Trends in Food Science & Technology 58 (2016) 55e68

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Trends in Food Science & Technology

journal homepage: http://www.journals.elsevier.com/trends-in-food-science-
and-technology

From market to food plate: Current trusted technology and

innovations in halal food analysis
Hamadah Nur Lubis a, 1, Noor Faizah Mohd-Naim b, c, 1, Nur Nazurah Alizul a,
Minhaz Uddin Ahmed a, *
a
Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam, Tungku Link Road, BE 1410, Brunei
Darussalam
b
Faculty of Medicine, Sir Alexander Fleming Building, Imperial College London, SW7 2AZ, United Kingdom
c
PAPRSB Institute of Health Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong, BE 1410, Brunei Darussalam

a r t i c l e i n f o a b s t r a c t

Article history: Background: The global halal industry is currently the fastest growing consumer segments in the world,
Received 31 January 2016 estimated to be worth over one trillion dollars. It has become a powerful market force with Muslims and
Received in revised form non-Muslims alike with future demand for halal food likely to remain strong. With halal products
7 August 2016
becoming mainstream consumer goods, incorrect certication of the halal brand on food products has
Accepted 27 October 2016
become a worryingly regular occurrence. In order to rapidly verify the halal authentication of food
products, sensitive, easy-to-use and reliable scientic method is especially required for halal food
screening.
Keywords:
Halal food
Scope and approach: In this review, we will explain what is considered as halal food before describing
Porcine existing methodologies and their limitations in detecting and identifying non-halal components in food
Pork products. We will also present important technological innovations with signicant potential as routine
Alcohol detection tools for halal analysis.
DNA and protein Key ndings and conclusions: The most commonly used molecular biomarkers during food analyses are
proteins and DNA. Although DNA is generally considered to be most appropriate for animal species
detection, it still presents numerous drawbacks. Nonetheless, with rapid development of modern
technology in biological analysis, a number of approaches incorporating latest biotechnological in-
novations with regards to halal authentication have emerged such as point-of-care diagnostics and
integration of assays with smartphones. It is clear that the evolution of the halal industry presents itself
as a lucrative market to be tapped into and needs to be supported by reliable and efcient screening
methods to ensure that food production is aligned with the halal principles.
2016 Elsevier Ltd. All rights reserved.

1. Introduction Islamic ideals that emphasise purity and cleanliness to promote

Halal food and beverage market is currently gaining momentum
and is predicted to be worth up to US$1.6tn and contribute up to
17.4% of the world food market by 2018 (Thomson Reuters, 2015).
This is only set to grow as the world's Muslim population, currently
estimated at 1.6bn, is expected to reach 2.2bn by 2030, making up
the core market of halal products. The halal principle is founded on
* Corresponding author.
E-mail addresses: minhaz.ahmed@ubd.edu.bn, minhazua@gmail.com
(M.U. Ahmed).
1
Note: Hamadah Nur Lubis and Noor Faizah Mohd-Naim have contributed
equally in this manuscript.
one's health and wholesomeness. Intriguingly, this also appeals to
non-Muslims consumers, who care about the governance and
sustainability of their food, particularly organic food partisans.
Although the concept of halal may be centuries old, the benets are
relevant to our current focus on conscientious eating and ethical
sourcing of safe food.
In recent years, however, halal food consumers are increasingly
concerned about the halal authenticity of their food. In particular,
pork meat substitution and the use of undeclared prohibited in-
gredients in food products are only some of the issues that plague
Muslim buyers. Monetary benets have encouraged the meat
processing industry to commit adulteration and fraud either by
substituting food ingredients for implicit alternatives that are

