Anal

ysi
sofMi
lk

Theterm“ mi lk”isalaytermf or“ cow’smilk.”I

tisacol loidalsuspensionofhy drophobic
fatmoleculessuspendedi nahy drophi
li
ccar ri
erofwat er,sugar s,
protein,andmi nerals.
Themai ncar bohydrat
einmi lki slactose;themai nproteiniscasei n(anal lergen)
;and
themaj orf atscont ai
nt hefatt yacidsoleicandpal mi t
ic.Inwhol emi lk,mostoft he
measurable aci dit
y comes f r om t he casein contentand t oal esserdegr ee the
phosphatecont ent.Animpor tantnuancet obeconsi der edist herelationshipbetween
pHandt i
tratableacidi
ty:pHmeasur esthest rengthoft heaci dsinasol uti
on,wher eas
ti
trat
ableaci dit
ymeasur esthet otalamountofaci dsinagi vensol ut
ion.
COMPOSI TIONALTESTS
1)FatDet
ermi
nat
ions
FatCont
ent
s
Thef atcont entinmi lkrangesf rom 2. 5t o6. 0%,wi thameanper centageof4. 0.Thef at
cont entofmi lkandsomeot herdai rypr oduct si seconomi cal l
yi mpor tantasi tcan
deter mi nepr i
ce.I ny earspast ,i twast hedet ermi nant;howev er ,incur r
entt imesot her
factor si ncl udingnut rit
ionalconsi der ationshav ecomei ntopl ay.Thef atoccur sasa
globul eencased i namembr ane.Thegl obul esar esuspended asanoi li n-wat er
emul si on whi ch can be di sr upt ed by shaki ng orot hermechani calmeans.The
membr aneencasi ngt hef atglobul esconsi st sofal i
pidbi l
ay ermembr ane
similart ot heapi cal membr aneofepi thel i
al cel l
s.
Chemi cal ly,fatsconsi stofagl y cer olbackbonet owhicht hr eef attyacidsar eesterified.
Thef at tyaci dsv ar yinchai nlengt handdegr eeofunsat uration.Themaj orf attyaci dsi n
mi l
kar e:
Palmat ic( 25%) ,
Oleic( 20%) ,
Stericandmy r
isti
c( 10%each)
Smal lamount sofbut yr
icacidar eal sopr esent( knownt ocausea“ vomit-
like”odor )and
cancont ribut et oar ancidf l
av orwheni tiscl eav edf rom t hegl ycerolbackbonebyt he
actionofani nherentl i
paseenzy mei nt hemi lk, orbymi crobial activi
ty.
IntheUni tedSt ates, t
hemostwi del yusedt ot al fatprocedur ef ordai ryproduct sisthe
a)Moj onni erf atmet hod.
Pr i
nci ple and Char acteristi
cs:Fati s ext ract ed wi t
h a mi xt ure ofet hylet herand
petrol eum et heri naMoj onnierf lask, andt heext r
actedf atisdr iedtoaconst antwei ght
andexpr essedasper centf atbywei ght.TheMoj onni ert esti sanexampl eoft he
discont inuoussol ventext r
act i
onmet hodanddoesnotr equi rer emov alofmoi stur e
from t hesampl e.Itcanbeappl iedt obot hliqui dandsol idsampl es.Ifpetroleum et heri s
usedt opur ifyt heext ractedf at, thismet hodi sv erysimi l
artot heRoese- Got tli
ebMet hod
(AOACMet hod905. 02)inbot hpr i
nci pleandpr act i
ce.TheMoj onni erfl
asksar eusednot
onlyf ort heMoj onni erandRoese- Got t
liebmet hods,butal sot odot hehy drolysis( aci d,
alkaline, orcombi nat ion)pr iort of atext ract ionandGCanal ysi st odet ermi nef atcont ent
andf attyaci dpr ofile.( Not et hatsomet imest het ermsMoj onni er,RoeseGot tli
eb,and
alkalinehy dr olysisar eusedi nt erchangeabl y.)
Itisi nv olved,t edi ous,and t ime- consumi ng;and,qui tev ariablei nt he numberof
extract ionsand
volumesofext ract ant( ethy lal cohol ,et hy let her ,and pet roleum et her ).Thel att
er
dependsont hepr oductandt hemet hodr equi r
esaspeci al“ Moj onni erf l
ask. ”Var ious
modi f i
cat ionsoft heMoj onni ermet hodhav ebeenpubl ishedi ncludingt hePennsy l
vania
Roccal ,Ger berPr ocedur es, and“ acidhy dr ol ysis.”
Anout lineoft hepr ocedur ei sasf ollows:( AOACMet hod989. 05)
Prepar ationofSampl e.Br ingt hesampl et oabout20◦ C; mixt opr epareahomogeneous
sampl ebypour i
ngbackandf or thbet weencl eanbeaker s.Pr ompt lywei ghormeasur e
thet estpor t i
on.I fl umpsofcr eam donotdi sper se, war mt hesampl einawat erbat hto
about38◦Candkeepmi xingunt i
liti shomogeneous,usi nga“ rubberpol i
ceman”i f
necessar ytor eincor por atet hecr eam adher ingt ot hecont ainerorst opper .Wheni tcan
bedonewi thouti nt er feringwi thdi sper saloft hef at ,coolwar medsampl est oabout20◦
Cbef oret r
ansf erri
ngt het estpor tion.
Procedur e:
1.To10gofmi lkinaMoj onni erf l
ask,add1. 5mLofNH4OH.Thebaseneut rali
zest he
sampl eanddi ssolv est hepr ot ein.
2.Af teraddi ng10mLof95%et hanol (t
opr eventgel for mation)andshakef or90s.
3.Add25mlofet hy let herandshakef or90s.Theet herdi ssolvest hel ipid.Cooli f
necessar y,andadd25mlofpet roleum et herandshakef or90s.Thepet roleum et her
remov esmoi sturef rom
theet hy l etherext ractanddi ssol vesmor enonpol arlipi d.
4.Cent rifugef or30sat600r pm.
5.Decantet hersol ut ionf r om t heMoj onni erf laski ntot hepr ev iouslywei ghedMoj onnier
fatdish.
6.Per for m secondandt hir dext ractionsi nt hesamemannerasf orthef i
rstext racti
on
descr ibedpr ev i
ousl y( et hanol ,et hy lether ,
pet roleum et her,cent ri
fugation, decant )
.
7.Ev apor atet hesol v enti nt hedi shont heel ect ri
chotpl ateat≤100◦ Ci nahood.
8.Dr yt hedi shandf att oaconst antwei ghti naf orcedai rov enat100◦ C±1◦ C.
9.Cool thedi sht or oom t emper atureandwei gh.
Calcul at i
on:
%Fat=100×[ (
wei ghtofdi sh+f at)−wei ghtofdi sh)−wei ghtofbl ank]/ weightof
sampl e

