnfod bv nrrow |n]) mIgrnfIng
profoIn sfrucfuros (IIguro 3.3) . Tronfmonf wIfh TT (Inno l) chnngos
fho sIow-mIgrnfIng bnnd fo n fnsfor-movIng profoIn bnnd. !nno 3,
confnInIng n snmpIo from fho poIIof obfnInod nffor ronnofIng nnd
confrIfugnfIon of unhonfod mIIk, domonsfrnfos fhnf fho mIgrnfIon
voIocIfv of fho sIow-mIgrnfIng sfrucfuro Is Incronsod (nrrow |c]).
Uhnpfor 3
43
!nhonfod mIIk Is composod of cnsoIn mIcoIIos nnd nnfIvo whov
profoIns. Arrow |n] IndIcnfos fho nnfIvo whov profoIns. UnsoIn
mIcoIIos In mIIk nro Inrgo coIIoIdnI pnrfIcIos wIfh n sIzo rnngIng from
20 fo 600 nm (l3, 32), mnInIv IInkod bv non-covnIonf InforncfIons.
Affor nddIfIon of SS fhoso nro dIsrupfod, rosuIfIng In monomors
(nrrow |n]) nnd dIsuIfIdo-IInkod poIvmors (nrrow |b]). Thoso poIvmors
cnnnof confnIn cnsoIn ofhor fhnn D
s2
- nnd N-cnsoIn, bocnuso of fho Inck
of cvsfoIno In fho ofhor cnsoIns. If hns boon shown provIousIv fhnf D
s2
-
cnsoIn Is found bofh ns n monomor nnd n dImor, whorons N-cnsoIn
oxIsfs ns n dIsuIfIdo-IInkod poIvmor (4, 29, 36, 3?) wIfh sIzos Inrgor
fhnn 500 kn. Tho nccurnfo moIocuInr woIghf couId nof bo
doformInod ns fho oxporImonfs woro porformod bv SS-IACI, whIch
hns n dofocfIon IImIf of 500 kn. Our rosuIfs domonsfrnfo fhnf fho
cnsoIn poIvmors bohnvo ns If fhov nro sIIghfIv Inrgor fhnn nggrognfos
wIfh n hvdrodvnnmIc dInmofor of 60 nm, whIch suggosf n roInfIvoIv
hIgh moIocuInr woIghf. !nno l shows fhnf In fho prosonco of TT fho
cnsoIn poIvmors nro dIsrupfod Info monomors; fhIs domonsfrnfos fhnf
fho cnsoIns nro dIsuIfIdo IInkod, whIch Is In ngroomonf wIfh fho
IIguro 3.3: SS goI oIocfrophorosIs of unhonfod mIIk (Inno 2), unhonfod mIIk
wIfh TT (Inno l), fho poIIof of unhonfod mIIk (Inno 3) nnd fho poIIof of mIIk
honfod nf ?0, ?5, 80, 85 nnd 90U (l0 mIn; Innos 4 fo 8 rospocfIvoIv) obfnInod
bv ronnofIng nnd confrIfugnfIon nnd dIssoIvod In SS-buffor. !nno 9 confnIns
whov profoIn nggrognfos wIfh n hvdrodvnnmIc dInmofor of 62 nm. IndIcnfod
In fho goIs nro fho bnnds corrospondIng fo cnsoIn monomors nnd nnfIvo whov
profoIns (nrrow |n]), fho cnsoIn poIvmors In unhonfod mIIk boforo nnd nffor
ronnofIng (nrrow |b] nnd |c], rospocfIvoIv) nnd fho cnsoIn-whov profoIn
poIvmors formod durIng honf fronfmonf (brnckof |d]).
Uhnpfor 3
44
IIfornfuro (29). !onnofIng of unhonfod mIIk cnusos n cIonr docronso In
sIzo of fho cnsoIn poIvmors, whIch Is IIkoIv duo fo ronnof hvdroIvsIng
N-cnsoIn Info n cnsoIn mncropopfIdo (UMI) nnd pnrn-N-cnsoIn, wIfh
sIzos of 6.8 nnd l2.3 kn, rospocfIvoIv (l8, 3l, 43). Tho UMI Is
roIonsod Info fho sorum whIIo fho pnrn-N-cnsoIn romnIns dIsuIfIdo-
IInkod fo fho cnsoIn mIcoIIo. SummnrIsIng, wo cnn concIudo fhnf
cnsoIn mIcoIIos nro nof puroIv nssocInfIon coIIoIds composod of
IndIvIdunI cnsoIns, buf fhnf dIsuIfIdo bonds bofwoon fho cnsoIns
confrIbufo sIgnIfIcnnfIv fo fho InfogrIfv of fho cnsoIn mIcoIIo.
