E-Nose Technology for Mycotoxin Detection in Feed: Ready for a Real Context in Field Application or Still an Emerging Technology?

Mycotoxin risk in the feed supply chain poses a concern to animal and human health, economy, and international trade of agri-food commodities. Mycotoxin contamination in feed and food is unavoidable and unpredictable. Therefore, monitoring and control are the critical points. Effective and rapid methods for mycotoxin detection, at the levels set by the regulations, are needed for an efficient mycotoxin management. This review provides an overview of the use of the electronic nose (e-nose) as an effective tool for rapid mycotoxin detection and management of the mycotoxin risk at feed business level. E-nose has a high discrimination accuracy between non-contaminated and single-mycotoxin-contaminated grain. However, the predictive accuracy of e-nose is still limited and unsuitable for in-field application, where mycotoxin co-contamination occurs. Further research needs to be focused on the sensor materials, data analysis, pattern recognition systems, and a better understanding of the needs of the feed industry for a safety and quality management of the feed supply chain. A universal e-nose for mycotoxin detection is not realistic; a unique e-nose must be designed for each specific application. Robust and suitable e-nose method and advancements in signal processing algorithms must be validated for specific needs.

Cold-Pressed Oilseed Cakes as Alternative and Sustainable Feed Ingredients: A Review.

Due to the increasing demand for alternative protein feed ingredients, the utilization of oilseed by-products in animal nutrition has been sought as a promising solution to ensure cheap and environmentally sustainable feedstuffs. This review aimed to summarize the nutritional value of six cold-pressed cakes (rapeseed, hempseed, linseed, sunflower seed, camelina seed, and pumpkin seed) and the effects of their inclusion in diet for ruminant, pig, and poultry on nutrient digestibility, growth and productive performance, and quality of the products. The presented results indicated that these unconventional feed ingredients are a good protein and lipid source and have a balanced amino acid and fatty acid profile. However, contradictory results of animal production performances can be found in the literature depending on the cake type and chemical composition, dietary inclusion level, animal category, and trial duration. Due to the substantial amount of essential fatty acid, these cakes can be efficiently used in the production of animal products rich in n-3 and n-6 polyunsaturated fatty acids. However, the utilization of cakes in pig and poultry nutrition is limited because of the presence of antinutritive factors that can deteriorate feed intake and nutrient utilization.

An Eight-Year Survey on Aflatoxin B1 Indicates High Feed Safety in Animal Feed and Forages in Northern Italy.

Aflatoxins (AFs) remain the main concern for the agricultural and dairy industries due to their effects on the performances and quality of livestock production. Aflatoxins are always unavoidable and should be monitored. The objective of this paper is to bring to light a significant volume of data on AF contamination in several animal feed ingredients in Northern Italy. The Regional Breeders Association of Lombardy has been conducting a survey program to monitor mycotoxin contamination in animal feeds, and in this paper, we present data relating to AFB1 contamination. In most cases (95%), the concentrations were low enough to ensure compliance with the European Union's (EU's) maximum admitted levels for animal feed ingredients. However, the data show a high variability in AF contamination between different matrices and, within the same matrix, a high variability year over year. High levels of AFs were detected in maize and cotton, especially in the central part of the second decade of this century, i.e., 2015-2018, which has shown a higher risk of AF contamination in feed materials in Northern Italy. Variability due to climate change and the international commodity market affect future prospects to predict the presence of AFs. Supplier monitoring and control and reduced buying of contaminated raw materials, as well as performing analyses of each batch, help reduce AF spread.

Integrated Mycotoxin Management System in the Feed Supply Chain: Innovative Approaches.

Exposure to mycotoxins is a worldwide concern as their occurrence is unavoidable and varies among geographical regions. Mycotoxins can affect the performance and quality of livestock production and act as carriers putting human health at risk. Feed can be contaminated by various fungal species, and mycotoxins co-occurrence, and modified and emerging mycotoxins are at the centre of modern mycotoxin research. Preventing mould and mycotoxin contamination is almost impossible; it is necessary for producers to implement a comprehensive mycotoxin management program to moderate these risks along the animal feed supply chain in an HACCP perspective. The objective of this paper is to suggest an innovative integrated system for handling mycotoxins in the feed chain, with an emphasis on novel strategies for mycotoxin control. Specific and selected technologies, such as nanotechnologies, and management protocols are reported as promising and sustainable options for implementing mycotoxins control, prevention, and management. Further research should be concentrated on methods to determine multi-contaminated samples, and emerging and modified mycotoxins.

Soybean Molasses in Animal Nutrition.

