1

SCEPTISM AMONG

MUSLIMS

WORLD

MUSLIM

POPULATION

2.2 BILLION, 2030

DEMAND

F O R

HALAL

PRODUCTS GELATIN-BASED PRODUCTS

SOURCES OF GELATIN

BOVINE POULTRYFISH PORCINE, 44%

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Halal Food Supply Chain

‘From Farm to Plate’ Concept

Storage & Distribution

Processing

Ingredients & Additives

Packaging

Storage

Transportation Consumption

FarmRaw material:*Animals*Plants

Handlinge.g. slaughtering

Processing/Operations Unit *Preliminary operation*Conversion operation*Preservation operation*Product development

Handling

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ISSUES IN HALAL FOODS

Halal concept is simple; however, the industry is becoming

more complex and sometimes confusing.

Due to breathtaking technological development today and

the diversification of sources acquired globally for

consumer products processing and production, numerous

number of processed products are available in the market.

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ISSUES IN HALAL FOODS

It is very challenging and increasingly difficult for Muslims to ensure the halal

status of products in the market.

This trend has raised concerns among Musllim

consumers regarding new processed food and

consumer products.

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AJINOMOTO’S MSG ISSUE - INDONESIA

In 2000, Indonesia facing the scandal involving

Ajinomoto’s MSG that is allegedly tainted with

pork enzymes.

The news of the discovery caused massive

protests in Jakarta and other Indonesian cities.

Ajinomoto recalled 3,000 tonnes of MSG

products from Indonesian shelves, the

company's factory and warehouses (Ciccone,

2004).

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PIG ENZYME IN CHICKEN - MALAYSIA

Protein hydrolysis enzymes derived

from pig once have caught Malaysian by

storm when the scandal threaten

Malaysian chicken industry.

It started when local tabloid reveal an

issue brought up by an email accuses

most of our country chicken and poultry

entrepreneur using the pig derived

enzyme as a supplement or substitutes

in poultry feed to enhance and shorten

the growth of the poultry.

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Eighty percent of gummies

derived from porcine gelatin

GELATIN CAPSULE

Utusan, 8 Mac 2011

Three of the 15 samples of local

pharmaceutical products using gelatin

capsules containing porcine DNA

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GLOBAL CHALLENGES TO HALAL ANALYSIS

More stringent monitoring system is

needed by Halal Authorities.

Analytical techniques become a major challenge for

authentication of halal

products.

Reliable state-of-the-art scientific methods are

required for analysis of non-halal components (e.g

porcine origin) in halal products.

Competent scientist and scientific methods are

needed.

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Cont...

Analysis should be able to reliably

identify origin of food components.

Sensitive and robust enough to be

applied to complex food/products

matrices.

Analysis based on certain identified

biomarkers;

- Oil/fat-based

- Protein-based

- DNA-based

- Metabolites-based

Muslims and Jews are prohibited to consume any porcine-based products (Regenstein et al., 2003)

The occurrence of BSE disease (FDA, 1997)

Gelatin allergic reaction(Sakaguchi et al., 2001; Kuehn et al., 2009)

Vegetarian lifestyle

Limited study has been done to determine the sourceof gelatin in processed foods;

- ELISA (Doi et al., 2009)

- SDS-PAGE (Nur Azira et al., 2014; Nur Azira et al., 2012)

- Liquid chromatography (Raraswati et al., 2014; Yilmaz et al., 2013)

- Spectroscopy (Xu et al., 2013)

Gelatin is used as an adulterant in edible bird’s nest (Lin et al., 2006; Goh et al., 2001)

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Problem statements

Laboratory analysis is needed to verify the gelatin sources used in the products. However, the method of analysis is still limiteddue to :

1. No reported study on development of analytical method for differentiation of bovine, porcine and fish gelatin, especiallyusing an RP-HPLC. The situation was referred to :-

i. Determination of amino acids in gelatin using 6-aminoquinolyl-n-hydroxysuccinimidyl carbamate as derivatizing reagent.ii. Using normalization technique in principal component analysis to grouping the amino acids according to different

gelatin sources.iii. Validated the method and estimated the uncertainty of amino acids in gelatin matrix based on ISO/IEC 17025

requirements.

2. No reported study on a detection level of non-halal gelatin adulterated in halal gelatin using RP-HPLC method.

3. No reported study on the procedure of extraction gelatin from the gelatin-based products such as paintball pellets,pharmaceutical capsules, gelatin dessert and gelatin gummies and differentiate the gelatin from various sources using the RP-HPLC and PCA.

