Seminar Report Molecular Approach in Authentication of Halal Food

7/24/2019 Seminar Report Molecular Approach in Authentication of Halal Food

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SEMINAR REPORT

BTE 3191

Molecular Approach in Authentication of Halal

Food

Muhaad Arif !in "uhairi 1##$%%&

Muhaad Nor 'a(ien !in Norra)hid 1###*$%

Super+i)or,Prof- .r- Ir/andi 0a)/ir

oordinator, .r Fa(ia Ad2ani !inti Ahad Fuad


2/15

Content Pag

e

Introduction 1Halal and Haram issues 1Electrophoresis

Approach

3

PR Approach 5LAMP Approach 9Conclusion 12Discussion 12


3/15

Introduction

ood is an important aspect !or human" #asicall$ it pro%ide us energ$

!or dail$ &or's" As modernit$ is going !or&ard( people comes &ith %ariet$ o!

!ood productions to meet the demands and desires( to ser%e meals &ith the

)est diet *ualit$( to promote to others the ne& disco%er o! !ood recipes( andto ma'e people happ$ en+o$ing the )eauti!ul o! taste"

or Muslims( special consideration o! ,Halal- status need to )e !ollo&ed

as a compulsor$ guideline !or !ood consumption" .he esta)lishment o! ,Halal-

status )$ Allah /"0"." is a great gi!t and a proo! o! Allah-s merc$ to His

creatures" .he )enets o! consuming ,Halal- !ood co%er the ph$sical(

spiritual( emotional and intellectual aspects" In uran( one %erse that

mentions a)out ,Halal- is !rom the second %erse o! /urah Al#a*arah( 4

man'ind( eat !rom &hate%er is on earth 6that is7 la&!ul and good and do not

!ollo& the !ootsteps o! /$aitan" Indeed( he is to $ou a clear enem$8" .here

are man$ other %erses mentioned )$ Allah /"0"." in uran a)out ,Halal-issue"

Also the restriction o! ta'ing ,Haram- !ood( in%erse o! ,Halal- is

supported )$ a hadith narrated )$ /a$$idina A)u #a'ar A" rom him(

asulullah /A0 said that 44.hat )od$ &ill not enter Paradise &hich has )een

nourished &ith Haram8" Hadith )$ #aiha*i" .hus !or Muslim( ,Halal- and

,Haram- concept is a crucial consideration to )e o)ser%ed and &ell

esta)lished in li!e to achie%e the ultimate pleasure o! Allah /"0"."

Halal and Haram issues

/urel$ there are man$ issues regarding ,Halal- and ,Haram- o! the !ood

made and manu!actured outside there" In Mala$sia( Department o! Islamic

De%elopment Mala$sia 6:A;IM7 shares statements a)out some issues on the

&e)site( &hich related to ,Halal- and ,Haram- !ood"

/ome o! the issues are the production o! ne& t$pes o! !ood( animal

)ased and imported !ood ingredients( slaughtering method( !ood additi%es(

!acilities(


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In the perspecti%e o! science( there are some possi)le and practical

&a$s to detect nonhalal constituents in the !ood sample" /cientists 'eep

&or'ing on nding eBcient approaches to trace nonhalal ingredient in order

to 'eep !ood in the mar'ets are %alid !or Muslim according to /hari-ah

compliance"

/ome techni*ues esta)lished and proposed )$ scientists are

1" Microscopic method"

2" Lipid)ased method" Anal$sis o! %olatile organic compound"


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In accordance to this pro+ect( the molecular approaches &e interested

&ith are PC( electrophoresis and LAMP" or the denitional purpose( the

term ,molecular- re!ers to operation in%ol%ing cell( DA( A( protein and

other %er$ small and )asic elements" ,Authentication- is a phrase related toidentication and conrmation"

Electrophoresis approach

a)e Stud2 0ournal,Identication pol$peptide )iomar'ers o! porcine s'in gelatin

)$ t&odimensional electrophoresis"


6/15

Preparation o! samples( isoelectric !ocusing 6IE7( /odium dodec$l sulphate

pol$acr$lamide gel electrophoresis 6/D/PA protein )iomar'ers in

5to 1K adulteration o! pig s'in gelatin" .he possi)le reason !or the presence

and a)sentee o! protein spots at certain le%el o! adulteration is due to

%ariation o! 2DE gel e@periment that aect the measured protein le%els"

ormali=ation step had )een per!ormed to minimi=e the e@perimental

%ariations"

?


