Replacing Cereal with Ultraprocessed Foods in Pig Diets Does Not Adverse Gut Microbiota, L-glutamate Uptake, or Serum Insulin.

BACKGROUND: Using ultraprocessed food (UPF) to replace traditional feed ingredients offers a promising strategy for enhancing food production sustainability. OBJECTIVE: To analyze the impact of salty and sugary UPF on gut microbiota, amino acids uptake, and serum analytes in growing and finishing pig. METHODS: Thirty-six Swiss Large White male castrated pigs were assigned to 3 experimental diets: 1) standard (ST), 0% UPF; 2) 30% conventional ingredients replaced by sugary (SU) UPF; and 3) 30% conventional ingredients replaced by salty (SA) UPF. The next-generation sequencing was used to characterize the fecal microbiota. Transepithelial electrical resistance and the active uptake of selected amino acids in pig jejuna were also evaluated. Data were enriched with measurements of fecal volatile fatty acids and serum urea, minerals, and insulin. All data analyses were run in R v4.0.3. The packages phyloseq, vegan, microbiome, and microbiomeutilities were used for microbiota data analysis. The remaining data were analyzed by analysis of variance using linear mixed-effects regression models. RESULTS: The UPF did not affect fecal microbiota abundance or biodiversity. The Firmicutes to Bacteroidetes ratio remained unaffected. SU-induced increase in the Anaerostipes genus suggested altered glucose metabolism, whereas SA increased the abundance of CAG-352 and p-2534-18B. No effects on fecal volatile fatty acids were observed. Assumptions of UPF negatively affecting small intestinal physiology were not supported by the measurements of transepithelial electrical resistance in pigs. Active amino acids uptake tests showed potential decrease in L-glutamate absorption in the SA compared with the SU diet. Blood serum analysis indicated no adverse effects on urea, calcium, magnesium, or potassium concentration but the SU group resulted in a lower blood serum insulin concentration at the time of blood collection. CONCLUSIONS: When incorporated at 30% into a standard growing finishing diet for pigs, UPF does not have detrimental effects on gut microbiota, intestinal integrity, and blood mineral homeostasis.

Gallic acid affects intestinal-epithelial-cell integrity and selected amino-acid uptake in porcine in vitro and ex vivo permeability models.

Gallic acid (GA) is widely used as a dietary supplement due to several health-promoting effects, although its effects on intestinal-epithelial-cell integrity and transport remain mostly unknown. The present study aims to clarify the effects of GA on tight junctions and intestinal nutrient uptake through in vitro and ex vivo models. Both intestinal porcine enterocyte cell line-J2 cells and porcine middle-jejunum segments were treated with 5 (T5), 25 (T25) and 50 (T50) µm GA and mounted in Ussing chambers to determine transepithelial resistance (TEER), claudin-1 (CLDN1), occludin (OCLN), zonula occludens-1 (ZO-1) protein (in tissues and cells) and mRNA (in cells) expression. In addition, uptake of l-glutamate (l-Glut), l-arginine (l-Arg), l-lysine (l-Lys) and l-methionine (l-Meth) together with cationic-amino-acid transporter-1 (CAT-1) and excitatory-amino-acid transporter-3 (EAAT3) expression was evaluated. No apoptosis was observed in GA-treated cells, but TEER and CLDN1 protein abundance was lower with T50 compared with untreated cells. l-Arg and l-Lys uptake was greater with T5 than with T25 and T50. Ex vivo, T50 decreased the TEER values and the protein levels of CLDN1, OCLN and ZO-1, whereas T5 and T25 only decreased CLDN1 protein expression compared with untreated tissues. Moreover, T25 increased l-Glut and l-Arg uptake, the latter confirmed by an increased protein expression of CAT-1. GA influences intestinal uptake of the tested cationic amino acids at low concentrations and decreases the intestinal-cell barrier function at high concentrations. Similarities were observed between in vitro and ex vivo, but different treatment times and structures must be considered.

Preliminary investigation on the impact of salty and sugary former foods on pig liver and plasma profiles using OMICS approaches.

