Simultaneous detection of mycotoxins and pesticides in human urine samples: A 24-h diet intervention study comparing conventional and organic diets in Spain.

Pesticides and mycotoxins, prominent chemical hazards in the food chain, are commonly found in plant-based foods, contributing to their pervasive presence in the human body, as evidenced by biomonitoring programs. Despite this, there is limited knowledge about their co-occurrence patterns. While intervention studies have demonstrated that organic diets can significantly reduce pesticide levels, their impact on mycotoxin exposure has been overlooked. To address this gap, this study pursued two objectives: first, to characterize the simultaneous presence of mycotoxins and pesticides in human urine samples by means of the control of the biomarkers of exposure, and second, to investigate the influence of consuming organic foods on these co-exposure patterns. A pilot study involving 20 healthy volunteers was conducted, with participants consuming either exclusively organic or conventional foods during a 24-h diet intervention in autumn 2021 and spring 2022 to account for seasonal variability. Participants provided detailed 24-h dietary records, and their first-morning urine samples were collected, minimally treated and analysed using LC-Q-ToF-MS by means of a multitargeted method in order to detect the presence of these residues. Results indicated that among the 52 screened compounds, four mycotoxins and seven pesticides were detected in over 25% of the samples. Deoxynivalenol (DON) and the non-specific pesticide metabolite diethylphosphate (DEP) exhibited the highest frequency rates (100%) and concentration levels. Correlations were observed between urine levels of mycotoxins (DON, ochratoxin alpha [OTα], and enniatin B [ENNB]) and organophosphate pesticide metabolites DEP and 2-diethylamino-6-methyl-4-pyrimidinol (DEAMPY). The pilot intervention study suggested a reduction in ENNB and OTα levels and an increase in ß-zearalenol levels in urine after a short-term replacement with organic food. However, caution is advised due to the study's small sample size and short duration, emphasizing the need for further research to enhance understanding of the human chemical exposome and refine chemical risk assessment.

NIR-HSI as a tool to predict deoxynivalenol and fumonisins in maize kernels: a step forward in preventing mycotoxin contamination.

BACKGROUND: Maize is frequently contaminated with deoxynivalenol (DON) and fumonisins B1 (FB1 ) and B2 (FB2 ). In the European Union, these mycotoxins are regulated in maize and maize-derived products. To comply with these regulations, industries require a fast, economic, safe, non-destructive and environmentally friendly analysis method. RESULTS: In the present study, near-infrared hyperspectral imaging (NIR-HSI) was used to develop regression and classification models for DON, FB1 and FB2 in maize kernels. The best regression models presented the following root mean square error of cross validation and ratio of performance to deviation values: 0.848 mg kg-1 and 2.344 (DON), 3.714 mg kg-1 and 2.018 (FB1 ) and 2.104 mg kg-1 and 2.301 (FB2 ). Regarding classification, European Union legal limits for DON and FB1 + FB2 were selected as thresholds to classify maize kernels as acceptable or not. The sensitivity and specificity were 0.778 and 1 for the best DON classification model and 0.607 and 0.938 for the best FB1 + FB2 classification model. CONCLUSION: NIR-HSI can help reduce DON and fumonisins contamination in the maize food and feed chain. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Detection and quantification of zearalenone and its modified forms in enzymatically treated oat and wheat flour.

An analytical method for the analysis of the mycotoxin zearalenone (ZEN) and its modified forms was developed. Sample preparation was performed based on a modified QuEChERS method combined with liquid chromatography coupled to a triple quadrupole mass spectrometry detection. The method was tested for linearity, precision, limits of detection and quantification and recoveries. The evaluation of the above-mentioned parameters was performed on oat flour. The method was applied to oat and wheat flours that were submitted to an amylolytic treatment (α-amylase and amyloglucosidase), similar to the one used in the cereal-based baby food production process. A decrease in ß-zearalenol (ß-ZEL) and ß-ZEL-14-sulfate of approximately 40% after 90 min incubation was observed, the other analytes did not show any significant changes. To our knowledge, this is the first method that approaches the identification and assessment of ZEN-sulfate derivates in a cereal matrix. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05683-6.

