European food safety research: An explorative study with funding experts' consultation.

The European research area exhibits considerable opacity and fragmentation in food safety research funding and organizational structures, impeding the exploitation of existing research potential across European countries. Given that food safety is inherently linked to the societal challenges of our time, identifying and removing existing barriers to research funding in this area is crucial. Towards investigating this matter, interviews were conducted with funding bodies from six European countries to assess key issues related to research funding in general and food safety in particular. Funding experts were then invited to a workshop to jointly discuss the challenges identified and explore strategies to address them. Evaluation of the food safety research funding situation in selected European countries revealed both convergences and significant differences among national funding bodies. Engaging with funding experts provided invaluable insights into the issues encountered with research funding, such as inadequate call management staff or insufficient research funds, culminating in a set of recommendations for action to remedy the situation.

Industrial-Scale Cleaning Solutions for the Reduction of Fusarium Toxins in Maize.

Grain cleaning is the most effective non-destructive post-harvest mitigation strategy to reduce high levels of mycotoxins on account of the removal of mold-infected grains and grain fractions with high mycotoxin content. In this study, the reduction in the concentration of some co-occurring Fusarium toxins in maize, namely deoxynivalenol (DON), zearalenone (ZEA) and fumonisins B1 and B2 (FBs), was evaluated at an industrial-scale level by mechanical removal (sieving and density separation) of dust, coarse, small, broken, shriveled and low-density kernels and/or optical sorting of defected kernels. Samples were dynamically collected according to the Commission Regulation No. 401/2006 along the entire process line. Mycotoxin analyses of water-slurry aggregate samples were performed by validated LC methods. Depending on the contamination levels in raw incoming maize, the overall reduction rates ranged from 36 to 67% for DON, from 67 to 87% for ZEA and from 27 to 67% for FBs. High levels of DON, ZEA and FBs were found in all rejected fractions with values, respectively, up to 3030%, 1510% and 2680%, compared to their content in uncleaned maize. Results showed that grain cleaning equipment based on mechanical and or optical sorting technologies can provide a significant reduction in Fusarium toxin contamination in maize.

Mycotoxin Analysis of Grain via Dust Sampling: Review, Recent Advances and the Way Forward: The Contribution of the MycoKey Project.

The sampling protocols for the official control of the levels of mycotoxins in foodstuffs are very costly and time-consuming. More efforts are needed to implement alternative sampling plans able to support official control, or to adapt the current ones. The aim of the research carried out within the European Horizon 2020 MycoKey project was to evaluate the applicability at industrial scale of the dust sampling approach to detect multiple mycotoxins in grains. To this end, two trials were performed on an EU industrial site: (i) control of the unloading of wheat from train wagons; (ii) control of the unloading of wheat from trucks. In line with previous studies, the MycoKey results indicated that dust sampling and mycotoxin analysis represent a fitness for purpose approach for non-destructive and rapid identification of wheat commodities compliant to the maximum permitted levels. Based on reviewed and newly generated results, this article discusses potential applications and limits of the dust sampling methodology, identifying future research needs.

Introduction to This Special Issue of Toxins: Application of Novel Methods for Mycotoxin Analysis.

Crop contamination by mycotoxins is a global problem that poses significant economic burdens due to the food/feed losses that are caused by reduced production rates; the resulting adverse effects on human and animal health and productivity; and the trade losses associated with the costs incurred by inspection, sampling, and analysis before and after shipments [...].

Monitoring Alternaria toxins in Italian food to support upcoming regulation.

The collection of occurrence data on Alternaria toxins in food and feed across the European countries is required since 2012 by the European Commission, endorsing the relevant scientific opinion by the EFSA CONTAM Panel. Within this framework, occurrence data for Alternaria toxins (Alternariol, Alternariol monomethyl ether, Tenuazonic acid, Tentoxin, and Altenuene) in 97 samples of cereal foods, tomato products, and sunflower seeds have been provided as requested by the Italian national monitoring programme (years 2017-2020). To this purpose, an LC-MS/MS method was set up and validated, obtaining fit for purpose sensitivity, recoveries (70-120%), repeatability (≤20%) and within laboratory reproducibility (≤26%). Occurrence data showed that oilseeds were the most contaminated food group with levels of Tenuazonic acid up to 16752 µg/kg and Tentoxin up to 570 µg/kg, whereas for the other mycotoxin/commodities combinations, the percentage of left censored data (below the limit of quantification) ranged from 74 to 100%.

Determination of Zearalenone and Trichothecenes, Including Deoxynivalenol and Its Acetylated Derivatives, Nivalenol, T-2 and HT-2 Toxins, in Wheat and Wheat Products by LC-MS/MS: A Collaborative Study.

