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Meat Science 90 (2012) 686689

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

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


productsHalal/Kosher authentication
Yasemin Demirhan a, Pelin Ulca a, Hamide Z. Senyuva b,
a
b

A&T Food Laboratory, Mega Center No. 29 34045, Istanbul, Turkey


FoodLife International, ODTU Teknokent Ikizler Binasi No. ara-1, 06531 Ankara, Turkey

a r t i c l e

i n f o

Article history:
Received 19 July 2011
Received in revised form 24 October 2011
Accepted 26 October 2011
Keywords:
Gelatine
Halal authentication
Kosher authentication
Real-time PCR

a b s t r a c t
A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine specic primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purication of
DNA from gelatine were successfully achieved using the SureFood PREP Animal system, and real-time PCR
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
food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of fourteen food products from Germany, two samples were found to contain porcine gelatine, whereas of
twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained
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 pharmaceutical 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 extraction with water, sterilisation, and drying. These processes are
not standardised and have effects on the properties of the nal gelatine product. In the nal 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 materialto-material.
In Europe, about 80% of edible gelatine is produced from pigskin,
but vegetarian, Halal and Kosher gelatine, prepared from seaweed,
sh bones or non-porcine sources, is also available (Boran and
Regenstein, 2010). Although gelatine must be labelled appropriately,
once it has been manufactured, puried and in commercial trade, it is
difcult to ensure its provenance or whether it has been inadvertently mixed at any point in the food chain. It is therefore important to
have methods available whereby pure gelatine can be checked to ensure 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).
0309-1740/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2011.10.014

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 identication rather than identication of gelatine. Polymerase chain reaction (PCR)-based methods have been the most successful in terms
of both specicity 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 puried 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 Schwgele,
2007). Using restriction fragment length polymorphism (RFLP)

Y. Demirhan et al. / Meat Science 90 (2012) 686689

analysis of a conserved region of the mt cytb DNA extracted from sausages, clear PCR products were produced on amplication (Aida et al.,
2007). For the same food products, using species-specic PCR, detection of a conserved region in the mt 12S ribosomal RNA (rRNA) gene
was employed as an alternative method. The extracted DNA was amplied by PCR targeting specic regions of the 12S rRNA gene of 387
base pairs (bp) from pork species. The species-specic PCR was
used for successful identication 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 PCRRFLP was reported for beef, pork,
buffalo, quail, chicken, goat, and rabbit species identication and
Halal authentication. PCR products of 359-bp were successfully
obtained from the cytb gene of these meats, and ve different specic
enzymes were identied as potential restriction endonucleases for
differentiation purposes (Murugaiah et al., 2009). Specic PCR amplication of a repetitive DNA sequence has been used for the identication of pork in processed and unprocessed food. A level of addition of
1% pork was detectable with 20 PCR amplication cycles and 0.005%
pork with 30 PCR amplication cycles (Calvo, Zaragoza, and Osta,
2001). A species-specic duplex PCR assay has been used for the simultaneous detection of pork and poultry meat species, again using
the mt cytb as target gene for pork. By amplication of DNA from
meat mixtures of two species, linear calibration was obtained using
uorescence intensities of PCR products for pork (149-bp) in the
range of 175%, with a sensitivity of 0.1% addition. In-house validation, using samples with known amounts of pork, gave a coefcient
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 identication of beef, pork, horse, mutton, chicken and turkey in processed
meat down to a level of 0.010.05% (Jonker, Tilburg, Gele, and De
Boer, 2008).
TaqMan real-time PCR using a bovine-specic primer pair for the
mt cyt b gene and a FAM-labelled mammalian-specic 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). Specic primers and TaqMan probes
have been designed for the mt ND2, ND5 and ATP 68 genes for donkey, pork and horse, respectively. Only one cross-reaction was observed between the horse species specic primer-probe set and
100 ng pork DNA at the cycle threshold (Ct) value of 33.01 (corresponding to 0.01 ng horse DNA). The assay enabled the detection of
0.0001 ng of template DNA from pure meat for each species investigated (Kesmen, Gulluce, Sahin, and Yetim, 2009).
Several species-specic PCR methods have been published to determine the origin of raw materials used in gelatine manufacture. A
bovine species-specic PCR primer set targeting the ATPase 8 subunit gene in bovine mt DNA was demonstrated to be suitable for detection of bovine material in gelatine. This PCR primer set was optimised using conventional and real-time PCR approaches. The
inclusion of bovine gelatine in pork or sh 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 gelatinecontaining 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 because this product seems to have been overlooked in terms of testing
methodology, and yet has a high potential for inadvertent adulteration with porcine material or mislabelling.

