Non-ruminant Processed Animal Proteins Research Articles

Tetracyclines in Processed Animal Proteins: A Monitoring Study on Their Occurrence and Antimicrobial Activity.

In 2013, the European Union (EU) lifted the feed ban restriction, authorizing the use of non-ruminant (NR) processed animal proteins (PAPs) as ingredient in aquafeed. A further relaxation is soon expected, and NR PAPs will be allowed in next future in poultry and pig feed, avoiding cannibalism. Other potential hazards linked to PAPs as raw material should be evaluated. Antibiotics administered along the lifecycle of animals may leave residue in tissues and bones and still be present in PAPs. This monitoring study aimed to determine tetracyclines (TCLs), known to cumulate in bones, in PAPs and their possible residual antibiotic activity (RAC). A sensitive Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS) method for the quantification of TCLs in PAPs was developed and applied to 55 PAPs from EU manufactures. Most PAP samples (n = 40) contained TCLs (concentrations 25.59 ÷ 456.84 µg kg−1). Among samples containing more than 25 µg kg−1 for at least three TCLs, three PAPs were chosen for RAC test before and after TCLs extraction procedure applying an in vitro acidic digestion: in two out of those three samples, RAC was observed after in vitro digestion. TCLs were determined in the digested PAPs (concentrations 26.07 ÷ 64.55 µg kg−1). The detection of TCLs in PAPs should promptly target the risk assessments of this unconsidered way of exposure to antibiotic residues.

Species discrimination of terrestrial processed animal proteins by laser-induced breakdown spectroscopy (LIBS) based on elemental characteristics

Description of the subject. This research presents a novel approach for species discrimination of terrestrial processed animal proteins (PAP) using laser-induced breakdown spectroscopy (LIBS) as an alternative technique for determining feed safety. Objectives. To explore the correlation between LIBS results and elemental composition. To assess the potential of LIBS spectroscopic methods for species discrimination of terrestrial PAP. Method. A total of 42 reliable terrestrial PAP samples of ovine, bovine, porcine, and poultry origin were analyzed and LIBS spectra were innovatively collected from the separated bone fraction. Partial least squares-discriminant analysis (PLS-DA) models were performed on the dataset. Results. Significant differences were found in the element compositions (carbon, nitrogen, hydrogen, calcium, sodium, potassium, magnesium, zinc, strontium and barium, p < 0.05) of PAP from different sources, which indicated a high potential for species discrimination. High-marked LIBS spectral characteristics (21 variables) related to six elements (calcium, sodium, magnesium, zinc, strontium and barium) for different species of terrestrial PAP samples were found out. PAP from the four tested species could be well classified with high sensitivity and specificity (> 0.90), while the ruminant and non-ruminant PAP could be thoroughly discriminated. For the discriminant analysis of ruminant-derived components in terrestrial PAPs, the detection of 10%-40% adulteration can be achieved and the correct rate is about 95%. Conclusions. This study demonstrates that LIBS can mostly exhibit the elemental composition of different terrestrial PAP, and can be a promising method for discriminating the different animal species or animal groups (ruminant/non-ruminant) involved in terrestrial PAPs.

Identifying animal species in NIR hyperspectral images of processed animal proteins (PAPs): Comparison of multivariate techniques

The use of PAPs in animal feed has several advantages over other feed ingredients, but requires rigorous and accurate control mechanisms that ensure the absence of ruminant meal. In order to differentiate between animal species while simultaneously offering the capacity to inspect PAPs in large volumes, a hyperspectral imaging (HSI) system operating in the NIR spectral range is proposed. This study investigates the sensitivity, specificity and other parameters with which HSI can discriminate between different animal species (ruminants, swine and poultry), making use of various classification methods. Diffuse reflectance spectra were acquired from 125 rendered meal samples in the 1000–1700 nm wavelength range; measured PAPs included particles of scale, hair, feather, blood, grease, skin, muscle and bone from both ruminant and non-ruminant animals, obtained in a rendering plant. Various classification methods were then applied to the dataset to determine the accuracy with which different animal species could be discriminated from each other. Support Vector Machine classification performed best in discriminating between animal species, with a sensitivity and specificity of around 90% and a Matthew's correlation coefficient of around 0.7 for non-ruminant species and higher than 0.95 for ruminant species. Other methods, such PLS-DA and Subspace Discriminant, also produced acceptable results and required less computational time. This study showed that spectral analysis of PAPs, based on diffuse reflectance spectroscopy, is a promising technique for differentiating between ruminant species and other terrestrial animal species. The technique may therefore offer accurate and fast analysis of large volumes of feed products, a necessary prerequisite for the lifting of the EU ban on non-ruminant processed animal proteins.

Peptidomic Approach to Developing ELISAs for the Determination of Bovine and Porcine Processed Animal Proteins in Feed for Farmed Animals.

