FLEISCHWIRTSCHAFT international 1/2017

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Fleischwirtschaft international 1_2017
Research &Development
Methods of differentiating animal species in foods –Status quo
An advantage here is the large sample weight (generally25g)provided
for in some protocols by comparison with e.g. PCR-based methods, as
this allows more representative sampling of large sample volumes.
The detection limits stated by the manufacturer or cited in literature
are in the range of 0.05 to 5%. However, the achievable level of sensitivity
depends stronglyonthe animal species, the nature of the meat component
used (muscle meat or inner organs) and the extent to which the food
to be examined has been processed. In the case of highlyheated foods,
the detection limit rises steeplydue to denaturation/destruction of the
target proteins, so that incorrect negative results in highlytreated (e.g.
canned) samples cannot be ruled out. In dry, scalded and cooked
sausages, a1%meat component is generallyidentified safely.
Furthermore, there are commercial methods which detect not onlyone
or afew species, but also relativelylarge, phylogeneticallyrelated
groups such as poultry or ruminants. In the latter case, anti-body-based
kits are offered that are reportedlysuitable for products which have
been heated to extremelyhigh temperatures (up to 150°C) and treated
under strong pressure, such as e.g. meat-and-bone meals. Heat-stable
target proteins named by kit manufacturers (e.g. Transia or Neogen) or
cited in literature are for example troponin I(CHEN,2002) or h-Caldesmon
(KIM,2004).
Ring trials have been conducted above all with kits for detecting
ruminants in highlyprocessed meat-and-bone meals (FUMIÈRE,2009; VAN
RAAMSDONK,2012). The desired detection limit of 0.1% (w/w) for ruminant
material heated up to 133°C/20 min/3 bar in compound feed has not
been reached with the available commercial kits so far.
In view of the differing composition of foods and different production
methods, the animal species detections using ELISA are therefore to be
classified as purelyqualitative.
Animal species differentiation
using LC-MS-MS
The development of mass-spectrometry methods for differentiating
animal species is as yet astill relativelynew technique that is encountering
increasing interest in research, and in isolated cases is already
being used in routine analyses.
In particular targeted proteomics, in other words the targeted massspectrometry
detection of enzymaticallygenerated marker peptides, is
becoming established as an alternative method of species identification.
For this it is first necessary to identify sequence polymorphisms (insertions,
deletions, amino acid exchanges) in the proteome that are specific
for the species to be identified. Identification of these peptides can
be carried out via databases. Frequentlythese databases are not complete,
however, so that experimental identification of these polymorphisms
by means of high-resolution mass spectrometry becomes necessary.Marker
peptides that contain the corresponding sequence polymorphisms
can then be detected sensitivelyand specificallythrough
mass spectrometry, even on routine equipment. Detection of horse or pig
in beef now manages this with detection limits of up to around 0.1%,
even in processed foods (VON BARGEN,2013, VON BARGEN,2014). The signal
conditions of corresponding marker peptides from homologous proteins
of the species to be differentiated can be used for relative quantification
of mixtures of different species (WATSON,2015). Alternative approaches to
animal species differentiation use the direct comparison of alarge
number of MS/MS spectra (spectral matching) in order to achieve differentiation
without prior identification of marker peptides (OHANA,2016).
This omits the need for partiallycostlyidentification of specific biomarkers,
but for each measurement of an unknown sample at least 2000
MS/MS spectra have to be generated here and compared with spectral
libraries in order to allow authentication.
Molecular biology methods for
differentiating fish species
Today, PCR sequencing with universal primers is the method of choice
for differentiating fish species (GRIFFITH et al., 2014). The mitochondrial
markers cytochrome b(cytb) and cytochrome coxidase subunit I(cox1)
are predominantlyused. While according to the publications, cytb was
preferred for fish species differentiation up to the year 2007 (TELETCHEA,
2009), agrowing trend in the direction of cox1can be noted. This is due
not least to the “International Barcode of Life (iBOL)” Initiative (HEBERT et
al., 2003). Mitochondrial gene markers satisfy the condition regarding
high interspecific and low intraspecific variability for reliable identification
(WARD et al., 2005). In view of approx. 33000 different fish species
(Fishbase), the identification represents amajor challenge. Various
universal M13-marked cox1primer cocktails for barcoding of fish were
first presented by the working group IVANOVA et al. (2007). In this study
the cox1-cocktail COI-3 was convincing regarding PCR and sequencing
success, so that within the context of the EU “Labelfish” project a
standard operating procedure (SOP) with this cocktail was developed
and validated in an international ringtrial (publication pending).
In addition, further mitochondrial markers are used, such as for example
the 16SrRNA gene which is to be classified more as conserved and is
used for confirmation of an unknown fish sample (REHBEIN and OLIVEIRA,
2012), or the variable control region gene used for clear differentiation of
closelyrelated fish species such as tuna fish (Thunnus spp.) (VIŇAS and
TUDELA,2009). Especiallywhen allocation of the fish species is rendered
more difficult for instance by hybridisations, introgressions and few SNP
(Single-Nucleotide-Polymorphism) differences, as in the case of tuna
fish species, it is advisable to conduct FINS (ForensicallyInformative-
Nucleotide-Sequencing) with avariable mitochondrial and anuclear
marker (VIŇAS and TUDELA,2009). The use of anuclear marker alone, such
as the intron-free rhodopsin gene 1(Rh1),shows limitations when differentiating
between species of the same genus, e.g. in the case of eels
(Anguilla spp.) or sturgeons (Acipenser spp.) (REHBEIN,2013).
Traditionallythe RFLP, SSCP or sequencing of an amplicon from the
16SrRNA gene are frequentlycarried out to detect mussels and crustaceans
(MARÍN et al., 2013;SCHIEFENHÖVEL and REHBEIN,2010). However, cox1
barcoding is also becoming increasinglymore popular for differentiating
between mussels and crustaceans by providing suitable primer systems
(LOBO et al., 2013;GELLER et al., 2013).
Until recently, Next-Generation Sequencing (NGS) techniques were
limited to the area of fish, molluscs and crustaceans in biodiversity
studies. To identify species on the basis of pyrosequencing, the research
group DE BATTISTI et al. (2013)developed techniques for diverse
fish species, while the research group ABBADI et al. (2016)developed
techniques for various mussel types. However, these techniques only
consider individuals and not mixtures. In the meantime, afew papers
have been published that describe the NGS method for identifying animal
and plant species in unknown mixed samples as well. There are NGS
approaches that carry out an analysis via the classic barcode sequence
regions and also non-targeted techniques (STAATS et al., 2016;RIPP et al.,
2014).
Quick methods
In order to achieve fast and high sample throughputs in species differentiation
of fisheries products, quick methods or screening applications
are increasinglybeing developed alongside the conventional PCR sequencing
method.
Real-Time PCR
While the developments in real-time PCR techniques for mussels (SÁNCHEZ
et al., 2014)and cephalopods (HERRERO et al., 2012;ESPIŇEIRA and VIEITES,
2012)have been very clearlystructured to date (there have been none
for crustaceans so far), in recent years anumber of different qualitativelyoriented
single, duplex and multiplex applications have been
published for relevant fish species. In this context we can name, for
example, the real-time PCR techniques for detecting the oilfish Lepidocybium
flavobrunneum and Ruvettus pretiosus (GIUSTI et al., 2016), the
European eel (Anguilla anguilla )byESPIŇEIRA and VIEITES (2016), as well as
sole (Solea solea)byHERRERO et al. (2014)and tuna fish (CHUANG et al.,
2012). Commercial real-time PCR kits are already available on the market
for various salmonid and gadoid species and for the European hake