Search

Microbial transglutaminase (TG) is an enzyme isolated on an industrial scale from Streptomyces mobaraensis. Technical TG, a formulated powder, is primarily used to restructure meat in the meat-processing industry, typically at a 1% concentration and is often referred to as “meat glue.” In the European Union, meat restructured with TG requires the indication “formed meat” on the label according to Regulation (EU) No 1169/2011. In order to detect food fraud like the undeclared TG usage in meat and meat products, a qualitative mass spectrometric method using specific tryptic marker peptides has been published in 2017. Here the successful inter-laboratory validation and first-time standardization of a proteomics method for food control is described, which was subsequently included into the Official Collection of Analysis Methods according to the German Food and Feed Code (§ 64 LFGB). Thirteen laboratories from governmental, academic, and private institutions participated in the study, whereas four laboratories did not meet the minimal quality criteria and therefore their results had to be excluded. Three different test materials containing between 0.2 and 2% technical TG as well as blank samples were produced and tested. The laboratories used triple-quadrupole mass spectrometers from several vendors as well as quadrupole time-of-flight instruments. The detection of TG was considered to be positive, if three mass transitions for the marker peptides VTPPAEPLDR (TG-1) and SPFYSALR (TG-2), each, showed a signal-to-noise ratio of at least 3. The level of detection LOD95% for the median laboratory with intermediate performance was 0.31%, the false-positive rate was 0% and the false-negative rate was 2.1%.

In the present study, we assessed if different legacy and novel molecular analyses approaches can detect and trace prohibited bovine material in insects reared to produce processed animal protein (PAP). Newly hatched black soldier fly (BSF) larvae were fed one of the four diets for seven days; a control feeding medium (Ctl), control feed spiked with bovine hemoglobin powder (BvHb) at 1% (wet weight, w/w) (BvHb 1%, w/w), 5% (BvHb 5%, w/w) and 10% (BvHb 10%, w/w). Another dietary group of BSF larvae, namely *BvHb 10%, were first grown on BvHb 10% (w/w), and after seven days separated from the residual material and placed in another container with control diet for seven additional days. Presence of ruminant material in insect feed and in BSF larvae was assessed in five different laboratories using (i) real time-PCR analysis, (ii) multi-target ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), (iii) protein-centric immunoaffinity-LC-MS/MS, (iv) peptide-centric immunoaffinity-LC-MS/MS, (v) tandem mass spectral library matching (SLM), and (vi) compound specific amino acid analysis (CSIA). All methods investigated detected ruminant DNA or BvHb in specific insect feed media and in BSF larvae, respectively. However, each method assessed, displayed distinct shortcomings, which precluded detection of prohibited material versus non-prohibited ruminant material in some instances. Taken together, these findings indicate that detection of prohibited material in the insect-PAP feed chain requires a tiered combined use of complementary molecular analysis approaches. We therefore advocate the use of a combined multi-tier molecular analysis suite for the detection, differentiation and tracing of prohibited material in insect-PAP based feed chains and endorse ongoing efforts to extend the currently available battery of PAP detection approaches with MS based techniques and possibly δ13CAA fingerprinting. 1. Introduction 2. Materials and methods 2.1. Feed preparation 2.2. Rearing of BSF larvae and sample preparation 2.3. Detection of bovine hemoglobin in the feeding media and in BSF larvae 2.3.1. Real time-PCR (laboratories A and B) 2.3.2. Multi-target UHPLC-MS/MS (laboratory A) 2.3.3. Protein-centric immunoaffinity LC-MS/MS (laboratory B) 2.3.4. Peptide-centric immunoaffinity LC-MS/MS (laboratory C) 2.3.5. Spectral library matching (laboratory D) 2.3.6. Stable isotope analyses (laboratory E) 3. Results and discussion 3.1. Black soldier fly larvae development 3.2. Detection of bovine hemoglobin powder in the feeding media and in BSF larvae 3.2.1. qPCR 3.2.2. LC-MS/MS-based approaches 3.2. 3 δ 13CAA fingerprinting method 4. Conclusions

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 nonruminant PAPs for 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, because 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/MS-based quantification. In combination with a previously published assay for species identification, we were able to confirm the species and tissue origin of six ring trial samples obtained in former PCR and microscopy proficiency tests. 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.