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High molecular weight protein

Obtained by the partial hydrolysis of collagentype I (skin and bone)

Mainly derived from mammals;Pig skin and cow hide due to availability and attainable quality(Gelatin Manufacturers of Europe, 2015; Balti et al., 2011)

The gelling properties and surface behaviour have justified its use in many processed products;

- Confectionery (jelly, gummy and marshmallow)- Pharmaceutical products (hard shell capsule)

(Schreiber and Gareis, 2007)

Gelatin

GELATIN

High molecular weight protein

Obtained by the partial hydrolysis of

collagen (no plant sources of collagen)

Mainly derived from mammals;

Pig skin and cow hide due to availability

and attainable quality

The gelling properties and surface

behaviour have justified its use in

processed products;

-Confectionery (jelly, gummy and marshmallow)

- Pharmaceutical products

Pig skin, 42.40%

Bovine Hides,

29.30%

Bones, 27.60%

Other, 0.70%

Gelatin World Market

2003

Gummies

Gelatin Dessert

A) FOOD GUMMIES Food gummies are composed of :

a) gelatin : 7% (200 Bloom) , 9% (250 Bloom)b) glucose syrup, sucrose : 73%c) flavour and colord) water : 5%

Gelatin is used to improve the chewiness (elasticity) of the end product.

B) Gelatin Dessert A typical gelatin dessert consist of:

a) Sucrose : 86.5% e) salt b) Gelatin (250 Bloom) : 9% f) flavourc) Fumaric acid : 2.5% g) colord) Sodium citrate : 1.2%

The gelatin concentration in finished product of:a) 230 -250 Bloom – 1.5 -2%b) 250 -270 Bloom - > 2.5%

Two types of capsules : a) Hard shell capsulesb) Soft shell capsules

The shells of hard gelatin capsule is composed of:a) gelatin : 30% d) dyesb) water : 65% e) opacifierc) surface active substance

The shells of soft gelatin capsule is composed of: a) gelatin and plasticizer (ratio 0.3-1.0 for very hard shell to 1.0-1.8 for very soft

shell). b) opacifierd :Titanium oxidec) chelating agent (iron) :< 15ppm d) flavouring : ethyl vanillin (0.15% ) , essential oil (2%) e) moisture content : 6-10 % f) sugar : 5%g) fumaric acid : 1 %

Paintballs are made entirely of non-toxic , food-grade ingredients.

It comprising of 2 parts : a shell and an inner filler. The inner filler composition comprises of :

a) polyethylene glycol (PEG 400 or 600 and/or 3350)b)dyes c) Opacifier (titanium oxide/flour)d)water

The shells consist of:a) gelatin : 45 – 47 % b) plasticizer (glycerin, non-crystallizing sorbitol) : 20-22%c) water : 30-32 %d) dyes e) opacifier

Edible bird’s nestGelatin

(adulterant)+ To increase weight

Porcine gelatin is added to increase the net weight of the EBN (Lin et al., 2006; Goh et al., 2001)

Difficult to detect due to similar color, appearance, taste and texture to the actual EBN

Need method for detection of EBN from adulterant

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DEVELOPMENT OF ANALYTICAL TECHNIQUES FOR HALAL PRODUCTS ANALYSIS

Fourier Transform Infrared (FTIR) spectroscopy

Electronic Nose (E-nose) technology

Differential Scanning Calorimetry (DSC)

Molecular Biology techniques (DNA, Electrophoresis, ELISA)

Chromatography (e.g. GC, HPLC, LC-MS/MS)

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MOLECULAR BIOLOGICAL TECHNIQUES

Protein-based techniques

Protein-based techniques is an alternative for DNA that is

degraded by the manufacturing process.

We develop a method and a kit of extracting gelatin protein from

collagen-containing raw materials and identifying the source of

the extracted gelatin protein.

The analysis of extracted protein by using SDS-PAGE can give

valuable information regarding its source by comparing it with

the original origin.

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SDS-PAGE

DENSITOMETER

Separating the extracted gelatin proteins to their molecular weights (Molecular weight differentiation ranging from 53 – 220 kDa is used as biomarker for the identification)

Sodium dodecyl sulphate - polyacrylamide gel electrophoresis

Porcine gelatin Bovine gelatin

Molecular weight polypeptides - biomarker

An immunological technique that involves a labelled enzyme (a protein that converts substrate into color product) to detect the presence of antigen-antibody interaction in a sample.