Concerning the increasing global demand for food and accumulation of huge amounts of biomass waste from the agro-food industry whose manipulation is usually inadequate, the potential of livestock to convert by-products as alternative feed ingredients into valuable proteins has been proposed as an outstanding option. Soybean molasses present a by-product of soybean protein concentrate production with low commercial cost but high nutritive and functional value. It is a rich source of soluble carbohydrates in the form of sugars and soybean phytochemicals. Therefore, this paper provides a review of published works about the production of soybean molasses, chemical composition, and nutritive value. In addition, the possibility of the application of soybean molasses in animal nutrition as a pelleting aid and functional feed ingredient is also discussed. Special attention is devoted to the influence of the inclusion of soybean molasses in the diets for ruminants, non-ruminants, and aquaculture on animal performance and health.

Reduce, Reuse, Recycle for Food Waste: A Second Life for Fresh-Cut Leafy Salad Crops in Animal Diets.

The world's population is growing rapidly, which means that the environmental impact of food production needs to be reduced and that food should be considered as something precious and not wasted. Moreover, an urgent challenge facing the planet is the competition between the food produced for humans and the feed for animals. There are various solutions such as the use of plant/vegetable by-products (PBPs) and former foodstuffs, which are the co/by-products of processing industries, or the food losses generated by the food production chain for human consumption. This paper reviews the by-co-products derived from the transformation of fresh-cut leafy salad crops. A preliminary nutritional evaluation of these materials is thus proposed. Based on their composition and nutritional features, in some cases similar to fresh forage and grasses, this biomass seems to be a suitable feedstuff for selected farm animals, such as ruminants. In conclusion, although the present data are not exhaustive and further studies are needed to weigh up the possible advantages and disadvantages of these materials, fresh-cut leafy salad crops represent a potential unconventional feed ingredient that could help in exploiting the circular economy in livestock production, thereby improving sustainability.

Decontamination of Mycotoxin-Contaminated Feedstuffs and Compound Feed.

Mycotoxins are known worldwide as fungus-produced toxins that adulterate a wide heterogeneity of raw feed ingredients and final products. Consumption of mycotoxins-contaminated feed causes a plethora of harmful responses from acute toxicity to many persistent health disorders with lethal outcomes; such as mycotoxicosis when ingested by animals. Therefore, the main task for feed producers is to minimize the concentration of mycotoxin by applying different strategies aimed at minimizing the risk of mycotoxin effects on animals and human health. Once mycotoxins enter the production chain it is hard to eliminate or inactivate them. This paper examines the most recent findings on different processes and strategies for the reduction of toxicity of mycotoxins in animals. The review gives detailed information about the decontamination approaches to mitigate mycotoxin contamination of feedstuffs and compound feed, which could be implemented in practice.

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.

Invited review: Dairy proteins and bioactive peptides: Modeling digestion and the intestinal barrier.

Dairy products are one of the most important sources of biologically active proteins and peptides. The health-promoting functions of these peptides are related to their primary structure, which depends on the parent protein composition. A crucial issue in this field is the demonstration of a cause-effect relationship from the ingested protein form to the bioactive form in vivo. Intervention studies represent the gold standard in nutritional research; however, attention has increasingly been focused on the development of sophisticated in vitro models of digestion to elucidate the mechanism of action of dairy nutrients in a mechanistic way and significantly reduce the number of in vivo trials. On the other hand, the epithelial intestinal barrier is the first gate that actively interacts with digestion metabolites, making the intestinal cells the first target tissue of dairy nutrients and respective metabolites. An evolution of the in vitro digestion approach in the study of dairy proteins and derived bioactive compounds is the setup of combined in vitro digestion and cell culture models taking into consideration the endpoint to measure the target organism (e.g., animal, human) and the key concepts of bioaccessibility, bioavailability, and bioactivity. This review discusses the relevance and challenges of modeling digestion and the intestinal barrier, focusing on the implications for the modeling of dairy protein digestion for bioactivity evaluation.

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.

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.

Mycotoxin Contamination in the EU Feed Supply Chain: A Focus on Cereal Byproducts.

