Meat Science 90 (2012) 686–689

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Meat Science
journal homepage: www.elsevier.com/locate/meatsci

Detection of porcine DNA in gelatine and gelatine-containing processed food

products—Halal/Kosher authentication
Yasemin Demirhan a, Pelin Ulca a, Hamide Z. Senyuva b,⁎
a
A&T Food Laboratory, Mega Center No. 29 34045, Istanbul, Turkey
b
FoodLife International, ODTU Teknokent Ikizler Binasi No. ara-1, 06531 Ankara, Turkey

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

Article history: A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine spe-
Received 19 July 2011 cific primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purification of
Received in revised form 24 October 2011 DNA from gelatine were successfully achieved using the SureFood® PREP Animal system, and real-time PCR
Accepted 26 October 2011
was carried out using SureFood® Animal ID Pork Sens kit. The minimum level of adulteration that could be
detected was 1.0% w/w for marshmallows and gum drops. A small survey was undertaken of processed
Keywords:
Gelatine
food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of four-
Halal authentication teen food products from Germany, two samples were found to contain porcine gelatine, whereas of
Kosher authentication twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained
Real-time PCR porcine DNA and thus was not Halal, and neither was the use of porcine gelatine indicated on the product
label.
© 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Gelatine is a highly processed protein, which is widely used as a
gelling and thickening agent (E441) in a variety of foodstuffs such
confectionary products, water-based desserts and in the pharmaceu-
tical industry e.g. in gel capsules for medicines. Gelatine is obtained
by hydrolysis of collagen, which is extracted from materials such as
bones, hides and skins from animal slaughterhouses (Karim and
Bhat, 2008). Gelatine production involves controlled acidic or basic
hydrolysis of connective tissue raw material, high temperature ex-
traction with water, sterilisation, and drying. These processes are
not standardised and have effects on the properties of the final gela-
tine product. In the final gelatine product, both proteins and nucleic
acids are highly degraded (Boran and Regenstein, 2010). Additionally,
the amount of DNA in gelatine is very low and differs from material-
to-material.
In Europe, about 80% of edible gelatine is produced from pigskin,
but vegetarian, Halal and Kosher gelatine, prepared from seaweed,
fish bones or non-porcine sources, is also available (Boran and
Regenstein, 2010). Although gelatine must be labelled appropriately,
once it has been manufactured, purified and in commercial trade, it is
difficult to ensure its provenance or whether it has been inadvertent-
ly mixed at any point in the food chain. It is therefore important to
have methods available whereby pure gelatine can be checked to en-
sure its authenticity and that it is free from cross-contamination with
⁎ Corresponding author. Tel./fax: + 90 312 2101060.
E-mail address: hamide.senyuva@foodlifeint.com (H.Z. Senyuva).
porcine gelatine. Equally the ability to test processed food products
for the presence of porcine gelatine is an essential requirement for
food control in Muslim or Jewish communities (Riaz and Chaudry,
2004).
Most published methods have focussed on meat species identifi-
cation rather than identification of gelatine. Polymerase chain reac-
tion (PCR)-based methods have been the most successful in terms
of both specificity and sensitivity of species detection. A review of
PCR-based methods applied to the authentication of meat products
cites some twenty-nine publications (Mafra, Ferreira, and Oliveira,
2008). Extraction of good quality DNA is an important pre-requisite
for PCR-based analysis and this can be a potential problem if there
has been extensive heat processing. For example, only poor quality
genomic DNA was extractable from bread and biscuits, although it is
not clear if this was because of high temperature degradation during
cooking or because lard, containing only small amounts of DNA, was
the target source of DNA (Aida, Che Man, Raha, and Son, 2007).
DNA has been isolated from meat and cheese using a standard CTAB
protocol and from milk using a Promega Wizard Magnetic kit and pu-
rified by Qiagen silicon spin columns (Zhang, Fowler, Scott, Lawson,
and Slater, 2007). With gelatine, despite both extensive heat and
chemical treatment, it has been demonstrated that it is possible
with nucleic acid binding columns or standard ethanol precipitation
to obtain template DNA. Analysis of the extracted DNA on agarose
gels was used to demonstrate that it had remained essentially intact
(Tasara, Schumacher, and Stephan, 2005).
A number of PCR approaches have been used to detect porcine
DNA in meat and meat products (e.g. Binke, Spiegel, and Schwägele,
2007). Using restriction fragment length polymorphism (RFLP)

