Development and Validation of an UHPLC-MS/MS Method for the Simultaneous Determination of 11 EU-Regulated Mycotoxins in Selected Cereals.

The availability of reliable sensitive multi-analyte methods for unambiguous determination of mycotoxins is crucial for ensuring food and feed safety, considering their adverse health effects and (co-)occurrence in various foods. Accordingly, a multi-mycotoxin confirmatory method for simultaneous determination of 11 mycotoxins regulated in cereals within the European Union (EU) using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was developed and in-house validated to fit the EU legislation requirements for analytical methods. A simple sample preparation was based on a solid−liquid extraction using a solvent mixture acetonitrile/water/formic acid (79/20/1, v/v/v) and a dilution of raw extract using water/acetonitrile/formic acid (79/20/1, v/v/v) before instrumental analysis. Average recoveries in all three validated cereal crop types (maize, wheat, and barley), spiked at multiple levels, were found acceptable for all analytes when matrix-matched calibration was used, ranging from 63.2% to 111.2% and also showing very good repeatability, with relative standard deviations below 20%. Matrix effect (SSE) evaluation revealed maize as the most complex of the three analyzed cereal matrices, with strong SSE (<50% and >150%) recorded for all 11 analyzed mycotoxins. An additional method verification was performed through successful participation in proficiency testing schemes, with the achieved z-scores generally in the acceptable range of −2 ≤ z ≤ 2. The obtained validation results demonstrated the suitability of the developed confirmatory multi-mycotoxin UHPLC-MS/MS method based on a dilute-and-shoot principle for the simultaneous determination of low concentrations of 11 EU-regulated mycotoxins in cereals, including aflatoxins B1, B2, G1 and G2, deoxynivalenol, fumonisins B1 and B2, zearalenone, T-2 and HT-2 toxins, and ochratoxin A.

Occurrence of Patulin and Polyphenol Profile of Croatian Traditional and Conventional Apple Cultivars during Storage.

Apples and apple-based products are among the most consumed fruits around the world. However, they are susceptible to infection with the fungi Penicilium expansum. In addition to the reduction of apple quality, secondary metabolism of this fungus produces a mycotoxin patulin that has a negative effect on human health. Currently, there is no available research in the literature on the resistance of Croatian traditional apple cultivars to contamination with P. expansum, and consequently, on the patulin content in apples and apple juice produced from those apples. Although the mechanism of apple resistance to fungal diseases has not yet been sufficiently investigated, some studies have shown that polyphenolic compounds have some impact on fungi growth. In order to contribute with new knowledge, this research deals with monitoring the growth of P. expansum on apples, patulin detection by LC/MS-MS, determination of polyphenol profile by validated HPLC method, and determining the effect of polyphenolic compounds on fungi growth and patulin production during apple storage. The results of this study have shown that Croatian traditional apple cultivars harvested from family farm Horvatic contain higher concentration of polyphenolic compounds and higher antioxidant activity. At the same time, they showed more resistance to infection by P. expansum than conventional ones. The higher content of dihydrochalcones and flavanols encouraged the biosynthesis of patulin in examined cultivars. However, the higher content of non-flavonoids such as 2-6 dimethoxybenzoic acid, 4-hydroxycinnamic acid and chlorogenic acid leads to decrease in content of patulin. In conclusion, it seems that content of polyphenols and patulin production are correlated.

Regulated Mycotoxin Occurrence and Co-Occurrence in Croatian Cereals.

A total of 209 samples of various cereal crops (maize, wheat, barley, rye and oats) grown in Croatian fields during 2016 and 2017 were collected to analyze and determine the occurrence and co-occurrence of EU regulated mycotoxins in cereals (AFB1, AFB2, AFG1, AFG2, DON, FB1, FB2, ZEA, T-2, HT-2 and OTA). The analysis, performed by a validated confirmatory LC-MS/MS method based on a dilute and shoot principle, highlighted Fusarium mycotoxins as the main contaminants, often co-occurring in samples from both years (50.0% in 2016 and 33.7% in 2017). DON was found to be the most frequent mycotoxin, present in 72.5% of the 2016 samples and 32.6% of the 2017 samples, while maize proved to be the most contaminated cereal type of both years with FUM as the most abundant mycotoxins, with an average concentration of 1180 µg/kg. Moderate temperatures with periods of high humidity favored the accumulation of DON in wheat samples instead of other Fusarium mycotoxins, while similar conditions favored maize contamination with FUM. A total of 8.3% of all the 2016 harvest samples and 7.9% of the 2017 harvest samples were assessed as non-compliant, containing mycotoxins in concentrations higher than the levels set by the EU legislation for food.