Thereagentbl
anki
smadewi t
h10gofwaterinsteadofmilk.
b)BabcockMethodforMi
lkFat(
AOACMet hod989. 04and989.
10)
I
ncont r
asttotheessent
ial
l
ygravi
met
ri
cMoj onniermethod,t
heBabcockmet
hodi
s
vol umet r i
c butexpr essed on a wei ghtbasi s.I templ oys a speci albot tl
e wi tha
graduat ed, const rictedneckorst em.
Pr i
nci ple
Int heBabcockmet hod,H2SO4 i saddedt oaknownamountofmi lkint heBabcock
bot tle.The sul furic aci d di gest s prot ein,gener ates heat ,and r el
eases t he f at.
Cent rifugat ionandhotwat eraddi ti
oni sol atef atf orquant ifi
cat i
oni nt hegr aduat ed
por tionoft het estbot t l
e.
Thef ati smeasur edv ol umet r
ical l
y,butther esulti sexpr essedasper centf atbywei ght.
Pr ocedur e
1.Accur atel ypi pet t
et hemi l
ksampl e(17. 6ml )int oaBabcockt estbot t
le
2.Addr eagentgr ade( 1. 82speci ficgravity)sul fur i
caci d( 17.
5ml )tot hebot tle,allowi ng
theaci dt of l
owgent l
ydownt heneckoft hebot tleasi tisbei ngsl owl yrotated.Theaci d
digest spr ot einst ol i
ber atet hef at .
3.Cent rifuget hemi xt uref or5mi nandl iqui dfatwi llrisei ntothecal ibratedbot t
leneck.
Thecent rifugemustbekeptat55–60◦ Cdur ingcent ri
fugat i
on.
4.Addhotwat ert obr ingl iquidf atupi ntot hegr aduat edneckoft heBabcockbot tle.
5.Thedi rectper cent ageoff atbywei ghti sr ead t ot henear est0. 05% f rom t he
graduat ionmar koft hebot tl
e.
Appl icat ions
TheBabcockmet hod, whi chisacommonof fi
cialmet hodf orthedet ermi nationoff atin
mi lk,t akesabout45mi nanddupl i
catet est sshoul dagr eewi thin0. 1%.TheBabcock
met hoddoesnotdet er minet hephosphol ipidsi nt hemi l
kpr oduct s.Itisnotappl icable
to pr oduct scont ai
ni ngchocol at eoraddedsugarwi thoutmodi f
icationbecauseof
char ri
ngofchocol ateandsugar sbysul fur icaci d.
c)Ger berMet hodf orMi lkFat
Pr i
nci ple
Thepr inci pleoft heGer bermet hodi ssimi lartot hatoft heBabcockmet hod, buti tuses
sul fur i
caci dandamy lal cohol .Thesul f uricaci ddi gest spr oteinsandcar bohy dr ates,
releasesf at, andmai nt ainst hef atinaliqui dst atebygener ati
ngheat .
Pr ocedur e
1.Tr ansf er10ml ofH2SO4at15–21◦Ci nt oaGer bermi lkbot tl
e.
2.Accur atel ymeasur emi lksampl e(11ml )intot heGer berbot tl
e,usingaGer berpi pet te.
3.Add1ml ofi soamy l alcohol tot hebott l
e.
4.Ti ght ent hest opperandmi xbyshaki ngt hebot tle.
5.Cent rifuget hebot t l
ef or4mi n.
6.Pl acet hebot t l
ei nawat erbat hat60–63◦ Cf or5mi nandt henr eadt hef atcont ent
from t he
graduat ionsont hebot tl
eneck.
Appl icat ions
TheGer bermet hodi scompar abl etotheBabcockmet hodbuti ssi mpl erandf asterand
haswi derappl i
cat iont oav ar i
etyofdai rypr oduct s.Thei soamy lalcoholgener all
y
prev
ent sthecharr
ingofsugarfoundwi
tht
her
egul
arBabcockmet
hod.Thi
stesti
smor
e
popularinEuropethanAmerica.