0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2011.10.014
 Y. Demirhan et al. / Meat Science 90 (2012) 686–689
analysis of a conserved region of the mt cytb DNA extracted from sau-
sages, clear PCR products were produced on amplification (Aida et al.,
2007). For the same food products, using species-specific PCR, detec-
tion of a conserved region in the mt 12S ribosomal RNA (rRNA) gene
was employed as an alternative method. The extracted DNA was am-
plified by PCR targeting specific regions of the 12S rRNA gene of 387
base pairs (bp) from pork species. The species-specific PCR was
used for successful identification of pork DNA, but the performance
of the method in terms of sensitivity was not reported (Che Man,
Aida, Raha, and Son, 2007).
A similar method utilising PCR–RFLP was reported for beef, pork,
buffalo, quail, chicken, goat, and rabbit species identification and
Halal authentication. PCR products of 359-bp were successfully
obtained from the cytb gene of these meats, and five different specific
enzymes were identified as potential restriction endonucleases for
differentiation purposes (Murugaiah et al., 2009). Specific PCR ampli-
fication of a repetitive DNA sequence has been used for the identifica-
tion of pork in processed and unprocessed food. A level of addition of
1% pork was detectable with 20 PCR amplification cycles and 0.005%
pork with 30 PCR amplification cycles (Calvo, Zaragoza, and Osta,
2001). A species-specific duplex PCR assay has been used for the si-
multaneous detection of pork and poultry meat species, again using
the mt cytb as target gene for pork. By amplification of DNA from
meat mixtures of two species, linear calibration was obtained using
fluorescence intensities of PCR products for pork (149-bp) in the
range of 1–75%, with a sensitivity of 0.1% addition. In-house valida-
tion, using samples with known amounts of pork, gave a coefficient
of variation from 4.1 to 7.6% (Soares, Amaral, Isabel Mafra, Oliveira,
and Beatriz, 2010). Real-time PCR has also been used for the identifi-
cation of beef, pork, horse, mutton, chicken and turkey in processed
meat down to a level of 0.01–0.05% (Jonker, Tilburg, Gele, and De
Boer, 2008).
TaqMan real-time PCR using a bovine-specific primer pair for the
mt cyt b gene and a FAM-labelled mammalian-specific cyt b probe
could quantitatively detect as little as 35 pg bovine DNA and showed
no cross-reaction with ovine, caprine or porcine DNA. The system was
used to measure bovine DNA in fresh and processed meat, milk and
cheese (Zhang et al., 2007). Specific primers and TaqMan probes
have been designed for the mt ND2, ND5 and ATP 6–8 genes for don-
key, pork and horse, respectively. Only one cross-reaction was ob-
served between the horse species specific primer-probe set and
100 ng pork DNA at the cycle threshold (Ct) value of 33.01 (corre-
sponding to 0.01 ng horse DNA). The assay enabled the detection of
0.0001 ng of template DNA from pure meat for each species investi-
gated (Kesmen, Gulluce, Sahin, and Yetim, 2009).
Several species-specific PCR methods have been published to de-
termine the origin of raw materials used in gelatine manufacture. A
bovine species-specific PCR primer set targeting the ATPase 8 sub-
unit gene in bovine mt DNA was demonstrated to be suitable for de-
tection of bovine material in gelatine. This PCR primer set was opti-
mised using conventional and real-time PCR approaches. The
inclusion of bovine gelatine in pork or fish gelatine could be detected
at levels of 0.1% by conventional PCR and 0.001% by light cycler PCR
after DNase I decontamination (Tasara et al., 2005). The viability of
testing pure gelatine by PCR was demonstrated, although the method
was not taken any further in terms of analysis of commercial gelatine-
containing food products.
In this paper we have deliberately adopted the approach of using