+HDWHGPLON
Honf fronfmonf of mIIk (IIguro 3.3, Innos 4 fo 8) nffocfs fho
oIocfrophorofIc mobIIIfv of fho profoIn sfrucfuros, InducIng sIowor-
mIgrnfIng profoIn sfrucfuros wIfh IncronsIng fompornfuro of honf
fronfmonf (IIguro 3.3, brnckof |d]). Tho chnngo In oIocfrophorofIc
mobIIIfv Is duo fo dIsuIfIdo InforncfIons bofwoon whov profoIns nnd N-
cnsoIn (l6). If Is possIbIo fhnf D
s2
-cnsoIn nIso roncfs wIfh whov
profoIns duo fo Ifs cvsfoIno rosIduo, buf fhIs hns nof vof boon roporfod
In fho IIfornfuro nnd wns nof sfudIod In fhIs pnpor. As nImosf nII N-
cnsoIn Is prosonf In Inrgo poIvmors (4, 29, 36, 3?) fho dIsuIfIdo
InforncfIons wIfh whov profoIns wIII mnInIv nffocf fho poIvmorIc
profoIn sfrucfuro (IIguro 3.3, nrrow c), whIch oxpInIns fho vorv Inrgo
profoIn sfrucfuros formod durIng honf fronfmonf. SummnrIsIng, honf
fronfmonf of mIIk cnusos whov profoIn nssocInfIon wIfh N-cnsoIn
prosonf In poIvmors whIch rosuIfs In vorv Inrgo profoIn sfrucfuros.
Tho rosuIfs do nof provIdo InformnfIon on fho dIsfrIbufIon of fho whov
profoIns on fho surfnco of fho cnsoIn mIcoIIo. ThIs wIII bo fho subjocf
of chnpfor 4 (4l).
Uhnpfor 3
45
AId-Indued ueIutIon
Iv nddIfIon of C! mIIk wns sIowIv ncIdIfIod nnd fho mobIIIfv
of fho pnrfIcIos wns monIforod bv WS, roprosonfod bv W

. IIguro 3.4
shows fho W

ncIdIfIcnfIon curvos of unhonfod mIIk nnd mIIk honfod nf

?0, ?5, 80, 85 nnd 90qU. !nhonfod mIIk shows n sfondv docronso of W

sfnrfIng nf pH 5.3 nnd ronchIng n mInImum nf pH 5.0, foIIowod bv n
shnrp Incronso. Tho sfnrf of fhIs Incronso Is horonffor cnIIod fho
goInfIon pH. MIIk honfod nf ?0qU shows n sImIInr bohnvIour fo
unhonfod mIIk, whIIo mIIk honfod nf ?5qU sfIII hns n mInImum In W

boforo fho onsof of goInfIon buf n sIIghfIv hIghor goInfIon pH. MIIk
honfod nf 80qU nnd hIghor fompornfuro hns much hIghor goInfIon pH
vnIuos nnd n sfondv docronso prIor fo fho goInfIon pH Is no Iongor
obsorvod.
IncronsIng honfIng fompornfuro cnusos n shIff fo hIghor
goInfIon pH. ThIs Is cnusod bv fho whov profoIn confIng of fho cnsoIn
mIcoIIos nnd fho prosonco of whov profoIn nggrognfos. Tho whov
profoIn confIng chnngos fho surfnco proporfIos of fho cnsoIn mIcoIIo
duo fo chnngos In fho IsooIocfrIc pH, I.o 4.6 for cnsoIn mIcoIIo fo 5.2
for E-Ig, whIch cnusos n docronso In oIocfrosfnfIc ropuIsIon nf hIghor
pH vnIuos nnd nn onrIIor sfnrf of goInfIon (l5, 23, 39). Tho mInImum
In W

prosonf In unhonfod mIIk Is duo fo fho coIInpso of fho hnIrv

brush of N-cnsoIn moIocuIos nf nppronchIng fho pH nf whIch fho
IIguro 3.4: AcIdIfIcnfIon frncos of skIm mIIk (W

vorsus pH) unhonfod (+) nnd

honfod nf ?0 (7), ?5 (|), 80 (), 85 (?) nnd 90U (z) ns monIforod bv WS.
