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8/16/2019 Isolation and Hydrolysis of Casein From Non Fat
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ISOLATION AND
HYDROLYSIS OF CASEINFROM NON FAT MILK
Acao, PraiseusAwat, Ann ChristineCoru!, Ma" Cristina
Esca#ona, Roe#io$ianan, A%irah
&M'
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Proteins
One o( the (our )io%o#ecu#es Po#*%eric+ the %ono%er is an a%ino aci A%ino acis are #in-e .ia Petie )ons Two t*es o( roteins/ $#o)u#ar an Fi)rous So%e (unctions o( rotein inc#ue/Re0u#ator*, Cata#*tic, Structura#,De(ensi.e, Stora0e, Transort, Recetor
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Mi#-
Co%%on (oo which has hi0h nutritiona# .a#ue 1 essentia# a%ino acis (oun in %i#- rotein Di2erent -ins o( %i#-/ Non3(at, S-i% Mi#-, Low
(at an ure (at Three roteins (oun in %i#-/ Casein,#acta#)u%in an #acta0#o)u#ins" A## are 0#o)u#arroteins"
NON-FAT MILK was used to prevent FATfrom 'contaminating' CASIN! Fats goa"ong wit# t#e protein iso"ation$#%dro"%sis
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Casein One o( the %a4or roteins in %i#- Most a)unant rotein in %i#- 5a#on0 with whe*6 Phoshorotein3 contains one or %ore hoshate
0rous attache in sie chains 5usu" serine orthreonine6
S#ow3i0estin0 rotein 5ro.ies a sustaine re#ease o(a%ino acis6
A %i7ture o( a#ha, )eta an -aa caseins (or% ac#uster ca##e mice""e which is resonsi)#e (or thewhite oa8ue aearance o( %i#-"
Mice""e 3 an* water3so#u)#e a00re0ate, sontaneous#*
an re.ersi)#*, (or%e (ro% a%hihi#e5ossesses )oth h*roho)ic an h*rohi#ice#e%ents6 %o#ecu#es"
I is at H :";
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O'TRALI?E@@
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MATERIALS5NO NEED6
0 Mi#- Ma0ic B Acetic aci H %eter N HSO: 5(or aci h*ro#*sis6 'a5OH6 5(or )ase h*ro#*sis6
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METHODOLO$Y0 %i#- ower
• A %L isti##e water• Stir war% to GC• Re%o.e (ro% the hot #ate
ar% %i#- so#ution• Note initial pH• A rowise o( B Acetic aci5HAc6 whi#e
stirrin0 unti# H :"; 5o not estro* the #u%s(or%e6
• Note the volume of HAc used• Let it stan
Casein• Decant• Dr* )etween #ter an tissue aers•
Compute for percent yield
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Yie# casein
Di.ie e8ua##* into two
Intact casein Casein• ra with
A#u%inu% (oi# an#a)e#
• Re(ri0irate
Intact rotein(or ne7t
%eetin0
• Cut in s%a## ieces• Put in %L
Er#en%e*er Jas-
AciH*ro#*sis
'aseH*ro#*sis
Actua# Yie# 50 Casein6
Theoretica# Yie# 50 Mi#-6
B
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•
A :%# o( N HSO:• P#u0 with cotton anco.er with a#u%inu%(oi# then #a)e#
• Note appearance• Autoc#a.e at Bsi (or
hoursAci
H*ro#*!ate• Di#ute with B%# isti##e
HO
• Trans(er into %L)ea-er
• Neutra#i!e )* ain0 Borce#ain satu#a o('a5OH6 then isso#.e a##o( 'a5OH6 )* heatin0 the
so#ution whi#e stirrin0, onot #et it )oi#
• Check pH using the pHmeter
• I( not *et neutra#i!e, arowise o( saturate'a5OH6
• Check pH again using the pH meter
• Fi#ter
Resiue5iscar6
Fi#trate Trans(er to
0rauatec*#iner, %a-eto %L
%LNeutra#i!eH*ro#*!ate
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Methoo#o0*
Non3(at %i#- was use so that itwou# )e easier to iso#ate casein aswho#e %i#- contains %ore roteins"
The %i#- was on#* war%e so as notto enature the %i#-"
Denature to cause the tertiar*
structure o( 5a rotein6 to un(o#, aswith heat, a#-a#i, or aci
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Methoo#o0*
Autoc#a.e This was one (or the heat3cata#*se
reaction" This was to (ree the a%ino acis in
hi0h ressure as the aci)asecata#*ses the reaction"
HI$H PRESS>RE FASTERHYDROLYSIS
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roup(Acid #%dro"%sis)
*efore Autoc"aving After Autoc"aving
B Casein i not
isso#.e+ Caseinieces with c#ear
