An Overview of the Current Analytical Methods for Halal Testing

Abstract

The objective of this paper is to review all the methods that have been developed in the authentication of halal food products, including those developed in our institute. The need for proper control and monitoring of authenticity of food is a serious matter to the authority and the food manufacturers. Strong commitment and continuous support from the government through various agencies would ensure the integrity of the food itself, in terms of both safety and quality. Islamic food laws are based on cleanliness, sanitation, and purity. Hence, the importance of establishing laboratories and using analytical techniques (methods) of authenticity in food for ensuring food safety and protecting consumers from fraud and deception as well as for product recall purposes. Laboratory data may help define the overall scope of work, levels of worker protection, and remediation and disposal methods. Instrumental methods in detection of contamination and/or adulterants in food would clarify any doubt to Muslims, and information can be disseminated for consumer transparency giving better trust and confidence to the authority.

Full text

Chapter 27
An Overview of the Current Analytical
Methods for Halal Testing
Irwandi Jaswir, Muhammad Elwathig S. Mirghani,
Hamzah M. Salleh, Noriah Ramli, Fitri Octavianti and Ridar Hendri
Abstract The objective of this paper is to review all the methods that have been
developed in the authentication of halal food products, including those developed in
our institute. The need for proper control and monitoring of authenticity of food is a
serious matter to the authority and the food manufacturers. Strong commitment and
continuous support from the government through various agencies would ensure
the integrity of the food itself, in terms of both safety and quality. Islamic food laws
are based on cleanliness, sanitation, and purity. Hence, the importance of estab-
lishing laboratories and using analytical techniques (methods) of authenticity in
food for ensuring food safety and protecting consumers from fraud and deception as
well as for product recall purposes. Laboratory data may help define the overall
scope of work, levels of worker protection, and remediation and disposal methods.
Instrumental methods in detection of contamination and/or adulterants in food
would clarify any doubt to Muslims, and information can be disseminated for
consumer transparency giving better trust and confidence to the authority.
Keywords Authentication Halal laboratory Halal products Instrumental
analysis Rapid method
I. Jaswir (&)M.E.S. Mirghani H.M. Salleh N. Ramli
International Institute for Halal Research and Training (INHART),
International Islamic University Malaysia (IIUM), Jalan Gombak,
53300 Kuala Lumpur, Malaysia
F. Octavianti
Faculty of Dentistry, Universiti Sains Islam Malaysia (USIM),
Level 15, Tower B, Persiaran MPAJ, Jalan Pandan Utama,
55100 Kuala Lumpur, Malaysia
R. Hendri
Faculty of Fisheries, Riau University,
Jl. Binawidya KM 12.5 Simpang Baru Pekanbaru, Riau, Indonesia
©Springer Science+Business Media Singapore 2016
S.K. Ab. Manan et al. (eds.), Contemporary Issues and Development
in the Global Halal Industry, DOI 10.1007/978-981-10-1452-9_27
291
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27.1 Introduction
Halal food in the contemporary food industry means that the food is of high quality
and safety and conforms to international standards such as food safety according to
Hazard Analysis and Critical Control Point (HACCP), and of course it should be
permitted under the Islamic Shariah law. It is very challenging and increasingly
difficult for Muslims to ensure the halal status of food in the market due to the
diversification of sources acquired globally for food processing and production.
This trend has raised concerns among Muslim consumers regarding processed food.
Adulteration of value-added food products involving the replacement of high-cost
ingredients with lower grade and cheaper substitutes can be very attractive and
lucrative for food manufacturers or raw material suppliers. Many fraudulent and
deception cases reported worldwide involve adulteration of haram ingredients in
halal food (especially porcine-based products). In other cases, non-halal contami-
nants got introduced in the final food products unintentionally.
Halal food is a sensitive and serious matter to Muslims. With many fraudulent
issues around and cases of unintentional non-halal contaminants in food, more
stringent monitoring should be established by the competent certification authori-
ties. Authentication and verification for halal have become one of the major chal-
lenges in the analysis of processed food. At present, very limited analytical methods
are available for halal food verification. Rapid, sensitive, reliable, and yet affordable
methods are urgently needed for halal food verification and for detection of
non-halal components (e.g., porcine origin) in food products.
The objective of this paper is to review all the methods that have been developed
in the authentication of halal food products, mostly in Malaysia, including those
developed in our institute.
27.2 Current Analytical Methods for Halal Food
Authentication
27.2.1 Gas Chromatography (GC)
Gas–liquid chromatography (GLC), or simply gas chromatography (GC), is a
common type of chromatography used in organic chemistry for separating and
analyzing compounds that can be vaporized without decomposition. Typical uses of
GC were for the determinations of non-halal ingredients in food or for the analysis
of toxicity, which makes the food non-toyyib, i.e., non-halal.