687

2. Materials and methods


2.1. Sample preparation
Twelve gelatine samples of known origin (bovine, porcine or seaweed) were obtained in powder or sheet form, and employed as reference 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 marshmallow together with porcine gelatine, and mixing to a ne 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 containing 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. Subsamples (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 product into a 1.5 ml reaction tube together with 20 mg of porcine gelatine. 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 containing gelatine (400 mg) using the Sure Food Prep Animal X kit
(CONGEN, R-Biopharm, Germany). Lysis buffer (1000 l) and Proteinase K (40 l) 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 lter
was placed in a receiver tube. The solution was transferred into spin
lter and centrifuged at 24,150g for 2 min. The spin lter was discarded. Binding buffer (200 l) was added to the ltrate, which was
vortexed thoroughly. The ltrate was transferred to a new spin lter
placed in a new receiver tube and centrifuged again at 24,150g for
2 min. After the ltrate was discarded, 550 l of pre-wash buffer
was added into the spin lter and centrifuged at 24,150g for 1 min.
This step was repeated twice. After discarding the lter, it was centrifuged for 2 min at 24,150g to remove wash buffer completely. A new
spin lter was placed in a new 1.5 ml receiver tube; 50 l of preheated elution buffer was pipetted directly onto the spin lter and incubated at room temperature for 3 min. Finally, it was centrifuged for
2 min at 16,770g and the puried DNA solution (50 l) was stored at
4 C.
2.3. PCR amplication
A pork reaction mixture containing specic primers and TaqPolymerase are supplied as part of the commercial test kit. The reaction 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
3.0
5.0
10.0

495.0
388.0
380.0
450.0

5.0
12.0
20.0
50.0

688

Y. Demirhan et al. / Meat Science 90 (2012) 686689

dispensing for replicate analysis. For each reaction a total of 25 l of


the above mixture was dispensed (containing 19.9 l pork reaction
mix, 0.1 l Taq-Polymerase (SureFood Animal ID Pork SENS Plus V
kit) and 5 l extracted DNA). Amplication was performed with a
real-time PCR (Eppendorf, Mastercycler ep Realplex2). The thermal
cycler followed the programme of initial denaturation at 95 C for
5 min to denature the DNA template completely, followed by 45 cycles of denaturation at 95 C for 15 s, and annealing and extension
at 55 C for 30 s. The proprietary computer software is designed to examine the uorescent intensity at 522 nm (FAM) and 553 nm (VIC). A
negative control was included in every run. A sample was deemed to
be pork positive, if the sample DNA showed amplication in the FAM
channel. A sample was deemed to be negative, if the sample DNA
showed no amplication in the detection system and the internal amplication control (inhibition control) of the sample was positive.

Table 2
Ct values of 100%, 10.0% and 1.0% pork gelatin. Results are shown for 6 replicate assays.
Ct values 100%
pork gelatine

Ct values 10.0%
pork gelatine

Ct values 1.0%
pork gelatine

Ct values 0.1%
pork gelatine

28.07
28.57
28.44
28.34
28.87
28.43

32.09
32.29
32.48
32.21
33.92
31.63

33.55
41.89
37.04
36.14
41.59
37.32

Ctcycle threshold value.


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

3. Results and discussion


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 detection of porcine DNA in pure gelatine and detection of porcine DNA in
food products containing gelatine. Replicate real-time PCR measurements 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 amplied with a reproducible Ct value. A similar approach was adopted to determine the sensitivity for the detection 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 marshmallows, 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 ve 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 nalised protocol set out above.