With the reintroduction of nonruminant processed animal proteins (PAPs) for use in aquaculture in 2013, there is a suitable alternative to replace expensive fish meal in fish feed. Nevertheless, since the bovine spongiform encephalopathy (BSE) crisis, the use of PAPs in feed is strictly regulated. To date, light microscopy and polymerase chain reaction are the official methods for proving the absence of illegal PAPs in feed. Due to their limitations, alternative methods for the quantitative species differentiation are needed. To address this issue, we developed and validated an 8-plex mass spectrometry-based immunoassay. The workflow comprises a tryptic digestion of PAPs and blood products in suspension, a cross-species immunoaffinity enrichment of 8 species-specific alpha-2-macroglobulin peptides using a group-specific antibody, and a subsequent analysis by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry for species identification and quantification. This workflow can be used to quantitatively determine the species origin in future feed authentication studies.

The breadth of applications of nanoparticles and the access to food-associated consumer products containing nanosized materials lead to oral human exposure to such particles. In biological fluids nanoparticles dynamically interact with biomolecules and form a protein corona. Knowledge about the protein corona is of great interest for understanding the molecular effects of particles as well as their fate inside the human body. We used a mass spectrometry-based toxicoproteomics approach to elucidate mechanisms of toxicity of silver nanoparticles and to comprehensively characterize the protein corona formed around silver nanoparticles in Caco-2 human intestinal epithelial cells. Results were compared with respect to the cellular function of proteins either affected by exposure to nanoparticles or present in the protein corona. A transcriptomic data set was included in the analyses in order to obtain a combined multiomics view of nanoparticle-affected cellular processes. A relationship between corona proteins and the proteomic or transcriptomic responses was revealed, showing that differentially regulated proteins or transcripts were engaged in the same cellular signaling pathways. Protein corona analyses of nanoparticles in cells might therefore help in obtaining information about the molecular consequences of nanoparticle treatment.

Silver nanoparticles are advertised as antimicrobial agents in a wide range of products. The majority of available studies suggest that silver nanoparticle toxicity is mainly caused by silver ions released from the particles. However, it remains challenging to distinguish between the effect of silver nanoparticles and silver ions. Here we used a combination of a short-term in vivo study in rats and an in silico-based toxicokinetic model to determine tissue distribution of administered ionic and nanoparticulate silver, and to estimate mixture ratios of the different silver species, namely primary nanoparticles, ions and secondary particles. Our data indicate that silver nanoparticles and silver ions are not or only marginally bioavailable after oral ingestion of a single, non-toxic dose. Experimental data on organ distribution after intravenous injection were accurately reflected by the predictions of the in silico model. Toxicokinetic modeling suggests systemic distribution of a major proportion of the injected ionic silver as de novo formed secondary nanoparticles, and the presence of such particles was proven by electron microscopy. The observation that silver ions form secondary particles, underlines the difficulties in distinguishing between particle- and ion-dependent effects of silver nanoparticles.

The ban of processed animal proteins (PAPs) in feed for farmed animals introduced in 2001 was one of the main EU measures to control the bovine spongiform encephalopathy (BSE) crisis. Currently, microscopy and polymerase chain reaction (PCR) are the official methods for the detection of illegal PAPs in feed. However, the progressive release of the feed ban, recently with the legalization of nonruminant PAPs for the use in aquaculture, requires the development of alternative methods to determine the species origin and the source (legal or not). Additionally, discussions about the need for quantitative tests came up, particularly if the zero-tolerance-concept is replaced by introducing PAP thresholds. To address this issue, we developed and partially validated a multiplex mass spectrometry-based immunoassay to quantify ruminant specific peptides in vegetal cattle feed. The workflow comprises a new sample preparation procedure based on a tryptic digestion of PAPs in suspension, a subsequent immunoaffinity enrichment of the released peptides, and a LC-MS/MS-based analysis for peptide quantification using isotope labeled standard peptides. For the very first time, a mass spectrometry-based method is capable of detecting and quantifying illegal PAPs in animal feed over a concentration range of 4 orders of magnitude with a detection limit in the range of 0.1% to 1% (w/w).