An analytical method for the simultaneous determination of trichothecenes-namely, nivalenol (NIV), deoxynivalenol (DON) and its acetylated derivatives (3- and 15-acetyl-DON), T-2 and HT-2 toxins-and zearalenone (ZEN) in wheat, wheat flour, and wheat crackers was validated through a collaborative study involving 15 participants from 10 countries. The validation study, performed within the M/520 standardization mandate of the European Commission, was carried out according to the IUPAC (International Union of Pure and Applied Chemistry) International Harmonized Protocol. The method was based on mycotoxin extraction from the homogenized sample material with a mixture of acetonitrile-water followed by purification and concentration on a solid phase extraction column. High-performance liquid chromatography coupled with tandem mass spectrometry was used for mycotoxin detection, using isotopically labelled mycotoxins as internal standards. The tested contamination ranges were from 27.7 to 378 µg/kg for NIV, from 234 to 2420 µg/kg for DON, from 18.5 to 137 µg/kg for 3-acetyl-DON, from 11.4 to 142 µg/kg for 15-acetyl-DON, from 2.1 to 37.6 µg/kg for T-2 toxin, from 6.6 to 134 µg/kg for HT-2 toxin, and from 31.6 to 230 µg/kg for ZEN. Recoveries were in the range 71-97% with the lowest values for NIV, the most polar mycotoxin. The relative standard deviation for repeatability (RSDr) was in the range of 2.2-34%, while the relative standard deviation for reproducibility (RSDR) was between 6.4% and 45%. The HorRat values ranged from 0.4 to 2.0. The results of the collaborative study showed that the candidate method is fit for the purpose of enforcing the legislative limits of the major Fusarium toxins in wheat and wheat-based products.

Application of an Integrated and Open Source Workflow for LC-HRMS Plant Metabolomics Studies. Case-Control Study: Metabolic Changes of Maize in Response to Fusarium verticillioides Infection.

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) represents the most powerful metabolomics platform to investigate biological systems. Reproducible and standardized workflows allow obtaining a meaningful biological interpretation. The purpose of this study was to set up and apply an open-source workflow for LC-HRMS plant metabolomics studies. Key steps of the proposed workflow were as follows: (1) experimental design, (2) sample preparation, (3) LC-HRMS analysis, (4) data processing, (5) custom database search, (6) statistical analysis, (7) compound identification, and (8) biochemical interpretation. Its applicability was evaluated through the study of metabolomics changes of two maize recombinant inbred lines with contrasting phenotypes with respect to disease severity after Fusarium verticillioides infection of seedlings. Analysis of data from the case-control study revealed abundance change in metabolites belonging to different metabolic pathways, including two amino acids (L-tryptophan and tyrosine), five flavonoids, and three N-hydroxynnamic acid amides.

Aflatoxin Reduction in Maize by Industrial-Scale Cleaning Solutions.

Different batches of biomass/feed quality maize contaminated by aflatoxins were processed at the industrial scale (a continuous process and separate discontinuous steps) to evaluate the effect of different cleaning solutions on toxin reduction. The investigated cleaning solutions included: (i) mechanical size separation of coarse, small and broken kernels, (ii) removal of dust/fine particles through an aspiration channel, (iii) separation of kernels based on gravity and (iv) optical sorting of spatial and spectral kernel defects. Depending on the sampled fraction, dynamic or static sampling was performed according to the Commission Regulation No. 401/2006 along the entire cleaning process lines. Aflatoxin analyses of the water-slurry aggregate samples were performed according to the AOAC Official Method No. 2005.008 based on high-performance liquid chromatography and immunoaffinity column cleanup of the extracts. A significant reduction in aflatoxin content in the cleaned products, ranging from 65% to 84% with respect to the uncleaned products, was observed when continuous cleaning lines were used. Additionally, an overall aflatoxin reduction from 55% to 94% was obtained by combining results from separate cleaning steps. High levels of aflatoxins (up to 490 µg/kg) were found in the rejected fractions, with the highest levels in dust and in the rejected fractions from the aspirator and optical sorting. This study shows that a cleaning line combining both mechanical and optical sorting technologies provides an efficient solution for reducing aflatoxin contamination in maize.

Critical Comparison of Analytical Performances of Two Immunoassay Methods for Rapid Detection of Aflatoxin M1 in Milk.

