Preliminary Feed Sedimentation Step for the Sensitive and Specific Detection of Processed Animal Proteins by Mass Spectrometry-Based Proteomics.

The circular economy is one of the main building blocks of the European Green Deal. In this context, the use of former foodstuffs containing ruminant gelatin was recently authorized in nonruminant feed. This minor modification makes it more challenging, if not impossible, to interpret the analytical results of the official control for animal proteins. The presence of ruminant DNA from authorized byproducts (i.e., milk and/or gelatin) may hide the use of prohibited byproducts. The objective of this work was to evaluate the use of sedimentation to increase the sensitivity and specificity of bovine-processed animal proteins (PAPs) detection by mass spectrometry-based proteomics. Both approaches (standard versus optimized method) were evaluated by UHPLC-MS/MS on various animal feeds and samples from an interlaboratory study. The optimized method was able to achieve the adulteration level below the level of 0.1% PAPs required by the European Commission. This approach presents a simple and economical solution to improve the method without the need for new equipment or expertise since it is already in place in the control laboratories.

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.

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.

Synchronous fluorescence spectroscopy for detecting blood meal and blood products.

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 (hemoglobin 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.