Monica Olsen

The presence of Aspergillus section Flavi and aflatoxins (AFs) in nut chains are of great concern due to the toxic effects of AFs on human and animal health as well as the economic losses during commercialisation. Extreme events related to climate change like increased temperature, severe rainfalls and droughts are modulating factors of fungal growth and mycotoxin production. The Expert Working Group (EWG) on nuts that worked under the frame of the Horizon 2020 Mycokey Project prepared this review based on discussions about the situation of contamination with AFs in nuts and the available strategies to reduce the impact of these toxins in the nut chains. The EWG selected as examples peanuts, pistachios and Brazil nuts, and discussed in the review the importance of nut value chains in the production, commercialisation and regulations that establish maximum levels for AFs. Also data on the Rapid Alert System for Food and Feed (RASFF) was considered and showed several notifications for mycotoxins, mainly AFs for nuts and nut products, such as pistachio nuts and dried mix nuts. Under a scenario of climate change, we elaborated strategies to prevent AFs contamination with emphasis on biocontrol and decision support systems. Also, the relevance of strategies to prevent and reduce both fungal infection and AFs accumulation in peanuts, Brazil nuts and pistachio are included as guidelines.

A simple and effective approach for HPLC-MS/MS based multi-mycotoxin analysis in human urine samples was developed by application of dried urine spots (DUS) as alternative on-site sampling strategy. The newly developed method enables the detection and quantitation of 14 relevant mycotoxins and mycotoxin metabolites, including citrinin (CIT), dihydrocitrinone (DH-CIT), deoxynivalenol (DON), fumonisin B₁ (FB₁), T-2 Toxin (T-2), HT-2 Toxin (HT-2), ochratoxin A (OTA), 2′R-ochratoxin A (2′R-OTA), ochratoxin α (OTα), tenuazonic acid and allo-tenuazonic acid (TeA + allo-TeA), zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (α-ZEL), and β-zearalenol (β-ZEL). Besides the spotting procedure, sample preparation includes enzymatic cleavage of glucuronic acid conjugates and stable isotope dilution analysis. Method validation revealed low limits of detection in the range of pg/mL urine and excellent apparent recovery rates for most analytes. Stability investigation of DUS displayed no or only slight decrease of the analyte concentration over a period of 28 days at room temperature. The new method was applied to the analysis of a set of urine samples (n = 91) from a Swedish cohort. The four analytes, DH-CIT, DON, OTA, and TeA + allo-TeA, could be detected and quantified in amounts ranging from 0.06 to 0.97 ng/mL, 3.03 to 136 ng/mL, 0.013 to 0.434 ng/mL and from 0.36 to 47 ng/mL in 38.5%, 70.3%, 68.1%, and 94.5% of the samples, respectively. Additional analysis of these urine samples with an established dilute and shoot (DaS) approach displayed a high consistency of the results obtained with both methods. However, due to higher sensitivity, a larger number of positive samples were observed using the DUS method consequently providing a suitable approach for human biomonitoring of mycotoxin exposure.

The determination of mycotoxin and metabolite concentrations in human and animal urine is currently used for risk assessment and mycotoxin intake measurement. In this study, pig urine (n = 195) was collected at slaughterhouses in 2012 by the Swedish National Food Agency in three counties representing East, South and West regions of Sweden. Urinary concentrations of four mycotoxins, (deoxynivalenol (DON), zearalenone (ZEA), fumonisin B₁ (FB₁), and ochratoxin A (OTA)), and four key metabolites, (deepoxy-deoxynivalenol (DOM-1), aflatoxin M₁ (AFM₁, biomarker of AFB₁), α-zearalenol (α-ZOL), and β-zearalenol (β-ZOL)) were identified and measured by UPLC-MS/MS. Statistically significant regional differences were detected for both total DON (DON + DOM-1) and total ZEA (ZEA + α-ZOL + β-ZOL) concentrations in pig urine from the three regions. These regional differences were in good agreement with the occurrence of Fusarium graminearum mycotoxins (DON + ZEA) in cereal grains harvested in 2011 in Sweden. There were no statistically significant differences in FB₁, AFM₁ and OTA urinary concentrations in pigs from the three regions. The overall incidence of positive samples was high for total ZEA (99–100%), total DON (96–100%) and OTA (85–95%), medium for FB₁ (30–61%) and low for AFM₁ (0–13%) in the three regions. Urinary mycotoxin biomarker concentrations were used to estimate mycotoxin intake and the level of mycotoxins in feeds consumed by the monitored pigs. The back-calculated levels of mycotoxins in feeds were low with the exception of seven samples that were higher the European limits.

Biomarker-based methods are being more and more used to assess dietary exposure of mycotoxins in a population. The aim of the present study was to perform an extended analysis of urinary multiple mycotoxin levels and associations with background characteristics and food groups. Exposure assessment calculations were performed on three urine mycotoxins as described below and the probable daily intake (PDI) was compared with the established tolerable daily intake (TDI) to uncover potential exposure risks. The study population consisted of 250 adults and 50 school children in grade five from two surveys conducted by the Swedish National Food Agency. Six mycotoxins (deoxynivalenol (DON), zearalenone (ZEA), fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), ochratoxin A (OTA), and nivalenol (NIV) and four metabolites (deepoxy-deoxynivalenol (DOM-1), aflatoxin M₁ (AFM₁), α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) were measured by an ultra-performance liquid chromatography tandem mass spectrometry based method (LC-MS/MS). OTA and DON were the most commonly occurring mycotoxins in urine of both adults and children, 51 and 63%, respectively in adults and 96 and 94%, respectively in children. A positive correlation was found between urinary NIV and total cereal consumption among adults. ZEA, α-ZOL, β-ZOL and FB₂ were significantly higher in females than males (P1] were below the TDI values except for DON exposure in adults, as reported previously, 1.3% of the volunteers were above the TDI.

Isolates ofPenicillium commune, Penicillium crustosum, Penicillium expansum, Penicillium roqueforti and Aspergillus versicolor, were inoculated on different food items (hard cheese, crème fraiche, tomato purée, apple and blueberry jam) and incubated at 15 °C for 14 days at 50% relative humidity (RH). After incubation the food samples were divided into 3 subsamples; A was 0-2 cm from the surface and including the fungal colony, subsample B was 2-4 cm and subsample C was the rest from >4 cm from the surface. The subsamples were analysed with a multianalyte method capable of identifying more than several hundreds of fungal metabolites. The outcome showed that mouldy food can contain a cocktail of bioactive secondary metabolites including mycotoxins and sometimes at high concentrations. Measurements of the diffusion of fungal metabolites from the colony on the surface (layer A) into the food (layer B and C) showed that the fungal metabolites do not diffuse more than 2 cm into the inner core of the hard cheese. On the other hand in more liquid foods, such as crème fraiche, fruit jams and tomato purée, the toxins diffused quite readily throughout the entire food sample. The levels of patulin found in the apple jam indicate that the tolerable daily intake for patulin may easily be exceeded even if the mouldy layer A is removed. This limited study calls for more similar studies to be performed to give risk managers a sound basis for advice to consumers.