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Conclusion

1> )iomar'ers gelatin pol$peptides o! porcine s'in &ere success!ull$

identied )$ 2DE" High resolution o! proteins separation in 2DE gel can )e

o)tained )$ e@traction o! protein !rom gelatin using )uer !ollo&ed )$

protein precipitation using cold acetone" 2DE method has the a)ilit$ todetect as lo& as 1K le%el o! adulteration in gelatin samples" .he data

represented can ser%e as )asis in identi!$ing the sources o! gelatin !or

authentication purposes"

PR Approach

PR )tand for pol2era)e chain reaction- It i) o)t /idel2 u)e techni4ue in the !iotechnolo52

field for an2 purpo)e e)peciall2 in .NA anal2)i) li6e food proce))in5- A) the nae indicate the

techni4ue u)e pol2era)e en(2e to add nucleotide and create a chain reaction or !a)icall2

aplif2 teplate .NA to create a cop2 )e5ent of it repeatedl2-

The fundaental of pol2era)e chain reaction con)i)t of 3 )tep- Fir)tl2 i) the denaturin5 )tep-

The .NA i) heated at it) eltin5 teperature u)uall2 at 9$7 for a!out 1 inute to # inute )o

that the dou!le )trand .NA /ill )plit apart- Ne8t i) the annealin5 )tep- In thi) )tep teperature i)

dropped to &$7 to $7 allo/in5 the prier to !ind to the copleentar2 )trand of the

teplate .NA- Finall2 i) the e8ten)ion )tep- The teperature in thi) )tep i) increa)ed to %$7 for

1 inute to # inute thi) allo/ the thero)ta!le pol2era)e to elon5ate the ne/ copleentar2

)tand fro the teplate .NA /hich )tart at the prier /here our tar5et )e4uence i) identif2

!efore the PR proce))-

Soe of the 6e2 eleent that ha) to !e ta6en /hen doin5 the PR i) eltin5 .NA teperature-Althou5h the denature teperature are u)uall2 9$7: different )pecie) or or5ani) ha) different

teperature for the .NA to denature- It i) al)o iportant to de)i5n the correct prier /hich can

!ind to the copleentar2 )trand of .NA near the tar5et )e4uence- .NA pol2era)e i) al)o the

6e2 pla2er in anufacturin5 ne/ .NA copie) u)uall2 Ta4 pol2era)e are u)ed /hich /a)

i)olated fro !acteria li+in5 in hot )prin5 called Theru) a4uaticu)-

5


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There are actuall2 different +ariation of PR for e8aple colon2 PR: ne)ted PR: Multiple8

PR: In+er)e PR: lon5 PR: Re+er)e Tran)cripta)e PR: Real tie PR and an2 ore-

Real Tie PR

Real tie PR /ill !e ela!orate in detail )ince one of our ca)e )tud2 of olecular anal2)i) on

halal food u)e) thi) ethod- A) the nae indicate thi) PR techni4ue i) )iilar to thecon+entional PR ethod the onl2 different i) that /e are a!le to onitor the aplification of

teplate .NA duplicated in real tie not at the end- It i) actuall2 onitor !2 the ei))ion of

fluore)cence durin5 each c2cle- The theroc2cle /hich denature: anneal: and elon5ate the .NA

teplate !a)icall2 i) connected to the )pectrofluoroeter /hich detect the inten)it2 of the

fluore)cence- Fluore)cence !ind to the .NA )o a) the nu!er of 5ene increa)e the nu!er of

fluore)cence /ill al)o increa)e- ;)uall2 the increa)e of fluore)cence i) ea)ured durin5 the

e8ponential pha)e of PR !2 4uantification-

There are 5enerall2 t/o t2pe of pro!e for the detection of fluore)cence /hich are non)pecific

detection and )pecific detection- Soe e8aple of non)pecific detection are S


9/15

Ta4Man i) a d2e /hich con)i)t of t/o part the 4uencher d2e and the reporter d2e- The reporter

d2e ha+e hi5h tendenc2 to eit photon) !ut )ince it i) di)tri!uted freel2 it i) al/a2) near the

