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See detailDevelopment of complementary methods for the detection of animal by-products in feedingstuffs
Lecrenier, Marie-Caroline ULiege

Doctoral thesis (2019)

Since the mid-1980s, several transmissible spongiform encephalopathies have been reported in humans and animals. Bovine spongiform encephalopathy was first identified in cattle in 1986 and, in subsequent ... [more ▼]

Since the mid-1980s, several transmissible spongiform encephalopathies have been reported in humans and animals. Bovine spongiform encephalopathy was first identified in cattle in 1986 and, in subsequent years in other animal species. Consumption by cattle of meat and bone meal containing carcasses of infected animals was incriminated. The use of such animal by-products was first banned in feed for ruminants and then, with few exceptions such as the use of fish meal for non-ruminants, extended to all feeds for farmed animal. Thanks to prevention and control efforts, a gradual lifting of the feed ban is now possible. The reintroduction of non-ruminant processed animal proteins into the feed of aquaculture animals was adopted in 2013. In this context of dynamic relaxation of the ban, the challenge remains the development of complementary methods or the adaptation of official methods aimed at refining the identification of feed materials. Indeed, the combination of light microscopy and PCR does not differentiate among certain animal by-products and this can make the results uninterpretable. This PhD thesis focused on (1) the evaluation of current legislation and available methods of analysis for the detection of animal proteins; (2) the development of a fluorescence in situ hybridisation method combining the advantages of microscopy (localisation and identification of type of tissues) and molecular biology techniques (identification of the species by DNA); (3) the identification of specific peptides from blood products by high resolution mass spectrometry and (4) the development of a sensitive and routinely applicable tandem mass spectrometry method paving a new way to the detection of processed animal proteins. The results obtained allowed to (1) highlight current and foreseen analytical gaps as part of the feed ban relaxation dynamics; (2) propose a method for a specific detection of processed bone particles of ruminant origin independently of contamination by other by-products such as milk powder; (3) identify 9 biomarker peptides of plasma powder of bovine origin and 7 biomarker peptides of bovine haemoglobin powder, 6 of which also allow the detection of blood meal of bovine origin and (4) develop a UHPLC MS/MS multi-target method allowing the simultaneous detection of several by-products of ruminant origin (blood meal, blood products and dairy products) at a limit of detection (LOD) of 0.1% (w/w). Finally, new analytical schemes incorporating the results of this thesis as well as potential sources of improvement for future analytical developments have been proposed. [less ▲]

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See detailSelection and evaluation of new biomarkers for ruminant plasma powder detection
Lecrenier, Marie-Caroline ULiege; Plasman, Lisa; Collard, Marie et al

Conference (2019, May)

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See detailSpecies-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra
Belghit, Ikram; Lock, Erik-Jan; Fumière, Olivier et al

in Animals (2019), 9(5), 222

Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as ... [more ▼]

Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as processed animal proteins (PAP). It is therefore of interest to develop methods for regulatory use, which unambiguously identify the species origin of insect-based ingredients. We performed (i) total protein quantification of insect samples using the traditional nitrogen-to-protein conversion factor of 6.25 and the sum of anhydrous amino acids, (ii) quantitative amino acid profiling and (iii) high-throughput tandem mass spectrometry to describe and differentiate 18 different commercial-grade insect meal samples derived from Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). In addition, we investigated and compared different protein extraction and digestion protocols for proteomic analysis. We found that irrespective of sample preparation, shotgun proteomics in combination with direct spectral comparison were able to differentiate insect meal according to their taxonomic classification. The insect specific spectral libraries created in the present work can in future be used to develop more sensitive targeted methods of insect PAP identification and quantification in commercial feed mixtures. [less ▲]

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See detailUHPLC-MS/MS method for sensitive, specific and simultaneous detection of bovine blood meal, blood products and milk products in compound feed
Lecrenier, Marie-Caroline ULiege; Dieu, M; Fumière, O et al

in Proceeding of Feed 2018 (2018, October 25)

Feed availability is one of the biggest challenges for the future. Solutions will be found by increasing the production efficiency and finding new sources without jeopardizing feed quality and safety ... [more ▼]