commercial test kits both for DNA extraction and for the real-time
PCR analysis. Although, there have been many very successful
methods published for detection of porcine DNA, there is a real
need for food control laboratories to apply these methods routinely
using commercially available kits. We have focussed on gelatine be-
cause this product seems to have been overlooked in terms of testing
methodology, and yet has a high potential for inadvertent adultera-
tion with porcine material or mislabelling.
687
2. Materials and methods
2.1. Sample preparation
Twelve gelatine samples of known origin (bovine, porcine or sea-
weed) were obtained in powder or sheet form, and employed as ref-
erence standards. Pure gelatine mixtures were prepared by extracting
and purifying the DNA from 500 mg of porcine gelatine and diluting
the resulting DNA solution with bovine DNA solution to obtain 10%,
1% and 0.1% mixtures. Mixtures of food products containing porcine
gelatine were individually prepared by grinding gum drops or marsh-
mallow together with porcine gelatine, and mixing to a fine powder.
The composition of these mixtures is shown in Table 1.
Forty-three samples of the soft and fruity chew confectionery
(gum drops), Turkish delight, jelly and marshmallows/cakes contain-
ing gelatine were obtained from markets in Turkey and Germany.
Samples were stored at −20 °C. Approximately, 300 g of each sample
was blended in the frozen state using a Waring blender (Torrington,
USA) to produce a powder, which was thoroughly mixed. Sub-
samples (400 mg) were taken for DNA extraction.
Spiking of the above retail food products with 5% porcine gelatine
was carried out by weighing a 380 mg amount of the composite prod-
uct into a 1.5 ml reaction tube together with 20 mg of porcine gela-
tine. The whole mixture (400 mg) was then taken for DNA extraction.
2.2. Extraction of DNA
DNA was extracted from pure gelatine or from food products con-
taining gelatine (400 mg) using the Sure Food® Prep Animal X kit
(CONGEN, R-Biopharm, Germany). Lysis buffer (1000 μl) and Protein-
ase K (40 μl) were added to 400 mg of sample and mixed by vortexing
(Fisherbrand ZX Wizard). The mixture was incubated at 65 °C for
1 hour in a thermomixer (Eppendorf, comfort) under continuous
shaking. At the end of the incubation, the solution was centrifuged
at 24,150g for 2 min (Eppendorf 5430). After centrifuging, a spin filter
was placed in a receiver tube. The solution was transferred into spin
filter and centrifuged at 24,150g for 2 min. The spin filter was dis-
carded. Binding buffer (200 μl) was added to the filtrate, which was
vortexed thoroughly. The filtrate was transferred to a new spin filter
placed in a new receiver tube and centrifuged again at 24,150g for
2 min. After the filtrate was discarded, 550 μl of pre-wash buffer
was added into the spin filter and centrifuged at 24,150g for 1 min.
This step was repeated twice. After discarding the filter, it was centri-
fuged for 2 min at 24,150g to remove wash buffer completely. A new
spin filter was placed in a new 1.5 ml receiver tube; 50 μl of pre-
heated elution buffer was pipetted directly onto the spin filter and in-
cubated at room temperature for 3 min. Finally, it was centrifuged for
2 min at 16,770g and the purified DNA solution (50 μl) was stored at
4 °C.
2.3. PCR amplification
A pork reaction mixture containing specific primers and Taq-
Polymerase are supplied as part of the commercial test kit. The reac-
tion mix, Taq-Polymerase (SureFood® Animal ID Pork SENS Plus V
kit) and extracted DNA were mixed in the ratio 9.95: 0.05: 2.5 for
Table 1
Composition of mixtures of gum drops/marshmallows mixed with various levels of
porcine gelatin.
Level of addition (%) Wt of food (mg) Wt of bovine gelatine (mg)
1.0 495.0 5.0
3.0 388.0 12.0
5.0 380.0 20.0
10.0 450.0 50.0
688 Y. Demirhan et al. / Meat Science 90 (2012) 686–689