To be suitable for GC analysis, a compound must have sufficient volatility and
thermal stability. If all or some of compound’s molecules are in the gas or vapor
phase at 400–450 °C or below, and they do not decompose at these temperatures,
GC can probably analyze the compound. Analysis of foods is concerned with the
assay of lipids, proteins, carbohydrates, preservatives, flavors, colorants, and texture
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modifiers, and also vitamins, steroids, drugs, pesticide residues, trace elements, and
toxins. Most of the components are non-volatile, and although high-pressure liquid
chromatography (HPLC) is now used routinely for much food analysis, GC is still
frequently used, for examples, derivatization of lipids and fatty acid to their methyl
esters (FAMEs), of proteins by acid hydrolysis followed by esterification (N-propyl
esters), and of carbohydrates by silylation to produce volatile samples suitable for
GC analysis.
GC analysis was used to detect the fatty acid composition. The lard differed from
cow fat in C20:0, C16:1, C18:3, and C20:1, and with chicken fat in C12:0, C18:3,
C20:0, and C20:1 fatty acids (DeMan 1999). Lard and chicken fats are significantly
different in the disaturated and triunsaturated triacylglycerols (TAGs). Marikkar
et al. (2002) had used GC to determine fatty acid composition of RBD palm oil and
a series of RBD palm oil samples adulterated with enzymatically randomized lard
(ERLD). There is a gradual decrease and increase in the amount of C16:0 and
C18:1 and C18:2, respectively, as adulterant is increased in concentration.
27.2.2 Gas Chromatography–Mass Spectroscopy (GC–MS)
It is similar as GC (above); however, it is more accurate, reliable, and fast since two
techniques (GC and MS) are integrated to form a single powerful method for
analyzing mixtures of chemicals. Nowadays, a GC–MS equipment is connected to a
computer and uses advance software that allows building a library of the structures
of targeted compounds to be analyzed.
27.2.3 High-Pressure Liquid Chromatography (HPLC)
HPLC is now used routinely for much food analysis. Modern HPLC has many
applications including separation, identification, purification, and quantification of
various compounds. The major advantage of HPLC is its ability to handle com-
pounds of limited thermal stability or volatility (Macrae 1988). Preparative HPLC
refers to the process of isolation and purification of compounds. Important is the
degree of solute purity and the throughput, which is the amount of compound
produced per unit time. This differs from analytical HPLC, where the focus is to
obtain information about the sample compound. The information that can be
obtained includes identification, quantification, and resolution of a compound
(Regnier 1983).
Chemical separations can be accomplished using HPLC by utilizing the fact that
certain compounds have different migration rates given a particular column and
mobile phase. Thus, the chromatographer can separate compounds from each other
using HPLC; the extent or degree of separation is mostly determined by the choice
of stationary phase and mobile phase. Purification refers to the process of separating
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or extracting the target compound from other (possibly structurally related) com-
pounds or contaminants. Each compound should have a characteristic peak under
certain chromatographic conditions. Depending on what needs to be separated and
how closely related the samples are, the chromatographer may choose the condi-
tions, such as the proper mobile phase, to allow adequate separation in order to
collect or extract the desired compound as it elutes from the stationary phase. The
migration of the compounds and contaminants through the column needs to differ
enough so that the pure desired compound can be collected or extracted without
incurring any other undesired compound.
In order to identify any compound by HPLC, a detector must first be selected.
Once the detector is selected and is set to optimal detection settings, a separation
assay must be developed. The parameters of the assay should be such that a clean
peak of the known sample is observed from the chromatograph. The identifying
peak should have a reasonable retention time and should be well separated from
extraneous peaks at the detection levels, which the assay will be performed. To alter
the retention time of a compound, several parameters can be manipulated such as
the choice of column, choice of mobile phase, and the choice of flow rate.
HPLC application to food analysis had been reviewed by Macrae (1988). Folkes
and Crane (1988) reported on the application of HPLC for carbohydrate analysis
such as low melting point sugars and oligosaccharides. Procedures are now well
established for quantitative determination of carbohydrates in foods and in many
cases have been adopted as standard methods. For complex lipids, those of low
volatility, and those whose chemistry is sensitive to elevated temperatures, HPLC is
the most useful technique. There is an extensive literature on the use of HPLC for
the determination of vitamins in food (Lambert et al. 1985; Christie and Wiggins
1978; Parrish 1980). Other applications for which HPLC seems to be suited are the
resolution of amino acids into their optically active enantiomers and the analysis of
peptides from the Edman degradation in which the amino-terminal residue is
labeled and cleaved from the peptide without disrupting the peptide bonds between
other amino acid residues.