Replacing cereals with food leftovers could reduce feed-food competition and keep nutrients and energy in the food chain. Former food products (FFPs) are industrial food leftovers no more intended for human but still suitable as alternative and sustainable feedstuffs for monogastric. In this study, omics approaches were applied to evaluate the impact of dietary FFPs on pig liver proteome and plasma peptidome. Thirty-six Swiss Large White male castrated pigs were randomly assigned to three dietary treatments [control (CTR), 30% CTR replaced with salty FFP (SA), 30% CTR replaced with sugary FFP (SU)] from the start of the growing phase (22.4 ± 1.7 kg) until slaughtering (110 ± 3 kg). The low number of differentially regulated proteins in each comparison matrix (SA/SU vs. CTR) and the lack of metabolic interaction indicated a marginal impact on hepatic lipid metabolism. The plasma peptidomics investigation showed low variability between the peptidome of the three dietary groups and identified three possible bioactive peptides in the SA group associated with anti-hypertension and vascular homeostasis regulation. To conclude, the limited modulation of liver proteome and plasma peptidome by the SA and SU diets strenghtened the idea of reusing FFPs as feed ingredients to make pig production more sustainable.

Sustainable pig diets: partial grain replacement with former food products and its impact on meat quality.

This study investigated the effects of salty and sugary former foodstuff products (FFPs) on the quality traits and meat composition of 36 male castrated pigs (Swiss Large White breed) as well as sensory characteristics of the loins. The animals were fed three different diets for both the growing (G) and finishing (F) phases: (1) a standard diet (ST), 0% FFPs; (2) a diet with 30% of sugary FFPs (e.g., chocolate, biscuits, cakes) as a replacement for traditional ingredients (SU); and (3) a diet with 30% of salty FFPs (e.g., bread, pasta, and breadsticks) as a replacement for traditional ingredients (SA). For a comprehensive assessment of meat quality, protein and fat content in the LD were analyzed. AA and FA profile were determined both in the LD and backfat. Meat quality traits such as pH and temperature, thawing, cooking and drip losses, and shear force have been evaluated. Then, pork loins have been assessed for sensory attributes by a trained sensory panel. The SA diet decreased 20:5 n-3 levels (P < 0.001) in the muscle and 22:5 n-3 levels (P < 0.05) in both muscle and backfat but increased (P < 0.05) the ratio of mono-unsaturated to saturated fatty acids compared to the ST group. Both the SU and SA diets elevated (P < 0.001) the n-6:n-3 fatty acids ratio compared to the ST diet. Dietary treatments did not affect other meat quality traits. Regarding sensory attributes, the loin from pigs fed with SU and SA diets were sweeter (P < 0.001). Loins of SA pigs were more tender (P < 0.001), had a more intense pork aroma (P < 0.001) and had more flavor (P < 0.01) compared to ST loins. Overall, the use of FFPs affected the fatty acid profile of pork while improving the sensory quality of the loins, with no negative effects observed on the technological and nutritional quality of the meat.

Pigs are ideal species to convert food losses, also named former food products (FFPs), into animal proteins. The present study investigated the impact of incorporating sugary and salty FFPs into the diets of growing and finishing pigs on meat quality and its sensory characteristics. Our study showed that, while technological meat quality remained largely unaffected, the dietary treatments led to slight alterations of meat and backfat fatty acids profile. Moreover, a panel test for sensory analyses revealed that loins from both the sugary and salty ingredients-fed pigs were perceived as sweeter, and loins from pigs fed salty ingredients were noted for increased tenderness, intense pork aroma, and flavor. Overall, FFPs inclusion into pig diets had no detrimental effects on technological or nutritional aspects of the meat, confirming their potential use as alternative animal feed.

Packaging contaminants in former food products: Using Fourier Transform Infrared Spectroscopy to identify the remnants and the associated risks.

Food waste and feed-food competition can be reduced by replacing traditional feed ingredients such as cereals, with former food products (FFPs) in livestock diets. These foodstuffs, initially intended for human consumption, are recovered, mechanically unpacked, and then ground. Despite this simple and inexpensive treatment, packaging contaminants (remnants) are often unavoidable in the final product. To maximize the exploitation of FFPs and to minimize the associated risks, packaging remnants need to be quantified and characterized. This study tested the efficacy of the Fourier Transform Infrared Spectroscopy coupled with an optical microscope (µFT-IR) in identifying packaging remnants in 17 FFP samples collected in different geographical areas. After a visual sorting procedure, presumed packaging remnants were analyzed by µFT-IR. The results showed significant differences (p < 0.05) between the FFPs in terms of the total number of foreign particles found (plastics, cellulose and aluminum remnants, ranging from 4 to 19 particles per 20 g fresh matter), and also regarding the number of cellulose and aluminum particles. These data clearly demonstrate the need for sensitive instruments that can characterize the potential contaminants in the FFPs. This would then help to reduce the overestimation of undesirable contaminants typical of simple visual sorting, which is currently the most common method.

