FLEISCHWIRTSCHAFT international 1/2017

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50 Fleischwirtschaft international 1_2017 Research &Development Methods of differentiating animal species in foods –Status quo By H.U. Waiblinger, D. Bartsch, J. Brockmeyer, C. Bruenen-Nieweler, U.Busch, I. Haase, A. Hahn, M. Haarmann, W.Hauser, I.Huber, K.D. Jany, N. Kirmse, S. Lindeke, K.Neumann, H. Naumann, A. Paschke, K. Pietsch, B.Pöpping, R. Reiting, U. Schroeder, F.Schwägele, M.G. Weller and J. Zagon Work on standardising methods in the fieldofanimal species differentiation has been intensified in Germanyinrecent years, not least due to the horsemeat scandal in 2013.Eventhough there are now hardly ever anypositive findings anymore in examinations to detecthorse adulterations in foods such as lasagne, animal species differentiation altogether ranks high in detecting adulteration of foods. This articletherefore summarises the current status of analytical techniques used in Germanywith standardisation at German level. It has been established by the working group “Biochemical and Molecular Biological Analytics” of the Lebensmittelchemische Gesellschaft (FoodChemistry Society within the German Chemical Society) with support of experts in the working group “Molecular biologytechniques for differentiating plant and animal species” (§ 64 of the German Food and Feed Code –LFGB) and the “Immunologyand molecular biology” task force of the food hygiene and food of animal origin working group (ALTS), both from Germany. Analytical methods for detecting animal species At present molecular biology techniques on the basis of real-time polymerase chain reaction (PCR) and DNA chips as well as immunological methods (e.g. ELISA) are mainlyused. In addition, applications of mass spectroscopy (LC-MS) techniques have been published (VON BARGEN,2013 and 2014;WATSON,2015;OHANA,2016). Over 20-year-old methods for analysing proteins on the basis of electrophoresis and isolelectric focusing exist in the official collection of analytical techniques (ASU L06.00-19,1990, ASU L01.00-39, 1995). These are now onlyapplied in isolated cases, but they currentlyrepresent the onlyofficial methods for animal species differentiation in milk and dairy products. The corresponding techniques for meat and meat products can be used for raw (PAGE) and heated (PAGIF) products. In most cases the detection limit is around 5%, which is why these methods are suitable for identifying non-declared admixtures upwards of this concentration. PCR-based and other molecular biology detection methods Polymerase chain reaction (PCR), aDNA amplification technique, represents asensitive technique for detecting animal species in foods. Species-specific genes encoded in the cell nucleus can be used for detection, as described for growth hormone genes (MEYER,1995a) and phosphodiesterase gene (LAUBE,2007) and, more recently, for other targets (DRUML,2015a und b; ROJAS,2011; MERZ,2016). Frequently, however, conserved sequence segments from the mitochondrial (mt) genome serve as target sequence, especiallyasegment of the gene of the cytochrome c oxidase subunit I(COI or cox1) (HEBERT,2003) and the cytochrome bgene (MEYER,1995b; MATSUNAGA,1999). However chromosomal coded segments may also be suitable for this, for instance the myostatin gene occurring in mammals and poultry (LAUBE,2007). Using “universal primers” that bind to such conserved sequences, it is possible to amplify DNA sequences in a cross-species manner.The animal species are then identified by sequencing, specific probe binding real-time PCR, melting point analysis or restriction fragment analysis. Animal species differentiation via DNA chip It is also possible to use acommerciallyavailable method on the basis of aDNA chip to screen for animal species, e.g. products of unknown composition (IWOBI,2011).Alongside the major mammal/livestock species, game (e.g. roe deer, red deer, kangaroo) and poultry species (e.g. goose, pheasant, ostrich, duck species) are also covered. The differentiation is based on species-specific differences in asequence of the mt 16SrRNA gene of the animal species. In PCR, biotinylated primers are used. In the subsequent hybridisation reaction, the corresponding biotinylated amplificates bind to species-specific oligonucleotide probes immobilised on achip. The detection is carried out via an enzyme substrate cascade using alkaline phosphatase coupled with streptavidin and subsequent colour reaction. The evaluation is carried out with the chip scanner and the associated software. The method is purelyqualitative and is suitable for mixtures. The detection limit is, case-depend, between 0.1% and 1%. Multiplex methods In 2008 and 2011,KÖPPEL et al. presented two real-time PCR methods for simultaneous species-specific determination of four different animal species, each: cattle, pig, turkey and hen (“All Meat”) (KÖPPEL,2008) and then cattle, pig, equidae and sheep (“All Horse”) (KÖPPEL,2011).Byusing calibrators or by calibrating with DNA extracts from materials of defined composition, the DNA-based methods were applied to quantify the quantity of the respective animal species (KÖPPEL,2012). In addition, the methods were trialled on (processed) meat products. The condition for using these methods with regard to percentage ratios is that no further animal species apart from the respective four animal species may be present. Amatrix-independent approach for simultaneous detection of different animal species, e.g. cattle, pig, equidae and sheep was published by IWOBI et al. (2015 and 2017). In these multiplex real-time PCR methods, the myostatin gene is used as areference gene in combination with animal species-specific standard series for quantifying the DNA ratios of the respective animal species. This method has already proven successful in meat products, dairy products and feedstuffs. Digital PCR Keywords » Animal species differentiation » Fish species » PCR » Standardisation Initial applications of the ‘digital PCR’ which allows standard-independent quantification at DNA level have been published (FLOREN,2015; CAI, 2014). Further development and testing for practicability are underway.