27.2.4 Microscopic Determinations (Microanalysis)
The scanning electron microscopy (SEM) and the transmission electron microscopy
(TEM) laboratory techniques offer a wide range of technologies. It enables expertise
to devise innovative procedures for the study of unusual samples.
The scanning electron microscope (SEM) is a one that uses electrons rather than
light to form an image. There are many advantages in using the SEM instead of a
light microscope. The SEM has a large depth of field, which allows a large amount
of the sample to be in focus at one time. The SEM also produces images of high
resolution, which means that closely spaced features can be examined at a high
magnification. Preparation of the samples is relatively easy since most SEMs only
require the sample to be conductive. The combination of higher magnification,
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larger depth of focus, greater resolution, and ease of sample observation makes the
SEM one of the most heavily used instruments in research areas today. It had been
used for the determination of non-halal leather in leather products (Mirghani et al.
2009). It also has the potential to be used for the determination of non-halal food
products.
27.2.5 Fourier Transform Infrared (FTIR) Spectroscopy
FTIR spectroscopy could be used to analyze food samples such as animal fats,
chocolate, cake, and biscuits for the presence of non-halal ingredients such as lard.
Analyses include characterizing and identifying the differences in FTIR spectra
profiles. FTIR spectroscopy with chemometric analysis offered rapid, simple, reli-
able, and environmentally friendly analytical technique that can detect and quantify
low level of lard-adulterated food samples (3–5 % detection limit). Spectroscopic
methods are an attractive option, fulfilling many analytical requirements such as
speed and ease of use. Of those, mid-infrared methods (Wilson and Goodfellow
1994) have recently been applied to the authentication of a range of materials,
including fruit purees (Defernez et al. 1995), jam (Defernez and Wilson 1995),
olive oil (Yoke Wah et al. 1994), and coffee (Briandet et al. 1996). Che Man and
coworkers have successfully used the FTIR spectroscopy in determining some
quality parameters in edible oils and fats such as iodine value (1999), free fatty
acids (1999a), anisidine value (1999b), peroxide value (2000), and aflatoxins in
groundnut and groundnut cakes (2001), and in detecting the presence of lard in
mixture with animal fats (2000). Al-Jowder et al. (1997) used the mid-infrared
spectroscopy for addressing certain authenticity problems with selected fresh meats
and reported about semiquantitative analysis of meat mixtures.
27.2.6 Electronic Nose (E-Nose) Technology
The new analytical electronic nose, the zNoseTM, is based on electronic sensor
technology, and for the first time, there is a vapor analyzer that performs flash
chromatography and VaporPrintTM imaging in seconds (Staples 2001). E-Nose
provides rapid, early identification and quantification of atmospheric changes
caused by chemical species to which it has been trained. E-Nose can also be used to
monitor cleanup processes after a leak or a spill. Studies have showed that E-Nose
can be used as a rapid detection of non-halal food contaminants in the food matrix
by characterizing simple and complex odors. These instruments could be used for
the authentication of halal food, non-halal items such as alcohol and intoxicating
materials and to some extent to detect whether the slaughtering of animals is
following the Islamic slaughtering, which is a purposeful act, the intention of which
is to take the life of the animal in order to use it as food. That could be, to some
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extent, detection of blood retention in the meat or determining the amount of iron
(Fe) mixed in the flesh.
Furthermore, the potential of E-Nose technology to sense the presence of
pathogens in humans can contribute to the early detection of diseases. Recently,
medical applications of electronic noses have been explored. The use of a novel
electronic nose to diagnose the presence of aflatoxins and other mycotoxins in food
or feeding stuff is of great potential. The relationship between electronic nose
analysis and sensory evaluation of vegetable oils during storage is studied by Shen
et al. (2001). Capone et al. (2001) has used the electronic nose for monitoring
rancidity of milk during its aging days. It was also used as a useful tool for
monitoring environmental contamination (Baby et al. 2000).
27.2.7 Differential Scanning Calorimetry (DSC)
Differential scanning calorimetry (DSC) is a thermoanalytical technique for mon-
itoring changes in physical or chemical properties of material by detecting the heat
changes. Thermogram profiles show the presence of mixed or added substances
such as lard in food sample. It also provides fast and accurate determination of lard
mixed with other oils or other animal fats.
DSC is an instrument that has been widely used in polymer science for a variety
of analyses. The advantages of DSC are that it works rapidly and simply, much
valuable information can be obtained by a single thermogram, and small sample can
yield accurate result (Wang, 1991). Based on DSC profile, melting point, cloud
point, and iodine value of palm oil could be determined quantitatively (Haryati
1999). Marikkar et al. (2001) reported about the detection of lard and randomization
lard as adulterants in refined bleached deodorized palm oil by DSC. Haryati (1999)
found that the difference in TG group composition in fats is reflected on the DSC
thermograms. Detection of animal body fat in ghee and butter using DSC has been
reported by Lambelet (1983), Lambelet et al. (1980), and Coni et al. (1994),
respectively.