Lupinus albus γ-Conglutin: New Findings about Its Action at the Intestinal Barrier and a Critical Analysis of the State of the Art on Its Postprandial Glycaemic Regulating Activity.

γ-Conglutin (γ-C) is the glycoprotein from the edible seed L. albus, studied for long time for its postprandial glycaemic regulating action. It still lacks clear information on what could happen at the meeting point between the protein and the organism: the intestinal barrier. We compared an in vitro system involving Caco-2 and IPEC-J2 cells with an ex vivo system using pig ileum and jejunum segments to study γ-C transport from the apical to the basolateral compartment, and its effects on the D-glucose uptake and glucose transporters protein expression. Finally, we studied its potential in modulating glucose metabolism by assessing the possible inhibition of α-amylase and α-glucosidase. RP-HPLC analyses showed that γ-C may be transported to the basolateral side in the in vitro system but not in the pig intestines. γ-C was also able to promote a decrease in glucose uptake in both cells and jejunum independently from the expression of the SGLT1 and GLUT2 transporters.

A Single Dose of Synbiotics and Vitamins at Birth Affects Piglet Microbiota before Weaning and Modifies Post-Weaning Performance.

Early-life microbial colonization is an important driver for the development and maturation of the gut. The present study aimed to determine whether a single-dose supplement given only at birth would improve piglet performance and modify their fecal microbiota during the suckling and post-weaning periods. At birth, piglets from eight litters received a supplement (SUP+) while piglets from six other litters received water (SUP-). All piglets were monitored until two weeks post-weaning, and fecal samples were collected on Day 16 of age and two weeks post-weaning (Day 39 ± 1). The supplementation resulted in an improvement of average daily gain during the whole experimental period, mainly due to a better growth and a reduction in the incidence of diarrhea in the post-weaning period. There were no differences in the abundance and diversity of the main taxa, although the supplementation increased the relative abundance of rare taxa, such as bacteria from the Saccharibacteria and Cyanobacteria phyla, and the Lentisphaeria class in the suckling period. In addition, at 16 days of age, SUP+ piglets had a more diverse core microbiota, with bacteria from the Lactobacillus genus being present in the core microbiota of SUP+ piglets and absent from SUP- piglets. Therefore, the enhanced growth performance and reduction in diarrhea seem to be related to changes in fecal microbiota during the suckling period rather than at two weeks post-weaning.

The Contribution of Dietary Magnesium in Farm Animals and Human Nutrition.

Magnesium (Mg) is a mineral that plays an essential role as cofactor of more than 300 enzymes. Mg in farm animals' and human nutrition is recommended to avoid Mg deficiency, ensure adequate growth and health maintenance. Mg supplementation above the estimated minimum requirements is the best practice to improve farm animals' performances (fertility and yield) and food products' quality, since the performance of farm animals has grown in recent decades. Mg supplementation in pigs increases meat quality and sows' fertility; in poultry, it helps to avoid deficiency-related health conditions and to improve meat quality and egg production by laying hens; in dairy cows, it serves to avoid grass tetany and milk fever, two conditions related to hypomagnesaemia, and to support their growth. Thus, Mg supplementation increases food products' quality and prevents Mg deficiency in farm animals, ensuring an adequate Mg content in animal-source food. These latter are excellent Mg sources in human diets. Sub-optimal Mg intake by humans has several implications in bone development, muscle function, and health maintenance. This review summarizes the main knowledge about Mg in farm animals and in human nutrition.

Sweet vs. Salty Former Food Products in Post-Weaning Piglets: Effects on Growth, Apparent Total Tract Digestibility and Blood Metabolites.