http://dx.doi.org/10.1016/j.tifs.2016.10.024
0924-2244/ 2016 Elsevier Ltd. All rights reserved.
56 H.N. Lubis et al. / Trends in Food Science & Technology 58 (2016) 55e68
cheaper or adding elusive substances to add product weight so that
the product appear to be of higher value. Methods for halal
authentication of food products are of paramount important to
ensure consumers protection.
Various reviews have previously been published in relation to
the analysis and detection methods of pork derivatives in food.
Rohman and Che Man (2008, 2012) described the analytical tech-
niques of studying porcine proteins while other reviews addition-
ally described other methods used to analyse porcine DNA in halal
food screening (Mursyidi, 2013; Nakyinsige, Man, & Sazili, 2012). In
this review, we will briey present current biological detection
techniques in the analysis of both porcine protein and DNA,
including their limitations. Furthermore, we will summarise com-
mercial kits currently available for the detection of porcine protein
and DNA developed for and targeted to halal consumers and testing
laboratories. More importantly, this review will highlight some
biological innovations reported in recent years, which have great
potential for their future development as halal screening tools in
order to overcome the drawbacks set by current biological detec-
tion methods.
2. What is halal?
Muslims consume foods that are deemed halal, which means
allowed, permitted and wholesome in Arabic (Khattak et al., 2011).
Food or drink that is permitted for consumption must be conrmed
by the Islamic law as revealed in the Quran or the tradition of
Prophet Muhammad (hadith). Descriptions of the type of foods that
are permissible for consumption are further specied in the
following sections.
3. Various food types and their halal classication
All food can be consumed by Muslims except those that are
made of or contain the following: carrion or dead animals, blood
(owing or congealed), swine and all its derivatives, animals
slaughtered in the name of any other than Allah (how Muslims refer
to their God) or not according to Islamic law, animals that were
killed accidentally or on purpose through means such as strangling
or beating, intoxicants (alcohol and drugs), carnivorous animals,
predator birds, and certain land animals (Khattak et al., 2011; Riaz
& Chaudry, 2003). The slaughtering procedure needs to be per-
formed by a sane Muslim who invokes Allah's name while
slaughtering and cut the animal's jugular vein in the neck with a
sharp knife. This is to allow the rapid draining of blood in order to
ensure the quickest death for the animal (Khattak et al., 2011).
4. Animal meat and blood
Meat that is permitted in Islam is those from domesticated
animals such as goat, sheep, cattle, buffalo and camel, which are all
ruminants possessing split hoof with padding in between. Mean-
while, meats from pigs, boars and swines, or any of their derivatives
are strictly forbidden. Birds such as chicken, duck, turkeys, pigeons
and quails, among others, are allowed. Prey birds with sharp claws
such as eagles, falcons and vultures are not permissible. Fresh, raw
meat from permitted animal is halal as long as the animal was
slaughtered accordingly and that the place of slaughter is not
contaminated with any pork meat or its derivatives (Hassan &
Lewis, 2014; Khattak et al., 2011). Meat can undergo various
treatments that may result in changes to its original taste, structure
and texture. During the processing of meat, adulteration can occur
whereby the meat is mixed or substituted with alternative meats
that are cheaper and more easily available, such as pork (Nakyinsige
et al., 2012). This becomes an added concern for Muslim consumers
when purchasing meat.
Consumption of blood or blood plasma is forbidden in Islam
(Hassan & Lewis, 2014; Khattak et al., 2011). Hence, any food
products made of or containing blood or blood plasma, such as
blood sausages, are prohibited and are not considered halal.
5. Fish and seafood
Generally, shes and seafood, such as molluscs and crustaceans,
are halal for Muslims consumption (Hassan & Lewis, 2014; Khattak
et al., 2011). Precautions only arise with processed seafood products
such as surimi, sh cakes, sh llets and sh balls, such that they
neither contain nor become contaminated with any non-halal
ingredients.
6. Food ingredients
Specic food ingredients play a major role in determining
whether a food product is halal or otherwise. Vegetable and its
derivatives are halal as long as they are not adulterated with non-
halal ingredients or intoxicating substances. Gelatin, enzymes and
lard are examples of common food ingredients that may become a
point of concern for Muslims.
Gelatin can be extracted from several sources including pig-
skins, bovine hides and splits, and bone materials from animals
including sh (Cai, Gu, Scanlan, Ramatlapeng, & Lively, 2012;
Elgadir, Mirghani, & Adam, 2013). Porcine gelatin has been the
preferred type in non-Muslims countries due to its cheaper price
and has been regularly used in place of beef gelatin to prevent mad
cow disease contraction (Elgadir et al., 2013). Enzymes are complex
organic molecules that can be derived from plants, animals and
microorganisms. Hence, it is important to note, should enzymes be
present in a product, the source of enzymes, and to ensure it is not
extracted from prohibited animals. Lard is a type of fat that is
derived from pork, and because pork is strictly forbidden in Muslim
diet all food products that contain lard must also be avoided.
7. Milk products
In the dairy industry, cheese-making traditionally requires a
substance called rennet that is used to coagulate milk curds into
cheese. Rennet is usually extracted from the stomach lining of
newly born ruminants including pig. However, thanks to the
advancement of science, cheese manufacturers nowadays have the
option of using microbial or fungal rennet as a substitute in cheese
making. The source of rennet used in cheese production must
therefore be stated on the package labelling to aid Muslim
consumers.
Yogurt, cultured milk, cream and sour cream are made from
processed milk. Milk is generally halal if it is produced from
permissible animals. However, other ingredients such as gelatin,
emulsiers, colourings, stabilisers and enzymes may be added,
usually to produce the desired texture, colour and to extend its
shelf-life. Provided that all other ingredients are obtained from
halal sources, the milk product is considered halal.
8. Beverages and alcohol-containing foods
In Islamic Law, according to both the Quran and the Prophet's
hadith, all intoxicants are forbidden, which includes alcoholic
drinks (Regenstein, Chaudry, & Regenstein, 2003). The general
hadith that discusses this stated that anything which when
consumed in a large quantity causes one to become drunk, then the
consumption of a small quantity of it is also haram. Alcohol itself is
not specically mentioned in the Quran and hadith. What is
 H.N. Lubis et al. / Trends in Food Science & Technology 58 (2016) 55e68
mentioned are wine (Al-Khamr in Arabic), which is a beverage
that contains alcohol derived from grapes and is forbidden in Islam
because it is considered an intoxicant (Riaz & Chaudry, 2003).
Eating, drinking and using products from this source are not
allowed. Synthetic alcohol, or one derived from grapes, may be
used in the production of food and drinks as solvent or as ingre-
dient to bring out specic avours in food (Al-Mazeedi, Regenstein,
& Riaz, 2013; Regenstein et al., 2003; Riaz & Chaudry, 2003). Some
halal-certication organisations do allow a small percentage of
alcohol to be used in food production as long as it is evaporated to a
nal level of not more than 0.5% in food ingredients, and 0.1e0.2%
in consumer products (Al-Mazeedi et al., 2013; Regenstein et al.,
2003; Riaz & Chaudry, 2003). Some halal-certifying bodies may
follow stricter guidelines, while others may have a higher cut off.
However, it is inaccurate to label every drink or food that con-
tains alcohol as unlawful. Traces amount of alcohol (usually in
quantities less than 0.1%) do exist naturally in fruits, fruit juices,
vegetables and breads, in the form of ethyl alcohol (or ethanol), as a
result of natural fermentation (Gunduz, Yilmaz, & Goren, 2013).
Because large quantities of natural alcohol derived from these
sources are not intoxicating, it is not considered haram. Most halal-
certifying bodies would accept small amounts of natural alcohol,
generally less than 0.1% and up to 0.5% (Riaz & Chaudry, 2003).
Again, this could be region-specic and consultations with the
proper authorities as well as end users are needed in order to
establish the specic acceptable guidelines.
9. Chocolates
Chocolates are made up of a variety of ingredients such as milk,
emulsiers, preservatives and gelatin. These additives could be
obtained either from plants or animals, therefore Muslim con-
sumers must be vigilant. Chocolates may also contain alcohol if it is
used during the processing step although alcohol may not be listed
as an ingredient, rendering the chocolate non-halal, even if the rest
of the ingredients do not necessarily derive from animal.
10. Pharmaceutical drug
Besides its utilisation in the food industry, gelatin is also used in
pharmaceutical and cosmetic industries due to its gelling and
thickening properties (Demirhan, Ulca, & Senyuva, 2012; Elgadir
et al., 2013). Therefore, it is also necessary for drugs, medicines