27.2.8 ELISA Technique
Enzyme-linked immunosorbent assay, also called ELISA, is a biochemical tech-
nique used mainly in immunology to detect the presence of an antibody or an
antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and
plant pathology, as well as a quality control check in various industries. ELISA
technique is relatively simple to perform. In halal industries, the ELISA technique
has been used for detection of pig derivatives qualitatively in the food samples,
such as sausages from various types of meat. The analysis yielded excellent results
for detection of pig derivatives in samples.
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27.2.9 Molecular Biology Approaches
Molecular biology techniques are commonly employed in research and service
laboratories around the world using the polymerase chain reaction (PCR) which is a
technique to amplify a single or few copies of a piece of DNA, as primer, across
several orders of magnitude, generating thousands to millions of copies of a par-
ticular DNA sequence.
Primers (short DNA fragments) containing sequences complementary to the
target region along with a DNA polymerase (after which the method is named) are
key components to enable selective and repeated amplification. As PCR progresses,
the DNA generated is itself used as a template for replication, setting in motion a
chain reaction in which the DNA template is exponentially amplified. PCR can be
extensively modified to perform a wide array of genetic manipulations. The PCR
technique can be used to verify, certify, and monitor most animal proteins and
related products for halal authentication efficiently and effectively as well as some
other consumer products such as the genetically modified organisms (GMOs).
Nucleic acids present in food are characteristic of the various biologic compo-
nents in complex products. Analysis of specific nucleic acids in food allows the
determination of the presence or absence of certain constituents in complex prod-
ucts or the identification of specific characteristics of single food components.
As DNA is a rather stable molecule, processed food is generally analyzed using
DNA-based method. Due to its specificity and rapidity, the polymerase chain
reaction (PCR) is the method of choice for this purpose.
PCR analysis of a food includes the following steps: isolation of DNA from the
food, amplification of the target sequences by PCR, separation of the amplification
products by agarose gel electrophoresis, estimation of their fragment size by
comparison with a DNA molecular mass marker after staining with ethidium bro-
mide, and finally, a verification of the PCR results by specific cleavage of the
amplification products.
A very convenient approach is to perform PCR amplification and verification in
one single run by using a target-specificfluorescent-labeled oligonucleotide probe
in a real-time PCR system. Real-time PCR requires more expensive laboratory
equipment, but allows the gel-free product detection without the need to open the
PCR tubes after amplification again. This approach is therefore less
time-consuming and labor-intensive. It implies a lower risk of contamination, and
there is no need to use mutagenic staining dyes such as ethidium bromide. With
real-time PCR, also highly accurate quantitative results can be obtained.
Many procedures of halal authentication using PCR have been developed, for
example, a method for species identification from pork and lard samples using
polymerase chain reaction (PCR) analysis of a conserved region in the mitochon-
drial (mt) cytochrome b (cyt b) gene. Genomic DNA of pork and lard was extracted
using Qiagen DNeasy
®
Tissue Kits and subjected to PCR amplification targeting
the mt cyt b gene. The genomic DNA from lard was found to be of good quality and
is used to produce clear PCR products on the amplification of the mt cyt b gene of
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approximately 360 base pairs. To distinguish between species, the amplified PCR
products were cut with restriction enzyme BsaJI resulting in porcine-specific
restriction fragment length polymorphisms (RFLPs). The cyt b PCR-RFLP species
identification assay yielded excellent results for the identification of pig species.
27.2.10 Conventional Chemical Testing
Traditional wet chemical testing has been used in many laboratories to determine
food quality. Many chemists rely on wet chemical methods; however, these
methods are considered to be non-environmentally friendly as many of these
chemicals are hazardous to living things as well as the environment. Testing of
packaging material and microbial testing are also important for any type of raw
material, food, or feeding stuff, and it is of great importance for packed food as it
can easily spread by local and/or international trading.
27.3 Conclusion
Research and development in halal food authentication are meant to help producers
and processors to verify the technical aspects of halal food production and certi-
fication of food ingredients and additives, as well as find alternatives to existing
non-halal or doubtful (shubhah or mashbooh) ingredients and food processing aids.
The scientific advices in food production especially in terms of halal interpretations
could also influence market potentials and business opportunities along the entire
halal food value chain. International Institute for halal research and Training
(INHART), IIUM, is committed to continuously conduct various researches in the
area of halal food authentication. This will enable us to consolidate and integrate the
opportunities to optimize resources and increase competitiveness to contribute
toward the goal to develop Malaysia as an international Hub for halal products and
services.
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