Uhnpfor 3
46
mIcoIIos fond fo noufrnIIfv (l9) nnd nIso fo chnngos In fho mIcoI duo
fo roIonso of cnIcIum phosphnfo nnd sorum cnsoIns. In mIIk honfod nf
80, 85 nnd 90qU fho goInfIon hns sfnrfod nIrondv nf pH vnIuos of 5.4
nnd hIghor, whIIo fho dosfnbIIIsnfIon of fho N-cnsoIn brush sfnrfod nf
pH 5.3 (unhonfod mIIk) nnd fhoroforo fho mInImum Is no Iongor
obsorvod.
In IIguro 3.5 fho goInfIon pH Is pIoffod vorsus fho porconfngo
of donnfurod E-Ig, fho porconfngo of E-Ig prosonf In soIubIo nggrognfos
nnd fho porconfngo of E-Ig nssocInfod wIfh fho cnsoIn mIcoIIos ns
doformInod In mIIk honfod nf fompornfuros rnngIng from ?0 fo 90qU.
In nII cnsos fho dnfn show n good IInonr corroInfIon wIfh fho goInfIon
pH.
Tho pH of goInfIon Is mnInIv doformInod bv fho nmounf of
donnfurod E-Ig, ns wns domonsfrnfod bv VnsbIndor of nI. (39).
Thoroforo, onIv fho porconfngo of donnfurod E-Ig wns pIoffod ns n
funcfIon of fho goInfIon pH. If npponrs fhnf fho goInfIon pH corroInfos
IInonrIv wIfh fho porconfngo of donnfurod E-Ig prosonf In honfod mIIk.
Tho fofnI nmounf of donnfurod E-Ig Is composod of nggrognfos nnd
profoIn nssocInfod wIfh fho cnsoIn mIcoIIo, whIch nro prosonf In n
consfnnf rnfIo In fho mIIks honfod nf pH 6.? nf ?0 fo 90qU. Thoroforo,
undor fhoso condIfIons If Is nof possIbIo fo sopnrnfo fho offocfs of fho
fwo groups of donnfurod whov profoIns. Tho IInonr corroInfIon
obsorvod In IIguro 3.5 domonsfrnfos fhnf fho chnngod goInfIon
IIguro 3.5: CoInfIon pH vorsus porconfngo of fofnI donnfurod E-Ig (), E-Ig
nssocInfod wIfh fho cnsoIn mIcoIIo (z) nnd E-Ig In whov profoIn nggrognfos ()
prosonf In skIm mIIk unhonfod nnd honfod nf ?0 fo 90U (l0 mIn).
Uhnpfor 3
4?
proporfIos of honfod mIIk nro compIofoIv cnusod bv whov profoIn
donnfurnfIon. If fho rnfIo bofwoon nggrognfos nnd nssocInfod whov
profoIns Is consfnnf, fho fofnI nmounf of donnfurod profoIn
doformInos fho goInfIon proporfIos.