Influence of Agronomic Factors on Mycotoxin Contamination in Maize and Changes during a 10-Day Harvest-Till-Drying Simulation Period: A Different Perspective.

Agronomic factors can affect mycotoxin contamination of maize, one of the most produced cereals. Maize is usually harvested at 18% moisture, but it is not microbiologically stable until it reaches 14% moisture at the drying plants. We studied how three agronomic factors (crop diversification, tillage system and nitrogen fertilization rate) can affect fungal and mycotoxin contamination (deoxynivalenol and fumonisins B1 and B2) in maize at harvest. In addition, changes in maize during a simulated harvest-till-drying period were studied. DON content at harvest was higher for maize under intensive tillage than using direct drilling (2695 and 474 µg kg-1, respectively). We found two reasons for this: (i) soil crusting in intensive tillage plots caused the formation of pools of water that created high air humidity conditions, favouring the development of DON-producing moulds; (ii) the population of Lumbricus terrestris, an earthworm that would indirectly minimize fungal infection and mycotoxin production on maize kernels, is reduced in intensive tillage plots. Therefore, direct drilling is a better approach than intensive tillage for both preventing DON contamination and preserving soil quality. Concerning the simulated harvest-till-drying period, DON significantly increased between storage days 0 and 5. Water activity dropped on the 4th day, below the threshold for DON production (around 0.91). From our perspective, this study constitutes a step forward towards understanding the relationships between agronomic factors and mycotoxin contamination in maize, and towards improving food safety.

Diversity and metabolomic characterization of Penicillium expansum isolated from apples grown in Argentina and Spain.

Apples (Malus domestica) are one of the most consumed fruits globally. It is a relevant crop in Argentina and Spain, and one of the main fruits for export and industrialization in these countries. Quality control of apples, fundamentally in the postharvest stage, is critical to prevent fungal diseases. The blue mould, caused by Penicillium expansum, is responsible for great economic losses due to the deterioration of the fruit and mycotoxin production. Many studies have characterized this pathogen; however, little is known about the differences between populations from distant geographical origins. The objective of the present study was to characterize two P. expansum populations, from Argentina and Spain, through morphological, metabolomic and molecular approaches, and to evaluate the existence of differences related to their geographical source. A total of 103 isolates, 53 from Argentina and 50 from Spain were studied. Their morphological features were consistent with the species description. The secondary metabolite profiles revealed low chemical diversity. All 103 isolates shared the production of 13 compounds, namely andrastins, aurantioclavine, chaetoglobosins, communesins, expansolides, roquefortine C and patulin. Penostatins and citrinin were produced by 102 and 101 isolates, respectively. A region of the ß-tubulin gene was selected to analyse the diversity of the P. expansum isolates. No substantial differences were observed between isolates of different geographical origins through morphology, patulin accumulation, secondary metabolite profiles and phylogenetic analysis. However, the analysis of polymorphisms revealed 29 haplotypes with a relative separation between isolates of both populations; 13 haplotypes contained Argentinean isolates, while Spanish isolates were separated into 16 haplotypes. The diversity indices of Shannon (H'=2.075; H'=2.402) and Simpson (SiD = 0.850; SiD = 0.895) for isolates from Argentina and Spain, respectively, indicated that the diversity of P. expansum is greater in Spain than in Argentina. This distribution could be explained both by the existence of haplotype exchange between both countries, with the ancestral haplotypes originating in Spain, and the subsequent adaptation to the environmental conditions or apples varieties grown in each region.

Deterministic and Probabilistic Dietary Exposure Assessment to Deoxynivalenol in Spain and the Catalonia Region.