Former food products (FFPs) have a great potential to replace conventional feed ingredients. This study aimed to investigate the possibility to partially replace standard ingredients with two different types of FFPs: bakery (FFPs-B) or confectionary (FFPs-C) FFPs and their effects on growth performances, feed digestibility and metabolic status in post-weaning piglets. Thirty-six post-weaning piglets were randomly assigned to three experimental diets (n = 12 per diet) for 42 days: a standard diet (CTR), a diet where 30% of standard ingredients were replaced by confectionary FFPs (FFPs-C) and a diet where 30% of standard ingredients were replaced by bakery FFPs (FFPs-B). Individual body weight and fecal dry matter were measured weekly. Feed intake (FI) was determined daily. Average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated. Fecal samples were collected daily for three days/week to determine apparent total tract digestibility of dry matter (ATTD). At day 0, 21 and 42, blood samples were collected from all the piglets. No significant differences (p > 0.05) between groups were found in growth performances and metabolic profile. However, ATTD in FFPs-B group was lower (p < 0.05) compared to the CTR group at the end of the experiment. This study confirmed the possibility to formulate homogeneous diets integrated with 30% of both categories of FFPs. Further investigations are needed to clarify the effects of bakery former food products on the digestibility of the diet.

Comparative Proteomics of Milk Fat Globule Membrane (MFGM) Proteome across Species and Lactation Stages and the Potentials of MFGM Fractions in Infant Formula Preparation.

Milk is a lipid-in-water emulsion with a primary role in the nutrition of newborns. Milk fat globules (MFGs) are a mixture of proteins and lipids with nutraceutical properties related to the milk fat globule membrane (MFGM), which protects them, thus preventing their coalescence. Human and bovine MFGM proteomes have been extensively characterized in terms of their formation, maturation, and composition. Here, we review the most recent comparative proteomic analyses of MFGM proteome, above all from humans and bovines, but also from other species. The major MFGM proteins are found in all the MFGM proteomes of the different species, although there are variations in protein expression levels and molecular functions across species and lactation stages. Given the similarities between the human and bovine MFGM and the bioactive properties of MFGM components, several attempts have been made to supplement infant formulas (IFs), mainly with polar lipid fractions of bovine MFGM and to a lesser extent with protein fractions. The aim is thus to narrow the gap between human breast milk and cow-based IFs. Despite the few attempts made to date, supplementation with MFGM proteins seems promising as MFGM lipid supplementation. A deeper understanding of MFGM proteomes should lead to better results.

Multivariate image analysis for the rapid detection of residues from packaging remnants in former foodstuff products (FFPs) - a feasibility study.

From a circular economy perspective, feeding livestock with food leftovers or former foodstuff products (FFPs) could be an effective option aimed at exploiting food leftover resources and reducing food losses. FFPs are valuable energy sources, characterised by a beneficial starch/sugar content, and also fats. However, besides these nutritional aspects, safety is a key concern given that FFPs are generally derived from packaged food. Packaging materials, such as plastics and paper, are not accepted as a feed ingredient which means that residues should be rigorously avoided. A sensitive and objective detection method is thus essential for an accurate risk evaluation throughout the former food production chain. To this end, former food samples were collected in processing plants of two different European countries and subjected to multivariate analysis of red, green, and blue (RGB) microscopic images, in order to evaluate the possible application of this non-destructive technique for the rapid detection of residual particles from packaging materials. Multivariate Image Analysis (MIA) was performed on single images at the pixel level, which essentially consisted in an exploratory analysis of the image data by means of Principal Component Analysis, which highlighted the differences between packaging and foodstuff particles, based on their colour. The whole dataset of images was then analysed by means of a multivariate data dimensionality reduction method known as the colourgrams approach, which identified clusters of images sharing similar features and also highlighted outlier images due to the presence of packaging particles. The results obtained in this feasibility study demonstrated that MIA is a promising tool for a rapid automated method for detecting particles of packaging materials in FFPs.

Potentials and Challenges of Former Food Products (Food Leftover) as Alternative Feed Ingredients.