and supplements to have proper halal certication to ensure that
animal derivatives, such as gelatin, have its source clearly stated in
the ingredient list. However, the use of substances derived from
pork in pharmaceutical drugs, in case of life-threatening afictions
in which no alternative drugs are available, may be allowed as they
are deemed life-saving. Muslim patients may be exempted from the
normal dietary law during the time of their illness.
In 2001, the World Health Organization (WHO) reported on the
Islamic legal scholars verdict when they convened on the topic The
Judicially Prohibited and Impure Substances in Foodstuff and
Drugs (the letter can be retrieved from http://www.immunize.org/
concerns/porcine.pdf). They have agreed that the transformation of
porcine products into gelatin alters them sufciently to make them
permissible for observant Muslims to take gelatin capsules-
packaged medicine and porcine-containing vaccines. However,
because different Muslim patients may have different in-
terpretations of Islam, this practice may be acceptable to some
patients but not others. Healthcare professionals, therefore, should
advice Muslim patients on locally available products and their
availability, and if any, of other suitable alternatives to the drug
treatment in question should the drug contain questionable
ingredient.
57
11. Importance of unambiguous halal certication of food
products
Due to the religious requirements stated above, additional layers
of concern and surveillance exist for Muslims when shopping for or
partaking food. Hence, ensuring that food is halal, with clear halal
labels before consumption, is highly crucial as consumers should
feel condent that all aspects of their food, from the ingredients to
the processing and handling, meet the appropriate requirements.
Although halal food industry is a growing food market globally,
trepidations regarding the halal status of food products still persist
today. This is due to the repeated discoveries of non-halal in-
gredients in food otherwise labelled as halal as a result of their
omission altogether from the ingredient list. Due to this recurring
concern, proper determination of halal and non-halal status of food
products become extremely essential and the status needs to
regularly reviewed. This can be performed by modern biological
tools and techniques.
12. Halal supervision
It is important to highlight that halal analysis and verication
need to be performed primarily at the production site before
manufactured products are shipped out in order to ensure no
contaminated product left the production site. It is also additionally
crucial for halal analysis and verication to be employed at loca-
tions downstream of production site, such as importing country
docks and distribution warehouses, to ensure tampering of prod-
ucts does not occur in transit. The food industry works closely with
various halal supervision agencies to obtain permission to use their
supervision agency's trademark symbol on their products based on
their credibility, recognition and acceptance (Motarjemi, Moy, &
Todd, 2014, pp. 490e491). This is a particularly important consid-
eration by various halal food importing countries such as Malaysia,
Saudi Arabia, Singapore and Brunei Darussalam, among others.
Therefore, hiring a reputable halal supervision agency and properly
trained on-site Halal supervisors are critical for any business
dealings involving halal products.
13. Modern biological approaches in halal food analysis
Through the development of biotechnology, techniques used for
halal analysis or pork derivatives detection have been booming,
with the use of hi-tech analytical equipment that provides fast,
robust and reliable detection and analysis. To date, two main
methods for meat species detection and identication in food are
routinely utilised, which are protein- and DNA-based method
(Ballin, Vogensen, & Karlsson, 2009).
Examples of techniques used in protein-based analyses are
those that employ antibodies or aptamers to recognize specic
protein epitopes such as enzyme-linked immunosorbent assay
(ELISA), or those that separates proteins in electric elds such as
isoelectric focusing (IEF) (Fig. 1), electrophoresis and chromatog-
raphy (Fig. 2A). Other methods such as mass spectrometry (Fig. 2A)
and Fourier transform infrared spectroscopy (FTIR) (Fig. 2B) will
also be discussed below (Ballin, 2010; Rodrguez, Garca, Gonza lez,
Herna ndez, & Martin, 2005). Protein-based methods, however, do
have their limitations in the detection of animal species from
cooked, baked or heat-treated food products. This is due to the
characteristic of proteins that tend to denature at high temperature,
limiting their usability for downstream identication steps
(Camma , Domenico, & Monaco, 2012; Nakyinsige et al., 2012).
DNA, on the other hand, is stable at higher temperature, can be
found in a majority of cells and is species-specic (Camma  et al.,
2012; Nakyinsige et al., 2012). This lends a hand in its extraction
58 H.N. Lubis et al. / Trends in Food Science & Technology 58 (2016) 55e68
Fig. 1. Enzyme-linked immunosorbent assays (ELISA) using antibodies, assays us-
ing aptamers and isoelectric focusing. (A) In indirect ELISA, antigen added to plate is
bound by primary antibody, which is then detected by enzyme-labelled secondary
antibody. Substrate is added and converted by the enzyme to detectable coloured
products. Sandwich ELISA utilises an additional layer of capture antibody before the
addition of antigen. (B) Aptamer can be used as an alternative to antibody in assays
analogous to the ELISA. (C) In isoelectric focusing assay, ampholyte solution is incor-
porated into a gel. Electric eld establishes a pH gradient and separate sample proteins
according to their pI or isoelectric point. The gel is stained to reveal the positions of
proteins within the sample.
from various tissues and identifying the origin of food component.
Several molecular techniques based on DNA analyses are species-
specic polymerase chain reaction (PCR) (Amaral, Santos, Melo,
Oliveira, & Mafra, 2014), real-time or quantitative PCR (qPCR)
(Soares, Amaral, Oliviera, & Mafra, 2013; Ulca, Balta, ag in, &
Senyuva, 2013) and polymerase chain reaction e restriction frag-
ment length polymorphism (PCR-RFLP) (Mane, Mendiratta, &
Tiwari, 2009) (Fig. 3). These techniques will be discussed in more
details in the following sections.
14. Protein-based analysis methods
14.1. Immunoassay
Indirect enzyme-linked immunosorbent assay (ELISA) and
sandwich ELISA (Fig. 1A) are the two most common forms of im-
munoassays employed for protein-based species detection. The
assays are based on the interaction between a specic animal
protein and an antibody which recognises it. ELISAs are widely used
due to their specicity and simplicity, and make use of both
monoclonal (MAbs) and polyclonal antibodies (PAbs). MAbs are
produced from hybridoma cells and are selectively specic to tar-
geted antigen. However, production of MAbs is time-consuming
and expensive. Polyclonal antibodies, on the other hand, are a
cheaper option but tend to recognize multiple epitopes, resulting in
non-specic binding.
Due to the tendency of proteins to denature under heat treat-
ment, target antigens of choice need to be thermostable in order to
withstand extreme food processing steps. For example, Chen and
Hsieh (2002) combined indirect ELISA and MAbs against pork
troponin I, a heat stable structural protein, to successfully distin-
guish between species in raw and processed food. Doi, Watanabe,
Shibata, and Tanabe (2009), on the other hand, investigated
porcine gelatin by employing sandwich ELISA using PAbs from
rabbits and goats immunised against bovine gelatin. Porcine gelatin
was observed at a concentration as low as 0.78 ng/ml from com-
mercial processed food. False positives, however, have been ob-
tained with cooked or boiled meats when using ELISA, potentially
due to the assay's affected sensitivity when test samples were
derived from multiple species.
14.2. Aptamers
An alternative method to detect the presence of porcine pro-
teins, other than utilising antibodies, is by the use of highly specic
aptamers. Aptamers are nucleic acids or peptides with appropriate
secondary structures that articially function as ligands to specic
target, much like antibodies. Nucleic acid aptamers are generated
against targets by selection from a large random sequence pool
through a process of Systematic Evolution of Ligands by Exponen-
tial Enrichment (SELEX). Possible aptamer candidates are repeat-
edly cycled with the targets and amplied to obtain an enriched
pool of nucleic acids that are of high afnity and specicity.
Following cloning and sequencing, aptamers specic to the target
are determined. Peptide aptamers selection can be made by yeast
two-hybrid system or biopannings (Song, Lee, & Ban, 2012).
A few studies incorporating aptamer-based analysis have been
conducted to develop strategies to ensure food safety (Fig. 1B).
Nadal, Pinto, Svobodova, Canela, and Osullivan (2012) had gener-
ated highly specic DNA aptamers against Lup an 1 food allergen.
Moreover, Maeng et al. (2012) constructed RNA aptamers targeting
cell wall components of three different foodborne pathogens. Upon
addition of uorescence-tagged RNA aptamers, RNA aptamer-
bacteria-uorophore complex were observed by uorescence mi-
croscopy (Maeng et al., 2012). Although no reports on aptamers
 H.N. Lubis et al. / Trends in Food Science & Technology 58 (2016) 55e68 59