ConIusIons
Honf fronfmonf of mIIk nf fompornfuros rnngIng from ?0 fo
90qU cnusos donnfurnfIon of whov profoIns of whIch E-Ig donnfuros nf
n hIghor rnfo fhnn D-Inc. Tho donnfurod whov profoIns form soIubIo
whov profoIn nggrognfos nnd whov profoIn confIng of fho cnsoIn
mIcoIIos. Af 90qU nggrognfos nro formod wIfh n sIzo of 60-l00 nm
confnInIng nbouf 25 of bofh whov profoIns, nnd nbouf 65 of E-Ig
nnd 50 of D-Inc hns nssocInfod wIfh fho cnsoIn mIcoIIo. Tho
nggrognfos show n rnfIo of D-Inc fo E-Ig whIch Is roprosonfnfIvo of fho
rnfIo In fofnI donnfurnfod whov profoIns In mIIk. Tho whov profoIn
confIng on fho cnsoIn mIcoIIo Is onrIchod In E-Ig. SInco N-cnsoIn In
mIIk Is prosonf In Inrgo poIvmorIc sfrucfuros, honf-Inducod
nssocInfIon of N-cnsoIn nnd whov profoIns cnusos vorv Inrgo poIvmorIc
profoIn sfrucfuros. Honf fronfmonf of mIIk cnusos n shIff fo hIghor
goInfIon pH vnIuos upon ncIdIfIcnfIon. Thoso goInfIon pH vnIuos
corroInfo IInonrIv wIfh fho fofnI nmounf of donnfurod whov profoIns,
fho nmounf of soIubIo nggrognfos nnd fho nmounf of whov profoIns
nssocInfod wIfh fho cnsoIn mIcoIIo. If Is oxpocfod fhnf fho whov
profoIn dIsfrIbufIon nffocfs fho goInfIon proporfIos; fhIs wIII bo fho
subjocf of chnpfor 4 (4l).
5HIHUHQFHV
l. AIfIng, A.U., !.J. Hnmor, U.C. do KruIf nnd !.W. VIsschors. 2000. IormnfIon
of dIsuIfIdo bonds In ncId-Inducod goIs of prohonfod whov profoIn IsoInfo. J.
AgrIc. Iood Uhom. 48: 500l.
2. Anomn, S.C., 2000. Iffocf of mIIk conconfrnfIon on fho IrrovorsIbIo fhormnI
donnfurnfIon nnd dIsuIfIdo nggrognfIon of E-IncfogIobuIIn. J. AgrIc. Iood Uhom,
48: 4l68.
3. Anomn, S.C., 200l. KInofIcs of fho IrrovorsIbIo fhormnI donnfurnfIon nnd
dIsuIfIdo nggrognfIon of D-IncfnIbumIn In mIIk snmpIos of vnrIous
conconfrnfIons. J. Iood ScI. 66: l: 2.
4. Annnn, W.. nnd W. Mnnson. l969. A frncfIonnfIon of fho Ds-cnsoIn compIox of
bovIno mIIk. J. nIrv !os. 36: 259.
5. Inrbuf, S. nnd I.A. IoogodIng. l993. Un2+-Inducod goInfIon of pro-honfod
whov profoIn IsoInfo. J. Iood Sc. 58: 4: 86?.
Uhnpfor 3
48
6. UorrodIg, M. nnd .C. nIgIoIsh. l996. Iffocf of fompornfuro nnd pH on fho
InforncfIons of whov profoIns wIfh cnsoIn mIcoIIos In skIm mIIk. Iood !os. Inf.
29: l: 49.
?. nnnonborg, I. nnd H.C. KossIor. l988. !oncfIon kInofIcs of fho donnfurnfIon
of whov profoIns In mIIk. J. Iood ScI.53: 258.
8. nnnonborg, H. nnd H.C. KossIor. l988. Iffocf of donnfurnfIon of E-
IncfogIobuIIn on foxfuro proporfIos of sof-sfvIo nonfnf voghurf. l. SvnorosIs.
MIIchwIssonschnff. 43: 632.
9. nnnonborg, I. nnd H.C. KossIor l988. Iffocf of donnfurnfIon of E-
IncfogIobuIIn on foxfuro proporfIos of sof-sfvIo nonfnf voghurf. 2. IIrmnoss nnd
fIow proporfIos. MIIchwIssonschnff. 43: ?00.
l0. nIgIoIsh, .C., !. vnn MourIk, M. UorrodIg. l99?. Honf-Inducod InforncfIons
of whov profoIns nnd cnsoIn mIcoIIos wIfh dIfforonf conconfrnfIons of D-
IncfnIbumIn nnd E-IncfogIobuIIn. J. AgrIc. Iood Uhom. 45: 4806.
ll. Hoorfjo, I., J. VIssor nnd I. SmIfs. l985. Sfrucfuro formnfIon In ncId mIIk goIs.
Iood MIcrosfrucfuro. 4: 26?.
l2. HIII, A.!. l989. Tho E-IncfogIobuIIn-k-cnsoIn compIox. Unn. Insf. Iood ScI.
