Multi-Mycotoxin Long-Term Monitoring Survey on North-Italian Maize over an 11-Year Period (2011-2021): The Co-Occurrence of Regulated, Masked and Emerging Mycotoxins and Fungal Metabolites.

Maize is considered one of the most susceptible crops to mycotoxin-producing fungi throughout the world, mainly belonging to the Fusarium spp. and Aspergillus spp. Maize is mainly used as animal feeds in Italy, as well as for human consumption, being essential for all the protected designation of origin (DOP) products. Our study investigated the occurrence of regulated mycotoxins in 3769 maize grain samples collected from 88 storage centers by the National Monitoring Network over an 11-year period (2011-2021). Moreover, an in-depth survey over a 4-year period, characterized by extremely different meteorological conditions, was conducted to investigate the co-occurrence of regulated, masked, and emerging mycotoxins. The survey confirmed that Fusarium spp. was the most frequent fungi and fumonisins were the main mycotoxins that were constantly detected in the different years and areas. Moreover, the areas characterized by high fumonisin levels were also the most prone to contamination by emerging mycotoxins produced by the same Fusarium species of the Liseola section. On the other hand, as a result of climatic changes, maize grains have also been affected by the increased frequency of aflatoxin accumulation. Deoxynivalenol, zearalenone, and other emerging mycotoxins produced by the same Fusarium species as the Discolor section occurred more abundantly in some areas in Northern Italy and in years characterized by predisposing meteorological conditions.

The Effect of Foliar Fungicide and Insecticide Application on the Contamination of Fumonisins, Moniliformin and Deoxynivalenol in Maize Used for Food Purposes.

The fungal ear rot of maize cultivated in temperate areas is mainly due to the Fusarium species. The use of insecticides against European Corn Borer (ECB) reduces the severity of fungal ear rot as well as the fumonisin (FB) and moniliformin (MON) levels in maize kernels at harvest, which in turn results in a lowering of their effect on deoxynivalenol (DON) control. However, the direct fungicidal control of ear rot has rarely been implemented for maize, and the first studies reported conflicting results on the reduction of mycotoxins. In the present experiment, field trials were carried out in North Italy over three growing seasons to study the effect of fungicide application timings on maize to control mycotoxins, considering the interaction of the application with the insecticide treatment, according to a full factorial split plot design. The mycotoxin content was determined through LC-MS/MS analysis. The field trials showed a significant reduction in ECB severity (75%), fungal ear rot severity (68%), Fusarium Liseola section infection (46%), FBs (75%) and MON (79%) as a result of the insecticide application for all the years, while the DON content increased by 60%. On the other hand, a fungicide application alone or applied in plots protected by an insecticide was never effective for the fungal symptoms, infection or mycotoxin content. The results confirm that a correct insecticide application to control ECB damage is the most effective agrochemical solution for the control of fungal ear rot, FBs and MON.

The Role of Nitrogen Fertilization on the Occurrence of Regulated, Modified and Emerging Mycotoxins and Fungal Metabolites in Maize Kernels.

The European Food Safety Authority is currently evaluating the risks related to the presence of emerging mycotoxins in food and feeds. The aim of this study was to investigate the role of soil fertility, resulting from different nitrogen fertilization rates, on the contamination of regulated mycotoxins and emerging fungal metabolites in maize grains. The trial was carried out in the 2012-2013 growing seasons as part of a long-term (20-year) experimental platform area in North-West Italy, where five different N rates, ranging from 0 to 400 kg N ha-1, were applied to maize each year. Maize samples were analyzed by means of a dilute-and-shoot multi-mycotoxin LC-MS/MS method, and more than 25 of the most abundant mycotoxins and fungal metabolites were detected. Contamination by fumonisins and other fungal metabolites produced by Fusarium spp. of the section Liseola was observed to have increased in soils that showed a poor fertility status. On the other hand, an overload of nitrogen fertilization was generally associated with higher deoxynivalenol and zearalenone contamination in maize kernels, as well as a higher risk of other fungal metabolites produced by Fusarium spp. sections Discolor and Roseum. A balanced application of N fertilizer, in accordance with maize uptake, generally appears to be the best solution to guarantee an overall lower contamination by regulated mycotoxins and emerging fungal metabolites.

Influence of H2O2-Induced Oxidative Stress on In Vitro Growth and Moniliformin and Fumonisins Accumulation by Fusarium proliferatum and Fusarium subglutinans.

