The Necessity of Laboratory Analysis to

Verify the Authenticity of Halal Products

Dr. Shuhaimi MustafaDeputy Director, Putra University

Malaysia

The Second Gulf Conference on Halal Industry and its Services22-24 January, 2013

Farwaniyah, State of Kuwait-Crown Plaza Hotel, Al

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Contents

� Background � Current issues in halal products� Halal analysis and authentication� Conclusion

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Halal – permissible by Shariah Law.

Thoyyib – Good and wholesome (quality , safe, nutritious, pure).

The needs for analysis

� Religious beliefs� Allergic� Fair-trade� Laidan (2011) stated that a particular Halal-certified

product cannot be genuinely guaranteed halal withoutbeing tested in halal laboratory.

� The following examples justify the need for analysis:� From 902 meat products tested; 15.9% cases for raw product

containing undeclared animal species and 22.9% cases forcooked products containing undeclared animal species.

� ~50% animal feeds contain undeclared animal species.

Challenges in halal analyses

� No pressure/requirement by relevant authorities.

� Industry wants faster halal certification process

� Complex analytical techniques.

� Lack of competent analysts.

� Undeclared ingredients and processing aids.

� Lack of biomarkers:

- oil/fat-based

- protein-based

- DNA-based

- metabolites-based

Some methods developed specifically for halal

Technique Target gene/sequence Reference

Conventional PCR 12S rRNA Dalmasso et al. (2004)

Taqman Real-time PCR Genomic Cai et al. (2012)

Molecular Beacon real time PCR Mitochondrial cytochrome b Mohd Yusop et al. (2012)

Commercial Real-time PCR Kit Unknown Demirhan et al. (2012)

Technique Sample Reference

FTIR Shortening Syahariza et al. (2005)

FTIR Gelatin Hashim et al. (2010)

FTIR + PLS Meatball Rohman et al. (2011)

FTIR + chemometrics Ham sausages Xu et al.(2012)

Technique Sample(s) Reference

DSC Cooking oils + lard Mansor et al. (2012)

Technique Sample(s) Reference

MALDI-TOF-MS, LC-MS Porcine gelatine Zhang et al. (2009)

HS-SPME-GC-MS Alcoholic beverage Garcia-Martin et al. (2010)

GCxGC-TOF-MS Lard Indrasti et al. (2010)

GC-MS Pork Nurjuliana et al. (2011)

Halal Products Analyses

Towards ISO 17025

Accreditation

Halal products analysis using Real-

Time PCR

� Reliable

� Sensitive

� 0.001ng DNA - SYBR Green

� 0.0001ng DNA - TaqMan

� 0.001% in actual sample

� Fast, specific and cheap.

1 step, 1 button, ~ 1 hour

Collect Sample

Place in

Test Module

Insert into Analyzer

On one touch

Yes/No Results

Red line : Internal control; blue line : beef sample (Negative Result)

Red line : Internal control; blue line : Cat food containing pork (Positive Result)

Red line : Internal control; blue line : no -template control

Animal feeds

PIG HAIR BRUSH-HIGH RESOLUTION STEREOSCOPIC ZOOM

MICROSCOPE

HIGH RESOLUTION STEREOSCOPIC ZOOM MICROSCOPE

ANIMAL BRUSH

FTIR SPECTRUM OF

DIFFERENT SYNTHETIC

BRUSH

FTIR SPECTRUM OF

DIFFERENT PIG HAIR

BRUSH

Amino acid analysis of gelatin using

HPLC and Chemometric approaches

Stages in analysis

Explanation

Acid hydrolysis � hydrochloric acid is the most widely used agent for hydrolyzing

proteins.

� the hydrolysis process occurred in 110OC within 24hrs

Pre-column derivatization

(Using AccQ tag Fluor reagent )

� using 6-aminoquinolyl-N- hydroxysuccinimidyl carbamate derivatives

Quantification separations by HPLC-FD

� All amino acid derivative separations required a gradient

chromatography due to the wide hydrophobicity range of the amino

acid side chains

� use fluorescence detection with excitation at 248nm and emission at

395nm.

PCA VIEW – SCORES PLOT

Projection of unknown samples

Gelatin capsules – Nitta (Japan), Gelita (india), Sterling (brazil) & Rousoullet (china).

Porcine samples

� Porcine (Merck)� Porcine (Fluka)� Porcine skin gelatin� Porcine(Dr.Oetker)� Porcine (Tessenderlon)

Alcohol in products

Static Headspace parameters

Parameter Value

Thermostat temperature (ºC) 70

Needle Temperature (ºC) 80

Transfer Line Temperature (ºC) 90

Vial Equilibration Time (min) 10.0

Sample Amount (ml) 5.0

Injection time (min) 0.6

Pressure (psi) 10.0

Pressurise time (min) 1.0

Loop Fill time (min) 0.3

Shaking high

GC/MS parameters

ParametersChromatographic column DB-WAX, Agilent – J&W122-7032

Carrier gas Helium, 1ml/min

Injector Split type, 250ºCSplit 10 :1

Oven temp. programming 40 ºC (4 min) - 20 ºC/min to 65 ºC (3 min)- 50 ºC/min up to 220 ºC (1 min).

DetectorMS SourceMS QuadElectron EnergyScan modeMS delay parameter

Mass Spectrometry230ºC180ºC70eVmz 30-4501.90 min

Standard and Sample preparation

Standard preparation GC Standard/ Sample preparation

Sample dilution (if required) 5mL of liquid std/sample was

pipetted into 20mL of headspace vial

5µL of internal standard was pipetted into the vials and capped

immediately

Run sample

Weighting accurately 1 gram of ethanol (purity> 99.9 %) in 100-

mL volumetric flask

Topped up with deionised water, mix well

A series of 7 dilutions (1 ppm –1000 ppm)

y = 0,0069x + 0,0696

R² = 0,9989

0,0000

1,0000

2,0000

3,0000

4,0000

5,0000

6,0000

7,0000

8,0000

0 200 400 600 800 1000 1200

ET

OH

/AC

N P

EA

K A

RE

A

ETHANOL CONCENTRATION (PPM)

LOW ETHANOL CALIBRATION CURVE

(1 - 1000 PPM)

y = 0,0001x - 0,0019

R² = 0,9982

0,0000

0,2000

0,4000

0,6000

0,8000

1,0000

1,2000

1,4000

1,6000

0 1000 2000 3000 4000 5000 6000 7000 8000 9000 1000011000

ET

OH

/AC

N P

EA

K A

RE

A

ETHANOL CONCENTRATION (PPM)

HIGH ETHANOL CALIBRATION CURVE

(1000 - 10 000 PPM)

Calculation of Ethanol Content

� By taking the equation y = 0.0001x - 0.0019, theamount of ethanol present in sample (ex : Sushidipping sauce) can be calculated.

Y = Peak area of Ethanol / Peak area ofAcetonitrile

x = Ethanol Concentration (ppm)� When y = 0.6204, x = 0.6204 + 0.0019/0.0001

= 6222.916 ppm or 0.6223%� Multiply by 10 (1 gm of original sample was diluted

with deionized water up to 10 ml )� So, original sample contains 6.223% w/v ethanol

(considered as HARAM).

Conclusion

�Halal should take into account beyond just

trust, social, spiritual, environmental, and

sustainability issues through document audit-

based and site visit approaches as practiced

by almost all halal certification bodies, but

must also be verified through laboratory

analysis.

�Are we ready?