Challenges related to the application of analytical methods to control insect meals in the context of European legislation.

Since their approval for use in aquaculture in 2017, processed insect proteins have been extensively studied for their nutritional quality in animal feed. This new type of meal is highly promising but requires, as for other products used in animal feed, strict sanitary control in accordance with European legislation. Within this legal framework, light microscopy and PCR remain the official methods but have some analytical limitations that other methods could overcome. This paper aims to provide an overview of the European legislation concerning use of processed insect proteins, but also to highlight the advantages and disadvantages of the official methods for their analysis. It also points out other analytical methods, which have already proved their worth for the analysis of processed animal proteins, which could be used as complementary methods.

Detection of Alphitobius diaperinus by Real-Time Polymerase Chain Reaction With a Single-Copy Gene Target.

Use of edible insects as an alternative source of proteins in food and feed is increasing. These last years, numerous companies in Europe have started producing insects for food and feed purposes. In the European Union, the use of edible insects for human consumption falls within Regulation (EU) No. 2015/2283 on novel foods. For feed, Commission Regulation (EU) 2017/893 authorizes seven insect species as processed animal proteins for aquaculture. Methods of authentication are required to check the conformity of the products. In this study, we propose a real-time polymerase chain reaction (PCR) method for the specific detection of the lesser mealworm (Alphitobius diaperinus), one of the species included in the shortlist of authorized insects. The selected target is the cadherin gene with a single-copy (per haploid genome) illustrated by our experimental evidence. The PCR test amplified a 134-bp fragment of the cadherin gene. The qualitative method was assessed toward several performance criteria. Specificity was checked against 54 insect species next to other animal and plant species. The sensitivity, efficiency, robustness, and transferability of the PCR assay were also successfully tested. Finally, the applicability of the test was assessed on real-life processed samples (industrial meals) of A. diaperinus. The study also showed that there seems to be a huge confusion on the correct labeling of the marketed mealworms. We did not succeed to get Alphitobius laevigatus samples. They all appeared to belong to the A. diaperinus taxon.

Official Feed Control Linked to the Detection of Animal Byproducts: Past, Present, and Future.

In the context of the expansion of the human population, availability of food, and in extension of animal feed, is a big issue. Favoring a circular economy by the valorization of byproducts is a sustainable way to be more efficient. Animal byproducts are an interesting source of feed materials due to their richness in proteins of high nutritional value. Prevention and control efforts have allowed a gradual lifting of the feed ban regarding the use of animal byproducts. Nevertheless, the challenge remains the development of analytical methods enabling a distinction between authorized and unauthorized feed materials. This Review focuses on the historical and epidemiological context of the official control, the evaluation of current and foreseen legislation, and the available methods of analysis for the detection of constituents of animal origin in feedingstuffs. It also underlines the analytical limitations of the approach and discusses some prospects of novel methods to ensure food and feed safety.

Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra.

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.

Development of real-time PCR tests for the detection of Tenebrio molitor in food and feed.

Insects are rich in proteins and could be an alternative source of proteins to feed animals and humans. Numerous companies have started the production of insects for feed purposes. In Europe, these processed animal proteins are not yet authorised by legislation as many questions still need to be answered concerning this 'novel food'. Authorisations will be possible when methods of authentication of the products are available. In this study we propose real-time PCR methods for the specific detection of the mealworm (Tenebriomolitor), one of the most widely used insects for food and feed production. Two PCR assays are proposed: the first based on the wingless gene and the second based on the cadherin gene. The PCR tests amplify fragments of 87 bp. These qualitative methods were tested according to several performance criteria. The specificity was tested on 34 insect species' DNA, but also on non-insect species including crustacean, mammals, birds and plants. The limit of detection was determined and was below 20 copies for the two PCR tests. The applicability of the tests was demonstrated by the analysis of real-life processed samples containing T. molitor.

Identification of Proteins and Peptide Biomarkers for Detecting Banned Processed Animal Proteins (PAPs) in Meat and Bone Meal by Mass Spectrometry.

The outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom in 1986, with processed animal proteins (PAPs) as the main vector of the disease, has led to their prohibition in feed. The progressive release of the feed ban required the development of new analytical methods to determine the exact origin of PAPs from meat and bone meal. We set up a promising MS-based method to determine the species and the source (legal or not) present in PAPs: a TCA-acetone protein extraction followed by a cleanup step, an in-solution tryptic digestion of 5 h (with a 1:20 protein/trypsin ratio), and mass spectrometry analyses, first without any a priori, with a Q-TOF, followed by a targeted triple-quadrupole analysis. Using this procedure, we were able to overcome some of the major limitations of the official methods to analyze PAPs, detecting and identifying prohibited animal products in feedstuffs by the monitoring of peptides specific for cows, pigs, and sheep in PAPs.

Novel approach for interlaboratory transfer of real-time PCR methods: detecting bovine meat and bone meal in feed.

The availability of robust methods for the species-specific detection of meat and bone meal (MBM) in compound feedingstuffs is an important prerequisite to enforce current and upcoming European legislation on the use of processed animal proteins in animal nutrition. Among possible methods, those based on DNA turned out to be a reliable tool for this aim, since DNA is a quite thermostable molecule able to resist severe heat treatments applied in the manufacturing of animal meals. The application of such methods by control laboratories implies that the method has been validated including an assessment of its robustness. Successful transferability between laboratories is considered an important robustness criterion of the method. However, corresponding guidelines regarding the design of such a study relevant to this field are missing. Here, we demonstrate the feasibility of an alternative concept that was applied to check for the transferability of a qualitative assay for the detection of banned MBM in feedingstuffs at trace level based on real-time PCR. The concept was based on an experimental nested design applying analysis of variance (ANOVA) that was conducted independently in two laboratories and which allows for establishing major factors influencing the result of analysis. Statistical assessment of the results confirmed the importance of the DNA extraction/purification step utilised, whereas the PCR step turned out to be a minor factor regarding the overall variability of the results. Furthermore, blind samples comprised of compound feed adulterated with MBM at 0.1% and blank compound feed were correctly classified as "positive" or "negative" samples, thus confirming fitness of purpose for the method. This approach can be of interest for other research groups working in the development of real-time PCR methods and in their use by control laboratories.

