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Application of Mass Spectrometry-Based Immunoassays for the Species- and Tissue-Specific Quantification of Banned Processed Animal Proteins in Feeds Andreas E. Steinhilber, Felix Florian Schmidt, Wael Naboulsi, Hannes Planatscher, Alicia Niedzwiecka, Jutta Zagon, Albert Braeuning, Alfonso Lampen, Thomas O. Joos, and Oliver Poetz Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04652 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 17, 2019
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Analytical Chemistry
Application of Mass Spectrometry-Based Immunoassays for the Species- and Tissue-Specific Quantification of Banned Processed Animal Proteins in Feeds Andreas E. Steinhilber1,3, Felix F. Schmidt1, Wael Naboulsi3, Hannes Planatscher3, Alicia Niedzwiecka2, Jutta Zagon2, Albert Braeuning2, Alfonso Lampen2, Thomas O. Joos1, and Oliver Poetz*1,3 1
NMI Natural and Medical Sciences Institute at the University of Tuebingen, 72770 Reutlingen, Germany German Federal Institute for Risk Assessment, Dept. Food Safety, 10589 Berlin, Germany 3 SIGNATOPE GmbH, 72770 Reutlingen, Germany 2
ABSTRACT: Processed Animal Proteins (PAPs) are considered as a sustainable protein source to improve the nutritional profile of feed for livestock and aquaculture. However, the use of these proteins is strongly regulated since the bovine spongiform encephalopathy (BSE) crisis. The reintroduction of non-ruminant PAPs for the use in aquaculture in 2013 has driven the need for alternative analytical methods to determine the species origin as well as the tissue source (legal or not). The current official methods, light microscopy and polymerase chain reaction, do not fulfill these requirements. Furthermore, future methods need to be quantitative, since the pending zero-tolerance-concept is planned to be replaced by accurate thresholds. Here we developed a 7-plex mass spectrometry-based immunoassay that is capable of quantifying 0.1% (w/w) ruminant PAP in feed in a tissue- and species-specific way. The workflow comprises a 2 h tryptic digestion of PAPs in suspension, an immunoaffinity enrichment of peptides and LC-MS/MSbased quantification. In combination with a previously published assay for species identification, we proved the authenticity of six official ring trial samples. The sensitive, quantitative, species- and tissue-specific character of the developed assays meets the requirements for new methods for PAP detection and can be used in future feed authentication studies.
Processed animal proteins (PAPs) are valuable ingredients for livestock and aquaculture feed 1. They increase the overall sustainability of the food chain, since they are produced from animal byproducts that are not intended for human consumption 2. PAPs are characterized by high levels of fat, proteins, minerals, and even essential vitamins for which reason they are considered to improve the nutritional value of feed for monogastric animals like chicken and pigs 1,3,4. Furthermore, they do not contain anti-nutrients, which are often present in plant-derived products limiting their use as feed supplements 5,6 . In aquaculture, the partial substitution of plant proteins with poultry PAPs in feed was shown 7. Also blood meal (BM), a special type of PAP, is increasingly used to substitute expensive fish meal in aquaculture feed 1. The prices for fish meal reached historic heights in 2014 as result of a higher feed demand in a rapidly growing aquaculture industry 8. Not only PAPs, but also lower processed products such as spray-dried hemoglobin meal (SDHM) and spray-dried plasma (SDP), as defined by regulation (EU) No. 142/2011 9, are increasingly used since they show a high digestibility 10,11. As a consequence of the outbreak of the bovine spongiform encephalopathy (BSE) crisis in 1986 in the UK, the use of animal proteins in feed became strictly regulated. One central measure to gain control over the crisis was the ban for mammalian-derived proteins in animal feed introduced with Commission Regulation (EC) No. 999/2001 12 and later expanded by amendment No. 1234/2003 13. The common procedure of
adding meat and bone meal (MBM) to cattle feed was prohibited by this regulation. Consequently, prion-contaminated cattle MBM, which was supposed to be the main vector of disease, was excluded from the food chain. As a result, the registered BSE cases decreased nearly down to zero in Europe including UK, the former hot spot of the BSE pandemic 14,15. To date, the transmission of BSE between non-ruminants is considered unlikely and BSE seems to be almost eradicated. In consideration of a sustainable feed chain the European Commission is currently working on the reintroduction of nonruminant PAPs in feed without bypassing the ban for intraspecies feeding 16. So far, fish meal in milk replacer for weaning calves was reauthorized in 2008 17, followed by the reintroduction of non-ruminant PAPs in feed for aquaculture in 2013 18, amended by seven defined insect species in 2017 19. PAPs from pig and poultry origin are planned the be legalized in near future 16. The progressive lifting of the feed ban demands for analytical methods with high sensitivity and specificity to prove the absence of ruminant material. Furthermore, minor cross-contaminations in the production chain during transport between slaughter houses, rendering plants and feed producers can never be completely eliminated. Against this background, producers as well as control laboratories call for quantitative accurate thresholds replacing the pending zero-toleranceconcept. The European Food Safety Authority (EFSA) currently works on a risk assessment for a safe PAP level in feed and the implementation of a threshold as “technical zero” on
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demand of the European Commission 20. As a consequence, future methods for PAP detection must be quantitative. If thresholds are implemented it can be strongly assumed that the required detection limit will be in the range of