Fusarium proliferatum and Fusarium subglutinans are common pathogens of maize which are known to produce mycotoxins, including moniliformin (MON) and fumonisins (FBs). Fungal secondary metabolism and response to oxidative stress are interlaced, where hydrogen peroxide (H2O2) plays a pivotal role in the modulation of mycotoxin production. The objective of this study is to examine the effect of H2O2-induced oxidative stress on fungal growth, as well as MON and FBs production, in different isolates of these fungi. When these isolates were cultured in the presence of 1, 2, 5, and 10 mM H2O2, the fungal biomass of F. subglutinans isolates showed a strong sensitivity to increasing oxidative conditions (27-58% reduction), whereas F. proliferatum isolates were not affected or even slightly improved (45% increase). H2O2 treatment at the lower concentration of 1 mM caused an almost total disappearance of MON and a strong reduction of FBs content in the two fungal species and isolates tested. The catalase activity, surveyed due to its crucial role as an H2O2 scavenger, showed no significant changes at 1 mM H2O2 treatment, thus indicating a lack of correlation with MON and FB changes. H2O2 treatment was also able to reduce MON and FB content in certified maize material, and the same behavior was observed in the presence and absence of these fungi, highlighting a direct effect of H2O2 on the stability of these mycotoxins. Taken together, these data provide insights into the role of H2O2 which, when increased under stress conditions, could affect the vegetative response and mycotoxin production (and degradation) of these fungi.

Commercial Organic Versus Conventional Whole Rye and Wheat Flours for Making Sourdough Bread: Safety, Nutritional, and Sensory Implications.

Organic farming is gaining a broad recognition as sustainable system, and consumer demand for organic products has increased dramatically in the recent past. Whether organic agriculture delivers overall advantages over conventional agriculture is, however, contentious. Here, the safety, nutritional, and sensory implications of using commercial organic rye, soft, and durum wheat flours rather than conventional-made sourdough bread have been investigated. Culture-dependent and culture-independent approaches were used to explore the microbial architecture of flours and to study their dynamics during sourdough propagation. Besides biochemical features, the main nutritional (amino acid content, asparagine level, and antioxidant activity) characteristics of sourdoughs were investigated, and their effect on the structural, nutritional, and sensory profiles of breads assessed. Overall, the organic farming system led to flours characterized by lower content of asparagine and cell density of Enterobacteriaceae while showing higher concentration of total free amino acids. Differences of the flours mirrored those of sourdoughs and breads. The use of sourdough fermentation guaranteed a further improvement of the flour characteristics; however, a microbial and sensory profile simplification as well as a slight decrease of the biochemical parameters was observed between breads with sourdough after one-cycle fermentation and 10 days of propagation.

Fate of regulated, masked, emerging mycotoxins and secondary fungal metabolites during different large-scale maize dry-milling processes.

The use of maize in the food chain could be mainly limited due to its contamination by mycotoxins. As scarce information is available, the current study is aimed at collecting new data on the co-occurrence and the fate of the most frequent masked, modified and emerging mycotoxins and other second fungal metabolites in maize food products and by-products. Three maize lots, obtained in different growing seasons, were processed using two different degermination processes, a dry-degermination system or a tempering-degermination one, in order to compare the interaction between mycotoxins and the dry-milling management system. Whole grain before and after cleaning, and all the products and the by-products were sampled twice for each lot and were subjected to a multi-mycotoxin LC-MS/MS analysis. More than 30 mycotoxins and other fungal metabolites, including masked or modified forms, co-occurred in all the maize milling fractions. Grain cleaning reduced all the detected fungal metabolites by 1.2-2 times, compared to the grain before cleaning. Animal feed flour showed the highest content of almost all the mycotoxins and fungal metabolites, with a consequent negative impact on animal health. Considering that for all the mycotoxins and fungal metabolites an inverse relationship with particle size was observed, flaking grits represented the healthiest maize products with the least contamination level, while the abatement was always lower for maize flour. Furthermore, the metabolites were variably redistributed in the maize fractions. The total aflatoxins, kojic acid, deoxynivalenol and its modified form, culmorin, and its associated forms, butenolide, fusaproliferin, fusaric acid, fusarinolic acid and, in some cases, zearalenone and its modified forms, and fusarin C were found to be concentrated significantly in the germ. Some of them also had a greater permanence in the maize food fractions and a weaker decontamination, both of which point to a higher risk of exposure for the end consumers. The co-occurrence of a such a high number of mycotoxins and fungal metabolites and their different fates during the dry-milling process have never been described before.