Deoxynivalenol (DON) remains one of the most concerning mycotoxins produced by the Fusarium genus due to the wide occurrence in highly consumed cereal-based food and its associated toxicological effects. Previous studies conducted in Spain and other European countries suggested that some vulnerable groups such as children could be exceeding the tolerable daily intakes. Thus, the aim of this study was to conduct a comprehensive and updated dietary exposure assessment study in Spain, with a specific analysis in the region of Catalonia. Cereal-based food samples collected during 2019 were analysed using liquid chromatography coupled to tandem mass spectrometry for multi-mycotoxin detection including DON and its main metabolites and derivatives. Consumption data were gathered from the nation-wide food surveys ENALIA and ENALIA2 conducted in Spain, and a specific survey conducted in Catalonia. The data were combined using deterministic and semi-parametric probabilistic methods. The results showed that DON was widely present in cereal-based food highly consumed in Spain and the Catalonia region. Exposure to DON among the adult population was globally low; however, among infants aged 3-9 years, it resulted in the median of 192 ng/kg body weight/day and the 95th percentiles of 604 ng/kg body weight/day, that would exceed the most conservative safety threshold for infants. Bread and pasta were the main contributing foodstuffs to the global exposure to DON, even among infants; thus, those foods should be considered a priority for food control or to develop strategies to reduce the exposure. In any case, further toxicological and epidemiological studies are required in order to refine the safety thresholds accounting for the sensitivity of the infant population.

Relevant Fusarium Mycotoxins in Malt and Beer.

Mycotoxins are secondary fungal metabolites of high concern in the food and feed industry. Their presence in many cereal-based products has been numerously reported. Beer is the most consumed alcoholic beverage worldwide, and Fusarium mycotoxins originating from the malted and unmalted cereals might reach the final product. This review aims to describe the possible Fusarium fungi that could infect the cereals used in beer production, the transfer of mycotoxins throughout malting and brewing as well as an insight into the incidence of mycotoxins in the craft beer segment of the industry. Studies show that germination is the malting step that can lead to a significant increase in the level of all Fusarium mycotoxins. The first step of mashing (45 °C) has been proved to possess the most significant impact in the transfer of hydrophilic toxins from the grist into the wort. However, during fermentation, a slight reduction of deoxynivalenol, and especially of zearalenone, is achieved. This review also highlights the limited research available on craft beer and the occurrence of mycotoxins in these products.

Occurrence and Dietary Exposure Assessment to Enniatin B through Consumption of Cereal-Based Products in Spain and the Catalonia Region.

Enniatin B (ENNB) is a mycotoxin produced by moulds from the Fusarium genera and its toxic effects are still not fully elucidated, hence a safe reference exposure value has not been established yet. ENNB is the most prevalent emerging mycotoxin and is widely found in cereal-based products, nevertheless, there are no comprehensive exposure assessment studies. For that reason, the aim of this study was to characterise the occurrence of ENNB and estimate the exposure of the Spanish and Catalan populations. A total of 347 cereal-based products were collected in 2019 and were analysed using liquid chromatography-tandem mass spectrometry. Consumption data were obtained from the national food consumption surveys (ENALIA) and a regional survey conducted in Catalonia. The global exposure was estimated using deterministic and probabilistic methods. The results showed a high occurrence of close to 100% in all foodstuffs, with a range from 6 to 269 µg/kg, and a strong correlation with the levels of deoxynivalenol. Children aged one-nine years were the most exposed, showing mean estimates in the range 308-324 ng/kg bw/day and 95th percentiles 697-781 ng/kg bw/day. This study stresses the need for further toxicological data to establish reference doses and conclude formal risk assessment, accounting for the co-occurrence with deoxynivalenol.

Near-infrared hyperspectral imaging for deoxynivalenol and ergosterol estimation in wheat samples.

The present study aimed to evaluate the use of hyperspectral imaging (HSI)-NIR spectroscopy to assess the presence of DON and ergosterol in wheat samples through prediction and classification models. To achieve these objectives, a first set of bulk samples was scanned by HSI-NIR and divided into two subsamples, one that was analysed for ergosterol and another that was analysed for DON by HPLC. This method was repeated for a second larger set to build prediction and classification models. All the spectra were pretreated and statistically processed by PLS and LDA. The prediction models presented a RMSEP of 1.17 mg/kg and 501 µg/kg for ergosterol and DON, respectively. Classification achieved an encouraging accuracy of 85.4% for an independent validation set of samples. The results confirm that HSI-NIR may be a suitable technique for ergosterol quantification and DON classification of samples according to the EU legal limit for DON.