Thet ur bidmet ricappr oachi sdescr i

bedi nt heSt andar dMet hodsf ort hePr oduct s.To
thedi lut edsampl e, EDTAi saddedt oel imi nat epr ot einpar t
icl
ei nt erfer ence.Thesampl e
i
saddedt oaFossMi l
kTest er,ort heequi valent ,t hel ightscat ter ingi smeasur ed
electroni callyandadi rectr eadouti sobt ained.Thepr ocedur ei sr api d,accur at e,and
preciseusi ngapr oper l
ycal ibratedi nst rument .Ot hercommonf att est ingmet hods
i
ncl ude t he Soxhl etext raction met hod wi th cal cul at i
ons bei ng der i
v ed as “ fatby
diff
er ence”si mi lart ot heMoj onniermet hod,onl yt hef atisext r
act edbyanal cohol
solventov erheat .
d)Fr eeFat tyAci ds
Fattyaci dsv ar yinl engt hbutmostar ecar boxy licaci dswi thl ongunbr anched“ tails”or
“chains”ofcar bonmol ecules.Theyar ecl assi fi
edgener i
callyasei t
herbei ng“ satur at ed”
or“ unsat urated, ”asi dent if
iedbyt hehy dr ogenat omsont hecar bonchai n.I ft hecar bon
atomsar ecompl etel yf il
ledwi t hhy dr ogenat oms[ wi tht heexcept ionoft hecar boxy li
c
acid( –COOH)f unct ionalgr oup] ,t
hent heyar econsi der ed“ sat urat ed. ”Unsat ur atedf at t
y
acidshav eatl eastonedoubl ebondbet weenadj acentcar bongr oups,sucht hatt he
normal “–CH2–CH2–”ofasat urated
chai nhasa“ –CH=CH–”( call
edanal kenef unct ional gr oup) .
Fattyaci dscanbeei therboundorunboundt oot hermol ecules,mai nlyt rigly cerides.I n
theirunboundst at e, theyar ecal l
ed“ fr
eef at t
yaci ds( FFA) .”
Theyar ecommonl yusedi nt hef oodi ndust ryasameasur eofr anci di ty,ast heyar e
pronet onor malaci d–baser eact i
onsoft hecar boxy lf unct i
ongr oupor“ aut o-oxi dat ion.”
Thef attyaci dwi llnat urallybreakdowni nt oi tsconst ituentpar ts,mai nlyhy dr ocar bons,
ketones, al
dehy des, andsmal l
eramount sofal cohol s.I nt heirpur ef or ms, ori npr oduct s
withahi ghf atcont ent ,theseoi l
sandf at sar eof tent reat edwi thchel at i
ngagent s, such
asci tricaci d, tor et ardt hisaut ooxidat ionpr ocess.
Thequi ckestandeasi estwayt ot estf orFFAi sbydi rectaci d- baset itrat i
onusi ngan
ether –f atext r action,andendpoi ntdet ermi nat ionbyaphenol pht hal ei ncol orchange.
Themai ndr awbackt ot hismet hodi st hatt heendpoi ntcanbedi ff i
cul tt odet er minej ust
byv i
sualmeans.Aut omat icsy stemst hatcandet ermi net hepKaendpoi ntar eamuch
mor er eliablemeans,butt heyar eof tencost ly.Howev er,thiscostdi ff er encecanof ten
beof fsetbyt hedecr easedt echni ciant i
met hatwoul dbespentont hemanualt itr ation
process.Thi sobv iousl yi sonl yf easi bl ef orl abor at or i
eswi thal ar gert hroughputof
sampl es.TheFFAcont entiscal cul atedasol eicaci d.Tot hel iqui df atsampl e,anet her
extract ion i s added,neut rali
zed et hanol -cont aining phenol pht hal ein.The sampl ei s
ti
tratedwi t
hst andar di zedsodi um hy droxi de.
Calcul at ion:
%Fr eeFat t
yAci d=[ mLNaOH×Nor mal ityofNaOH×28. 2]/wei ghtofsampl e
e)Per oxi deVal ue
The  per oxi dev al ue  isdef i
nedast heamountof  peroxi de  oxy genper1ki logr am off ator
oil.
Int hei ni tial,orpr imar y,per iodofl ipi doxi dation,theper oxidesorl i
poper oxi desar e
formed.Thef reer adi cal sr eactwi thi odi def or mingf reei odi newhi chi st itratedwi t
h
standar di zedsodi um t hiosul fat eusi ngast archi ndicat or.Thev aluei sexpr essedas
mi l
li
equi valent sofper oxideperki logr am off at.
Calcul at i
on:
Peroxi deVal ue=[ mLS2O3(−2)×Nor mal ity×1000]/gr amssampl e.
Thedegr adat i
onofhy dr oper oxi desl eadst ot hef ormat i
onofnonr adi calssuchas
aldehy desandket oneswhi char eby product soft het ermi nalphase.Hy dr olyti
cr anci dit
y
appear st o bet hel eastunder stood l ipid pat hway .Howev er ,iti sunder stood t hat
tri
gly cer idesar ebr okendownv iahy dr ol ysisofest erl inkagesbyl i
paseenzy mes.Thi s
react ioni nt ur nleadst ot hef ormat ionofFFA.Thecont ribut ingf actorsofbot hoxi dat i
ve
andhy dr oly ti
cpat hway scont ribut et ot het ast e,smel l,andt ext ur
eoft hepr oduct .
Theor et ically ,lipid oxi dat i
on can occuras soon as i tl eav es t he pr oduct ion l i
ne.
Mi l
li
equi valent sperki l
ogr am ofper oxi dear et estedusi ngi odi deandat itration.FFAi s
alsot est edusi ngat i
trat i
on.Bot hr esul tsgi vesomei ndi cat i
ont ohow muchoxi dation
hasoccur redbet weent het i
met hepr oductwasmanuf act uredandt het i
mei tr eaches
thel abor at or y.
2)Tot alSol ids
Thewat ercont entofaf oodexi stsi nt hr eef orms.
1.Fr eewat er:Thi swat err etainsi tsphy sicalpr opertiesandt husact sast hedi sper sing
agentf or
colloidsandt hesol v entf orsal ts.
2.Adsor bedwat er :Thi swat eri shel dt ight l
yori soccl udedi ncel lwal lsorpr otopl asm
andi shel dt i
ght lyt opr otei ns.
3.Wat er of hy dr ation:Thi s wat er i s bound chemi cally,f or exampl e,l act ose
monohy dr at e;also
somesal tssuchasNa2SO4·10H2O.
Freewat er,absor bedwat er( incel l
sandboundt opr ot eins) ,andchemi callybound
(wat erofhy drat i
on)wat er.Ther ear emanymet hodsf ordet er miningt hemoi sture
cont entofaf ood.Thef or m ofwat eri nt hef ood, i
tsconcent ration, thedesi redaccur acy
and pr eci sion oft he anal y sis,and t he anal y ti
calt i
me t or esul tar e al li mpor tant
consi der at i
onst ot akei nt oaccount .Sampl ecol l
ectioni si mpor tantasmoi stur ecanbe
l
ostorgai nedi ft hepr opersampl ingt echni quesandcol lect i
oncont ainersar enotused.
Theheadspacei nt hecont ainermustbekeptt oami nimum.Themoi sturecont entofa
foodi si mpor tantt ot hemanuf act ur erasi ti ssubj ectt oast andar dofi dent i
ty ,a
cust omer ’sspeci ficat ion,t heshel fst abili
tyoft hepr oduct( driedandconcent rated
mi l
ks) ,andi nsomei nst ancest hequal it
ycont r
olpr act i
cesi nt hepl ant .Af tert he
moi st ur ei sr emov ed, av aluer ef er r
edt oast otal soli
dsi sobt ained.
Calcul at i
on:
%Tot al Sol i
ds=100%−%Moi sture
Anot her“ t
otalval ue”usedi ndai ryindust r
yist het ot al solids-not-f
at.
Calcul at ion:
%Tot al Sol i
ds=100%−( %Moi sture+%Fat )
3)Moi st ure
Moi st urev al
uesf indutili
tyinpr oductspeci ficati
onsandst andardsofi dent i
ty.Per cent
moi st uredet ermi nati
onsi nt hedai ryi ndustryar er atherpr oductspeci fi
cwhensel ecting
◦
themet hod.Themostcommonpr ocedur eempl oy sav acuum ov endr yingat100Cf or
about5hour s.Cheeses,dr iedmi l
ks,casei npowder s,anddr yi cecr eam mi xesf allinto
thiscat egor y.
Fordr iedwheyandwheypr oduct s,t hetemper atur ei sr educedt o65◦ Candt hedr ying
periodext endedt o16hour s.Fl uidmi l
ksr equi r
eapar ti
aldr ying( usuallyov erast eam
bath)pr iortov acuum dr ying.Thegener almet hodol ogyi nvolvesahomogenat ioni na
mechani calblender ,weighingt hesampl eonat arr edanal yt
icalbalancei nadi sposabl e
alumi num pan wi t
h a cov er( to av oid spl att
er ing) ,heat i
ng,desi ccation,and r e-
wei ghi ng. Withsomepr oduct s,ot herpr ocedur escanbeused,suchast hef orced-
draftov enmet hod, t
hemi crowav eov en,orot hermul ticomponent
procedur es(lactomet r
icori nf ared).
Precaut ionforhi ghmoi sturesampl esi nov en:
Foodsampl est hathav ehi ghmoi sturecont entsar eusual l
ydr iedi nt wost agest o
prev ent" spatteri
ng"oft hesampl e,andaccumul ationofmoi stureint heov en.Spat tering
i
st hepr ocesswher ebysomeoft hewat erjumpsoutoft hef oodsampl edur i
ngdr yi
ng,
carry i
ngot herf oodconst it
uent swi thi t
.Forexampl e,mostoft hemoi stureinmi lki s
remov edbyheat ingonast eam bat hpr iortocompl et i
ngt hedr yi
ngi nanov en.