Cost effective, high sensitivity, specific and easy to operate.

Synthetic peptide is one of the popular antigen for ELISA development.

It enables the produced antibodies to be targeted with fine specificity to small regions of the protein.

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Enzyme – linked immunosorbent assay (ELISA)

Substrate

Primary antibody

Secondary antibodyconjugate

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Objective

To identify the potential marker peptides of porcine gelatin by combination of sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS PAGE) and

peptide mass fingerprinting (PMF)

IDENTIFICATION OF POTENTIAL BIOMARKERS OF PORCINE GELATIN

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80ᵒC

60ᵒC

40ᵒC

RT Heat-treated gelatin

Products-containing gelatin

125 kDapolypeptide band

SDS-PAGE

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PMF

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List of immunogens

Rabbit immunization

Peptide antigen Polyclonal antibodies

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Objective

To develop and evaluate the efficiency of pAbs (pAb 1, 2 and 3) for determination of gelatin sources in raw materials, pharmaceutical and confectionery products by

competitive indirect ELISA

COMPETITIVE INDIRECT ELISA FOR GELATIN SOURCE DETERMINATION IN COMMERCIALLY PROCESSED PRODUCTS

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ELISAs performance

Based on specificity and sensitivity, pAb2 was selected for further evaluation

This method is useful as a screening approach to reduce the time and cost prior to verification with a highly sophisticated method.

70 commercial products (56 confectionery products and 14 pharmaceutical capsules)

Protein extraction Acetone precipitation

Confectionery products8.9% may be mislabeled as the ingredient label

declared agar and carrageenan as their constituent

Pharmaceutical capsulesNo false-positives results

Competitive indirect ELISA (pAb2)

Analysis of commercial samples

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General conclusion

Antibody Sample/ELISA format

pAb1 -

pAb2 Confectionery, pharmaceutical capsules (Competitive indirect ELISA)Edible bird’s nests(Non-competitive indirect ELISA)

pAb3 Edible bird’s nests(Competitive indirect ELISA)

The developed ELISAs were successful in overcoming the EBN authentication problem of porcine gelatin.

In regard to halal authentication requirements, the developed ELISAs are useful as a screening approach to reduce the time and cost prior to verification with a highly sophisticated method.

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List of Publications

Journals:

1. Nur Azira, T., Amin, I., Shuhaimi, M. and Muhajir, H. (2015). Enzyme immunoassay for detection of porcinegelatin in edible bird’s nest. Food Additives and Contaminants – Part A. 32: 1023 – 1028.

2. Nur Azira, T., Amin, I., Shuhaimi, M. and Muhajir, H. (2016). Determination of porcine gelatin in edible bird’snest by competitive indirect ELISA based on anti-peptide polyclonal antibody. Food Control. 59: 561 – 566.

3. Nur Azira, T., Amin, I., Shuhaimi, M. and Muhajir, H. (2016). Development of anti-peptide enzyme-linkedimmunosorbent assay for determination of gelatin in confectionery products. International Journal of FoodScience and Technology. 51: 54 – 60.

4. Nur Azira, T., Amin, I., Shuhaimi, M. and Muhajir, H. Determination of gelatin sources using anti-peptidepolyclonal antibodies. Under review, Analytical Methods.

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CHROMATOGRAPHY

High Performance liquid chromatography Q-Trap LCMS/MS System

Differentiation of bovine, porcine and fish gelatin using amino acids

analysis incorporated with normalization technique in principal

component analysis.

Detection on primary and secondary amines

Bovine gelatin

Porcine gelatin

Fish gelatin

Bovine gelatin

Porcine gelatin

Fish gelatin

Cumulative Eigenvalue = >95%

PC1 = > 50%

Bovine gelatin

Fish gelatin

Porcine gelatin

Bovine gelatin

Porcine gelatin

Fish gelatin

Results : PC1 = 63 %: Cumulative Eigenvalue = >95%

Detection of adulteration between

porcine and bovine gelatin using RP-

HPLC method coupled with principal

component analysis.

Porcine

at 0.05, 0.1, 0.5, 1.0, 3.0, 5.0, 9.0,12.0, 15.0, 20.0% (w/w)

Bovine

Porcine

Fish

EXPECTATION

(0.05 % w/w of Porcine Gelatin)

Total variances : 96.7 % PC1 : 85 %

(0.05, 0.1, 0.5,1.0, 3.0, 5.0, 9.0, 12.0, 15.0, 20.0 % w/w

of porcine gelatin) Total variances : 95.2 % PC1 : 65 %

Total variances : 96.4 %PC1 : 74 %

Scores Plot Total variances of cumulative eigenvalue :

95.5% PC1 : 67 % The paintball pellet shells were located in

bovine gelatin group.