TochnoI. J. 22: 2: l20.
l3. HoIf, U., A.M. KImbor, I. Irookor nnd J.H. IronfIco. l9?8. Avorngo rndII of
bovIno cnsoIn mIcoIIos. XX-InfornnfIonnI-nIrv-Uongross; I, 234.
l4. Hongsprnbhns, I. nns S. Inrbuf. l99?. IrofoIn nnd snIf offocfs on Un
2+
-
Inducod coId goInfIon of whov profoIn IsoInfo. J. Iood ScI. 62: 2: 382.
l5. Horno, .S. nnd U.M. nvIdson. l993. InfIuonco of honf fronfmonf on goI
formnfIon In ncIdIfIod mIIks. IrofoIn nnd fnf gIobuIo modIfIcnfIon, In
IrocoodIngs of II SomInnr. 26?.
l6. Jnng, H.. nnd H.I. SwnIsgood. l990. IsuIfIdo bond formnfIon bofwoon
fhormnIIv donnfurod E-IncfogIobuIIn nnd N-cnsoIn In cnsoIn mIcoIIos. J. nIrv
ScI. ?3: 900.
l?. do KruIf, U.C., M.A.M. Hoffmnnn, M.I. vnn MnrIo, I.J.J.M. vnn MII, S.I.I.M.
!oofs, M. VorhouI nnd I. Zoon. l995. CoInfIon of profoIns from mIIk. Inrndnv
Iscuss. l0l: l85.
l8. do KruIf, U.C. nnd I.I. ZhuIInn. l996. N-cnsoIn ns n poIvoIocfroIvfo brush on
fho surfnco of cnsoIn mIcoIIos. UoIIoIds nnd Surfncos A. ll?: l5l.
l9. do KruIf, U.C. l99?. SkIm mIIk ncIdIfIcnfIon. J. UoIIoIds Inforfnco ScI. l85: l9.
20. !nw, A.J.!., .S. Horno, J.M. Innks nnd J. !onvor. l994. Honf-Inducod
chnngos In fho whov profoIns nnd cnsoIns. MIIchwIssonschnff. 49: 3: l25.
2l. !nw, A.J.!. nnd J. !onvor. l99?. Iffocf of mIIk profoIn conconfrnfIon on fho
rnfos of fhormnI donnfurnfIon of whov profoIns In mIIk. J. AgrIc. Iood Uhom.
45: 4255.
22. !nw, A.J.!. nnd J. !onvor. 2000. Iffocf of pH on fho fhormnI donnfurnfIon of
whov profoIns In mIIk. J. AgrIc. Iood Uhom. 48: 6?2.
23. !ucov, J.A., U. Tof Too, I.A. Munro nnd H. SIngh. l99?. !hooIogIcnI proporfIos
nf smnIl (dvnnmIc) nnd Inrgo (vIoId) doformnfIons of ncId goIs mndo from
honfod mIIk. J. nIrv !os. 64: 59l.
24. !ucov, J.A., I.A. Munro nnd H. SIngh. l998. !hooIogIcnI proporfIos nnd
mIcrosfrucfuro of ncId mIIk goIs ns nffocfod bv fnf confonf nnd honf fronfmonf.
J. Iood ScI. 63: 660.
Uhnpfor 3
49
25. oh, I., T. !Ichnrdson nnd !.K. Uronmor. l989. !ndIoInboIIIng sfudv of fho
honf-Inducod InforncfIons bofwoon D-IncfnIbumIn, E-IncfogIobuIIn nnd N-cnsoIn
In mIIk nnd In buffor soIufIons. J. Iood ScI. 54: 4: 889.
26. OIdfIoId, .J., H. SIngh nnd M.W. TnvIor. l998. AssocInfIon of E-IncfogIobuIIn
nnd D-IncfnIbumIn wIfh fho cnsoIn mIcoIIos In skIm mIIk honfod In nn uIfrn-
hIgh fompornfuro pInnf. Inf. nIrv J. 8: ?65.
2?. OIdfIoId, .J., H. SIngh, M.W. TnvIor, K.. Ionrco. 2000. Honf-Inducod
InforncfIons of E-IncfogIobuIIn nnd D-IncfnIbumIn wIfh fho cnsoIn mIcoIIo In
pH-ndjusfod skIm mIIk, Inf. nIrv J. l0: 509.