4uencher d2e /hich !loc6 the reporter fro eittin5 photon)- .urin5 anneal proce)) the d2e

/ill attach to the .NA )trand a) the Ta4 Pol2era)e elon5ate the .NA it /ill cut of the reporter

d2e fro the 4uencher d2e- Freein5 the reporter d2e /ill a6e it eit photon) and increa)e the

inten)it2 of fluore)cence- Soe of the ain ad+anta5e of thi) ethod i) that it i) +er2 )pecifichence it doe) not detect non)pecific PR product- ;nfortunatel2 it i) ore difficult to de)i5n:

)oetie the Ta4Man pro!e) do not 4uench efficientl2 and it i) ore e8pen)i+e-

Ta4Man )eparate 4uencher d2e fro reporter d2e )o fluore)cence can !e eitted,

a)e Stud2 0ournal, Anal2)i) of Por6 Adulteration in oercial Bur5er Tar5etin5 Porcine

Specific Mitochondrial 2tochroe B =ene !2 Ta4Man Pro!e RealTie Pol2era)e hain

Reaction

Thi) ournal e8plain a!out the u)e of )pecie))pecific prier and Ta4Man pro!e to detect and

aplif2 1$9!p of )/ine tc2! 5ene- Fir)tl2 )aple /a) collected in triplicate in three differentda2)- Soe of the )aple collected /a) fre)h ra/ u)cle ti))ue of pi5: co/: )heep: 5oat: deer:

and hen- Al)o fi+e fi)h )pecie) cichlid: )had: )hrip: tuna and cuttlefi)h- oercial !ur5er

containin5 por6 al)o purcha)e- Beef and chic6en !ur5er fro Mc.onald and F /a) al)o

!ou5ht a) )aple-

ali!ration of different percenta5e of por6 /a) i8ed /ith !eef !ur5er to )ee the fluore)cence

)i5nal re)pon)e for $-$1: $-1:1 and 1$C containation of por6 eat in !eef !ur5er-

Thi) te)t /ant to )ho/ ho/ efficient and accurate i) the techni4ue in detectin5 por6

containation in food- T/o pair of prier u)e are S/c2t!F and S/c2t!R /hich !a)icall2 tar5et

the 1$9!p fra5ent of )/ine c2t! 5ene- The Ta4Man pro!e u)ed /a) S/c2t!T4M con)i)tin5 ofFAM and 3IAB6FD: an addition of "EN pro!e /a) added a) the 4uencher )ince the pro!e i)

4uite lon5 thi) help reduce the !ac65round )i5nal in aplicon detection-

Re)ult of Fluore)cent profile of PR product aplif2, A and . ?fro porcine )pecific@ B i)

anial and i) fi)h ?fro endo5enou) PR )2)te@,


10/15

Re)ult 4uantification c2le 4 of porcine PR and endo5enou) PR,

Re)ult of fluore)cence profile of PR product o!tain fro !eef !ur5er /ith por6 adulteration A

?1$$C@: B ?1$C@: ?1C@: . ?$-1C@ and E ?$-$1C@ and F ?$C@


11/15

All thi) re)ult +erif2 ho/ accurate i) Ta4Man pro!e techni4ue in detectin5 )/ine c2t! 5ene- E+en

at containation of $-$1C of por6 the pro!e can detect it-

oop Mediated I)otheral Aplification ?AMP@ Approach

AMP i) al)o an aplification techni4ue for i)otheral nucleic acid- It differ fro PR

techni4ue )ince it doe) not need the chan5e in teperature for denature: anneal and elon5ate-

In)tead the teperature reain con)tant a!out $7 to & 7- The pol2era)e u)ed are hi5h

)trand di)placeent acti+it2 and replication acti+it2- The prier u)ed identif2 di)tinct re5ion

on tar5et 5ene )o different prier) are de)i5n- Thi) cau)e hi5h )pecificit2 of tar5et )e4uence-

The product of aplification can !e detect /ith the na6ed e2e !ecau)e of the increa)e 4uantit2 of

p2ropho)phate precipitate a !2product of aplification- So reaction can !e onitor in real tie

!2 fluore)cence d2e li6e S


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.NA i) aplif2 u)in5 the AMP techni4ue notice loop i) for at the end,

a)e Stud2 0ournal, .e+elopent of a Rapid Method for the +i)i!le .etection of Por6 .NA inhalal Product) !2 oopMediated I)otheral Aplification-