Fig. 2. Chromatography-mass spectrometry and Fourier transform infrared spectroscopy (FTIR). (A) Gas chromatography (GC) or liquid chromatography (LC) can be coupled to
a mass spectrometer to separate metabolites from food products. Sample is injected into a GC or LC column and is retained by the column based on the specic physical properties
of the metabolites within the sample. This results in different metabolites exiting the column and entering the mass spectrometer at different times. The abundance of ions at
specic masses is measured relative to the retention time (the time sample was introduced into column). The area under a chromatographic peak corresponding to a metabolite at a
specic mass and retention time equates to the amount of the metabolites present. Mass spectra can be compared to others in a metabolite database to identify specic metabolites.
(B) FTIR is used to obtain infrared spectrum of food sample using an interferometer. Infrared beam from a source is presented to the sample and enters an interferometer. The
resulting interferogram leaves the interferometer after spectral encoding. The beam enters the sample and the unique frequencies of energy of the sample are absorbed. Finally, the
beam enters the detector to measure the interferogram signal, which becomes digitised and sent to a computer. Fourier transformation is performed and the nal infrared spectrum
is produced.
60 H.N. Lubis et al. / Trends in Food Science & Technology 58 (2016) 55e68

Fig. 3. Polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (RFLP) and quantitative PCR. (A) Before PCR reaction can be performed, DNA is
extracted from food sample. DNA is used as templates for PCR reaction and loaded into a thermocycler along with species-specic primer and DNA polymerase. Heating cycle
separates DNA, allowing primers to bind to complementary target region. DNA polymerase synthesises new DNA strands that is subsequently used as template for further
duplication rounds, inducing a chain reaction. DNA template is exponentially amplied to obtain millions of DNA copies for further manipulation. In a conventional PCR, ampli-
cation products are separated by electrophoresis and the separation prole visualised using DNA stains. In a PCR-RFLP assay, PCR products are rst cleaved by restriction enzyme(s)
to obtain restriction fragments before separation by electrophoresis. (B) In quantitative PCR, amplication of target DNA is monitored quantitatively in a real-time PCR machine. PCR
products are detected using uorescent dyes or uorescently-labelled DNA probes. Amplication plot can be obtained from which the presence and abundance of particular DNA
sequence can be extracted.
 H.N. Lubis et al. / Trends in Food Science & Technology 58 (2016) 55e68
generated for porcine or other non-Halal food contaminants has
been recorded thus far, the potential to utilise this technology for
such use is highly promising.
14.3. Isoelectric focusing
Isoelectric focusing (IEF) is an electrophoretic method by which
proteins are separated based on their isoelectric points (pIs)
(Fig. 1C). The presence of pH gradient established by the addition of
carrier ampholytes is important when separating proteins using
this technique. Application of electricity move proteins through the
pH gradient towards the cathode or anode until they eventually
stop upon reaching the position where the pH is equal to its pI. At
this point, diffusion of protein to a region of lower or higher pH will
force it to migrate back to the cathode or anode, respectively. This
causes the proteins to condense into sharp bands at their specic pI
values, producing specic protein patterns (Iowa State University,
2015; AES, n.d).
In the event that IEF is unable to distinguish between identical
patterns due to derivatisation of samples from closely related
species, two-dimensional electrophoresis (2-DE) can be utilised to
aid identication. Following the differences between the molecular
weights and pI of the protein samples, proteins from beef, pork and
selected poultry species could be effectively differentiated
(Montowska & Pospiech, 2013). Samples from both raw and cooked
meat products were further subjected to mass spectrometry and in
silico proteomic analysis.The relative stability of species-specic
proteins, in relation to degradation due to aging or food process-
ing, is analysed. However, electrophoretic tests are tedious and the
use of proteomics tools for protein analysis would require speci-
alised skills and intensive knowledge of the area, requiring specic
highly skilled personnel.
14.4. Chromatography and mass spectrometry
Another analytical technique to detect proteins in food products
is by liquid (LC) or gas chromatography (GC), whereby sample is
separated into its separate components via interaction between a
liquid or gas mobile phase, respectively, which slowly lters down
through the solid stationary phase. This methodology focuses on
comparison of published protein proles for successful species
identication (referred by Ballin et al. 2009). Oftentimes, food
analysis combines chromatography with mass spectrometry (MS).
MS can be used to analyse proteins by virtue of their molecular
weight and amino acid sequence. Proteomic approaches using MS
are robust and reliable for highly processed meat authentication. In
comparison to DNA extraction, proteins and peptides extraction
protocols can be standardised. In addition, whereby short DNA
sequences could denature upon meat processing, primary struc-
tures of peptides are relatively resistant to harsh conditions.
14.5. Electric nose
In order to detect the presence of pork in meat and meat sau-
sages, Nurjuliana, Man, Mat Hashim, and Mohamed (2011) used an
electric nose based on surface acoustic wave sensor. It is a device
that analyses specic chemical makeup of an odour. GC-MS with a
headspace analyser for aroma proling was also employed to
identify the components contributing to the pork avour. The
electric nose successfully discriminated between samples qualita-
tively with the analysis completed in 15s. Requiring less than ve
grams of sample, analysis by electric nose is rapid, accurate, low
cost, and environmentally-friendly for detection of porcine-based
61
ingredients in food.
14.6. Mass spectrometry soft ionisation techniques
Prior to analysis using mass spectrometry, soft ionisation tech-
niques such as matrix-assisted laser desorption ionisation (MALDI)
and electrospray ionisation (ESI) could be performed (Sentandreu
& Sentandreu, 2014). These techniques allow molecules in the
samples to remain relatively intact during the ionisation process,
which can prove advantageous during the analysis step.
Montowska, Rao, Alexander, Tucker, and Barrett (2014) used
ambient desorption electrospray ionisation MS (DESI-MS) and
liquid extraction surface analysis MS (LESA-MS) to distinguish
skeletal muscle proteins of beef, pork, horse, chicken and turkey
meat from sample mixtures. Following peptidomics analysis from
data obtained by DESI-MS and LESA-MS, the most abundant skel-
etal muscle proteins were correctly identied and classied,
proving that these techniques have great potential for species-
specic analysis and differentiation of skeletal muscle proteins
(Montowska et al., 2014). In addition, by using a multiple reaction
monitoring (MRM) method, von Bargen, Dojahn, Waidelich,
Humpf, and Brockmeyer (2013) were the rst to use rapid and
sensitive mass spectrometry technique to detect the presence of
raw horse and pork in raw beef. The species-specic biomarker
peptides were identied by Fourier transform mass spectrometry
using shotgun proteomic approach and selected after referring to a
biomarker peptides database. With enhanced sensitivity, they were
able to detect pork contamination in beef for as low as 0.13% (von
Bargen et al., 2013) with the method later optimised in processed
food samples (von Bargen, Brockmeyer, & Humpf, 2014).
14.7. Fourier transform infrared spectroscopics and chemometrics
Infrared spectroscopy is a technique that uses infrared to anal-
yse the materials that make up a sample, allowing qualitative
identication of sample components. When the bonds of the atoms
of the material vibrate, the frequencies of vibrations are recorded in
the form of absorption peaks, which become the infrared spectrum
of the sample. This spectrum represents the sample's ngerprint
as no two compounds can possess the same spectrum due to the
difference in the combination of atoms from one compound to
another. Fourier transform infrared (FTIR) spectrometry employs
Fourier transformation, a mathematical technique used to obtain
intensity plot of each individual frequency recorded. The Fourier
transformation is performed by a computer and the resulting
spectrum used for analysis (Thermo Nicolet Corporation, 2001).
FTIR spectroscopy can be further combined with chemometrics in
order to identify and discriminate substituents of food samples. It is
a reliable, rapid and economic technique, which has been used as a
diagnostic tool in the food industry.
Rohman, Sismindari, Erwanto, and Man (2011) employed FTIR
spectroscopy to detect and quantify pork adulteration in beef