4)AddedWat er:
The boilingpoi ntelev ati
on  i
sa  coll
igati
vepr operty,whichmeanst hatitisdependenton
thepr esenceofdi ssol v
edpar ti
clesandt heirnumber ,butnottheiridentity
.Itisanef f
ect
oft hedi l
uti
onoft hesol venti nt hepr esenceofasol ute.Itisaphenomenont hat
happensf oral lsolutesi nal lsolutions,eveni nidealsoluti
ons,anddoesnotdependon
anyspeci f
icsol ute–solv entinteract i
ons.Theboi lingpointelevati
onhappensbot hwhen
the sol ute i s an  el
ectrolyte, such as v arious salts, and a nonel ectr
olyte.
In t
hermody nami c terms,t heor iginoft heboi l
ingpointelevationis entropic andcanbe
explainedi nt ermsoft he vaporpr essure or 
chemi calpotential
 oft hesol vent.Inbot h
cases,t heexpl anationdependsont hef actthatmanysol utesar eonl ypr esenti nthe
l
iquid phase and do notent eri ntot he gas phase ( exceptatext remel y high
temper atures).
Puti nv aporpr essur et erms,al iquidboilsatt het emperaturewheni tsv aporpr essure
equalst hesur roundi ngpr essur e.Fort hesol vent,thepresenceoft hesol utedecr eases
i
t svaporpr essur ebydi lution.
Whenasol utei saddedt owat ert heboi lingpoi nti si ncreasedandt hef reezi ngpoi ntis
decr eased.I nt hel at terinst ance,t hi spr inciplei sut i
lizedt odet ectwat ert hathasbeen
addedt omi lkt oi ncr easei tsv olume.Thus,i fwat eri saddedt omi lk,t hewat er -
soluble
subst ances( pr imar ilyinor gani csal t
sandl actose)ar edi lutedandt hef reezi ngpoi ntis
i
ncr easedov ert henor malav erageof0. 517◦ C.TheAOAC( 2006)hasst andar dizedt his
procedur eusi ngacy roscopeandi n
thi
smet hod, somet oleranceshav ebeeni ncor por atedt oaccountf orav ar ietyoff actors
thatcani nfluencet hev aluesobt ained.
5)Pr ot einDet ermi nat ionsi nDai ryPr oduct s
Allpl antand ani malcel l
shav est ruct ur alandbi ol ogicallyf unct ionalpr oteins.The
buildingbl ocksoft hesepr ot ei nsconsi stof20al phaami noaci ds[ RCH-NH2- COOH]
thatar el inkedt hr oughapept i
debr idge.Pr ot ei nsar ei mmensel yi nter est ingast hey
hav et heabi l
ityt of ormv erycompl exmol ecul es,andar et ypi callyr epr esent edi nf our
differentf or ms:pr i
mar y,secondar y,t er tiary,andquat ernar y.Mi lkpr ot eins,suchas
casei n,l act albumi n,orl actogl obul in,cont ainv ar yingamount sofami noni t r
ogenand
aromat icami noaci ds.Anal yticalpr ocedur esf orpr otei ncont enti ndai ryf oodsi nvolve
the det er mi nat ion of ni t
rogen ( i.e.,Kj eldahl ),dy e-binding ( Ami do Bl ack) ,and
spect r oscopi cpr ocedur es.Themostcommonl yusedmet hodi st heKj eldahl becauseof
arel at i
v elyshor ttimet or esul tandl ow- cost .
Milkpr oteinscont ai nasl i
ght lyl oweramountofni trogeni nt hei rpr ot eins,andaf actor
of6. 38i susedi nt hecal culation.
6)Lact osei nDai ryPr oduct s
Lact osei st hepr inci palsugarf oundi nmostofal lmammal ianmi lk,wi thonl ytwot o
threeexcept i
ons,whi char enotger manet ot hisdi scussi on( i
.e.,t heseal i
onandt he
duck- bi ll
edpl at ypusdonotpr oducenot iceabl el act osei ntheirmi lk) .Iti sadi sacchar ide
composedoft hemonosacchar idesl -gl ucoseandl -
gal act ose.Dai ryst artercul turesof
yest ery earspl itt hel act oseandf erment ed t hegl ucosewhi let hegal act osewoul d
accumul atei nt hepr oduct .Today ’
sst ar terscanbesel ected( mut at ed)t of er mentbot h
monosacchar ides.Lact osel ackst hesweet eningpoweroft hepl antsugarsucr ose.I n
recentt imes,conf ect i
oner shav eadded subst ant ialamount sofl act oset o cer t
ain
product sasi tenhancesst abi l
ityand i mpr ov esy ield.Thi shaspr ov ided t hedi ary
i
ndust rywi tht heoppor tunityt out i
lizeanot herf ract ionofmi lkasi def rom dr iedwhey ,
casei n,but terf at,andot hercommer ci alf ract i
ons.Thechemi calanal y sest oquant it
ate
the l act ose ar e qui tei nvolv ed and t edious and r equi re sophi sticat ed equi pment .
Polar imet ric, gr av imet r
ic, i nf rared, enzy mat ic, and hi gh per for mance l iquid
chromat ogr aphy( HPLC)met hodshav ebeengr eat lyadv ancedi nthepast10y ears.The
polar imet ricandHPLCmet hodsar eout linedi nSt andar dMet hodsf ort heExami nation
ofDai ryPr oduct s.Thef ormeri sr elat ivelyst rai ghtfor war d;howev er ,apol arimet eris
requi redandt hedi sposaloft hemer cur yusedi nt heassayr equi resspeci alhandl ing
andat tent ion.TheHPLCpr ocedur esar ei ndeedi nv ol ved,butar et hest andar di nt he
i
ndust ryast her ear enospeci alhandl ingordi sposalconcer nsbey ondt hoseofany
normal“ wetchemi st ry’labor atory.HPLCassay sof f
ertheaddedbenef i
toft esti
ngf or
additi
onalsugarf ract i
onsi nt hesampl e,suchasdext rose,f ructose,sucr ose,and
mal t
ose.
a)HPLCPr ocedur e:
A5. 00-gsampl eisdi ssol v edorext ractedbyst i
rri
ng,heating,and/ orshakingi nwater.
Thesol utionusedf oranal y sisisusual lya5% sol uti
onorsuspensi onofsampl e,and
contains 10% acet oni tr
ilei n 100 mL ofaqueous sol uti
on.The ext r
acti sf i
lt
ered,
chromat ogr aphedbyHPLC, andt hesugar si dentifi
edandquant itatedbycompar i
sonof
thesampl echr omat ogr am wi t
ht hatoft hesugarst andards.Spi kingofsomesampl es
maybeusef ulini dent ifying peaks.St andar d soluti
onscont ainf ructose,dextrose,
mal t
ose, l
act ose,andsucr ose.Si nglestandar dsorcombi nati
onst andardsmaybeused.
b)Pol arimet r
icProcedur e
From not es
7)Det ect ionofAdul terant si nMi l
k
Qualitycont r
oltest sf ormi l
kar ev eryi mpor t
antt oassur eadul t
erantf r
eemi l
kf or
consumpt ion.Adul ter ationofmi lkreducest hequal i
tyofmi l
kandcanev enmakei t
hazardous.  Adulter ant sl i
kesoap,aci d,st arch,tablesugarandchemi calslikeformalin
maybeaddedt ot hemi lk. Mostoft hechemi calsusedasadul terantsar epoisonous
andcauseheal thhazar ds.  Adul t
erantsar emai nlyaddedt oincr easet heshel fli
feof
milk.Some oft he pr eser vatives li
ke aci d and f ormali
ni s added t ot he mi l
k as
adulterant s,therebyi ncr easi ngt hest or
ageper i
odofmi lk.Gener ally,waterisaddedt o
themi lkt oi ncreaset hev ol umecont entoft hemi l
k. Someoft hecommonadul t
erants
foundi nmi lkandt hei rdet ect i
onar ediscussed.

