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Detection of Porcine DNA in Gelatine and Gelatine-Containing Processed Food Products-Halal - Kosher Authentication
Detection of Porcine DNA in Gelatine and Gelatine-Containing Processed Food Products-Halal - Kosher Authentication
Meat Science
journal homepage: www.elsevier.com/locate/meatsci
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
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
Table 1
Composition of mixtures of gum drops/marshmallows mixed with various levels of
porcine gelatin.
Level of addition (%)
Wt of food (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
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
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.
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 (%)
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
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
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.
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