meatball. Furthermore, Xu, Cai, Cui, Ye, and Yu (2012) used FTIR
spectrometer to obtain the spectral data of halal and non-halal
Chinese ham sausages. Another pork discrimination test using
FTIR was the analysis of lard in meatball broth (Kurniawati,
Rohman, & Triyana, 2014). Their samples were scanned using
FTIR spectrometer and different peaks could be discerned corre-
sponding to beef fat and lard. Lard, having a higher unsaturation
degree, has a higher peak intensity than beef fat. Using the principal
component analysis (PCA) plot, no lard was identied in the com-
mercial samples tested (Kurniawati et al., 2014).
62 H.N. Lubis et al. / Trends in Food Science & Technology 58 (2016) 55e68
15. DNA-based analysis methods
15.1. PCR
In species-specic PCR amplication of DNA, species-specic
primers are used to amplify certain DNA sequence of the animal
species. The PCR products are then analysed using agarose gel
electrophoresis to ascertain species differentiation (Soares, Amaral,
Mafra, & Oliveira, 2010, Fig. 3A).
Alternatively, before subjecting PCR products to agarose gel
electrophoresis as the nal conformational analysis, restriction
fragment length polymorphism (RFLP) can also be performed
whereby PCR products are digested with restriction enzymes prior
to the electrophoresis step (Fig. 3A). RFLP have few advantages such
as inexpensiveness with no oligonucleotide synthesis necessary
beforehand. An example of a study utilising this assay is by Ali,
Hashim, Mustafa, and Man (2012b) in which RFLP was performed
to selectively detect the presence of porcine DNA at traces amount
of 0.0001 ng and 0.01% pork in a ternary mixture of pork, beef and
wheat our. Even though species-specic PCR techniques can
provide relatively rapid, specic and highly sensitive species
detection from cooked or processed food products, it doesn't pro-
vide any information on the quantity of the species detected that
can greatly aid the deliberation of whether the contamination is
intentional or otherwise. This can be achieved by using real-time
PCR or quantitative PCR (qPCR) (Fig. 3B). During qPCR, products
after each amplication cycle can be monitored with the aid of
uorogenic probes or intercalating dyes. Since the uorescence
emitted can be automatically measured and is directly proportional
to the amount of products, the need for post-PCR analysis can be
obviated (Nakyinsige et al., 2012).
Various qPCR analysis strategies have been able to identify
porcine DNA from both raw and processed food. Ali et al. (2012a)
employed qPCR using species-specic primers and TaqMan
probes to successfully detect 0.01e100% pork contamination in beef
meatballs with high accuracy and precision. Their data was further
veried using RFLP. Additionally, detection of pork from raw meat
was achieved by Yusop, Mustafa, Man, Omar, and Mokhtar (2012)
by using a molecular beacon probe and the amplication of 119
bp fragment of the mitochondrial cytochrome b gene. The porcine
DNA detection limit and the sensitivity of detection were 0.0001 ng
and 0.1% w/w pork in meat mixtures, respectively. Moreover, Soares
et al. (2013) successfully identied and quantied as low as 5 pg of
porcine DNA from pork-poultry meat mixtures by using qPCR and
SYBR Green I intercalating dye. In addition, commercial qPCR kits
can also be utilised in the detection of contaminating species in
food. By using porcine-specic primers in the kit, 1.0% w/w porcine
DNA adulteration was detected in gelatin and gelatin-containing
food (Demirhan et al., 2012). Moreover, Ulca et al. (2013) were
able to detect less than 0.1% porcine DNA from meat mixture and
identify the correct meat species as mentioned in the product
labelling.
PCR-based DNA analyses do have some disadvantages, such as
the cost and time needed for DNA extraction. Extraction of DNA
could take up to 6 h depending on the sample composition, which
makes it a rather slow methodology. In addition, DNA is susceptible
to degradation in processed food, consequently causing difculty in
tracing the targeted DNA potentially giving rise to false-negatives
(Chiter, Forbes, & Blair, 2000).
15.2. Isothermal amplication
Isothermal amplication is a DNA amplication technique that
doesn't require cyclically changing the temperature as in PCR-based
amplication, thus alleviating the need to use expensive thermal
cyclers (Notomi et al., 2000; Safavieh et al., 2016). Several types of
isothermal amplication have been described such as loop-
mediated isothermal amplication of DNA (LAMP), transcription-
mediated amplication (TMA), nucleic acid sequence-based
amplication (NASBA), helicase-dependent amplication (HDA)
as well as many others (Karami, Gill, Motamedi, & Saghania, 2011;
Yan et al., 2014).
For halal detection analysis, LAMP, rst introduced by Notomi
et al. (2000), is widely used due to its highly specic and sensi-
tive gene amplication steps (Fig. 4). LAMP requires six primers for
six distinct sequences to amplify the target DNA of a single species.
The six primers include two loop primers, loop forward (LF) and
loop backward (LB); two inner primers, forward inner primer (FIP)
and backward inner primer (BIP); and two outer primers; forward
outer primer (F3) and backward outer primer (B3) (Ahmed, Nahar,
Safavieh, & Zourob, 2013; Barkway, Pocock, Vrba, & Blake, 2011).
The output of LAMP reaction can be observed by naked eyes
through various means. Liu et al. (2012), for example, subjected the
LAMP end products through gel electrophoresis and ethidium
bromide staining to observe the expected DNA bands. The SYBR
Green dye could also be used to indicate a successful LAMP reaction
by observing the colour change from orange to green after the
addition of the dye at the end of the reaction (Tie, Chunguang,
Xiaoyuan, Xinghua, & Xinhui, 2012). Lim, Teh, and Thong (2013)
employed real-time turbidimeter to measure the turbidity of the
LAMP reaction at 650 nm, whereby a positive reaction would give a
turbidity reading of 0.1 within 60 min. Another indicator dye, hy-
droxyl naphthol blue (HNB), can also be used and added to the
LAMP reaction mixture. The colour change from violet to sky blue
would indicate that the reaction is positive (Barkway et al., 2011).
Ahmed, Hasan, Hossain, Saito, and Tamiya (2010) utilised LAMP
reaction and the detection of amplied DNA electrochemically us-
ing carbon screen printed chip in order to perform species-specic
authentication of pork, chicken and bovine meat in about 60 min
(Ahmed et al. 2010). Later, Roy, Rahman, et al. (2016) and Roy, Wei,
et al. (2016) have used new sensitive loop primers for LAMP based
pork detection on printed graphene and carbon biochips (Roy et al.
2016a; 2016b).
15.3. Analysis of alcohol in food products
Other than the absence of pork, halal foods must also be alcohol-
free. Alcohol, especially ethyl alcohol or ethanol, is commonly used
in food manufacturing or as by-products during food processing.
Therefore, like porcine detection, it is equally crucial to have
analytical detection methods for alcohol in food products. Although
many articles were published describing a range of techniques used
for alcohol detection, this review will briey focus on studies that
employ amperometric biosensor for its detection. Alcohol
biosensor has a higher selectivity and specicity compared to
spectrometric and chromatographic methods, which require
expensive equipment and complex sample pre-treatment (Das &
Goswami, 2013).
Two types of enzymes are investigated using two types of
alcohol biosensors: alcohol dehydrogenase (ADH) and alcohol ox-
idase (AOX) for ADH-based and AOX-based amperometric bio-
sensors, respectively. Different types of electrodes can be used in
amperometric biosensors such as multiwalled carbon nanotubes or
modied glassy carbon electrodes. Das and Goswami (2013) was
able to detect ethanol at as low as 5  10 6 M by using multiwalled
carbon nanotubeseNaon (MWCT-Nf) matrix, which upon
enzyme immobilisation, was encapsulated with polyethylenimine
(PEI) on gold electrode (AuE). In addition, Kuswandi, Irmawati,
Hidayat, and Ahmad (2014) developed a simple and easy-to-use
dip-stick with an incorporated detector that detected the
Fig. 4. Loop-mediated isothermal amplication (LAMP) of DNA. The LAMP reaction employs several primers to target six distinct regions on the double-stranded DNA. The
reaction is initiated upon binding of FIP to the F2c region of the DNA. DNA polymerase extends the DNA from the FIP. The primer F3 binds to its complementary region on the DNA to
displace the newly synthesised DNA. Analogous reaction is performed by BIP and B3. Resulting displaced strands form dumbbell structures. FIP and BIP bind to these dumbbell
structures to synthesise a new strand that forms a loop due to the complementarity of the regions. Elongation of the loop structure forms double-stem DNA loops that displaces the
original strand, which can reform its dumbbell structure and undergo another round of amplication. BIP and FIP anneal to the double-stem DNA loops to prime further strand
displacement. Repeated rounds of these steps amplify target DNA to produce DNA structures of various sizes.
64 H.N. Lubis et al. / Trends in Food Science & Technology 58 (2016) 55e68