The European Commission (EC) wants to reintroduce nonruminant processed animal proteins (PAPs) safely into the feed chain. This would involve replacing the current ban in feed with a species-to-species ban which, in the case of nonruminants, would only prohibit feeding them with proteins from the same species. To enforce such a provision, there is an urgent need for species-specific methods for detecting PAPs from several species in animal feed and in PAPs from other species. Currently, optical microscopy and the polymerase chain reaction are the officially accepted methods, but they have limitations, and alternative methods are needed. We have developed immunoassays using antibodies raised against targets which are not influenced by high temperature and pressure. These targets were identified in a previous study based on an experimental approach. One optimized competitive ELISA detects bovine PAPs at 2% in plant-derived feed. The detection capability demonstrated on blind samples shows a good correlation with mass spectrometry results.

Multi-laboratory evaluation of a PCR method for detection of ruminant DNA in commercial processed animal proteins

In 2013, the European Union (EU) allowed the inclusion of non-ruminant processed animal proteins (PAP) in fish feeds. Plans also exist to allow non-ruminant PAP in poultry and pig feeds but to keep intraspecies recycling forbidden (e.g. poultry to poultry or pig to pig). However, due to the fear of transmissible spongiform encephalopathy (TSE), PAP from ruminant species remains illegal to use in feeds. To ensure feed safety, nations across Europe need to safeguard that commercial fish feeds do not contain PAP from ruminant species. In this work we report a multi-laboratory study on the detection of ruminant PAP in commercial PAP feed ingredients. A total of 19 non-ruminant PAP, provided by the European Fat Processors and Renderers Association (EFPRA), were analyzed for ruminant content with the European Union Reference Laboratory (EURL)-validated PCR assay by three labs and compared to the result obtained with an immunoassay-based method (MELISA-TEK). Ruminant PAP was detected in five of the 19 non-ruminant PAP samples with both methods, but there were methodological and multi-laboratory differences for the PCR method. Compared to the results with the EU-reference laboratory, 3 false-negatives were found by the two other laboratories. We speculate that the discrepancies seen for some of the samples determined with the PCR method rely on the homogenization and grinding step, which is not described in detail in the EURL Standard Operating Procedure (SOP), shift in the normal distribution of Ct-values near the cut-off of the PCR method, or PCR inhibition. In conclusion, this study demonstrates the presence of ruminant DNA in commercially available non-ruminant PAP by use of two methods for the detection of PAP in commercial feed ingredients, and highlights the importance of thorough homogenization prior to DNA extraction and assessment of normal distribution of Ct-values for successful PCR detection at low ruminant DNA contamination levels.

Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools

The rapidly growing aquaculture industry drives the search for sustainable protein sources in fish feed. In the European Union (EU) since 2013 non-ruminant processed animal proteins (PAP) are again permitted to be used in aquafeeds. To ensure that commercial fish feeds do not contain PAP from prohibited species, EU reference methods were established. However, due to the heterogeneous and complex nature of PAP complementary methods are required to guarantee the safe use of this fish feed ingredient. In addition, there is a need for tissue specific PAP detection to identify the sources (i.e. bovine carcass, blood, or meat) of illegal PAP use. In the present study, we investigated and compared different protein extraction, solubilisation and digestion protocols on different proteomics platforms for the detection and differentiation of prohibited PAP. In addition, we assessed if tissue specific PAP detection was feasible using proteomics tools. All work was performed independently in two different laboratories. We found that irrespective of sample preparation gel-based proteomics tools were inappropriate when working with PAP. Gel-free shotgun proteomics approaches in combination with direct spectral comparison were able to provide quality species and tissue specific data to complement and refine current methods of PAP detection and identification. SignificanceTo guarantee the safe use of processed animal protein (PAP) in aquafeeds efficient PAP detection and monitoring tools are required. The present study investigated and compared various proteomics workflows and shows that the application of shotgun proteomics in combination with direct comparison of spectral libraries provides for the desired species and tissue specific classification of this heat sterilized and pressure treated (≥133°C, at 3bar for 20min) protein feed ingredient.

Species identification of processed animal proteins (PAPs) in animal feed containing feed materials from animal origin

Since June 2013 the total feed ban of processed animal proteins (PAPs) was partially lifted. Now it is possible to mix fish feed with PAPs from non-ruminants (pig and poultry). To guarantee that fish feed, which contains non-ruminant PAPs, is free of ruminant PAPs, it has to be analysed with a ruminant PCR assay to comply with the total ban of feeding PAPs from ruminants. However, PCR analysis cannot distinguish between ruminant DNA, which originates from proteins such as muscle and bones, and ruminant DNA, which comes from feed materials of animal origin such as milk products or fat. Thus, there is the risk of obtaining positive ruminant PCR signals based on these materials. The paper describes the development of the combination of two analysis methods, micro-dissection and PCR, to eliminate the problem of ‘false-positive’ PCR signals. With micro-dissection, single particles can be isolated and subsequently analysed with PCR.