......................................................................................................... Fleischwirtschaft international 1_2017 51 Research &Development Sensitivity PCR-based methods for detecting animal species generallyachieve detection limits in the range of 0.1% in compound foods (LAUBE,2007; KÖPPEL,2012). In particular when using universal primer systems, it is possible that in mixtures of different animal or fish species, the target fragments are amplified to different extents of efficiency, depending on the species. This can adverselyaffect the sensitivity of the detection in certain cases (PIETSCH and WAIBLINGER,2010). Quantification Under the conditions described above, multiplex real-time PCR techniques make it possible to quantify species-specific DNA sequences and to determine the ratios of these DNA sequences to each other (IWOBI et al., 2016). When considering the question of whether acorrelation exists between DNA ratios and weight ratios of animals in mixed samples, the following aspects are among those which must be taken into account: in fatty tissue, compared to meat (muscle tissue), there is generallyless (around 30%) DNA. Offal such as liver and heart contain 10 to 25 times as much DNA (originating from mitochondria and the cell nucleus) as muscles (SCHWÄGELE,2003). When using PCR systems based on target sequences from mtDNA, it must also be taken into consideration that the number of mitochondria in animal cells can fluctuate very stronglyfor example depending on the animal species and tissue (SCHWÄGELE,2003). In fish roe, mtDNA is the prevailing DNA form (REHBEIN,2003). ELISA methods Immunochemical methods such as e.g. Sandwich ELISA (Enzyme Linked Immunosorbent Assay) or LFD (Lateral Flow Device; Dipsticks) are also frequentlyused in routine work. Generallykits provided by commercial suppliers are used. For example, detection methods for the main livestock species in raw or heated products such as pig/wild boar, cattle/ bison, sheep/goat or equidae (horse, mule, donkey etc.) and deer as a game species are available. The test kits detect animal species-specific proteins in both, unheated and heated meat products. German standard methods for animal species differentiation Tab.: Overview of the status quo in standardisation of animal differentiation analyses (except for isoelectric focusing) Designation Method Matrix Animal Remarks/Characteristics/Results species §35L06.00-47 ELISA heated meat products cattle Detection limit =2%;noquantitative data §64L08.00-61 Multiplex real-time PCR sausage products cattle Qualitative to semiquantitative technique: > 1% (DNA%) reliablyexceeded at 2.3 wt%; > 5% (DNA%) at 12.2 wt% pig Qualitative to semiquantitative technique: > 1% (DNA%) reliablyexceeded at 0.2 wt%; > 5% (DNA%) at 1wt% chicken Qualitative to semiquantitative technique: > 1% (DNA%) reliablyexceeded at 1.5 wt%; > 5% (DNA%) at 7.8wt% turkey Qualitative to semiquantitative technique: > 1% (DNA%) reliablyexceeded at 1.9 wt%; > 5% (DNA%) at 7.8wt% §64L08.00-62 Multiplex real-time PCR sausage products cattle Qualitative to semiquantitative technique: > 1% (DNA%) reliablyexceeded at 3.2 wt%; > 5% (DNA%) at 6.8 wt% pig Qualitative to semiquantitative technique: > 1% (DNA%) reliablyexceeded at 0.9 wt% sheep Qualitative to semiquantitative technique: > 1% (DNA%) reliablyexceeded at 1.1 wt%; > 5% (DNA%) at 5.3 wt% equidae Qualitative to semiquantitative technique: > 1% (DNA%) reliablyexceeded at 4.6 wt%; > 5% (DNA%) at 8.5 wt% (animal species: horse) §35L06.00-47 ELISA heated meat products pig Detection limit =0.2 to 0.5 %; no quantitative data §35L06.00-47 ELISA heated meat products poultry Detection limit =0.2 to 0.5 %; no quantitative data §64L06.26/27-2 PCR-RFLP fullypreserved meat horse qualitative technique, specificallyfor animal species horse; detection limit 1wt% §35L11.00-7 PCR (cytb) – RFLP fish (raw/heated) fish 36 fish species from the hake, eel, sardine, salmon/trout and flatfish families tested §64L10.00-12 PCR (cytb) – Sequencing §64L12.01-3 PCR (16S rRNA) – Sequencing fish (raw/heated) Legend: §64/§35=Methodinthe Official Collection of Analysis Techniques fish 6coded samples of plaice, sole, witch flounder, turbot, common dab and Pacific dab tested crustacean products crustaceans 6coded samples of black tiger shrimp, scampi/ Norway lobster, edible crab, Rosenbergii shrimp, northern deep water shrimp and white tiger shrimp tested Source: WAIBLINGER et al. FLEISCHWIRTSCHAFT international 1_2017
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