Aflatoxin B1 (AFB1) is a secondary metabolite produced by some Aspergillus spp. fungi affecting many crops and feed materials. Aflatoxin M1 (AFM1), the 4-hydroxylated metabolite of AFB1, is the main AFB1-related compound present in milk, and it is categorized by the International Agency for Research on Cancer (IARC) as a "group 1 human carcinogen". The aim of this work was to evaluate and compare the analytical performances of two commercial immunoassays widely applied for the detection of AFM1 in milk, namely strip test immunoassay and enzyme linked immunosorbent assay (ELISA). Assay validation included samples at AFM1 levels of 25, 50, 75 ng/kg and blank samples (AFM1 < 0.5 ng/kg). With respect to a screening target concentration (STC) of 50 ng/kg the two assays showed cut-off values of 37.7 ng/kg and 47.5 ng/kg for strip test and ELISA, respectively, a false suspect rate for blanks <0.1% (for both assays) and a false negative rate for samples containing AFM1 at levels higher than STC, of 0.4% (for both assays). The intermediate precision (RSDip) was <32% for the strip test and <15% for the ELISA. Method verification through long-term intra-laboratory quality control (QC) measurements confirmed the results from the validation study. Furthermore, a satisfactory correlation of the results obtained with both immunoassays and the AOAC Official Method 2000.08 was obtained for the analysis of cow milk samples naturally contaminated with AFM1 at levels within "not detected" (< 0.5 ng/kg) and 50 ng/kg. Finally, the extension of the scope of the strip test method to goat and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation.

In Vitro Fumonisin Biosynthesis and Genetic Structure of Fusarium verticillioides Strains from Five Mediterranean Countries.

Investigating the in vitro fumonisin biosynthesis and the genetic structure of Fusarium verticillioides populations can provide important insights into the relationships between strains originating from various world regions. In this study, 90 F. verticillioides strains isolated from maize in five Mediterranean countries (Italy, Spain, Tunisia, Egypt and Iran) were analyzed to investigate their ability to in vitro biosynthesize fumonisin B1, fumonisin B2 and fumonisin B3 and to characterize their genetic profile. In general, 80% of the analyzed strains were able to biosynthesize fumonisins (range 0.03-69.84 µg/g). Populations from Italy, Spain, Tunisia and Iran showed a similar percentage of fumonisin producing strains (>90%); conversely, the Egyptian population showed a lower level of producing strains (46%). Significant differences in fumonisin biosynthesis were detected among strains isolated in the same country and among strains isolated from different countries. A portion of the divergent FUM1 gene and of intergenic regions FUM6-FUM7 and FUM7-FUM8 were sequenced to evaluate strain diversity among populations. A high level of genetic uniformity inside the populations analyzed was detected. Apparently, neither geographical origin nor fumonisin production ability were correlated to the genetic diversity of the strain set. However, four strains from Egypt differed from the remaining strains.

Fluorescence Polarization Immunoassay for the Determination of T-2 and HT-2 Toxins and Their Glucosides in Wheat.

T-2 and HT-2 toxins and their main modified forms (T-2 glucoside and HT-2 glucoside) may co-occur in cereals and cereal-based products. A fluorescence polarization immunoassay (FPIA) was developed for the simultaneous determination of T-2 toxin, HT-2 toxin and relevant glucosides, expressed as sum. The developed FPIA, using a HT-2-specific antibody, showed high sensitivity (IC50 = 2.0 ng/mL) and high cross-reactivity (100% for T-2 toxin and 80% for T-2 and HT-2 glucosides). The FPIA has been used to develop two rapid and easy-to-use methods using two different extraction protocols, based on the use of organic (methanol/water, 90:10, v/v) and non-organic (water) solvents, for the determination of these toxins in wheat. The two proposed methods showed analytical performances in terms of sensitivity (LOD 10 µg/kg) recovery (92-97%) and precision (relative standard deviations ≤13%), fulfilling the criteria for acceptability of an analytical method for the quantitative determination of T-2 and HT-2 toxins established by the European Union. Furthermore, the methods were then validated in accordance with the harmonized guidelines for the validation of screening methods included in the Regulation (EU) No. 519/2014. The satisfactory analytical performances, in terms of intermediate precision (≤25%), cut-off level (80 and 96 µg/kg for the two methods) and rate of false positives (<0.1%) confirmed the applicability of the proposed methods as screening method for assessing the content of these toxins in wheat at the EU indicative levels reported for T-2 and HT-2 toxins.

Performance Evaluation of LC-MS Methods for Multimycotoxin Determination.