Feed availability is one of the biggest challenges for the future. Solutions will be found by increasing the production efficiency and finding new sources without jeopardizing feed quality and safety. Animal by-products are an interesting source of feed materials. These materials are rich in proteins of high nutritional value and have an economic interest since their non-use results to a logical loss of gains. However, since the mad cow disease crisis, their use has been strictly regulated. In 2013, non-ruminant processed animal proteins (PAPs) were reauthorised in aquafeed but ruminant PAPs remain forbidden. Official controls are based on a combination of light microscopy and PCR. But sometimes these methods are unable to distinguish some feed materials. The objective of this work was to develop a sensitive method using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the specific detection of bovine blood-derived products and milk powder in feed. This method has the advantage to be species and tissue specific. Peptide biomarkers identified in previous studies were used. The sample preparation and the analytical method were designed to provide a fast, simple and powerful method suitable for routine. Proteins were extracted in a buffer containing 200 mM TRIS-HCl pH 9.2, 2 M urea followed by trypsin digestion and purification with tC18 SPE (Waters). Analyses were performed by liquid chromatography (Acquity UHPLC system, Waters) coupled with a triple quadrupole mass spectrometer (Xevo TQS, Waters). Labelled peptides were used as internal standard in order to compare the results independently of the retention time variation due to matrix effect. Various commercial aquafeed batches artificially adulterated at levels of 0.1 % to 1 % (w/w) with bovine blood meal, bovine blood products or milk powder were analysed in order to assess the influence of matrix and to evaluate sensitivity and specificity of the method. The method was able to detect all adulterants at 0.1 % level in all matrices. This makes the method suitable for application in feed control and offers an innovative and complementary solution for the simultaneously identification of authorised and unauthorised animal by-products such as PAPs. [less ▲]

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See detailSynchronous fluorescence spectroscopy for detecting blood meal and blood products
Lecrenier, Marie-Caroline ULiege; Baeten, Vincent; Taira, Aurélien et al

in Talanta (2018), 189

Fluorescence spectroscopy is a powerful method for protein analysis. Its sensitivity and selectivity allow its use for the detection of blood meal and blood products. This study proposes a novel approach ... [more ▼]

Fluorescence spectroscopy is a powerful method for protein analysis. Its sensitivity and selectivity allow its use for the detection of blood meal and blood products. This study proposes a novel approach for the detection of hemoglobin in animal feed by synchronous fluorescence spectroscopy (SFS). The objective was to develop a fast and easy method to detect hemoglobin powder and blood meal. Analyses were carried out on standard reference material (hemoglobin and albumin) in order to optimize SFS method conditions for hemoglobin detection. The method was then applied to protein extracts of commercial feed material and compound feed. The results showed that SFS spectra of blood meal and blood products (he- moglobin powder and plasma powder) could be used to characterize hemoglobin. Principal component analysis (PCA) applied to area-normalized SFS spectra of artificially adulterated samples made it possible to define a limit of detection of hemoglobin powder or blood meal of 0.5–1% depending on the feed material. The projection in the PCA graphs of SFS spectra of real commercial compound feeds known to contain or to be free from blood- derived products showed that it was possible to discriminate samples according to the presence of hemoglobin. These results confirmed that SFS is a promising screening method for the detection of hemoglobin in animal feed. [less ▲]

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See detailMass Spectrometry based Approaches for Analysing Proteins in Processed Food and Feed
Raes, M; Planque, M; Marbaix, H et al

in Clinics in Oncology (2018), 3(1485),

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See detailMass spectrometry for detection of ruminant animal by-products in poultry feed
Lecrenier, Marie-Caroline ULiege; Henrottin, Jean; Dieu, Marc et al

in First PhD student day: 2018, May 18th, Liège, Sart Tilman (2018, May 18)

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See detailA mass spectrometry method for sensitive, specific and simultaneous detection of bovine blood meal, blood products and milk products in compound feed
Lecrenier, Marie-Caroline ULiege; Planque, Mélanie; Dieu, Marc et al

in Food Chemistry (2018), 245

Feed sustainability is one of the biggest challenges for the next few years. Solutions have to be found that take feed quality and safety into account. Animal by-products are one valuable source of ... [more ▼]

Feed sustainability is one of the biggest challenges for the next few years. Solutions have to be found that take feed quality and safety into account. Animal by-products are one valuable source of proteins. However, since the bovine spongiform encephalopathy (BSE) crisis, their use has been strictly regulated. The objective of this study was to propose a routine, sensitive and specific method using ultra-high performance liquid chromatography coupled to tandem mass spectrometry for the detection of blood-derived products and milk powder in feed. Contaminated aquafeeds were analysed in order to evaluate the sensitivity and specificity of the method. This new method meets both selectivity and sensitivity (0.1% (w/w)) requirements imposed by the European Commission for animal proteins detection methods. It offers an innovative and complementary solution for the simultaneously identification of authorised and unauthorised animal by-products such as processed animal proteins (PAPs). [less ▲]

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See detailRoutine mass spectrometry applied to aquafeed and transfer to poultry feed
Lecrenier, Marie-Caroline ULiege; Berben, Gilbert; Fumière, Olivier et al