Thi) ournal u)e the ethod of AMP to detect por6 containation: the techni4ue +i)i!l2

identif2 por6 .NA in eat product- pi5 )pecific prier /ere de)i5ned accordin5 to the

itochondrial .N1 5ene )e4uence- The prier u)e are inner prier ?FIP and BIP@ and outer

prier ?F3 and B3@- B)t .NA pol2era)e /a) u)ed and aplification of .NA /a) done under

i)otheral teperature at $ to & de5ree el)iu)- A) .NA i) aplif2 p2ropho)phate ion i)

produced thu) colour can !e +i)i!le in pre)ence of calcein and confirin5 por6 containation-

There are te)ted carried out- Fir)tl2 AMP techni4ue /a) te)t on different eat /hich are

cattle: !uffalo: )heep: 5oat: do5: ra!!it: rat: chic6en: duc6: fi)h and pi5- Thi) te)t the )pecificit2of the techni4ue- Secondl2: por6 detection /a) carried out on &$n5: &n5: $-&n5: &$p5: &p5: $-&p5:

&$f5: &f5: $-&f5 of por6 )aple to )ee ho/ accurate the detection i)- Thirdl2: por6 i) i8 /ith

!eef /ith $C: $-$1C: $-1C: 1C: 1$C and &$C of containation- a)tl2: the por6 /a) heated in

different /a2 in /ater at *$7 for 3$ inute and $ inute: in /ater at 1$$7 for 3$ inute and

$ inute: autocla+e at 1#17 for 3$ inute and icro/a+e heated- Thi) i) to )ee /hether .NA

/ill denature and di)inte5rate and can AMP detect it-

1>


13/15

ane 1 to 13 ?pi5: cattle: !uffalo: )heep: 5oat: fo8: do5: ra!!it: rat: chic6en: duc6: fi)h and no

teplate control@,

ane 1 to 9 por6 )aple ?&$n5: &n5: $-&n5: &$p5: &p5: $-&p5: &$f5: &f5: $-&f5 and NT@,

ane 1 to por6 i8ed /ith !eef ?&$C: 1$C: 1C: $-1C: $-$1C: $C and NT@,

ane 1 to * heatin5 proce)) of por6?/ater at *$7 for 3$ inute: /ater at *$7 for $ inute:

/ater at 1$$7 for 3$ inute: /ater at 1$$7 for $ inute: autocla+ed at 1#17 for 3$ inute:

fried for #$ inute: icro/a+e for #$ inute: ra/ and NT-@

The re)ult clearl2 )ho/ ho/ effecti+e i) AMP ethod in detectin5 por6 adulteration- It i)

)pecific )ince it onl2 detect por6 )aple: the accurac2 i) up to $-&p5 )aple: it detect all the

i8ture of por6 /ith !eef e+en at onl2 $-$1C containation and finall2 detect all the por6

under5one different heatin5 ethod /hich i) no )urpri)e )ince .NA i) a )ta!le )u!)tance-

11


14/15

onclu)ion

'e can conclude that all thi) techni4ue olecular are relia!le in detectin5 por6 adulteration in

food- The ain thin5 in anal2)i) i) the rate of containation detection !2 thi) ethod of cour)e

real tie PR and AMP triuph o+er electrophore)i)- Real tie PR i) ore coplicated than

AMP in ter of co)t and ener52 u)a5e !ecau)e of the proce)) /hile AMP i) 4uite )iple

!ecau)e the detection can !e )een /ith the na6ed e2e and not uch co)t and ener52 needed to

perfor the anal2)i)- Althou5h in ter of functionalit2 PR i) ore di+er)e !ut for 4uic6

anal2)i) to 5et re)ult AMP i) definitel2 !etter

Duic6: efficient: accurate and cheap i) our 6e2 in choo)in5 the !e)t anal2)i) ethod /hich i)

AMP ethod-

Reference

Ali: M-: Ha)hi: ;-: .hahi: T-: Mu)tafa: S-: Man:


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Huan5: D-: Gu: T-: 'an5: =-: Huan5: 0-: Gia: H-:

Documents

Seminar Report Molecular Approach in Authentication of Halal Food