The co-occurrence of regulated mycotoxins in foods and feeds, together with modified ("masked") and emerging mycotoxins, has been increasingly reported worldwide in recent years. Therefore, sensitive, accurate, and validated methods for the simultaneous determination of these hazardous contaminants in different matrices are highly demanded to fulfil regulatory requirements and to carry out reliable surveillance programs. In these last years, LC-MS methodologies for multimycotoxin screening and/or quantification are being routinely used in control laboratories. However, to date, only one European Standard for multimycotoxin determination is based on LC-MS (EN 16877:2016). The need for standardized LC-MS methods for multimycotoxin determination has been highlighted by regulatory authorities and scientific advisory bodies, including the U.S. Food and Drug Administration and the European Commission. The European Committee for Standardization (CEN) has issued calls for tender for the development of standardized LC-MS methods for mycotoxins in food and animal feeding stuffs. As deliverables, some LC-MS based methods for multimycotoxin determination are currently under approval as European Standards. In addition, the European Commission has recently established specific criteria with which screening methods for mycotoxins, including LC-MS methods, have to comply for use for regulatory purposes. Validation procedures by single-laboratory and collaborative trials have been defined. This paper provides insights and advances on guidelines and tools for performance evaluation of LC-MS methods intended for quantitative determination and for semiquantitative screening of multimycotoxins. In particular, performance criteria set in the European Union and the United States are critically overviewed, and expectations, needs, and future challenges relevant to LC-MS methods for multimycotoxin determination are also discussed.

Evaluation of Mycotoxin Screening Tests in a Verification Study Involving First Time Users.

(AFB1) in maize and wheat using LFD and LC-HRMS, respectively. The results of analyses were used to calculate intermediate precision (RSDip, covering the inter-analyst variability in preparing the analytical samples and the precision under repeatability conditions) cut-off values and false suspect rates. RSDip ranged from 6.5% to 30% for DON, and from 16% to 33% for AFB1. The highest obtained variances were associated with the AFB1 analyses due to working with much lower mass fractions. The rate of false suspect results were lower than 0.1% for all tested methods. All methods showed a fit-for-purpose method performance profile, which allowed a clear distinction of samples containing the analytes at the screening target concentration (STC) from negative control samples. Moreover, the first time users obtained method performances similar to those obtained for validation studies previously performed on the screening methods included in the training course.

In-house validation and small-scale collaborative study to evaluate analytical performances of multimycotoxin screening methods based on liquid chromatography-high-resolution mass spectrometry: Case study on Fusarium toxins in wheat.

A strong trend toward using highly selective mass spectrometry technologies for screening of multiple mycotoxins has been observed in recent years. In the present study, the process of validation of a multimycotoxin screening method based on liquid chromatography-high-resolution mass spectrometry method is presented. The method was intended for the simultaneous screening of the major Fusarium toxins (deoxynivalenol, 3- and 15-acetyl deoxynivalenol, T-2 and HT-2 toxins, zearalenone, enniatins A, A1, B, and B1, and beauvericin) in wheat. The sample preparation protocol was based on a double extraction (methanol followed by acetonitrile/water mixture) and purification through solid-phase extraction C18 column. To provide insights for full exploitation of the potential of the double-stage high-resolution mass spectrometry detection, a full-scan acquisition event followed by a sequence of 5 fragmentation events (variable data-independent acquisition) was set for mycotoxin detection, the latter to be exploited for confirmatory purposes. Method analytical performances were evaluated through in-house validation and small-scale interlaboratory study, designed according to Commission Regulation 519/2014/EU, setting performance requirements for screening methods for mycotoxins. Screening target concentrations were close to European Union maximum permitted or indicative levels. The in-house validation provided the precision of the response under repeatability conditions and the intermediate precision (both resulting lower than 30%), the cutoff value, and the rate of false suspect results for negative (free of the mycotoxin of interest) samples, which resulted lower than 0.1% in all cases. The collaborative study provided reproducibility and laboratory independent cutoff values. Analysis of reference materials proved method trueness and suitability for screening of the major Fusarium mycotoxins in wheat. Finally, the applicability of the full-scan/variable data-independent acquisition detection approach was successfully tested on a set of naturally contaminated wheat samples, where 2 characteristic product ions could be detected for all identified mycotoxins even at levels in the low µg/kg range.

Occurrence of deoxynivalenol and deoxynivalenol-3-glucoside in durum wheat from Argentina.

The occurrence of deoxynivalenol, 3- and 15-deoxynivalenol and deoxynivalenol-3-glucoside in 84 durum wheat samples, from the Argentinean main growing area, was investigated during 2012/13 and 2013/14 using LC-MS/MS. Deoxynivalenol was found in all samples at concentrations varying between

Multiplex Dipstick Immunoassay for Semiquantitative Determination of Fusarium Mycotoxins in Oat.