Conference (2018, April)

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See detailENTOFÔR project -From waste to resource
Lecrenier, Marie-Caroline ULiege; Fumière, Olivier; Berntssen, M.H.G. et al

in Book of abstracts of the 8th International Symposium on Recent advances in food analysis (2017, November)

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See detailUltra-High Performance Liquid Chromatography coupled to tandem Mass Spectrometry for the detection of animal by-products in feed
Lecrenier, Marie-Caroline ULiege; Planque, Mélanie; Dieu, Marc et al

in Book of abstracts of the 8th International Symposium on Recent advances in food analysis (2017, November)

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See detailSynchronous fluorescence spectroscopy for detecting blood meal and blood products in feed
Abbas, Ouissam; Lecrenier, Marie-Caroline ULiege; Baeten, Vincent

Poster (2017, September)

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See detailEffect of grinding on bone detection by light microscopy: Study results
Lecrenier, Marie-Caroline ULiege; Veys, Pascal; Planchon, Viviane et al

Conference (2017, June)

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See detailDetection of blood and milk in aquafeed using UHPLC MS/MS
Lecrenier, Marie-Caroline ULiege; Veys, Pascal; Fumière, Olivier et al

Conference (2017, April)

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See detailSpecific detection of blood derived products in animal feed using UPLC-MS/MS
Lecrenier, Marie-Caroline ULiege; Dieu, Marc; Veys, Pascal et al

in Abstracts of lectures and posters of the 11th Conference RME 2016 (2016, November)

Blood derived products are valuable animal products used in feed for their nutritional value and their positive effects on growth and health. Nevertheless, since the BSE crisis, their use is strictly ... [more ▼]

Blood derived products are valuable animal products used in feed for their nutritional value and their positive effects on growth and health. Nevertheless, since the BSE crisis, their use is strictly regulated. Blood meal and blood products (hemoglobin powder and plasma powder) of bovine origin are totally prohibited. Blood meal and blood products of porcine origin are authorised in aquafeed, whereas only blood products are allowed to be used in feed intended for other non-ruminant. The detection of the type of protein and the species of origin is therefore crucial to ensure feed safety. With the current official methods for the detection of PAPs, light microscopy and PCR, it is not always possible to specifically identify this type of protein source. The objective of our work was to set-up a routine mass spectrometry method for a qualitative detection of blood meal and hemoglobin powder of bovine and porcine origin in feed. The method was based on the detection of species-specific peptides biomarkers identified in a previous study by a non-targeted approach. All biomarkers were peptides of α and β hemoglobin subunits. Proteins were extracted using a TCA/acetone protein precipitation protocol followed by purification with a 2-D Clean-Up Kit (GE Healthcare, USA). After an in-solution trypsin digestion step, analyses were performed by liquid chromatography (Acquity system, Waters, UK) coupled with a triple quadrupole mass spectrometer (Xevo TQS, Waters, UK) with electrospray ionisation. The acquisition and processing of data was carried out by MassLynx software (v. 4.1, Waters) and multiple reaction monitoring (MRM) design was made using the open-source software Skyline (https://skyline.gs.washington.edu/labkey/project/home/software/Skyline/begin.view). Reference hemoglobin powder was used to select MRM transitions for each peptide biomarkers and to optimise their collision energy. Commercial feed material and compound feed containing or free from blood derived products were then analysed. Selected transitions were present in materials or feed known to contained blood derived product of the same origin and absent in the others. Artificially contaminated feed with various contamination levels were also analysed in order to evaluate the influence of matrix composition and to experimentally determine the limit of detection. A first estimation of the LOD was around 0.05 % w/w which was below the LOD imposed by the EC for animal proteins detection method. [less ▲]

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See detailBlood meal and blood products detection using Synchronous fluorescence spectroscopy
Lecrenier, Marie-Caroline ULiege; Abbas, Ouissam; Taira, Aurélien et al

in Abstracts of lectures and posters of the 11th Conference RME 2016 (2016, November)

In Europe, ruminant processed animal proteins (PAPs) and blood products are not allowed to be used in feed for farmed animal. In contrast, blood meal and blood products of porcine origin are both ... [more ▼]