Effects of Durum Wheat Cultivars with Different Degrees of FHB Susceptibility Grown under Different Meteorological Conditions on the Contamination of Regulated, Modified and Emerging Mycotoxins.

The enhancement of Fusarium head blight (FHB) resistance is one of the best options to reduce mycotoxin contamination in wheat. This study has aimed to verify that the genotypes with high tolerance to deoxynivalenol could guarantee an overall minimization of the sanitary risk, by evaluating the contamination of regulated, modified and emerging mycotoxins on durum wheat cvs with different degrees of FHB susceptibility, grown under different meteorological conditions, in 8 growing seasons in North-West Italy. The years which were characterized by frequent and heavy rainfall in spring were also those with the highest contamination of deoxynivalenol, zearalenone, moniliformin, and enniatins. The most FHB resistant genotypes resulted in the lowest contamination of all the mycotoxins but showed the highest deoxynivalenol-3-glucoside/deoxynivalenol ratio and moniliformin/deoxynivalenol ratio. An inverse relationship between the amount of deoxynivalenol and the deoxynivalenol-3-glucoside/deoxynivalenol ratio was recorded for all the cvs and all the years. Conversely, the enniatins/deoxynivalenol ratio had a less intense relationship with cv tolerance to FHB. In conclusion, even though the more tolerant cvs, showed higher relative relationships between modified/emerging mycotoxins and native/target mycotoxins than the susceptible ones, they showed lower absolute levels of contamination of both emerging and modified mycotoxins.

Elevated CO2 Impact on Common Wheat (Triticum aestivum L.) Yield, Wholemeal Quality, and Sanitary Risk.

The rising atmospheric CO2, concentration is expected to exert a strong impact on crop production, enhancing crop growth but threatening food security and safety. An improver wheat, a hybrid, and its parents were grown at elevated CO2, e[CO2] in open field, and their yield and rheological, nutritional, and sanitary quality were assessed. For all cultivars, grain yield increased (+16%) and protein content decreased (-7%), accompanied by a reduction in dough strength. Grain nitrogen yield increased (+24%) only in ordinary bread making cultivars. e[CO2] did not result in significant changes in phenolic acid content and composition, whereas it produced a significant increase in the deoxynivalenol content. Different responses to e[CO2] between cultivars were found for yield parameters, while the effect on qualitative traits was quite similar. In the upcoming wheat cropping systems, agronomic practices and cultivar selection suited to guarantee higher nitrogen responsiveness and minimization of sanitary risk are required.

Development and Comparison of Two Multiresidue Methods for the Determination of 17 Aspergillus and Fusarium Mycotoxins in Cereals Using HPLC-ESI-TQ-MS/MS.

Cereals can be contaminated by several mycotoxins, whose co-presence may represent an undervalued risk for humans and animals. Maize and wheat are the most contaminated cereals and in temperate areas could be affected in field conditions by several Fusarium and Aspergillus infections. To date, only B-fumonisins (FBs), aflatoxins (AFs), zearalenone (ZEA), deoxynivalenol (DON) and T-2 and HT-2 toxins have been regulated in cereals in European Union. The other fungal metabolites, are commonly referred to as "emerging" and "masked" mycotoxins, and more information on their occurrence in combination with the regulated mycotoxins, are needed to design combined toxicological and exposure assessments. This research intends to develop and compare two multiresidue HPLC-ESI-TQ-MS/MS methods for the simultaneous determination of the main regulated, emerging and masked mycotoxins in maize and wheat, among which: FB1, FB2, DON, ZEA, AFB1, AFB2, AFG1, AFG2, moniliformin (MON), deoxynivalenol-3-glucoside (DON-3-G), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV), enniatins A, A1, B, B1 (ENNA, ENNA1, ENNB, ENNB1). The extraction was performed for both methods using a mixture of CH3CN/H2O/CH3COOH (79/20/1, v/v/v), while the dilution/purification was carried out through two different procedures: (1) by the "dilute-and-shoot" technique diluting 1:2 the filtered extract with CH3CN/H2O/CH3COOH (20/79/1, v/v/v) to reduce the matrix effect; (2) using the Oasis® PRiME HLB clean-up columns. The analysis was carried out using CH3OH and H2O both acidified with 0.1% of CH3COOH as eluents. The injection volume was 20 µL and the flow rate 200 µL min-1. The analysis of two reference material (maize and wheat), was performed to evaluate the trueness and precision of the two methods by matrix-matched calibration curves. For all the regulated mycotoxins analyzed by both methods, the range of recovery percentage established by the Regulation (EC) No. 401/2006 was respected, except for ZEA by using the Oasis® PRiME HLB clean-up columns. Nevertheless, the results suggest that the Oasis® PRiME HLB clean-up columns, could be a valid alternative to the dilute-and-shoot method, although an additional cost for the clean-up has to be considered. In conclusion, both two analytical methods considerably reduce the analytical time and costs and therefore result to be promising and applicable for high-throughput routine multi-mycotoxins analysis by the use of a TQ.