Inter-laboratory validation study of two immunochemical methods for detection of processed ruminant proteins

In order to facilitate safe re-introduction of non-ruminant processed animal proteins (PAPs) in aqua feed, two immunoassays have been tested in an interlaboratory study for their capability to detect ruminant PAPs processed under European conditions. The sensitivity of the MELISA-TEK assay was improved by applying a specific extraction kit. Six approved blank pork and poultry samples were adulterated to produce 15 samples spiked at 0.5%, 1.0% and 2.0% with ruminant material, sterilised at either 133°C or 137°C. Fourteen participants investigated the 6 blanks and 15 spiked samples, making 21 samples for the final test. For both assays specificity and sensitivity were at 97% or higher. Concordance and accordance were higher than 95% with one exception. The results indicate that both assays provided correct results at 0.5% and higher for the detecting ruminant PAPs (sterilised at 133°C) in non-ruminant PAPs. Given the 2% upper limit of ruminant PAPs in non-ruminant PAPs for avoiding an increase in BSE incidents, these methods are fit for monitoring non-ruminant PAPs intended for aqua feed.

The Re-Authorization of Non-Ruminant Processed Animal Proteins in European Aqua feeds

Today, the global community is challenged to meet the pressing food needs of a growing population with finite natural resources. Fish and seafood have always been considered as an important part of human diet and it has long been recognized as a health-promoting food for human nutrition [1]. Globally, fish provides about 4.3 billion people with almost 15% of their average per capita intake of animal protein, with the global annual per capita consumption having doubled since the 1960s [2]. This change mainly was due to rising living standards, population growth, rapid urbanization and opportunities for trade and transformations in food distribution. This has led to a growing demand for proteic food products, in particular fish and meat. Fish consumption is expected to continue rising due to an expected increased demand, which is driven by population and income growth and dietary diversification. The European Union (EU) is by far the largest single market for imported fish products, with imports reaching US$47.0 billion in 2012 representing 36% of total world imports [3]. The dependence of the EU on imports for its fish consumption is growing, underlying the positive trend in consumption, but also evidences the constraints within the EU on further expansion of supply. In this respect, the current reform of its Common Fisheries Policy aims to rebuild its fish stocks, as well as boosting its aquaculture production.

Evaluation of a Commercial ELISA for Detection of Ruminant Processed Animal Proteins in Non-Ruminant Processed Animal Proteins

Abstract Due to a growing aquaculture industry, demand for high-quality proteins for aquatic feeds is increasing. Non-ruminant processed animal proteins (PAPs) have shown great potential for this purpose. Safe reintroduction of non-ruminant PAPs in aqua feed requires methods that can discriminate ruminant and non-ruminant PAPs at contamination levels at or below 2%. Because the official European Union method lacks species specificity, the performance of MELISA-TEK™ Ruminant, a commercial immunoassay, combined with the MELISA-TEK High Sensitivity Sample Extraction kit was evaluated. Various non-ruminant PAPs spiked with ruminant PAPs (processed at 133, 137, 141, and 145°C) were analyzed. Results showed an overall specificity of 99%, indicating no cross-reaction with non-ruminant PAPs. The sensitivity of the assay strongly depended on both processing temperature and proportion of muscle fibers of the ruminant PAPs. Overall sensitivity of samples with 1 and 2% ruminant PAPs was 92 and 100%, respectively. For ruminant PAPs processed at 133 and 137°C, the sensitivity was 100% for both 1 and 2% ruminant spikes. Overall accuracies were 96 and 99% for 1 and 2% ruminant spikes, respectively. In conclusion, the MELISA-TEK Ruminant assay showed satisfactory results, which makes it a suitable candidate method to enable safe reintroduction of non-ruminant PAPs in aqua feed.

Scientific Opinion on the revision of the quantitative risk assessment (QRA) of the BSE risk posed by processed animal proteins (PAPs)

The cattle Bovine Spongiform Encephalopathy (BSE) risk posed by bovine derived Processed Animal Proteins (PAPs) in feed was estimated, the diagnostic methods and their sensitivity to detect animal proteins in feed and compared different risk assessment methods for animal proteins in feed was reviewed. It was concluded that the current global limit of detection for PAPs in feed is still considered to be 0.1%. Data related to BSE monitoring and PAP production in the European Union (EU) were considered. A model (EFSA QRA PAP model) was developed to study the magnitude of the total BSE infectivity in PAPs and in ruminant feed under a certain scenario assuming some specific cross-contaminations. On the basis of the 2009 BSE surveillance data in the EU, assuming a 0.1% contamination (the limit of detection for PAP in feed) with non-ruminant PAPs and according to the EFSA QRA PAP model, the total BSE infectivity load that could enter in cattle feed per year in the EU would be equivalent to 0.2 Cattle oral Infectious Dose 50% (Co ID50) (9 x 10-5 - 1.3 CI95%) (that would mean that less than one additional BSE infected cattle could be expected in the EU cattle population per year with an upper 95% confidence). The specific scenario described by the model and the related assumptions and uncertainties are discussed in this scientific opinion.