28. InrnoII-UIunIos, I., Y. Knkudn, J.M. doMnn nnd I. UnzzoIn. l988. CoInfIon
profIIos of voghurf ns nffocfod bv honf fronfmonf of mIIk. J. nIrv ScI. ?l: 582.
29. !nsmusson, !.K. nnd T.I. Ioforson. l99l. IurIfIcnfIon of dIsuIfIdo-IInkod D
s2
-
nnd N-cnsoIn from bovIno mIIk. J. nIrv !os. 58: l8?.
30. !oofs, S.I.I.M., U.C. do KruIf. l994. A modoI for fho donnfurnfIon nnd
nggrognfIon of E-IncfogIobuIIn. Iur. J. IIochom. 226: 883.
3l. !oIIomn, H.S. UnsoIn AssocInfIon nnd mIcoIIo formnfIon, In Advnncod nIrv
UhomIsfrv, voIumo l. IrofoIns, odIfod bv I.I. Iox, IIsovIor ScIonco IubIIshors,
IngInnd. lll.
32. SchmIdf, .C., I. WnIsfrn nnd W. IuchoIm. l9?3. Tho sIzo dIsfrIbufIon of
cnsoIn mIcoIIos In cow's mIIk. ofh. MIIk nIrv J. 2?: l28
33. SIngh, H.S. l993. Honf-Inducod InforncfIons of profoIns In mIIk. IrofoIn nnd
fnf gIobuIo modIfIcnfIons-II somInnr, spocInI Issuo 9303. l9l.
34. SIngh, H., M.S. !oborfs, I.A. Munro nnd U.T. Too. l996. AcId-Inducod
dIssocInfIon of cnsoIn mIcoIIos In mIIk: offocf of honf fronfmonf. J. nIrv ScI.
?9: l340.
35. SmIfs, I. nnd J.H. vnn Irouworshnvon. l980. Honf Inducod nssocInfIon of E-
IncfogIobuIIn nnd cnsoIn mIcoIIos. J. nIrv !os. 4?: 3l3.
36. SwnIsgood, H.I., J.!. Irunnor nnd H.A. !IIIovIk. l964. IhvsIcnI pnrnmofors of
N-cnsoIn from cow`s mIIk. IIochomIsfrv. 3: l6l6.
3?. TnIbof, I. nnd .I. Wnugh. l9?0. MIcoIIo formIng chnrncforIsfIcs of monomorIc
nnd covnIonf poIvmorIc N-cnsoIns. IIochomIsfrv. 9: 280?.
38. TuInIor, !. An oxocoIIuInr poIvsncchnrIdo nnd Ifs InforncfIon wIfh profoIns.
Ih.. Iss. !nIvorsIfv of WngonIngon, WngonIngon, ofh.
39. VnsbIndor, A.J., I.J.J.M. vnn MII, A. Iof nnd U.C. do KruIf. 200l. AcId-
Inducod goInfIon of honf fronfod mIIk sfudIod bv IffusIng Wnvo Spocfroscopv.
UoIIoIds nnd Surfncos I. 2l: 245. Uhnpfor 6 In fhIs fhosIs.
40. VnsbIndor, A.J., A.U. AIfIng, !.W. VIsschors nnd U.C. do KruIf. Toxfuro of ncId
mIIk goIs: formnfIon of dIsuIfIdo cross-IInks durIng ncIdIfIcnfIon, submIffod for
pubIIcnfIon In fho Inf. nIrv J. Uhnpfor ? In fhIs fhosIs.
4l. VnsbIndor, A.J. nnd U.C. do KruIf. UnsoIn-whov profoIn InforncfIons In mIIk
honfod nf pH 6.3-6.9. mnnuscrIpf In propnrnfIon. Uhnpfor 4 In fhIs fhosIs.
42. vnn VIIof, T. nnd U.J.A.M. KoofoIs l995. Iffocf of prohonfIng of mIIk on fho
sfrucfuro of ncIdIfIod mIIk goIs. ofh. MIIk nIrv J. 49: 2?.
43. WnIsfrn, I. nnd !. Jonnoss. l984. nIrv UhomIsfrv nnd IhvsIcs, John WIIov
nnd sons, Inc, !SA.