Mycotoxins represent a risk to the feed supply chain with an impact on economies and international trade. A high percentage of feed samples have been reported to be contaminated with more than one mycotoxin. In most cases, the concentrations were low enough to ensure compliance with the European Union (EU) guidance values or maximum admitted levels. However, mycotoxin co-contamination might still exert adverse effects on animals due to additive/synergistic interactions. Studies on the fate of mycotoxins during cereal processing, such as milling, production of ethanol fuels, and beer brewing, have shown that mycotoxins are concentrated into fractions that are commonly used as animal feed. Published data show a high variability in mycotoxin repartitioning, mainly due to the type of mycotoxins, the level and extent of fungal contamination, and a failure to understand the complexity of food processing technologies. Precise knowledge of mycotoxin repartitioning during technological processes is critical and may provide a sound technical basis for feed managers to conform to legislation requirements and reduce the risk of severe adverse market and trade repercussions. Regular, economical and straightforward feed testing is critical to reach a quick and accurate diagnosis of feed quality. The use of rapid methods represents a future challenge.

State of the art in feedstuff analysis: a technique-oriented perspective.

The need for global feed supply traceability, the high-throughput testing demands of feed industry, and regulatory enforcement drive the need for feed analysis and make extremely complex the issue of the control and evaluation of feed quality, safety, and functional properties, all of which contribute to the very high number of analyses that must be performed. Feed analysis, with respect to animal nutritional requirements, health, reproduction, and production, should be multianalytically approached. In addition to standard methods of chemical analysis, new methods for evaluation of feed composition and functional properties, authenticity, and safety have been developed. Requirements for new analytical methods emphasize performance, sensitivity, reliability, speed, simplified use, low cost for high volume, and routine assays. This review provides an overview of the most used and promising methods for feed analysis. The review is intentionally focused on the following techniques: classical chemical analysis; in situ and in vitro methods; analytical techniques coupled with chemometric tools (NIR and sensors); and cell-based bioassays. This review describes both the potential and limitations of each technique and discusses the challenges that need to be overcome to obtain validated and standardized methods of analysis for a complete and global feed evaluation and characterization.

Effect of Escherichia coli lipopolysaccharide on u-PA activity and u-PA and u-PAR RNA expression in a bovine mammary epithelial cell line.

It is well known that the plasminogen-activating (PA) system plays a key role in the bovine mammary gland during tissue remodelling. However, the modulation of the PA cascade after bacterial infections needs to be elucidated. This study examined the effects of Escherichia coli lipopolysaccharide (LPS) on cell viability, the modulation of cell-associated u-PA activity, and the regulation of u-PA and u-PA receptor (u-PAR) RNA expression using the BME-UV1 bovine mammary epithelial cell line. LPS did not affect cell viability, but induced an increase in u-PA activity, with the maximum response after 6 h of incubation. Moreover, u-PA and u-PAR mRNA expression were both up-regulated in BME-UV1 cells after 3 h of incubation with LPS. These data indicated that E. coli LPS led to an increase in u-PA activity and RNA expression of u-PA and u-PAR in BME-UV1 cells, thus strengthening the role of the PA system during pathological processes.

Use of the electronic nose as a screening tool for the recognition of durum wheat naturally contaminated by deoxynivalenol: a preliminary approach.

Fungal contamination and the presence of related toxins is a widespread problem. Mycotoxin contamination has prompted many countries to establish appropriate tolerance levels. For instance, with the Commission Regulation (EC) N. 1881/2006, the European Commission fixed the limits for the main mycotoxins (and other contaminants) in food. Although valid analytical methods are being developed for regulatory purposes, a need exists for alternative screening methods that can detect mould and mycotoxin contamination of cereal grains with high sample throughput. In this study, a commercial electronic nose (EN) equipped with metal-oxide-semiconductor (MOS) sensors was used in combination with a trap and the thermal desorption technique, with the adoption of Tenax TA as an adsorbent material to discriminate between durum wheat whole-grain samples naturally contaminated with deoxynivalenol (DON) and non-contaminated samples. Each wheat sample was analysed with the EN at four different desorption temperatures (i.e., 180 °C, 200 °C, 220 °C, and 240 °C) and without a desorption pre-treatment. A 20-sample and a 122-sample dataset were processed by means of principal component analysis (PCA) and classified via classification and regression trees (CART). Results, validated with two different methods, showed that it was possible to classify wheat samples into three clusters based on the DON content proposed by the European legislation: (a) non-contaminated; (b) contaminated below the limit (DON < 1,750 µg/kg); (c) contaminated above the limit (DON > 1,750 µg/kg), with a classification error rate in prediction of 0% (for the 20-sample dataset) and 3.28% (for the 122-sample dataset).

Nutrition-based health: cell-based bioassays for food antioxidant activity evaluation.