a)Mi
croor
gani
sm:

 
   
  
  
 Mi l
kmaycont ainsomehar mf ulmi croor ganismsl i
kebact eri
aal ongwi th 
 some
potentiallybenef icialmi crobes.Mi crobiologicalanal ysi
s ofmi l
ki s carr
ied outt o
determi ne t he degr ee ofbact eri
alcont ami nat i
on in mi lkand   t o understand the
chemi cal changesbr oughtinmi l
kasar esultofmi crobialaction.
 Past euri
zati
oni sdone
todest roysuchhar mf ulbact eri
a. I fpasteur i
zat i
onofmi l
ki snotcar r
iedoutpr operl
y
therewi llbepr esenceofl ar
gercountofbact er iainthemi l
k. Met hyl
enebl ueReduct i
on
testisusedt odetectt hepr esenceofbact er
iai nmi lk.Thist estwor ksont hepr i
ncipl
e
thatthemet hy l
enebl ueindicatori spr esentinanoxi dizedfor m,buti nthepr esenceof
bacteria,leadst ot her eductionoft hisindicatori nacompar ativ
elyshor tspanoft ime. 
Thebl uecol ordev elopedonaddi ti
onoft hei ndicatort othemi l
kwi llchanget owhi t
e
colorwi t
hi nashor tper i
odi ndicatest hepr esenceofbact eri
ai nt hemi l
kandt hus
denotesi mpr operpast euri
zation.
Nor mal floraofmi lki s
 Ent erococcusf aecali
s
 St rept occusl actus
 Lact obacill
ussp.
 Candi daal bicans( yogur t
)
I
ncreasingt
henumberofbact eri
alf
lor
awill
reducet
hecolourofmethyl
enebl
uemore
rapi
dlyduetoincreasingconsumpti
onofoxygen.Thespeedofreducti
onofmet
hyl
ene
bluecol
ourisdirectl
yproport
ionalt
othenumberofbact
eriapr
esentinmil
ksampl
e.