presence of ethanol using immobilised AOX/polyaniline (PANI) lm.
Enzymatic reaction between ethanol and AOX oxidised the PANI
lm, which could be visually monitored by the change in the colour
of the dip-stick from green to blue. The biosensor had the detection
limit of 0.001% alcohol with reproducibility of 1.6% and a shelf life of
up to 7 weeks at 4  C (Kuswandi et al., 2014).
Detection of alcohol in complex food samples, however, is yet to
be established and developed. Thus far, the alcohol detector dis-
cussed above only used vapour ethanol, alcoholic beverage such as
beer, or ethanol solution. However, these detectors do provide us
with important platforms for future development of alcohol bio-
sensors to be used in food mixtures. In addition, it may also be
necessary to determine whether the presence of alcohol in food
and drink is due to its natural occurrence or its deliberate addition
during the manufacturing process. Thus far, no tests have been able
to distinguish between the two and this could be a future focus in
the halal analysis eld.
15.4. Analysis of porcine protein and DNA using commercially
available products
Conveniently, a variety of commercial kits that investigate
porcine protein and DNA in food products have been developed,
both for consumers and testing laboratories. Table 1 summarises
and compares several kits in terms of their detection format, time
needed for assay completion and their limit of detection.
Kits that detect porcine protein, such as HalalTest by Capital
Biotech or Pork Detection Kit by Xema (Table 1), are convenient and
straightforward to use, making them useful for end-users of foods,
pharmaceutical and cosmetic products in the market. They make
use of immunochromatographic strips that can detect the presence
of pork in various products. In principle, the strip test detects
specic protein derived from pork, which binds to xed antigen on
the strip resulting in a positive signal. Detection result can be ob-
tained in as short as 5 min, with detection level as low as 0.05%.
However, because the test strip is a paper-based detection method,
the sample needs to be in a liquid form before the test can be
carried out. Therefore, solid food must be dissolved in warm water
prior to testing, which could potentially dilute down the protein of
interest resulting in misinterpretation of data. Additionally, the test
strip might not be an appropriate choice to screen viscous samples
such as cream, paste and lotion. Moreover, porcine proteins may
have denatured under intense heat during the processing step of
specic products rendering them undetectable by the kit. This
could lead to false-negative results that may inspire the product to
be mistakenly labelled as halal.
Alternatively, pork proteins can be analysed by employing
colorimetry using kits such as Biokits Pork (RAW) by Neogen Corp
or ELISA-TEK Raw Meat Species Kit by ELISA Technologies Inc. The
concept of colorimetry in food analysis is similar to that employed
by the immunochromatographic strip test. Unlike the strip test,
however, a positive result could only be detected using a plate
reader. Additionally, it can only provide qualitative result and the
amount of antigen needed for detection is affected by the condition
and the content of the food sample. An impure food sample would
require a higher amount of antigen for positive identication of
possible contamination.
Unlike protein, DNA is more likely to survive extreme temper-
atures during product processing and can be isolated from various
sample types, making them more likely to be detected. PCR and
real-time PCR kits are more reliable and accurate compared to test
strips as they are able to detect minute presence of porcine. Un-
fortunately, PCR and real-time PCR kits involve tedious and labo-
rious works such as sample preparation, DNA isolation, PCR and
data analysis. They also require the use of laboratory instruments
such as incubator, microcentrifuge, and thermal cycler, the cost of
which would be tens and thousands of dollars. This make PCR and
real-time PCR kits not only unusable by direct consumers, but are
also substantially more expensive compared to strip tests that cost
as low as USD$6 per strip. Therefore, halal testing using these kits
can only be performed in a proper testing laboratory.
Nevertheless, it is important to recognize the issue that locally
run tests by consumers could lead to erroneous claims about
products, in case of false-positives. However, as long as the

