Isolation and Hydrolysis of Casein From Non Fat

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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2

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


3/23

3

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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4

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 :";


5/23

5

O'TRALI?E@@


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6

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


10/2310

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


12/23

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


14/2314

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


15/2315

Resu#ts

+ercent ,ie"d

$rou B ;1

$rou &"

$rou & ;"B

$rou : B

$rou ;"B


16/2316

Resu#ts

+ercent ,ie"d

$rou ;

$rou :"&

$rou ;";

$rou 1 ":

$rou B


17/2317

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"


18/2318

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"


19/2319

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"


20/23

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-,

Documents

Isolation and Hydrolysis of Casein From Non Fat