Former food products (FFPs) are foodstuffs that, even though they are nutritious and safe, have lost their value on the human consumption market for different reasons, such as production errors leading to broken or intermediate foodstuffs, surpluses caused by logistical challenges of daily delivery, or any other reason. The nutritional features of FFPs include carbohydrates, free sugars, and possibly also fats. FFPs tend to have been processed through various technological and heat treatments that impact the nutrients and the kinetics of digestion, as well as animal response and, particularly, gastro-intestinal health. This review integrates some of the most recently published works about the chemical composition, nutritional value, digestibility and glycaemic index of ex-foods. In addition, a view on the relationship between the use of FFPs and safety issues and their effects on pigs' intestinal microbiota are also given.

Punicalagin increases glutamate absorption in differentiated Caco-2 cells by a mechanism involving gene expression regulation of an EAAT3 transporter.

This study investigates, for the first time, the ability of punicalagin to modulate intestinal glutamate uptake by upregulation of the expression of one of its transporters present on the enterocyte membrane. The use of an Ussing chamber revealed an increase in glutamate transport in differentiated Caco-2 cells after punicalagin treatment for 24 h. This cell line constitutively expresses two glutamate transporters: EAAT1 and EAAT3. In response to punicalagin, the expression of EAAT3 was increased, at both mRNA and protein levels, but not that of EAAT1. Transfection with EAAT3-targeting siRNA specifically altered basal and induced EAAT3 gene expression, decreasing the positive effect of punicalagin on glutamate uptake. These data confirmed the involvement of EAAT3 in increasing glutamate uptake by enterocytes after punicalagin treatment.

Influence of Traditional vs Alternative Dietary Carbohydrates Sources on the Large Intestinal Microbiota in Post-Weaning Piglets.

In this study, common cereal grains were partially replaced by former foodstuffs products (FFPs) in post-weaning piglets' diets, to investigate how these alternative ingredients influence the faecal microbiota in the post-weaning period. Twelve post-weaning piglets were housed for 16 days in individual pens and were then fed two diets: a standard wheat-barley-corn meal diet and a diet containing 30% FFPs, thus partially substituting conventional cereals. The growth performance was monitored and faecal microbiota was characterized by the next generation sequencing of the 16S rRNA gene. The results showed no detrimental effects on growth performance when FFPs were used. However, the FFP diet decreased the bacterial richness and evenness in the large intestine, while minor differences were observed in the taxa composition. The core microbiota composition was only slightly affected, and no differences between the two groups in the gut microbiota composition at the phylum level over time were observed. Thus, although these results should be interpreted with caution, as they are case-specific, FFPs can be potentially used as alternative carbohydrate sources in post-weaning piglets, but further investigations are necessary to clarify their impact on gut health when used for a longer period.

Effect of Supplementing Hydrolysable Tannins to a Grower-Finisher Diet Containing Divergent PUFA Levels on Growth Performance, Boar Taint Levels in Back Fat and Intestinal Microbiota of Entire Males.

A retrospective data analysis suggested that the levels of boar taint compounds depend on the polyunsaturated fatty acid (PUFA) level of the adipose tissue (AT) being significantly greater in the unsaturated AT. In addition, we recently reported that hydrolysable tannins (HTs) offered to entire males (EMs) reduce skatole and, to a greater extent, indole levels in the AT. Thus, the objective of the study was to determine the impact of HTs and a high dietary level of PUFA on growth performance and board taint compounds in EMs. In addition, the interaction between PUFA and HTs on gut microbiota and its link to intestinal skatole and indole production was investigated. At 25 kg BW, 44 EM originating from 11 litters were randomly assigned within litter to four dietary treatments. Two basal grower (25-60 kg BW) and finisher (60-105 kg BW) diets containing either 2% soy oil (H = high PUFA level) or 2% tallow (L = low PUFA level) were formulated. The H and L diets were either supplemented (H+/L+) or not (H-/L-) with 3% chestnut extract containing 50% HTs. The pigs had ad libitum access to the diets and were slaughtered at 170 days of age. The microbiota composition was investigated through the 16S rRNA gene sequences obtained by next-generation sequencing (Illumia MiSeq platform, San Diego, CA, USA) and analyzed with a specific packages in R, version 3.5.0. Regardless of the PUFA content, the EMs fed the H+ diets were 2% (p < 0.01) less feed efficient overall. This was due to the slower (p = 0.01) growth in the finisher period despite similar feed intake. Carcass characteristics were not affected by the diets. Regardless of HT feeding, the PUFA level in the AT of the H pigs was 10% greater (p = 0.05) than in the L pigs. The indole level tended (p = 0.08) to be 50% lower in the H+ group. Surprisingly, the pigs that were fed diet H- had greater skatole levels than those fed diet L-, with intermediate skatole levels in the H+ and L+. Independent of the PUFA level, the HTs decreased bacteria abundance and qualitatively affected the microbiota composition. In conclusion, these data do not confirm that boar taint compound levels were related to PUFA levels in the AT. However, HTs can be considered to be a promising alternative to conventional antibacterial additives, with no detrimental effects on pig gut health and with appealing properties for reducing the synthesis of the main components of boar taint.