b)Det ectionandQuant ifi

cat ionofCaneSugari nMi lk
Thecommonsugarpr esenti nmi lkisl act ose.Thef atcont entoft hemi lkismor e
compar edt ot hepr ot eincont ent.Tabl esugarl ikesucr osei saddedt ot hemi lkt o
i
ncr easet hecar bohy dr atecont entoft hemi lkandt hust hedensi tyofmi lkwi llbe
i
ncr eased.Sot hemi lkcannow beadul ter atedwi t
hwat erandi twi llnotbedet ected
dur i
ngt hel actomet ert est. Ket osesugarwi llreactwi t
ht her esor cinolt ogi vear ed
color edpr ecipitat e,indi catingt hepr esenceofTabl esugari nmi lk.
Sucr osei sabsenti nmi l
kandi tspr esencei nmi lkindi cat eadul terat i
on.Pr esenceof
sucr osei nmi l
kcanbedet ermi nedbyt hef ol lowingmet hod.
Qual itati
v eMet hod:Modi fiedSel i
wanof f’sMet hod
Fruct osei ncanesugar( sucr ose)r eactswi thr esorcinol inHCl togiv eredcol our.
Seliwanof f’
st est  i
sa compound t estwhi ch separ at es al dose and ket ose sugar s.
Ket osesar edif ferent iatedf r
om al dosesbymeansoft hei rket one/ aldehy deusef ul
ness.
Ifthesugarcont ainsaket onebunch,i tisaket ose.I ft hesugarcont ainsanal dehy de
bunch,i ti sanal dose.Thi st estismuchl ikeBi al’
st est .Thi stestdependsont her ule
that,whenheat ed,ket onesar emor equi ckl ydr iedoutt hanal doses.I tisnamedaf ter
TheodorSel iwanof f,thesci ent ifi
cexper t,whoper f
or medt het est.Att hepoi ntwheni tis
addedt oasol ut ioncont ainingket ones,ar edshadi ngi sf ramedqui cklygi vingaposi tive
test.Att hepoi ntwhenaddedt oanel ementcont ainingal doses,asl owerf r
ami ngpi nk
l
ighti sseen.Let ’smov ewi thsel i
wanof ft estt odistingui shsucr osef rom f ructose.
Seliwanof f’
sTestPr inci ple:
Seliwanof fTesti sat estt osepar ateketosugar sst r
uct ureAl dosugar s.Ket osugar sget
driedouti nthenear nessofconcent ratedaci dst oy ieldf urfuralsort heirsubsi diaries
whi chr eactwi thr esor cinoli nSel i
wanof fr eagentt oy ieldacher ry-redhuecompl exas
the posi
ti
veresul
t.A cherry
-red shadi
ng shows t
he nearness ofketohex
oses.
Ketopent
osesy
iel
dblue-
greenseri
eswhi l
eal
dosesanddi
saccharidesgi
venoshadi
ng
change.

Wi t
hthistest,f
ructosewillbringaboutcher
ryredshading,whil
et healdosesugarsl i
ke
glucosewillgiv
eanegat i
ver esul
twithnoredshadingshowi ngupi ntheseri
es.Inany
case,ifthewar mingi snotasi ti
sexpected(whichismor et han5mi nut
es),aldose
sugarwi l
lcreateapi nkshadi ng.Whil
ethesucrose(abl endoff r
uctoseandgl ucose)
willcr
eateacher r
yr edshadingasar esul
tofthefr
uctoseinit
.

Reagent

A.Resor
cinolSol
uti
on(
0.5%)
:Wei
gh0.
5gofresor
cinoli
nabout40mlofdi
sti
l
ledwat
er.
Add35mlofconcent r
atedHCl(
12N)toi
tandmakeupt hevol
umeto100mlusing
di
sti
ll
edwater.

Note:Ther
esor
cinol
flakesshoul
dbewhi
tei
ncol
our
.
Pr
ocedure

Take1mlofmi l
kinat esttube.Add1mlofResor ci
nolSol
uti
onandmix.Placethetube
i
nboil
ingwaterbathfor5mi n.Withdr
awthetubeandobservethecol
our.Appearance
ofdeepredcolourindicatespr esenceofsucrose,oraketosesugar.Inpur emi l
k
sampl
esnosuchr edcol orisdevelopedandsampleremainswhit
einnature.Theli
mit
ofdet
ecti
onofmethodi s0.1%.

Quani
tat
iveMet
hodf
orsugaradul
ter
ati
on:

Sucr
osecontentcan bedeter
mined byanyofthe f
oll
owi
ng met
hods:Vol
umet
ri
c
method(
Lane-Eynon)andPol
ari
metr
icmethod.

Ti
tr
ati
onMet
hods
The Lane- Eynon method i s an exampl e ofa  tr
it
rati
on method ofdet ermining the
concentrationofreducingsugar sinasampl e.Abur etteisusedtoaddt hecarbohy dr
ate
solut
ionbei nganalyzedt oaf laskcontaini
ngaknownamountofboi l
ingcoppersul f
ate
solut
ion and a  methyl
ene  bl
ue indicator.The r educing sugarsi nt he carbohy dr
ate
solut
ionr eactwiththecoppersul f
atepresentint heflask.Onceal lthecoppersul f
atein
solut
ionhasr eacted,anyf urtheradditi
onofr educingsugar scausest heindicatorto
changef rom bluetowhi te.Thev ol
umeofsugarsol uti
onr equi
redt oreacht heendpoi nt
i
sr ecorded.Ther eactioni snot  
stoi
chemet r
ic,whi chmeanst hatiti snecessar yto
prepareacal i
brat
ioncur vebycar ryi
ngoutt heexper imentwi t
haser iesofst andard
solut
ionsofknowncar bohy drateconcentrat
ion.