Food science has progressively evolved and now there are wide evidences that foods have biological activities that are beyond their classical nutritional value. In this field, the antioxidant activity of pure compounds, food, feed, and dietary supplements has been extensively studied and numerous analytical approaches and assay models have been developed, involving various systems from simple chemical assays to animal models and human studies. This article is an overview of different cell-based models that have been used for testing the antioxidant properties of food, feed, and dietary supplements. Advantages, drawbacks, and technical problems to develop and validate suitable, robust, and high-throughput cell-based bioassays for screening food antioxidant activity will be discussed.

Alpha-tocopherol counteracts the cytotoxicity induced by ochratoxin a in primary porcine fibroblasts.

The aims of the current study were to determine the half-lethal concentration of ochratoxin A (OTA) as well as the levels of lactate dehydrogenase release and DNA fragmentation induced by OTA in primary porcine fibroblasts, and to examine the role of α-tocopherol in counteracting its toxicity. Cells showed a dose-, time- and origin-dependent (ear vs. embryo) sensitivity to ochratoxin A. Pre-incubation for 3 h with 1 nM α-tocopherol significantly (P < 0.01) reduced OTA cytotoxicity, lactate dehydrogenase release and DNA damage in both fibroblast cultures. These findings indicate that α-tocopherol supplementation may counteract short-term OTA toxicity, supporting its defensive role in the cell membrane.

Differential expression and secretion of alpha1-acid glycoprotein in bovine milk.

alpha1-Acid glycoprotein (AGP) is a lipocalin that is produced mainly by the liver and secreted into plasma in response to infections and injuries. In this study, we evaluated AGP isoforms that can be detected in bovine milk. We found that milk-AGP content is made up of at least two isoform groups, a low MW group (44 kDa) that is produced in the mammary gland (MG-AGP), and a higher MW group (55-70 kDa), that is produced by somatic cells (SC-AGP). Identical SC-AGP isoforms can be found both in milk and blood PMN cells. Analysis of the mammary tissue cDNA showed that the sequence of the MG-AGP isoform is identical to that of plasma AGP. Each group contains several proteins with different MWs and different isoelectric points, as shown by 2D-electrophoresis. The glycosylation patterns of these isoforms were analysed by means of specific lectin binding, to evaluate the degree of sialylation, fucosylation and branching. The MG-AGP glycan pattern was identical to plasma AGP produced by the liver. Several differences were detected, however, between plasma and SC-AGP isoforms, the most evident being the strong degree of fucosylation and the elevated number of di-antennary glycans in SC-AGP. Immunohistochemistry showed that AGP is found in all tissues that make up the mammary gland, but that it is most likely produced for the main part by the alveoli.

Effects of retinoids on proliferation and plasminogen activator expression in a bovine mammary epithelial cell line.

Effects of two natural (retinol and retinoic acid, RA) and one synthetic N-(4-hydroxyphenyl) retinamide (4-HPR) retinoids on proliferation and expression of urokinase-plasminogen activator (u-PA) by bovine mammary epithelial cells were examined. The BME-UV1 established bovine mammary epithelial cell line was used as a model system. All retinoids tested (retinol, RA and 4-HPR) were effective inhibitors of cell proliferation. When cells were cultured in the absence of fetal bovine calf serum (FBCS), inhibition occurred at concentrations as low as 1 nM for all retinoids tested. The effect of retinoids on cell proliferation was not dose-related when cells were cultured in the absence of FBCS. All retinoids (retinol, RA, 4-HPR), when used in the range 1 nM-10 microM (noncytotoxic concentrations), were equally effective and had identical inhibition patterns. Inhibition of cell proliferation by RA was apparent by 6 h and was higher after 24 h in culture. In contrast, when cells were cultured in the presence of FBCS, the effect of RA and retinol on cell proliferation was dose-related. RA and retinol inhibited cell proliferation (P<0.01) when added to the culture medium in concentrations as low as 10 nM and 100 nM, respectively. 4-HPR was inhibitory (P<0.01) in concentrations as low as 1 nM. Higher concentrations of 4-HPR in the range 1 nM-1 microM had no further effect on cell proliferation. None of the retinoids tested, when added to cultures in the presence or absence of FBCS, could completely arrest cell proliferation at noncytotoxic concentrations. RA at 1 microM inhibited (P<0.05) insulin or IGF-I-induced cell proliferation but had no effect (P>0.05) on u-PA mRNA levels or u-PA activity. Furthermore, RA inhibited cell proliferation in the presence of FBCS but had no effect (P>0.05) on u-PA mRNA levels. Thus, retinoids are effective inhibitors of bovine mammary epithelial cell proliferation and this growth inhibition does not seem to correlate with any changes in u-PA mRNA or u-PA activity.