Fumonisin Distribution in Maize Dry-Milling Products and By-Products: Impact of Two Industrial Degermination Systems.

In temperate areas, the main limitation to the use of maize in the food chain is its contamination by B-series fumonisins (FBs) during cultivation. Since the content of this group of mycotoxins may be distributed unevenly after milling, the aim of this study was to compare the distribution of FBs in maize fractions derived from two industrial dry-milling processes, that is, a dry-degermination (DD) system and a tempering-degermination (TD) system. Grain cleaning reduces FBs by about 42%. The germ of the two degermination processes showed a similar FB content of kernel after cleaning. Conversely, an animal feed flour resulted in a FB content that was two times higher than whole grain before cleaning. A significant FB reduction was observed in the milling fractions in both processes, with a higher reduction in the TD system than in the DD one. The average decontamination respect to uncleaned kernels in the DD process was 50%, 83% and 87%, for maize flour, break meal and pearl meal, respectively, while it was 78%, 88% and 94% in the TD process for small, medium and flaking grits, respectively. Among the milling fractions, the flaking grits with the highest particle size resulted in the highest FB reduction.

Application of Fungicides and Microalgal Phenolic Extracts for the Direct Control of Fumonisin Contamination in Maize.

Fungicides and, for the first time, microalgal phenolic extracts (MPE) from Spirulina sp. and Nannochloropsis sp. were applied on maize culture media under field conditions to evaluate their ability to minimize Fusarium species development and fumonisin production. An in vitro assay against F. verticillioides was carried out using maize grains as the culture medium. An open-field experiment was carried out in Northwest Italy under natural infection conditions. The compared treatments were factorial combinations of two insecticide treatments (an untreated control and pyrethroid, used against European Corn Borer), four antifungal treatments (an untreated control, MPE from Spirulina sp., MPE from Nannochloropsis sp., and a synthetic fungicide), and two timings of the application of the antifungal compounds (at maize flowering and at the milk stage). The MPE compounds were capable of inhibiting fumonisin production in vitro more efficiently than tebuconazole. Insecticide application reduced the infection by Fusarium species and subsequent fumonisin contamination. However, fumonisins in maize fields were not significantly controlled by either fungicide or MPE application.

Antineoplastic drugs determination by HPLC-HRMS(n) to monitor occupational exposure.

The purpose of this study was to develop a simple, direct, multiresidue highly specific procedure to evaluate the possible surface contamination of selected antineoplastic drugs in several hospital environment sites by using wipe test sampling. 5-fluorouracil (5-FU), carboplatin (C-Pt), cyclophosphamide (CYC), cytarabine (CYT), doxorubicin (DOX), gemcitabine (GEM), ifosfamide (IFO), methotrexate (MET), and mitomycin C (MIT) belong to very different chemical classes but show good ionization properties under electrospray ionization (ESI) conditions (negative ion mode for 5-FU and positive ion mode in all other cases). HPLC (high performance liquid chromatography) coupled with HRMS (high resolution mass spectrometry) appears to be the best technique for direct analysis of these analytes, because neither derivatization nor complex extraction procedure for polar compounds in samples is requested prior the analysis. Sample preparation was limited to washing wipes with appropriate solvents. Chromatographic separation was achieved on C18 reversed phase columns. The HPLC-HRMS/MS method was validated in order to obtain robustness, sensitivity and selectivity. LLOQ (lower limit of quantitation) values provided a sensitivity good enough to evidence the presence of the drugs in a very low concentration range (<1 pg/cm(2) ). The method was applied for a study of real wipe tests coming from many areas from a hospital showing some positive samples. The low quantitation limits and the high specificity due to the high resolution approach of the developed method allowed an accurate description of the working environment that can be used to define procedural rules to limit working place contamination to a minimum. Copyright © 2015 John Wiley & Sons, Ltd.