In Europe, ruminant processed animal proteins (PAPs) and blood products are not allowed to be used in feed for farmed animal. In contrast, blood meal and blood products of porcine origin are both authorised in aqua feed, whereas only porcine blood products are allowed to be used in feed intended for other non-ruminants. Besides official methods (light microscopy and PCR), complementary methods are developed in order to refine the by-products identification. By-products derived from blood are one of these products for which additional information are needed. Indeed, these prohibited materials sometimes cannot be distinguished from those authorised (e.g. milk powder) using official methods. The aim of this work was to develop a fast and easy method to detect blood meal and blood products. This study was based on the detection of hemoglobin in animal feed by synchronous fluorescence spectroscopy (SFS). To achieve this goal, preliminary tests were carried out on reference material (hemoglobin and albumin purchased from Sigma Aldrich) in order to determine appropriate conditions (solvent) and parameters (offset values) for hemoglobin detection. Selected settings were then applied to analyse protein extracts of commercial feed material derived from blood. The results obtained on blood meal and blood products (hemoglobin powder and plasma powder) showed fluorescence spectral bands that characterise hemoglobin. In order to determine whether the method was fit for the purpose, real commercial compound feeds known to contain or to be free from blood products or blood meal were analysed. The Principal Component Analysis (PCA) applied to these spectra showed that it was possible to discriminate samples containing hemoglobin from those that do not contain. This result confirmed that SFS is a promising screening method for the detection of hemoglobin in animal feed. [less ▲]

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See detailDetection of blood derived products in feed using targeted triple-quadrupole MS
Lecrenier, Marie-Caroline ULiege; Dieu, Marc; Veys, Pascal et al

Conference (2016, September)

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See detailIdentification of specific bovine blood biomarkers with a non-targeted approach using HPLC ESI tandem mass spectrometry
Lecrenier, Marie-Caroline ULiege; Marbaix, Hélène; Dieu, Marc et al

in Food Chemistry (2016), 213

Animal by-products are valuable protein sources in animal nutrition. Among them are blood products and blood meal, which are used as high-quality material for their beneficial effects on growth and health ... [more ▼]

Animal by-products are valuable protein sources in animal nutrition. Among them are blood products and blood meal, which are used as high-quality material for their beneficial effects on growth and health. Within the framework of the feed ban relaxation, the development of complementary methods in order to refine the identification of processed animal proteins remains challenging. The aim of this study was to identify specific biomarkers that would allow the detection of bovine blood products and processed animal proteins using tandem mass spectrometry. Seventeen biomarkers were identified: nine peptides for bovine plasma powder; seven peptides for bovine haemoglobin powder, including six peptides for bovine blood meal; and one peptide for porcine blood. They were not detected in several commercial compound feed or feed materials, such as blood by-products of other animal origins, milk-derived products and fish meal. These biomarkers could be used for developing a species-specific and blood-specific detection method. [less ▲]

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See detailDéveloppement de méthodes complémentaires pour la détection des protéines animales: Focus sur la spectrométrie de masse
Lecrenier, Marie-Caroline ULiege; Veys, Pascal; Berben, Gilbert et al

Scientific conference (2015, November)

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See detailBovine blood biomarkers as a way of processed animal proteins detection in feedingstuffs
Lecrenier, Marie-Caroline ULiege; Marbaix, Hélène; Veys, Pascal et al

in Book of abstracts of the 7th International Symposium on Recent advances in food analysis (2015, November)

The prohibition of using animal by-products in feedingstuffs depends on two factors: their nature defined by the tissue/cell type and the species of origin, and on their destination (pets, fur animals or ... [more ▼]

The prohibition of using animal by-products in feedingstuffs depends on two factors: their nature defined by the tissue/cell type and the species of origin, and on their destination (pets, fur animals or other farmed animals). Proteomics is particularly well-suited to the purpose of PAPs detection as it is a tissue and species-specific method. The aim of this study was the identification and the selection of specific peptide biomarkers using tandem mass spectrometry for the detection of bovine blood products and blood meals in animal feed. Twenty-nine samples of blood meals and blood products (plasma or haemoglobin powder) of porcine, poultry and bovine origin as well as three milk products and two fish meals were analysed using a Q TOF mass spectrometer. Vegetal feed samples adulterated with 1% or 10% of bovine plasma powder, haemoglobin powder or blood meal were also analysed to evaluate the applicability of the method. Four proteins of interest were highlighted: Alpha-2-macroglobulin, apolipoprotein A-1, serotransferrin and haemoglobin (α and β chains). From these proteins, sixteen peptides were identified as potential bovine blood biomarkers in feedingstuffs. Nine of them could be used for the detection of plasma powder and seven of them for haemoglobin powder or blood meal. The evaluation of these peptides by a search against NCBInr database revealed that some of them could also be used to detect other ruminant bloods such as ovine or caprine ones. These preliminary results are promising. Efforts are now focused to improve the protocol in order to increase the sensitivity of the method as regards the selected proteins. [less ▲]

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