dispensing for replicate analysis. For each reaction a total of 25 μl of Table 2

the above mixture was dispensed (containing 19.9 μl pork reaction Ct values of 100%, 10.0% and 1.0% pork gelatin. Results are shown for 6 replicate assays.

mix, 0.1 μl Taq-Polymerase (SureFood® Animal ID Pork SENS Plus V Ct values 100% Ct values 10.0% Ct values 1.0% Ct values 0.1%
kit) and 5 μl extracted DNA). Amplification was performed with a pork gelatine pork gelatine pork gelatine pork gelatine
real-time PCR (Eppendorf, Mastercycler ep Realplex2). The thermal 28.07 32.09 33.55 –
cycler followed the programme of initial denaturation at 95 °C for 28.57 32.29 41.89 –
5 min to denature the DNA template completely, followed by 45 cy- 28.44 32.48 37.04 –
28.34 32.21 36.14 –
cles of denaturation at 95 °C for 15 s, and annealing and extension
28.87 33.92 41.59
at 55 °C for 30 s. The proprietary computer software is designed to ex- 28.43 31.63 37.32 –
amine the fluorescent intensity at 522 nm (FAM) and 553 nm (VIC). A
Ct—cycle threshold value.
negative control was included in every run. A sample was deemed to – Indicates that porcine gelatine was not detectable at the % level of addition.
be pork positive, if the sample DNA showed amplification in the FAM
channel. A sample was deemed to be negative, if the sample DNA
showed no amplification in the detection system and the internal am- 3. Results and discussion
plification control (inhibition control) of the sample was positive.
3.1. Detection of porcine DNA in pure gelatin

2.4. Determination of the sensitivity of the assay
The sensitivity of the assay was measured in terms of both detec-
tion of porcine DNA in pure gelatine and detection of porcine DNA in
food products containing gelatine. Replicate real-time PCR measure-
ments were made of extracted DNA from gelatine spiked with 0.1,
1, and 10% and the limit of detection was taken as being the lowest
amount that could be amplified with a reproducible Ct value. A simi-
lar approach was adopted to determine the sensitivity for the detec-
tion of porcine gelatine spiked into samples of gum drops and
marshmallows, which had been found to be negative by real-time
PCR.
2.5. Determination of the reliability of the assay
The reliability of the assay was tested by spiking samples of gum
drops and marshmallows that were established as not containing
any porcine DNA. For gum drops, spiking was carried out at 0.0%,
0.5%, 3.0% and 5.0% addition of porcine gelatine and for marshmal-
lows, addition was made at levels of 0.0%, 0.1% 0.5%. 3.0% and 5.0%.
All samples, including unspiked gum drops and marshmallows were
analysed as five blind replicates making a total of 45 determinations.
2.6. Analysis of survey samples
Details of all survey samples from Germany and Turkey were
recorded and the packaging was retained for reference. All samples
were analysed in replicate and also spiked with 5% porcine gelatine,
following the finalised protocol set out above.
In the first set of experiments, two samples of gelatine, one desig-
nated as bovine and another as Halal, were tested and both confirmed
to be negative for pork by real-time PCR. In contrast, two samples
supplied as being porcine gelatine were both found to be clearly pos-
itive, and a mixture of 10.0% porcine in bovine gelatine was also clear-
ly positive. Samples of porcine gelatin gave Ct values from 28.1 to
28.8, whilst samples of gelatine containing 10.0% porcine gelatine
gave Ct values from 31.6 to 33.9. These experiments carried out as
six replicates clearly indicated that good quality DNA could be reliably
extracted from gelatine, and that the pork characteristic cytb-
sequence could be sensitively amplified and detected.
Experiments were then conducted taking various mixtures of bo-
vine DNA with additions of 10.0%, 1.0% and 0.1% porcine DNA (DNA
from gelatine used in this study). These results are shown in Table 2
where it can be seen that samples of bovine DNA containing 0.1% por-
cine DNA were all found to be negative. However, at the 1.0% level of
addition of porcine DNA, Ct values between 33.5 and 41.8 were
obtained. Although within the six replicates there was some variabil-
ity, a 1.0% addition (w/w) could, nevertheless, be reliably detected
and this was thus taken to be the effective limit of detection. Typical
Ct curves for detection of porcine DNA at different levels, by real-
time PCR are shown in Fig. 1. Thus, for any sample of gelatine desig-
nated as Halal, if any cross-contamination of raw materials such as in-
corporation of some pig skins had occurred during manufacture, the
test would be sufficiently sensitive to detect 1.0% adulteration. Al-
though this commercial test kit appears to be less sensitive than the
beef method reported by Tasara et al. (2005), this could be due to
the different gelatine types used and the differences in the extraction
methods. In reality high sensitivity is not really a necessity for Halal/
Kosher testing and a 1.0% cut-off provides a pragmatic limit for
testing.