Table 1
Examples of commercially available kits for halal (porcine) screening.

Commercial Name Supplier Detection Format Detection Limit Time (min) For Direct
Consumers

HalalTest Capital Biotech Immunochro- 5 ppm (food); 5e10

matography 0.5 ppm (fats, blood);
0.02% (alcohol)
Pork Detection Kit Xema Immunochro- 0.1% 5e10
matography
Porcine Detection Kit Perkin Elmer Immunochro- 0.05% (raw); 15e20
matography 0.5% (processed)
Biokits F.A.S.T. Pig Neogen Corporation Immunochro- 1% 30
matography
Biokits Pork (RAW) Neogen Corp Colorimetric 1% ~50 X
ELISA-TEK Raw Meat Species Kit ELISA Technologies Inc Colorimetric 1% ~50 X
Biokits Pork (CKD) Neogen Corp Colorimetric 1% ~150 X
Pork (Pig) ELISA Kit Abnova Corp Colorimetric 5 u/ml ~180 X
ELISA-TEK Cooked Meat Species Kit ELISA Technologies Inc Colorimetric 1% ~210 X
RapidFinder Pork ID Kit Life Technologies Real-time PCR 0.01% (fresh or minimally processed ~60 (qPCR X
meat) only)
RealLine Food Kit e Pork Detect Bioron GmnH Real-time PCR 1-5 copies of DNA ~60 (qPCR X
only)