A multiplex competitive antibody-based assay in a dipstick format for the simultaneous detection of major of Fusarium mycotoxins in oats is described. Ground oats sample is extracted with a mixture of methanol and water. Sample extract is diluted and directly analyzed by a multiplex dipstick. The test kit employs a microwell of reagents containing antibodies linked to gold particles and a dipstick made up of a nitrocellulose membrane were specific capture lines are located. In the presence of oats extract each antibody binds the corresponding mycotoxin before starting to run vertically on the dipstick in the direction of the capture lines. In 10 min red lines rise from the background on the dipstick. Results are interpreted by an optical reader measuring the ratio between each test line and a control line located on the top of the strip.

Determination of T-2 and HT-2 Toxins in Oats and Oat-Based Breakfast Cereals by Liquid-Chromatography Tandem Mass Spectrometry.

This protocol specifies an accurate and sensitive method for the determination of T-2 and HT-2 toxins content in oats and oat-based foods using liquid chromatography with tandem mass spectrometry detection. T-2 and HT-2 toxins are extracted from the test material with a mixture of acetonitrile and water. The filtered extract is dried, reconstituted with a mixture of methanol and water, then purified on a polymeric solid-phase extraction cartridge. Toxins are finally eluted from the column with methanol and quantified by reversed phase high performance liquid chromatography with tandem mass spectrometry detection.

Formulation of yeast-leavened bread with reduced salt content by using a Lactobacillus plantarum fermentation product.

A Lactobacillus plantarum fermentation product (Bio21B), obtained after strain growth (14h) in a wheat flour-based medium, was applied in the bread-making process as taste enhancer, in order to obtain a yeast-leavened bread with reduced salt content (20% and 50%) with respect to a reference bread (REF) not containing the fermentation product. Sensory analysis indicated that the Bio21B bread with salt reduced by 50% had a pleasant taste similar to the salt-containing bread (REF). l-Glutamate and total free amino acid content did not differ between REF and Bio21B breads, while the acids lactic, acetic, phenyllactic, 4-OH-phenyllactic and indole-3-lactic were present only in Bio21B breads. Moreover, the presence of several umami (uridine monophosphate, inosine monophosphate, adenosine, and guanosine) and kokumi (γ-l-glutamyl-l-valine) taste-related molecules was ascertained both in REF and in Bio21B breads. Therefore, a possible role of the acidic molecules in compensating the negative perception of salt reduction can be hypothesized.

Occurrence of Fusarium langsethiae and T-2 and HT-2 Toxins in Italian Malting Barley.

T-2 and HT-2 toxins are two of the most toxic members of type-A trichothecenes, produced by a number of Fusarium species. The occurrence of these mycotoxins was studied in barley samples during a survey carried out in the 2011-2014 growing seasons in climatically different regions in Italy. The percentage of samples found positive ranges from 22% to 53%, with values included between 26 and 787 µg/kg. The percentage of samples with a T-2 and HT-2 content above the EU indicative levels for barley of 200 µg/kg ranges from 2% to 19.6% in the 2011-2014 period. The fungal species responsible for the production of these toxins in 100% of positive samples has been identified as Fusarium langsethiae, a well-known producer of T-2 and HT-2 toxins. A positive correlation between the amount of F. langsethiae DNA and of the sum of T-2 and HT-2 toxins was found. This is the first report on the occurrence of F. langsethiae-and of its toxic metabolites T-2 and HT-2-in malting barley grown in Italy.

Toward Harmonization of Performance Criteria for Mycotoxin Screening Methods: The EU Perspective.

Screening methods are defined as methods that are used to detect the presence of a substance or class of substances at the level of interest. These methods must have the capability of high sample throughput when being used to screen large numbers of samples for potential noncompliant results. Before using a screening method for practical applications, its fitness for the intended purpose needs to be demonstrated. This is normally achieved by conducting a validation study, comparing method performance against predefined criteria. Official guidelines recently established by the European Union for the evaluation of fitness-for-purpose performance parameters of screening methods to be used for the detection of mycotoxins in foods are presented and discussed herein. Practical applications of this evaluation scheme for single- and interlaboratory validation studies, as well as relevant information on screening method performances are reviewed, with emphasis on the impact of mycotoxin contamination in real samples on the fitness-for-purpose of the screening test. Lastly, validation follow-up is discussed in terms of extension of the scope of the method (increasing the range of application in terms of mycotoxin/matrix combinations), method implementation and verification, and evaluation of the method's applicability to modified mycotoxins.