Penicillium expansum Impact and Patulin Accumulation on Conventional and Traditional Apple Cultivars.

Penicillium expansum is a necrotrophic plant pathogen among the most ubiquitous fungi disseminated worldwide. It causes blue mould rot in apples during storage, transport and sale, threatening human health by secreting patulin, a toxic secondary metabolite that contaminates apples and apple-derived products. Nevertheless, there is still a lack of sufficient data regarding the resistance of different apple cultivars to P. expansum, especially ancient ones, which showed to possess certain resistance to plant diseases. In this work, we investigated the polyphenol profile of 12 traditional and 8 conventional apple cultivar and their resistance to P. expansum CBS 325.48. Eight polyphenolic compounds were detected; the most prominent were catechin, epicatechin and gallic acid. The highest content of catechin was detected in 'Apistar'-91.26 mg/100 g of fresh weight (FW), epicatechin in 'Bobovac'-67.00 mg/100 g of FW, and gallic acid in 'Bobovac' and 'Kraljevcica'-8.35 and 7.40 mg/100 g of FW, respectively. The highest content of patulin was detected in 'Kraljevcica' followed by 'Apistar'-1687 and 1435 µg/kg, respectively. In apple cultivars 'Brcko', 'Adamcica' and 'Idared', patulin was not detected. Furthermore, the patulin content was positively correlated with gallic acid (r = 0.4226; p = 0.002), catechin (r = 0.3717; p = 0.008) and epicatechin (r = 0.3305; p = 0.019). This fact indicates that higher contents of gallic acid, catechin and epicatechin negatively affected and boost patulin concentration in examined apple cultivars. This can be related to the prooxidant activity of polyphenolic compounds and sensitivity of P. expansum to the disturbance of oxidative status.

Mycotoxins, Pesticide Residues, and Heavy Metals Analysis of Croatian Cereals.

Cereals are still one of the most important food and feed sources, thus determining cereal's safety, i.e., compliance with legislation, is extremely important. As systematic investigations of nowadays unavoidable secondary fungal metabolites and other common legally regulated contaminants occurrence in Croatian cereals are still lacking, this research aims to monitor the contamination levels of nation-wide crops by mycotoxins, pesticide residues, and heavy metals by employing UHPLC-MS/MS, GC-MS/MS, and atomic absorption spectrometer (AAS) validated analytical methods. The most common secondary fungal metabolites were found to be Fusarium mycotoxins, with DON being the most occurring present in 73.7% of the samples. At least one pesticide residue was found in 331.8% of the samples, and Hg and Cd were the most occurring heavy metals. A total of 8.5% of the samples was non-compliant to the European Union (EU) legislation for food regarding the found mycotoxins concentrations, 4.5% regarding pesticide residues and none regarding heavy metals. The unusual presence of certain pesticide residue and heavy metal indicates the importance of systematic control of the contaminant presence, in order to gather enough occurrence data for proper risk assessment that these contaminants represent for the consumer's health.

Antiaflatoxigenic effect of fullerene C60 nanoparticles at environmentally plausible concentrations.

Increased interest in fullerene C60 and derivatives in recent years implies an intensification of their environmental spread. Yet, the potential risks for living organisms are largely unknown, including the interaction of C60 with fungal organisms. This may be especially relevant for mycotoxigenic fungi since C60 may both scavenge and produce reactive oxygen species (ROS), and oxidative stress induces mycotoxin production in fungi. Therefore, this study examined effects of environmentally plausible concentrations of C60 (0, 10, 50, and 100 ng/mL) on Aspergillus flavus growth and aflatoxin production in culture media. In addition, ROS-dependent oxidative stress biomarkers-thiobarbituric acid reactive substances (TBARS), reduced and oxidised glutathione ratio, superoxide dismutase isoenzymes, catalase, glutathione peroxidase, and glutathione reductase were determined in mycelia. Nanoparticles of fullerene C60 (nC60) did not exhibit strong antifungal activity against A. flavus. At the same time, nC60 caused an antiaflatoxigenic effect at 10-100 ng/mL, and 50 ng/mL unexpectedly enhanced aflatoxin production. The TBARS content, reduced and oxidised glutathione ratio, and copper, zinc superoxide dismutase activity suggest that 10 ng/mL nC60 exerted antioxidative action and reduced aflatoxin B1 production within fungal cells. Detected prooxidative effects of 50 ng/mL fullerene exceeded cellular defenses and consequently enhanced aflatoxin B1 production. Finally, the results obtained with 100 ng/mL nC60 point to prooxidative effects, but the absence of increase in aflatoxin output may indicate additional, presumably cytotoxic effects of nC60. Thus, a range of rather low levels of nC60 in the environment has a potential to modify aflatoxin production in A. flavus. Due to possible implications, further studies should test these results in environmental conditions.