Progenus TagPro Pig Quantication Progenus Real-time PCR 0.0001% 120 X
Kit
Porcine Detection Kit Agilent Real-time PCR 300 fg <180 X
CarnoCheck Kit Greiner Bio-One GmBH Microarray (DNA Chip) 0.25e1% ~180 X
PKL Porcine PCR Detection Kit Profound Kestrel Species-specic PCR 5 pg 180e240 X
Laboratories
Ron's food kit e pig detect Bioron GmbH PCR 0.01e0.1% <400 X
(extended)
 H.N. Lubis et al. / Trends in Food Science & Technology 58 (2016) 55e68
shortcomings and reliability of each test, kit or technology is being
properly acknowledged, these tests could still be valuable as an
extra assurance for consumers. Should samples be found to be
positive for pork or alcohol, samples should be sent to a lab for
conrmation in order for complimentary tests to be run by tech-
nical experts before any positive claim about specic products are
announced to the public.
16. Future outlook
16.1. Point-of-care diagnostics
Point-of-care (POC) diagnostic refers to on-site diagnosis using
technology that produces real-time detection results for direct and
immediate interpretation without reliance on expensive and
complex laboratory equipment (Jung, Han, Choi, & Ahn, 2015; Park,
Zhang, Lin, Wang, & Yang, 2011). POC testing is available in different
platforms, such as lateral ow strips, lab-on-a-chip system or
microuidic analysis system (Ahmed, Saaem, Wu, & Brown, 2014,
2015). In order for POC diagnostics to be used in halal food anal-
ysis, the detection system should be reliable and affordable for
direct consumers. Due to the zero tolerance for porcine in halal
food, a system's analytical porcine detection must be highly sen-
sitive such that minute contamination is detectable with few-to-
none false negatives, and highly specic such that only porcine
species is identiable with few-to-none false positives. Since POC
diagnostic is to be performed on-the-spot, the detection system
must be user-friendly with simplied protocols so as to be acces-
sible to consumers with limited knowledge on biological analysis
(Jung et al., 2015). Moreover, the diagnostic technique must give out
results fairly rapidly so that users can perform the testing and
obtain the result quickly. The use of POC in halal analysis will
certainly expand the accessibility and practicality of such analysis
to direct consumers.
16.2. Nanobiotechnology
The interactions between biomolecules resulting in chemical
analytical information are the crux of molecular diagnostics and
bioanalytical testing. While normally performed in laboratories,
there has been increased focus to perform such diagnosis outside
the lab at the point of care (Bier, 2012). Nanobiotechnology, an
application of nanotechnology in life sciences, makes use of the
combined knowledge of biological science and material science to
fabricate point-of-care diagnostic or testing devices (Jewett &
Patolsky, 2013). Using nanobiotechnology, bioanalysis takes place
at a nano scale and only require a very small amount of sample
(Lim, Yoshikawa, Tamiya, Yasin, & Ahmed, 2014; Lim & Ahmed,
2015, 2016). The application of nanobiotechnology in the aspect
of food processing has already been incorporated in the food in-
dustry. Halal molecular detection method combined with nano-
biotechnology approaches could be an attractive avenue to explore
in the near future, such as the use of small biosensor chips in
identifying the presence of porcine or alcohol in minute food
sample. Although the use of nanoparticle-based detection is
currently not widely used, the potential of nanotechnology with
regards to rapid detection of non-halal materials is pertinent.
16.3. Next generation sequencing
Next-generation sequencing (NGS) is developed to overcome
the drawbacks of rst-generation Sanger sequencing, which are
time-consuming and expensive. NGS enables researchers to analyse
the genome, transcriptome, or epigenome of any organisms
(Illumina, 2015), and revolutionises genetics analysis (Mardis,
65
2007). NGS platform can be used to sequence the whole genome
of an organism, allowing for specic species identication. Coghlan
et al. (2012) used NGS technology for the identication of animals
and plants contaminants in traditional Chinese medicines. By tar-
geting the p-loop region of trnL gene and mitochondrial 16S rRNA
gene, amplicon sequences were generated, identied and
compared (Coghlan et al., 2012). Tillmar, Dell'Amico, Welander, and
Holmlund (2013) similarly targeted 16S rRNA to investigate the
reliability of NGS technology. By analysing mixtures of DNA from
different species using NGS, they were able to distinguish over
99.9% of mammal species. The sensitivity of the system was highly
profound as their experiment had proved that NGS was able to
detect multiple donors within a mixture containing minor com-
ponents that was as little as 1% of the total DNA (Tillmar et al.,
2013). Although most NGS are performed in laboratories with fa-
cilities specialising in intensive high-throughput sequencing tech-
nologies, the development of bench top next generation sequencers
such as Ion Torrent from Life Technologies and MiSeq from Illumina
has enabled researchers to explore NGS more affordably. However,
consumable products for NGS assay remained costly, with most
items licensed. The assay also requires the use of highly trained
personnel working in a specialised laboratory facility, rendering it
not currently ideal for routine halal analysis.
16.4. Digital PCR
The sensitive quantitation technique of qPCR has been the
analytical method of choice for species detection in food products
for many years. More recently, another type of PCR has been
employed as an alternative to qPCR called digital PCR, which has
been mostly utilised in the clinical eld and genetic analysis. In
contrast to qPCR, digital PCR provides absolute quantication of
target DNA without relying on a calibration or standard curve,
obviating the need for standard samples (Hayden et al., 2013; Kim,
Jeong, & Cho, 2014). Instead, unknown sample is compartmental-
ised into several thousands of minute droplets prior to amplica-
tion with each droplet considered as an individual reaction. If the
target DNA is present in a droplet, the compartment will uoresce
and labelled as positive. The amount of target DNA in the sample is
then quantied by comparing the number of positive compart-
ments to the total number of compartments using the Poisson
statistical method (Bharuthram, Paximadis, Picton, & Tiemessen,
2014; Floren, Wiedemann, Brenig, Schtz, & Beck, 2015). Due to
its sample partitioning nature, digital PCR is more sensitive than
qPCR due to its ability in amplifying DNA from a limited amount of
sample or sample with low copy number. Digital PCR, however, is
more expensive and time-consuming (Yang, Paparini, Monis, &
Ryan, 2014). Nevertheless, the benets offered by digital PCR may
justify further research in establishing its use as a highly sensitive
halal detection system. With technology constantly evolving, the
cost and the processing time for the use of digital PCR will assuredly
decrease as improvement to the technique is introduced.
16.5. Integration with smartphones
Modern smartphones are equipped with cameras that use
complementary metal oxide semi-conductor (CMOS) image sensor
technology to capture high quality and high denition photographs
and videos. The advance internal core processor and the availability
of wireless internet or Bluetooth connectivity additionally allow
faster photographic data transfer and sharing. These sophisticated
features of smartphone enable it to become powerful equipment
for point-of-care diagnosis, performing clinical testing away from
laboratories (Grudpan, Kolev, Lapanantnopakhun, & McKelvie,
2015). It makes it possible for patients in remote areas to use
66 H.N. Lubis et al. / Trends in Food Science & Technology 58 (2016) 55e68
smartphones for health check-ups with the data sent to physician
for monitoring and diagnosis (Roda et al., 2014).
Built-in cameras in smartphones could be used in the laboratory
as alternatives to expensive and bulky data recorder instruments.
For example, when attached to customised optical cradle, a
smartphone camera could function as a spectrometer and be used
as digital light sensor in ELISAs (Long et al., 2014). Alternatively,
smartphone has been used as uorescence spectrometer for
uorescence-based biological assays to detect specic microRNA
sequences (Yu, Tan, & Cunningham, 2014). Moreover, Barbosa,
Gehlot, Sidapra, Edwards, and Reis (2015) developed MCFphone, a
portable detection system which uses a smartphone to recognize
the presence of prostate specic antigen (PSA) by colorimetric and
uorescence quantitative sandwich immunoassay detection. With
the ability to detect PSA concentrations lower than clinical range of
PSA for prostate biopsy, the device could potentially be employed to
screen prostate cancer development without the use of laboratory
equipment (Barbosa et al., 2015).
Besides acting as reliable and useful colorimeter and uores-
cence spectrometer, smartphone could also be deployed as a
biosensor to image and quantify lights from biochemiluminescence
(BCL) assays (Roda et al., 2014). Recently, Lebiga, Fernandez, and
Beskok (2015) designed a disposable light shielded paper-plastic
microuidic device that was able to detect H2O2 at a concentra-
tion of 250 nM. Following a chemiluminescence reaction between
H2O2 and another reagent via plastic microuidic channels, the net
photon emission from the reaction is detected by a smartphone,
demonstrating the great potential of combining
chemiluminescence-based sensing system with smartphone inte-
gration as POC diagnostic tool (Lebiga et al., 2015).
Yet another use of smartphone as POC diagnostic tool is as a
potentiostat using its audio socket, eliminating the need for the
traditionally more bulky and expensive hardware (Delaney,
Doeven, Harsant, & Hogan, 2013). Potentiostatic control was ach-
ieved by connecting the smartphone to a sensor through the audio
jack and consequently playing a specic sound le, producing an
output voltage up to 1.77 V. Because smartphone is more or less
ubiquitous in our current daily lives and can be used for different
types of applications to detect specic targets, it is entirely possible
to integrate the use of smartphone for alcohol, DNA or protein
detection for halal food analysis in the future.
16.6. Rapid screening techniques
As part of consumers rights, it is important to disclose in details
the composition of specic products. Religious concerns placed a
certain pressure on the halal analysis and verication workow to
produce results more quickly. We have mentioned a couple of lab
analysis techniques in which the results can be obtained in a matter
of minutes. The combination of FTIR spectroscopy and chemo-
metrics (Xu et al., 2012), for example, produces results in just two
minutes per sample, while the electric nose analysis can be attained
in just 15 s (Nurjuliana et al., 2011). A couple of the halal test kits we
have referred to such as the HalalTest by Capital Biotech and the
Pork Detection Kit by Xema asserted an analysis time of between 5
and 10 min only. Moving forward, more similarly rapid as well as
high throughput techniques should be developed in order to in-
crease analysis efciency to meet the time-sensitive demand of
halal verication.
17. Conclusion
In this review, a considerable variety of studies from recent
years describing different methods employed in halal authentica-
tion analysis in food products have been briey presented. These
reports have shown that animal species validation through DNA
analysis is of great interest and is currently the most suitable
biomolecule for highly specic detection of contaminated food. To
date, PCR can be considered a conventional technique that offers
reliable and sensitive results for identication of miniscule amount
of DNA of different species. However, it does have its limitation, for
example in processing target DNA highly comprised of shorter
amplicons, which results in possible cross-species amplication
and false-positives. Moreover, PCR analyses are currently only
possible in proper testing laboratories and remain inaccessible to
product consumers. Furthermore, traditional protein-based detec-
tion methods using antibodies have been known to have a few
drawbacks such as inconsistent antibody manufacturing that leads
to generation of irreproducible data. In this case, the use of
aptamers could be a more effective approach due to their more
standardised production procedure. It is clear that current trusted
technologies in halal certication analysis can be improved upon
for increased reliability, sensitivity, specicity and faster processing
time. Nonetheless, the advancement of technologies within the
past decade have witnessed the development of several novel
methods with great potentials in detecting contamination in food
products, such as the development POC diagnostic tools that are
affordable, easy-to-use and sensitive as well as the integration of
smartphone into assays.
With the Muslim population showing rapid growth, the halal
food industry is set to expand, further emphasising the importance
of proper halal certication of food products. Western countries
with minority Muslim populations have started to tap into this
trillion dollars industry. Countries such as Brazil, Australia, USA and
France are among the top halal food exporters to Muslim countries
as stated in a 2014e2015 report commissioned by the Dubai
Chamber of Commerce (Thomson Reuters, 2015). Moreover, coun-
tries such as USA, UK, France and Belgium have taken the initiative
to produce and commercialise kits for halal analysis. Clearly, there
is great demand for measures that can assist Muslims with the
religious obligation of ensuring that the food they consume is halal.
Hand in hand, formal halal certication and the technology that can
verify and substantiate this certication will ensure compliance to
Islamic Law as well as generate trust amongst consumers.
Acknowledgements
Minhaz Uddin Ahmed would like to acknowledge the nancial
support for the project provided by Brunei Research Council
(Grant# BRC-10) from Economic and Planning Development, Prime
Minister's Ofce of Negara Brunei Darussalam. Hamadah Nur Lubis
would like to thank Universiti Brunei Darussalam (UBD) and Min-
istry of Education, Brunei Darussalam for the opportunity to un-
dertake Masters by Research course at UBD.
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