This presentation was given by Associate Professor Shuahaimi Mustafa, Universiti Putri Malaysia, at the Vita Foods Conference in Hong Kong, 2-3 September 2014

1. DETECTION OF NON-HALAL INGREDIENTS
Shuhaimi Mustafa
Halal Products Research Institute (IPPH), UPM
3-4 SEPTEMBER 2014
AsiaWorld-Expo, Hong Kong

2. Contents
Background
Current issues in halal products
Halal analysis and authentication
Conclusion

3. حلالاً طيبا Halal – permissible by Shariah Law. Haram – prohibited by Shariah Law. Thoyyib – Good and wholesome (quality, safe, nutritious, pure).

4. Muslims cannot consume the following:
•pork or pork by products,
•animals that were dead prior to slaughtering,
•animals not slaughtered properly or not slaughtered in the name of Allah,
•blood and blood by products,
•alcohol,
•carnivorous animals,
•birds of prey,
•land animals without external ears

5. The needs for analysis
Religious beliefs
Allergic
Fair-trade
Laidan (2011) stated that a particular Halal-certified product cannot be genuinely guaranteed halal without being 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 for cooked products containing undeclared animal species.
~50% animal feeds contain undeclared animal species.

9. Meat-forgery scandals

13. Status of some food additives/ingredients
Food additive/ingredients
Description
Status
Riboflavin
Color
Haram if from pork liver/kidney
Lycopene
Color
Doubtful, may be mixed with gelatin
Butylated hydroxyanisole (BHA)
Antioxidant
Doubtful, if animal fat is used as carrier
Glycerol
Sugar alcohols
Doubtful, if it is from animal
Mono-and diglycerides of fatty acids
Emulsifiers
Doubtful, if it is from animal

14. 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

18. 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)
Some methods developed specifically for halal analysis

19. Halal Products Analyses MS ISO/IEC 17025:2005 Accredited
Scope of Accreditation
Material/Products tested
Type of test
Method
Collagen, gelatin capsule
Amino acid profiling
HPLC
DNA (meat and meat- based products)
Porcine specific DNA marker
HaFYSTM

20. HaFYSTM: 1 step, 1 button, ~ 1 hour
Collect Sample
Place in
Test Module
Insert into Analyzer
On one
touch
Yes/No Results

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

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

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

24. Animal feeds

25. 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.

26. PCA VIEW – SCORES PLOT

27. Projection of unknown samples
Gelatin capsules – Nitta (Japan), Gelita (india), Sterling (brazil) & Rousoullet (china).

28. Porcine samples
Porcine (Merck)
Porcine (Fluka)
Porcine skin gelatin
Porcine(Dr.Oetker)
Porcine (Tessenderlon)

29. Alcohol in products (GC/MS method)

30. 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
10000
11000
ETOH/ACN PEAK AREA
ETHANOL CONCENTRATION (PPM)
HIGH ETHANOL CALIBRATION CURVE (1000 - 10 000 PPM)

31. Calculation of Ethanol Content
By taking the equation y = 0.0001x - 0.0019, the amount of ethanol present in sample (ex : Sushi dipping sauce) can be calculated.
Y = Peak area of Ethanol / Peak area of Acetonitrile
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).

32. Alcohol content in beverages
Beers (3-10%)
Wines (8-14%)
Fortified wines (16-22%)
Fruit juices (<0.1%)

34. 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.