Fig. 1. PCR amplification plots for samples of gelatine containing 100%, 10.0% and 1.0% pork gelatin.
 Y. Demirhan et al. / Meat Science 90 (2012) 686–689 689

Table 3
Ct values for different amounts of porcine gelatine spiked into marshmallows and gum drops. Results are shown for 5 replicate assays.

Addition porcine gelatine (%) Ct values porcine gelatine in marshmallow Addition porcine gelatine (%) Ct values porcine gelatine in gum drops

0 – – – – – 0 – – – – –
0.1 – – – – – 0.1 – – – – –
0.5 36.86 37.63 36.13 38.92 37.76 0.5 – – – – –
3.0 32.62 33.39 33.59 34.08 31.26 3.0 35.78 34.41 37.74 37.18 37.74
5.0 24.08 28.16 29.12 29.24 29.27 5.0 – 29.10 31.89 34.41 32.44

Ct—cycle threshold value.

– Indicates that porcine gelatine was not detectable at the % level of addition.

3.2. Detection of porcine DNA in gelatin-containing food products Table 4

Results of survey of samples obtained from Turkey and Germany shown as numbers of
positive and negative samples.
The sensitivity of the assay was also established for the detection
of porcine gelatine spiked into retail samples of gum drops and Products Total no. of Porcine negative Porcine positive
marshmallows shown to be negative by real-time PCR. In both food samples

products, a 1.0% addition of gelatine could be detected, consistent Turkey Germany Turkey Germany Turkey Germany
with the results for spiked gelatine. The reliability of the assay was Marshmallow/cake 17 – 16 – 1 –
established from blind replicate analysis of negative gum drops and Gum-drops 11 11 11 9 – 2
marshmallows and blind analysis of replicate samples spiked at levels Jelly 3 – 3 – – –
Turkish delight 1 – 1 – – –
above and below the detection limits. No false positives were
Total 32 11 31 9 1 2
detected, but one false negative sample was obtained giving a false
negative rate of 2.0% for the assay. The results of these tests to estab-
lish the reliability of the assay in real food products are shown in
Table 3. Turkey) for the supply of SureFood® kits used for the development
and validation work reported in this paper.
3.3. Survey of retail food products from Germany and Turkey
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Acknowledgement

The authors gratefully acknowledge the support of CONGEN Bio-

technologie GmbH (Berlin, Germany) and Sincer Dis Ticaret (Izmir,