Progressive Pearling of Barley Kernel: Chemical Characterization of Pearling Fractions and Effect of Their Inclusion on the Nutritional and Technological Properties of Wheat Bread.

Two hulled barley varieties have been sequentially pearled for one to eight cycles, each with 5% removal. The derived fractions were analyzed for their bioactive compound content. The dietary fiber (DF) decreased from the external to the internal layers, whereas ß-glucans showed an inverse trend. Deoxynivalenol contamination was concentrated in the outer layers. The total antioxidant activity (TAA) was higher in the 15-25% fractions, which were used to prepare bread. Five mixtures of refined wheat flour, with an increasing replacement of this pearled barley fraction, were compared with a control for the bioactive compound content, as well as for the rheological and physical bread properties. The inclusion of pearled fractions with up to a 10% substitution leads to a clear enhancement of the DF and TAA, with only minor detrimental effects on the physical parameters. Selected byproducts of barley pearling could be proposed as functional ingredients for bakery products rich in DF and TAA.

Role of the European corn borer (Ostrinia nubilalis) on contamination of maize with 13 Fusarium mycotoxins.

The European corn borer (ECB) plays an important role in promoting Fusarium verticillioides infections and in the consequent fumonisin contamination in maize grain in temperate areas. The objective of this study was to evaluate whether the ECB feeding activity could also affect the occurrence of emerging mycotoxins in maize kernels. During the 2008-10 period, natural infestation of the insect was compared, in field research, with the protection of infestation, which was obtained by using an entomological net. The ears collected in the protected plots were free from ECB attack, while those subject to natural insect attacks showed a damage severity that varied from 10% to 25%. The maize samples were analysed by means of an LC-MS/MS-based multi-mycotoxin method, which led to the detection of various metabolites: fumonisins (FUMs), fusaproliferin (FUS), moniliformin (MON), bikaverin (BIK), beauvericin (BEA), fusaric acid (FA), equisetin (EQU), deoxynivalenol (DON), deoxynivalenol-3-glucoside (DON-3-G), zearalenone (ZEA), culmorin (CULM), aurofusarin (AUR) and butenolide (BUT). The occurrence of mycotoxins produced by Fusarium spp. of Liseola section was affected significantly by the ECB feeding activity. The presence of ECB injuries increased the FUMs from 995 to 4694 µg kg(-1), FUS from 17 to 1089 µg kg(-1), MON from 22 to 673 µg kg(-1), BIK from 58 to 377 µg kg(-1), BEA from 6 to 177 µg kg(-1), and FA from 21 to 379 µg kg(-1). EQU, produced by F. equiseti section Gibbosum, was also increased by the ECB activity, by 1-30 µg kg(-1) on average. Instead, the content of mycotoxins produced by Fusarium spp. of Discolor and Roseum sections was not significantly affected by ECB activity. As for FUMs, the application of a strategy that can reduce ECB damage could also be the most effective solution to minimise the other mycotoxins produced by Fusarium spp. of Liseola section.

Moniliformin analysis in maize samples from North-West Italy using multifunctional clean-up columns and the LC-MS/MS detection method.

A fast clean-up method has been developed to purify maize extracts and to detect moniliformin (MON) in maize samples from North-West Italy over a four-year period (2008-2011). The method is based on the use of MycoSep® 240 Mon clean-up columns (Romer Labs®). Samples were extracted using acetonitrile/water (84:16, v/v), and the extracts were purified with previously described clean-up columns. The liquid chromatography-tandem mass spectrography (LC-MS/MS) analysis has been carried out by means of hydrophilic interaction chromatography (HILIC), combined with negative electrospray mass spectrometry. The method has a recovery of 76-91% (relative standard deviation, RSD%: 6-14%), a limit of detection (LOD) of 1 µg kg⁻¹ and a limit of quantification (LOQ) of 4 µg kg⁻¹. Naturally contaminated maize (108 samples) was analysed for MON content. The average percentages of positive samples was 93% with the following ranges (µg kg⁻¹): 33-2606 (2008);