In the rst set of experiments, two samples of gelatine, one designated as bovine and another as Halal, were tested and both conrmed
to be negative for pork by real-time PCR. In contrast, two samples
supplied as being porcine gelatine were both found to be clearly positive, and a mixture of 10.0% porcine in bovine gelatine was also clearly 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 cytbsequence could be sensitively amplied and detected.
Experiments were then conducted taking various mixtures of bovine 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% porcine 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 variability, 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 realtime PCR are shown in Fig. 1. Thus, for any sample of gelatine designated as Halal, if any cross-contamination of raw materials such as incorporation of some pig skins had occurred during manufacture, the
test would be sufciently sensitive to detect 1.0% adulteration. Although 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 amplication plots for samples of gelatine containing 100%, 10.0% and 1.0% pork gelatin.

Y. Demirhan et al. / Meat Science 90 (2012) 686689

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

0
0.1
0.5
3.0
5.0

36.86
32.62
24.08

37.63
33.39
28.16

36.13
33.59
29.12

38.92
34.08
29.24

37.76
31.26
29.27

Addition porcine gelatine (%)

Ct values porcine gelatine in gum drops

0
0.1
0.5
3.0
5.0

35.78

34.41
29.10

37.74
31.89

37.18
34.41

37.74
32.44

Ctcycle 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


The sensitivity of the assay was also established for the detection
of porcine gelatine spiked into retail samples of gum drops and
marshmallows shown to be negative by real-time PCR. In both food
products, a 1.0% addition of gelatine could be detected, consistent
with the results for spiked gelatine. The reliability of the assay was
established from blind replicate analysis of negative gum drops and
marshmallows and blind analysis of replicate samples spiked at levels
above and below the detection limits. No false positives were
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 establish the reliability of the assay in real food products are shown in
Table 3.

Table 4
Results of survey of samples obtained from Turkey and Germany shown as numbers of
positive and negative samples.
Products

Marshmallow/cake
Gum-drops
Jelly
Turkish delight
Total

Total no. of
samples

Porcine negative

Porcine positive

Turkey

Germany

Turkey

Germany

Turkey

Germany

17
11
3
1
32

11

11

16
11
3
1
31

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


References
The results of a small survey of retail products are shown in
Table 4. Of 11 retail products purchased in Germany, 2 samples
were found to contain porcine gelatine, with the remaining 9 samples
being negative. The 2 positive samples had Ct values of 30.04 and
43.00. Of a total of 32 samples from Turkey, 31 samples were found
to be negative. However, one product (cake covered with gelatine)
from the Turkish retail market was found to have a Ct value of 36.3
clearing indicating that this product contained porcine DNA. This
product was therefore not Halal and the labelling failed to indicate
the use of porcine gelatine. Spiking of all samples with 5.0% porcine
gelatine conrmed that the test was functioning adequately, although
one false negative was detected for a sample that remained negative
even after spiking. Whilst this survey was very limited in scope, the
clear discrimination between positive and negative retail samples of
differing compositions shows its robustness. The detection of a sample in Turkey, which was not Halal, clearly shows the need for further
surveillance of retail gelatine-containing foods and possible regulatory action by the authorities.
4. Conclusions
Using a commercial DNA extraction kit and a commercial realtime PCR kit it has been demonstrated that porcine DNA can be reliably detected in gelatine at a level below 1.0% w/w. It has also been
shown that porcine DNA can be detected in a variety of gelatinecontaining confectionary products at the levels of 1.0% w/w at a
false negative rate of 2.0% gelatine (all data for the gelatines used in
this study). In a small survey, one retail product from Turkey was
found to contain porcine gelatine whereas the consumer expectation
was that this was a Halal product.
Acknowledgement
The authors gratefully acknowledge the support of CONGEN Biotechnologie GmbH (Berlin, Germany) and Sincer Dis Ticaret (Izmir,

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