Thedi sadvantagesoft hismet hodare(i

)t heresult
sdependont hepreciser eact
ion
ti
mes, temperaturesandr eagentconcentr
ationsusedandsot heseparametersmustbe
caref
ullycontroll
ed;(i
i)i
tcannotdi st
ingui
shbet weendiff
erentty
pesofr educingsugar ,
and( i
ii
)itcannotdi rectl
ydet erminetheconcent r
ati
onofnon- reduci
ngsugar s,(iv
)i t
i
s sucsepti
ble 
t oint
erfer
encef rom ot
hertypesofmol ecul
esthatactasreducingagent s.

Appl
ythef
act
or0.
95f
orcal
cul
ati
ngt
hesugarcont
ent
.

c)Det
ect
ionandQuant
if
icat
ionofSt
archi
nMi
lk

Milkcontai
nsr el
ativelylargeamountoff at.Addi
ti
onofcar bohydr
atetomil
kincreases
i
tssolidcontent.Ther ebyr educi
ngtheamountoff atpresentinthemil
k.Star
chisone
suchcomponentt hati saddedt oadult
erat
emi l
k.Thetesttodetectst
archi
nmi l
kuses
i
odinesoluti
on,addi ti
onofwhi chtur
nst hemilksol
uti
ont oblueblackcol
orduet othe
for
mat i
onofst arch–I odocompl ex,i
nthepresenceofstar
ch.
Quali
tati
vemet hod
Reagent
A.Iodi
neSol uti
on:Di ssolve2.6gofi odineand3gofpot assium i
odi
deinasuffici
ent
quant it
yofwat erandmakeupt o200ml .
Procedur e
Takeabout5mlofmi lkinat estt ube.Br ingt oboi li
ngcondi t
ionandal l
owt het estt ube
to coolt or oom t emper at ure.Add 1- 2 dropsofi odine solut
ion tot he testt ube.
Dev elopmentofbl ue col ouri ndi catespr esence ofst arch which di sappear s when
sampl ei sboi ledandr eappear soncool ing.Thel i
mi tofdetecti
onofmet hodis0. 02%.
Quant i
tat i
veDet ermi nati
onofSt ar chi nMi lk
Iftestf orst ar chisposi t
iv e,quant it
ativeest imat i
onofst archi st obecar ri
edoutf or
determi nat ionofSNF( soli
dnonf at )inmi l
ksampl e.Thesampl eofmi lki scur dledwi th
alcohol ,andmadef reef rom l actosewhi chi snat urall
ypresentinmi lk.Thepr ecipitated
starchi swashedwi th50% al coholt of reei tfrom l act
ose.Thepr ecipitat
edst archi s
hydrol ysedt oconv erti ti
ntor educi ngsugar s.Reduci ngsugarisdet erminedbyLaneand
Eynonmet hodandmul ti
pliedwi th0. 9togett hest archcontentinmi lk.
Whathappenedt ost archwhenal coholi saddedi nit?
Whenet hanoli sadded,whi chi sl esspol art hanwat er,itlower sthesol ubilit
yand
potent i
alhy dr ogen bonds oft he  starch ,because t he wat eris mor e attracted t o
the alcohol  thant he  starch, the  star ch i
sdehy dratedbyt he 
alcohol andpr ecipit
at es
Reagent s:
A.Et hanol (98%) .

B.10%sodi
um hy
droxi
de.

C.Sodi um carbonate.
Procedur e
Wei ghappr oximately25gsampl ei na250ml beaker.Add20ml ofet
hanol t
ocur dlethe
milk.Filt
erthepr eci
pitat
eonaf ilt
erpaperandwasht heprecipitatewith50%et hanolt i
l
l
thepr ecipit
ateisf r
eef r
om lactose/sugari
.e.whent hewashi ngsgiveanegat ivetest
withresor ci
nol.Transfertheprecipit
atetoa500mlf laskwi t
habout200mlwat erand
add10mlconcent rat
eHClt ohy droly
sethestarchbyr ef
luxi
ngi naboi l
ingwat erbat h
for2.5hour s.Coolandneut ral
isewi th10% sodium hy dr
oxideandsodi um car bonate
towardst heendusi ngli
tmuspaper .Makeupt o500mlwi thwat er.Shakewel landf i
l
ter
i
fnecessar y.Det
er minereduci
ngsugarbyLaneandEy nonmet hod.Cal culatestarchas
fol
lows:
%st arch=%r educingsugarx0. 9

d)Det ect
ionofAddedUr eai nMilk
Ureai sanat uralconsti
tuentofmi l
kandi tformsamaj orpartoft henon-protei
n
ni
trogenofmi l
k.Ureaconcent rati
oni nmi l
ki sv ar
iabl
ewi thinherd.Ureacontentin
naturalmilkvariesfr
om 20mg/ 100mlt o70mg/ 100ml .Howev er
,ureacontentabove
70mg/ 100mli nmi l
kindicatesmi l
kcont ai
ning‘addedur ea’.Theadditi
onofur eato
milkcanbedet ectedbyusi ngpara-dimethyl
ami nobenzaldehyde(DMAB) .Thi
smet hod
i
sbasedont hepr i
ncipl
et hatureaformsay ell
owcompl exwi t
hDMABi nalowaci di
c
solut
ionatr oom temperature.