Correlation Loadings Plot The paintball pellet shells were correlated

to Hyp, Pro, Val, Ile and Leu (non-polarside chains amino acids)

Paintball pellet shells

PHARMACEUTICAL CAPSULES

SCORES PLOT The capsules were located in a group of

bovine gelatin. Total variances : 95.5%, 95.3%, 96.7% PC1 : 63.2%, 66.3%, 67.2%

1. As Sauda2. Nashmir3. Halagel4. Shaklee Lecithin5. Nigella Minsyam

1. Baraka2. Capsugel3. Sofilex4. Cod liver oil5. Scott vitamin

1. Donna2. Hovid3. Hurix4. Vacontil5. Ca Carbonate6. Teong Huat

Scores Plot Total variances of cumulative eigenvalue :

97.7% PC1 : 73.6 % The Porcine Torpac capsules were located

in porcine gelatin group.

Scores Plot Total variances of cumulative eigenvalue :

97.5% PC1 : 75.9 % The Mix Torpac capsules were located in

between the group of bovine and porcinegelatin group.

Scores Plot Total variances of cumulative eigenvalue :

95.8% PC1 : 80 % The veggie-capsule were located outside the

Hotelling T2 ellipse (outside gelatin zone)

Correlation Loadings Plot The amino acids were not correlated

to the veggie-capsule

Gelatin zone

SCORES PLOT Two types of gelatin dessert can be differentiated

according to the source of gelatin. The flavored gelatin dessert were separated from

the centre of data, but in the same region to theirrespective gelatin.

The scores plot were influenced by the content ofgelatin in the flavored gelatin dessert.

Total variances : 96.6%PC1 : 74.6%

The lower content of gelatin in the gelatin desserts had great influences to the distance and correlation of amino acids to their respective group of gelatin.

SCORES PLOT Two types of gelatin gummies can be

differentiated according to the source ofgelatin.

Total variances : 95%PC1 : 67.2%

CORRELATION LOADINGS PLOT Bovine gelatin gummies :

Hyp, Gly, Ala, Met and Ile. Porcine gelatin gummies :

Asp, Pro, Arg, Tyr, Lys, Leu and Phe

SCORES PLOT Bovine and porcine gelatin gummies were

located on the upper and lower part ofthe negative side of PC1 and weresymmetrical to their respective gelatinalong the PC1.

CORRELATION LOADINGS PLOT Different amino acids were correlated to

gelatin gummies and their respectivegelatin group.

Total variances : 95.2%PC1 : 56.1%

Bovine and porcine gelatin gummies can be differentiated from the fish gelatin. However, bovine and porcine gelatin gummies were separated from their respective group of gelatin.

• There are several methods have been developed to detect the presence of non-halal substances in foods and non-foods.

• Some of the methods are useful as screening tools to reduce the time and costprior to verification with a highly sophisticated method.

• The methods are helpful to ensure the integrity of industry in declaring thesource of non-halal substances.

• The development of analytical methods is not static, thus more works arewarranted in the future.

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Concluding Remarks

• Gelatin from different sources which having large similarities in physicochemical properties can bedistinguished using a classical method of amino acids analysis incorporated with PCA.

• A database from the collected data of analysis can be used as a reference to verify the source ofgelatin and authenticate the adulteration among gelatin from different sources.

• Modification on the classical method of analysis has successfully discriminated the porcine andbovine gelatin that used in the gelatin-based commercial product. However, it is recommended todevelop a new database for bovine, porcine and fish gelatin that used in different matrices ofproduct.

• This quantitative method was very useful as an alternative method for halal productsauthentication via laboratory testing.

• Universiti Putra Malaysia

• Prime Minister’ s Department Malaysia

• JAKIM

• Ministry of Science, Technology

and Innovation (MOSTI)

• Ministry of Higher Education

Acknowledgements

Acknowledgements

DR AZILAWATI MOHD ISMAIL

DR NUR AZIRA TUKIRAN

RAJA MOHD HAFIDZ RAJA NHARI

AISYAH ZAFIRAH MD DALI,

AISYAH BINTI ZULKARNAIN,

PENGEKALAN 3 PENSIJILAN ISO UPM

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Terima Kasih | Thank You