Tracing food packaging contamination: an electronic nose applied to leftover food.

The use of former food products (FFPs) as alternative feed ingredients in farm animal diets has several benefits. In fact, FFPs are a way of converting losses from the food industry into ingredients for the feed industry. FFPs are produced from packaged food and in general, they are recognised as safe. Packaging materials are not accepted as a feed ingredient according to Regulation (EC) No 767/2009, which imposes a rigorous evaluation of possible residues. A sensitive and objective detection method is therefore essential for an effective risk evaluation. Six FFP samples were subjected to electronic nose analysis. Each sample was assessed under three conditions: as-received, cleaned and experimentally-spiked. Packaging remnants were also quantified using a stereomicroscope. Data were analysed via principal component analysis using SPSS software. Although the stereomicroscopy analysis showed a low content of packaging remnants in as-received samples, the electronic nose was able to differentiate between cleaned, as-received and spiked samples. However, this method was not effective when different FFPs were analysed together. In the view of the limitations of this method, it can be concluded that the electronic nose can be considered an objective and sensitive method for the detection of packaging remnants in FFPs composed of the same matrix.

Combining E-Nose and Lateral Flow Immunoassays (LFIAs) for Rapid Occurrence/Co-Occurrence Aflatoxin and Fumonisin Detection in Maize.

The aim of this study was to evaluate the potential use of an e-nose in combination with lateral flow immunoassays for rapid aflatoxin and fumonisin occurrence/co-occurrence detection in maize samples. For this purpose, 161 samples of corn have been used. Below the regulatory limits, single-contaminated, and co-contaminated samples were classified according to the detection ranges established for commercial lateral flow immunoassays (LFIAs) for mycotoxin determination. Correspondence between methods was evaluated by discriminant function analysis (DFA) procedures using IBM SPSS Statistics 22. Stepwise variable selection was done to select the e-nose sensors for classifying samples by DFA. The overall leave-out-one cross-validated percentage of samples correctly classified by the eight-variate DFA model for aflatoxin was 81%. The overall leave-out-one cross-validated percentage of samples correctly classified by the seven-variate DFA model for fumonisin was 85%. The overall leave-out-one cross-validated percentage of samples correctly classified by the nine-variate DFA model for the three classes of contamination (below the regulatory limits, single-contaminated, co-contaminated) was 65%. Therefore, even though an exhaustive evaluation will require a larger dataset to perform a validation procedure, an electronic nose (e-nose) seems to be a promising rapid/screening method to detect contamination by aflatoxin, fumonisin, or both in maize kernel stocks.

Gravimetric quantitative determination of packaging residues in feed from former food.

Valorisation of former foodstuff products (FFP) in feed is part of a long-term strategy for sustainability. An approach to valorise FFP outside the waste value chain is their use as an alternative source of feed materials, with a subsequent optimisation of the environmental impact of products. In the current practice of food production, food packaging is provided to ensure the maintenance of food quality and safety during transport and storage. One of the problems of reusing FFP is how to deal with packaging materials or remains that can become residues in the feed. The aim of this study is to propose a fast and sensitive gravimetric method, fit for routine official controls, for the determination of packaging residues in feed. The developed method can briefly be summarised as: (1) visual selection of the undesired ingredients which can be identified as remnants of packaging materials; (2) weighing of the selected materials; (3) defatting; (4) dehydration; (5) final weighing; and (6) reporting of weight and percentage. Moreover, the method has been validated through the determination of some of the parameters listed in Council Regulation 2004/882/EC (i.e., specificity, limit of quantification (LOQ), recovery, repeatability, within-laboratory reproducibility and measurement uncertainty).

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.