#i8ui
'#ac- So#i
& Chun-*, Does notisso#.e easi#*
'#ac- so#ution withthin resiues on the
e0es o( the Jas- anso%e are Joatin0
C#ear #i8ui withwhite so#is
'#ac- So#i
C#ear #i8ui with
white c#u%s o( whitecasein
Dar- )rown with
white aer3thin)rown casein
1 C#ear so#ution withwhite so#is
C#ear so#ution withwhite so#is
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•
A %L o( )oi#in0isti##e HO• A "0 o( 'a5OH6• P#u0 with cotton an
co.er with a#u%inu%(oi# then #a)e#
•ar% 0ent#* toisso#.e a## 'a5OH6
• Note appearance• Autoc#a.e at Bsi (or
hours
'aseH*ro#*!ate
• Di#ute with B%# isti##e HO• Trans(er into %L )ea-er• Neutra#i!e )* ain0 B%L o(
B;N HSO: rowise
• Check pH using the pHmeter
• I( not *et neutra#i!e, arowise o( N HSO: unti#h
• Check pH again using the pH meter
• Fi#ter
Resiue5iscar6
Fi#trate Trans(er to
0rauatec*#iner, %a-eto %L
%LNeutra#i!eH*ro#*!ate
'aseH*ro#*sis
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roup(*ase #%dro"%sis)
*efore Autoc"aving After Autoc"aving
C#a* crea%* whiteso#i
Ye##ow oran0e withwhite reciitate
: C#ou*, #i0ht *e##owso#ution with #u%s
C#ou*, #i0ht *e##owwith s%a## #u%s
;
Dar-ish Ye##ow Mi#- tea co#or )rown
B
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Resu#ts
+ercent ,ie"d
$rou B ;1
$rou &"
$rou & ;"B
$rou : B
$rou ;"B
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Resu#ts
+ercent ,ie"d
$rou ;
$rou :"&
$rou ;";
$rou 1 ":
$rou B
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Discussion
Isoe#ectric Point 5I6 an Protein So#u)i#it* The pI o( a rotein is the p at w#ic# protein is"east so"u."e an as a resu#t it can )e easi#*cau0ht in a %i7ture or so#ution" The H where a %o#ecu#e has not net char0e"
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Discussion
Isoe#ectric Preciitation
A reciitate is a eosit o( so#i artic#es sett#eout o( a so#ution" The %i#- containin0 the rotein casein has an initia# )asic
H ran0in0 (ro% / to 0 (/!/)" Acetic aci wasae (or it to reach the roteins isoe#ectric oint, whichis at H :";" Durin0 the aition o( the aci 5onates HQ6, the ne0ati.echar0es on the outer sur(ace o( the %ice##e areneutra#i!e 5the hoshate 0rous are rotonate6, anthe neutra# rotein reciitates" The casein eties an %ice##e structures )eco%eistur)e or enature to (or% si%#er structures which)eco%e 0e#atinous" Casein was o)taine )* reachin0 its I"
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Discussion
Aci'ase H*ro#*sis o( Protein5Casein6 H*ro#*sis is the s#ittin0 or )rea-in0
o( a su)stance into s%a##er ieces withthe use o( water" Protein h*ro#*sis %a* in.o#.een!*%es or che%ica# rea0ents an inthis case, we use either an aci 3HSO: or a )ase 3 'a5OH6 (or s#ittin0roteins into s%a##er, a%ino acis"
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Aci H*ro#*sis
>ti#i!es su#(uric aci 5HSO:6 Less a%ino acis are estro*e H*ro#*sis that is (re8uent#* use
o.er )ase h*ro#*sis IMPORTANT A"A" DESTROYED IS Tr%ptop#an to 1MIN (*"ac2
pigment after autoc"ave)
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'ase H*ro#*sis
Destro*s %ore a%ino acis than acih*ro#*sis there(ore it is #ess(re8uent#* use" 5Tar0ets S, T, C anR6
>ti#i!es 'a5OH6 5)ariu% h*ro7ie6 IMPT A"A" estro*e/ R Arginine to 13A and O3NITIN
5No h*sica# chan0es6
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Neutra#i!ation
Aci h*ro#*sis/ 'a5OH6 'ase h*ro#*sis/ N HSO: Su"furic Acid 4 *arium %dro5ide
6 *arium Su"fate (Sa"t) (or%e inthe h*ro#*!ate (or (urthercharacteri!ation o( A"A" in EPT
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Re(erences A%hihi#e" 5n""6 Collins English Dictionary – Complete and Unabridged" 5B11B, B11:,
B11, , &6" casein" 5n""6 Mi##er3Keane Enc*c#oeia an Dictionar* o( Meicine, Nursin0, an
A##ie Hea#th, Se.enth Eition" 5&6" Retrie.e Sete%)er B& B(ro% htt/%eica#3ictionar*"the(reeictionar*"co%casein
C#ar-,