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.

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.

Collaborative study on the effect of grinding on the detection of bones from processed animal proteins in feed by light microscopy.

Bone fragments are essential structures for the detection of processed animal proteins (PAPs) in feed by light microscopy for official controls according to Annex VI of European Union Regulation EC/152/2009. The preparation of samples submitted for analysis requires a grinding step to make them suitable for microscopic slide preparation and observation. However, there are no technical guidelines set down for this step despite the fact that it can lead to an increase in bone numbers due to fragmentation. This was demonstrated by an in-house study carried out by the Irish National Reference Laboratory (NRL) for animal protein detection. The present collaborative study investigated the possible effects of three different grinding conditions on the final result for a feed adulterated with 0.05 and 0.01% (w/w) of PAP. The microscopic analysis either combined or not with an Alizarin Red staining was carried out by 10 different laboratories. The results demonstrated that although a large variation in the numbers of bone fragments was noted, five of the six different grinding/staining combinations applied at two levels of PAP adulteration did not significantly (at p = 0.05) differ from one another. The only exception occurred when grinding the feed containing 0.05% of PAP with a rotor mill equipped with a 0.5-mm sieve and combined with a staining which resulted in a greater number of bone fragments by forced fragmentation. Overall, the impact of the grinding/staining combinations on the final results was shown to be negligible when considering the regulatory limit of detection (LOD) requirement for the method and the current rules of implementation of the light microscopic method. From a total of 180 analyses carried out on the feed matrix containing 0.05% of PAP no false-negative result was observed, and at a level of 0.01% PAP only 10 false-negative results occurred.

Light microscopy with differential staining techniques for the characterisation and discrimination of insects versus marine arthropods processed animal proteins.

The aim of this study was to evaluate the use of light microscopy with differential staining techniques for the discrimination of insect material from marine arthropods - classified as fishmeal. Specifically, three samples of single-species insect material, Hermetia illucens (HI), Bombyx mori (BM) and Tenebrio molitor (TM), and two samples of marine arthropods, shrimp material and krill, were analysed and compared after staining by two reagents to enhance fragment identification. Alizarin Red (AR) and Chlorazol Black (CB), which react respectively with calcium salts and chitin, were tested for their potential efficacy in distinguishing between insect and marine materials. Results indicated that AR failed to stain HI, BM and TM materials. By contrast, the three insect species materials tested were stained by CB. When shrimp fragments and krill were considered, AR and CB stained marine materials reddish-pink and light blue to black, respectively. By combining these results, it can be suggested that CB staining may efficiently be used to mark insect materials; AR does stain shrimp fragments but does not stain the tested insect material, indicating a possible approach for discriminating between insects and marine arthropods. However, since the present study was performed on pure materials and a small set of samples, possible implementation of this technique still needs to be confirmed in complex matrices such as compound feed.

Specific localization and measurement of hydrogen peroxide in Arabidopsis thaliana cell suspensions and protoplasts elicited by COS-OGA.

H2O2 acts as an important signaling molecule during plant/pathogen interactions but its study remains a challenge due to the current shortcomings in H2O2-responsive probes. In this work, ContPY1, a new molecular probe developed to specifically detect H2O2 was used to study the elicitation of Arabidopsis thaliana cells by a complex of chitosan oligomers (COS) and oligogalacturonides (OGA). The comparison of cell suspensions, protoplasts of cell suspensions and leaf protoplasts treated with different inhibitors gave indications on the potential sources of hydrogen peroxide in plant cells. The relative contribution of the cell wall, of membrane dehydrogenases and of peroxidases depended on cell type and treatment and proved to be variable. Our present protocol can be used to study hydrogen peroxide production in a large variety of plant species by simple protocol adaptation.

Validation of the boronate sensor ContPY1 as a specific probe for fluorescent detection of hydrogen peroxide in plants.

Studying the implication of hydrogen peroxide in biological processes in plants remains a challenge due to the current shortcomings of H2O2-responsive probes. The use of ContPY1, a new fluorescent probe, which is highly selective and sensitive for H2O2, was investigated. To validate the use of ContPY1 on plants, we have generated protocols employing cells suspensions and leaves, and measured specifically H2O2 production by plants using spectrofluorometry and microscopy.

Interaction network of antimicrobial peptides of Arabidopsis thaliana, based on high-throughput yeast two-hybrid screening.

One mechanism used by plants to respond to infection is the production of antimicrobial peptides (AMPs). In addition to a role in defence, AMPs seem to have other biological functions. Furthermore, the number of cysteine-rich AMP-like peptides appears to have been underpredicted in plant genomes. Such peptides could be involved in plant defence and/or in other biological processes. Here we generated an interaction network between 15 AMPs/AMP-like peptides and ca. 8000 other Arabidopsis thaliana proteins (AtORFeome2.0) and found 53 putative novel interactions. These interactions involve five transcription factors, a subunit of the COP9 signalosome, a heat shock protein, a MAP kinase kinase, a thioredoxin and 4 uncharacterized proteins.

An overview of the legislation and light microscopy for detection of processed animal proteins in feeds.

From the first cases of bovine spongiform encephalopathy (BSE) among cattle in the United Kingdom in 1986, the route of infection of BSE is generally believed by means of feeds containing low level of processed animal proteins (PAPs). Therefore, many feed bans and alternative and complementary techniques were resulted for the BSE safeguards in the world. Now the feed bans are expected to develop into a "species to species" ban, which requires the corresponding species-specific identification methods. Currently, banned PAPs can be detected by various methods as light microscopy, polymerase chain reaction, enzyme-linked immunosorbent assay, near infrared spectroscopy, and near infrared microscopy. Light microscopy as described in the recent Commission Regulation EC/152/2009 is the only official method for the detection and characterization of PAPs in feed in the European Union. It is able to detect the presence of constituents of animal origin in feed at the level of 1 g/kg with hardly any false negative. Nevertheless, light microscopy has the limitation of lack of species specificity. This article presents a review of legislations on the use of PAPs in feedstuff, the detection details of animal proteins by light microscopy, and also presents and discusses the analysis procedure and expected development of the technique.