Qual
it
ati
veMet
hod
Thismet hodisbasedonthepr i
nci
plet
hatur
eaf
ormsayel
lowcomplexwi
thDMABina
l
owaci dicsolut
ionatroom t
emperatur
e.
Reagent
A.DMABr eagent(1.
6%,w/v):
Dissol
ve1.6gDMABin100mlet
hylal
cohol
andadd10
ml concentr
ateHCl .
Procedure

Mix1mlofmi lkwit
h1mlof1. 6%DMABr eagent
.Dist
incty
ell
owcol ourisobser
vedin
mil
kcontai
ningaddedurea.Thecont
rol(
normalmi l
k)showsasl i
ghtyell
owcolourdue
t
opresenceofnatur
alur
ea.Theli
mitofdetect
ionofmethodis0.2%.

Quant i
tati
veEst imati
onofUr eai
nMi l
k
Ureaisanat uralconst
it
uentofmi l
kandi spresenttotheextentof70mgper100ml( 700
ppm) .Thet estbasedont heuseofpar a-di
methy l
aminobenzaldehydecanbeusedf orthe
esti
mat ionofur eainmil
kafterpr
ecipi
tati
onofmi l
kprot
einsusi
ngt r
ichl
oroacet
icacid.
Reagent s/Appar at
us
A.p-Dimet hylami nobenzal
dehyde(DMAB)sol uti
on:Di
ssolve1.6gDMABi n100mlet hy
l
al
coholandadd10mlconcent r
ateHCl .Ther eagenti
sst abl
efor1mont h.Preparenew
standardcur vewi t
heachnewbat chofreagent.

B.Phosphat e Buffer pH 7.
0:Dissolv
e 3.
403 g anhydr
ous pot
assium di
hydr
ogen
or
thophosphate (KH2PO4) and 4. 355 g anhy
drous dipot
assi
um monohy dr
ogen
or
thophosphate(K2HPO4)separ
atel
yin100mlofdist
il
ledwater
.Combinesol
uti
onsand
di
l
uteto1l i
tr
ewithwat er.

C.Tri
chlor
oacet
icaci
d(TCA)24%,
w/v:Fr
eshl
ypr
epar
ed.24.
0gTCAi
sdi
ssol
vedi
ndi
sti
l
led
waterandvol
umemadeupt o100ml.

D.Di
lut
ingReagent:Equalv
olumesof24%TCAandphosphat
ebuf
fer(
pH7.
0)ar
emi
xedt
o
makethedil
uti
ngreagent.

E.UreaStandar
dSoluti
on:(a)Stocksolut
ion:5mg/ml .Di
ssolve5±0.001greagentgrade
ureainwateranddil
uteto1l i
tr
ewi t
hwater.(b)Worki
ngsol ut
ion:
–Pipet
te2,4,6,
8,10, 12,
14,16,18and20mlst ocksoluti
oninto250mlv olumetri
cf l
askanddil
utet
ov ol
umewi t
h
phosphat
ebuf f
er.(
c)Referencesoluti
on-Usestandardsoluti
oncont
aini
ng1.0mgur ea/5
ml asreferencest
andard.St
oreatl
essthan24°
C.Thereagenti
sstabl
efor1week.
1.2.4.
2.2.Apparat
us
A.Spect r
ophotometer–Inst
rumentwit
hmaximum bandwidth2.4nm at420nm,wi
th1cm
cell
s

B.What
manf
il
terpaper
:Gr
ade42.

C.Funnel
s.

D.Testt ubes.
Procedur e
Prepar ationofst andar dcur v e
Pipette5mlal i
quot sofwor kingst andar dsol utionsi nto20x150mm ( 25ml )t estt ubes
andadd5mlDMABsol utiont oeach.Pr eparer eagentbl ankof5mlbuf ferand5ml
DMABsol ution.Shaket ubest hor oughl yandl etst andf or10mi nutes.ReadAi n1cm
cellat420nm wi thr eagentbl ankatzer oA.Pl otAagai nstconcent rationur eaPl ot
shoul dbest raightl i
ne
Estimat ion
10mlofmi l
ksampl ei smi xedwi th10mlofTCAt opr ecipitatet hepr oteinsandf il
tered
usingWhat man42f ilterpaper .5ml off il
trateist hent r
eat edwi t
h5ml ofDMABr eagent
todev el
opt hecol our.Bl anki spr epar edbyt aking5mlofdi l
utingr eagentandt reat i
ng
with5mlofDMABr eagent .Theopt icaldensi t
yoft hey ellowcol ouri smeasur edat420
nm.Fr om st andar dcur vetheamountofur eai nmi l
ki scalcul ated.
5)Aci ds:
Gener allyaci dsl ikeBenzoi caci dandSal icyli
caci di susedasapr eserv ativei nf ood
i
ndust r y
.Iti saddedt omi lkt opr eserv eandt husi ncr easet heshel flifeofmi l
k.Pr esence
oftheseaci dscanbedet ect edbyaddi ngconc. sulphur icaci d  andf erri
cchl oride, which
whenr eact swi thbenzoi caci dandsal icyli
caci dt ogi v
ebuf fcoloredandv ioletcol ored
reactionpr oduct s. 
6)Soap:
Soapi saddedt omi lktoi ncr easet hef oami ngofmi l
kandt hust ohav et hickmi l
k. 
Addi t
ionofsuchchemi calswi llcauseheal thpr obl em especi all
yr elatedt ost omachand
kidney s.Soapcanbedet ect edbyaddi ngphenol pht halei
ni ndicatort ot headul terated
mi l
k.Api nkcol orwi llbeobser vedi fsoapi spr esentast heal kaliwi l
lbeneut r
alizedby
theaci dityoft hemi lkwhenphenol pht haleini ndicat orisadded.
7)For mal in:
Formal i
ni sapr eserv ati
veandcanpr eser vemi l
kf orlongper i
odoft i
me.Duet oitshi gh
toxicit
y ,iti sconsi der ed t o causel i
v erand ki dneydamage.  For mal inr eact swi th
Sulphur icaci dandf er ri
cchl or i
det ogi veapur plecol oredr ingatt hej unct ionoft hemi l
k
l
ay ers, t
her ebyi ndicatingt hepr esenceoff ormal inadul teratedi nmi lk.