Fullerol C60(OH)24 nanoparticles and mycotoxigenic fungi: a preliminary investigation into modulation of mycotoxin production.

Increased use of fullerols in various fields and expected increase of their environmental spread impose the necessity for testing fullerol nanoparticles (FNP) effects on microbiota. There is little information available on the interaction of mycotoxigenic fungi and FNP, despite FNP having a great potential of modifying mycotoxin production. Namely, FNP exhibit both ROS-quenching and ROS-producing properties, while oxidative stress stimulates mycotoxin synthesis in the fungi. In order to shed some light on the extent of interaction between FNP and mycotoxigenic fungi, the effects of fullerol C60(OH)24 nanoparticles (10, 100, 1000 ng/mL) on mycelial growth, aflatoxin production and oxidative stress modulation in an aflatoxigenic strain of Aspergillus flavus (NRRL 3251) during 168 h of incubation in a liquid culture medium were examined. FNP slightly reduced mycelial biomass weight, but significantly decreased aflatoxin concentration in media. Lipid peroxide content, superoxide dismutase, catalase and glutathione peroxidase activities suggest that FNP treatments hormetically reduced oxidative stress within fungal cells in turn suppressing aflatoxin production. These findings contribute to the assessment of environmental risk and application potential of fullerols.

Antifungal and antiaflatoxigenic activities of coumarinyl thiosemicarbazides against Aspergillus flavus NRRL 3251.

The antifungal and antiaflatoxigenic effects of two series of coumarinyl thiosemicarbazides on Aspergillus flavus NRRL 3251 were studied. Fungi were grown in YES medium for 72 h at 29 °C in the presence of 0, 0.1, 1, and 10 µg mL-1 of coumarinyl thiosemicarbazides: one series with substitution in position 7 and another with substitution in position 4 of the coumarin core. Dry mycelia weight determination was used for antifungal activity estimation, while the aflatoxin B1 content in YES media, determined by the dilute and shoot LC-MS/MS technique, was used for the antiaflatoxigenic effect estimation. Standard biochemical assays were used for oxidative status marker (TBARS, SOD, CAT, and GPX) determination in A. flavus NRRL 3251 mycelia. Results show that 7-substituted-coumarinyl thiosemicarbazides possess a better antifungal and antiaflatoxigenic activity than 4-substituted ones. The most prominent substituted compound was the compound 3, N-(4-chlorophenyl)-2-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetyl)hydrazine-1-carbothioamide, which completely inhibited aflatoxin production at the concentration of 10 µg mL-1. Oxidative stress response of A. flavus exposed to the selected compounds points to the modulation of oxidative stress as a possible reason of aflatoxin production inhibition.

Soil chemicals properties and wheat genotype impact on micronutrient and toxic elements content in wheat integral flour.

AIM: To determine impact of soil chemical properties and different wheat genotypes in Croatia on micronutrient and toxic elements content in wheat integral flour. METHODS: Research was conducted and soil samples were collected from two different production areas in the Republic of Croatia: Ovcara and Dalj. Besides soil samples, grain samples of four different Croatian wheat genotypes were also collected and analyzed. In total, 40 samples of soil and 40 samples of wheat grain were analysed for total (aqua regia) and plant available (EDTA extraction) heavy metal content of Fe, Mn, Zn, Cu, Pb, Cd RESULTS: Determined soil pHKCl ranged from 5.63 to 6.25 at Ovcara and from 6.95 to 7.37 at Dalj sampling sites. The highest total concentration of heavy metals in soil were determined for Fe, followed by Mn, Zn, Cu, Pb and the lowest total concentration was recorded for Cd. The highest EDTA concentrations in soil were determined for Mn, than followed by Fe, Cu, Pb, and the lowest EDTA concentration was recorded for Cd. The highest concentration in integral wheat flour was found for Fe, than lower for Mn, Zn, Cu, Pb and the lowest concentration was found for Cd. If consumers in Croatia used daily 203 g of bread made of integral flour, they would take 2.31 to 8.44 µg Cd daily, depending on soil and wheat genotype. CONCLUSIONS: The analysed soil and winter wheat genotypes have significant impact on potential daily intake of toxic and essential heavy metals by integral flour or bread.