Detection of ruminant meat and bone meals in animal feed by real-time polymerase chain reaction: result of an interlaboratory study.

The commercialization of animal feeds infected by prions proved to be the main cause of transmission of bovine spongiform encephalopathy (BSE). Therefore, feed bans were enforced, initially for ruminant feeds, and later for all feeds for farmed animals. The development and validation of analytical methods for the species-specific detection of animal proteins in animal feed has been indicated in the TSE (Transmissible Spongiform Encephalopathies) Roadmap (European Commission. The TSE (Transmissible Spongiform Encephalopathy) roadmap. URL: http://europa.eu.int/comm/food/food/biosafety/bse/roadmap_en.pdf, 2005) as the main condition for lifting the extended feed ban. Methods based on polymerase chain reaction (PCR) seem to be a promising solution for this aim. The main objective of this study was to determine the applicability of four different real-time PCR methods, developed by three National expert laboratories from the European Union (EU), for the detection and identification of cattle or ruminant species in typical compound feeds, fortified with meat and bone meals (MBM) from different animal species at different concentration levels. The MBM samples utilized in this study have been treated using the sterilization condition mandatory within the European Union (steam pressure sterilization at 133 degrees C, 3 bar, and 20 min), which is an additional challenge to the PCR methods evaluated in this study. The results indicate that the three labs applying their PCR methods were able to detect 0.1% of cattle MBM, either alone or in mixtures with different materials such as fishmeal, which demonstrates the improvement made by this technique, especially when compared with results from former interlaboratory studies.

Effective PCR detection of animal species in highly processed animal byproducts and compound feeds.

In this paper we present a polymerase chain reaction (PCR)-based method for detecting meat and bone meal (MBM) in compound feedingstuffs. By choosing adequate DNA targets from an appropriate localisation in the genome, the real-time PCR method developed here proved to be robust to severe heat treatment of the MBM, showing high sensitivity in the detection of MBM. The method developed here permits the specific detection of processed pig and cattle materials treated at 134 degrees C in various feed matrices down to a limit of detection of about 0.1%. This technique has also been successfully applied to well-characterised MBM samples heated to as high as 141 degrees C, as well as to various blind feed samples with very low MBM contents. Finally, the method also passed several official European ring trials.

Discriminating animal fats and their origins: assessing the potentials of Fourier transform infrared spectroscopy, gas chromatography, immunoassay and polymerase chain reaction techniques.

The objective of the reported study was to assess the abilities of various methods to differentiate the sources of fats used in feedstuff formulations. The main target was the identification of tallow (ruminant fat) and its differentiation from non-ruminant fats. Four different techniques were compared in terms of their suitability for enforcing existing and upcoming legislation on animal by-products: (1) Fourier transform infrared spectroscopy (FT-IR) applied to fat samples, (2) gas chromatography coupled with mass spectrometry (GC-MS) to determine fatty acid profiles, (3) immunoassays focusing on the protein fraction included in the fat, and (4) polymerase chain reaction (PCR) for the detection of bovine-specific DNA. Samples of the different fats and oils as well as mixtures of these fats were probed using these analytical methods. FT-IR and GC-MS differentiated pure fat samples quite well but showed limited ability to identify the animal species or even the animal class the fat(s) belonged to; no single compound or spectral signal that could permit species identification could be found. However, immunoassays and PCR were both able to identify the species or groups of species that the fats originated from, and they were the only techniques able to identify low concentrations of tallow in a mixture of fats prepared by the rendering industry, even when the samples had been sterilised at temperatures >133 degrees C. Fats used in animal nutrition come mainly from the rendering industry, thereby confirming the suitability of PCR and immunoassays for their identification. However, neither of these latter techniques was able to detect "premier jus" tallow, representing the highest quality standard of fat with extremely low protein concentration.

Identification on commercialized products of AFLP markers able to discriminate slow- from fast-growing chicken strains.

The European chicken meat market is characterized by numerous quality marks: "Label de Qualité Wallon" in Belgium, "Label Rouge" in France, denominations of geographical origin, organic agriculture, etc. Most of those certified productions have specifications requiring the use of slow-growing chicken strains. The amplified fragment length polymorphism (AFLP) technique has been used to search molecular markers able to discriminate slow-growing chicken strains from fast-growing ones and to authenticate certified products. Two pairs of restriction enzymes (EcoRI/MseI and EcoRI/TaqI) and 121 selective primer combinations were tested on individual DNA samples from chicken products essentially in carcass form that were ascribed as belonging to either slow- or fast-growing strains. Within the resulting fingerprints, two fragments were identified as type-strains specific markers. One primer combination gives a band (333 bp) that is specific for slow-growing chickens, and another primer pair generates a band (372 bp) that was found to be characteristic of fast-growing chickens. The two markers were isolated, cloned, and sequenced. The effectiveness and the specificity of the two interesting determinants were assessed on individuals of two well-known strains (ISA 657 and Cobb 500) and on commercialized products coming from various origins.