Tri-octahedral bentonites as potential technological feed additive for Fusarium mycotoxin reduction.

In 2009 the EU Regulation 386/2009 established a new functional group of feed additives called "substances for reduction of the contamination of feed by mycotoxin". Later, di-octahedral bentonite (1 m558) was authorised, as an anti-aflatoxin additive, being the only additive of this group authorised to date. This work aims to demonstrate the effectiveness of other bentonites, such as tri-octahedral bentonites, versus Fusarium-mycotoxins, since very few adsorbents have proved their effectiveness in relation to this group of mycotoxins. For this purpose, 7 bentonites (six of them tri-octahedral) and 7 commercial adsorbents, added at 0.02% (w/v), were assayed in an in vitro adsorption experiment using two simulated gastro-intestinal (GI) juices (pH 1.3 and 6.8) versus zearalenone (ZEN: 0.1-5 mg/L), fumonisin B1 (FB1: 1-10 mg/L) and deoxynivalenol (DON: 2-10 mg/L). Mycotoxin adsorption data were fitted to Langmuir and Freundlich isotherms. In vitro adsorption experiments showed that ZEN and FB1 (in the latter case only in acid medium) were partially adsorbed, while the adsorption of DON was negligible. Moreover, the increase of adsorbent dose (up to 0.20%, w/v) significantly improved the in vitro adsorption of ZEN and FB1, reaching >90% of adsorption. The present work proposes the use of some tri-octahedral bentonites as feed additives for Fusarium-mycotoxin reduction.

Fusarium mycotoxins in total mixed rations for dairy cows.

Mycotoxins produced by certain fungal species of the Fusarium genus are frequently found as contaminants in cereals and feedstuffs. Fumonisins (FBs), deoxynivalenol (DON) and zearalenone (ZEN) are of special concern relative to animal health and productivity. The aim of this work was to analyse the levels of Fusarium mycotoxin contamination in samples of total mixed rations (TMRs) for dairy cows. To accomplish this analysis, an HPLC-MS/MS multi-mycotoxin method was developed and validated. The relation between the formulation of TMR samples and the presence of mycotoxins was also studied. From February 2016 to January 2018, a total of 193 TMR samples for dairy cows collected from farms located in different areas of Spain were analysed for the presence of FBs, ZEN, DON and their metabolites. In total, 112 samples (58%) were contaminated with at least one mycotoxin, and 38 samples (20%) presented more than one mycotoxin. FBs were the mycotoxins most frequently found (34% positive samples). DON was detected in 17% of samples, and ZEN was detected in 16% of samples. Among the metabolites analysed, only deoxynivalenol-3-glucoside (DON-3-Glc) and 15-acetyldeoxynivalenol (15-ADON) were detected. The levels of all the Fusarium mycotoxins studied were always below the values recommended by the European Commission for feedstuffs. The wide variety of ingredients used in the formulation of the analysed samples made it difficult to reach definite conclusions, although it seemed that some cereal silages and concentrates such as cereals or compound feed used as ingredients of the TMR may be related to the presence of mycotoxins.

Formation of patulin-glutathione conjugates induced by pulsed light: A tentative strategy for patulin degradation in apple juices.

Patulin is a toxic mycotoxin usually associated with apple products. Due to its unhealthy effects for humans, its content is regulated by the food safety authorities. The removal or degradation of this mycotoxin in contaminated apple juices has been studied with different approaches with uneven effectiveness. However, a strategy based on the chemical reaction between patulin and glutathione (GSH), in order to generate the conjugates that are formed during cell detoxification process, is an innovative approach yet to be evaluated. In this work, the formation of patulin-GSH conjugates activated by the application of pulsed light treatments and catalyzed by Fe2+ ions was evaluated. The study of patulin degradation and effect of the GSH/Fe2+ molar ratio showed that a molar ratio of 5 allows an adequate catalytic effect of the metal ions. In addition, mono-substituted patulin-glutathione adducts were identified as the main type of generated conjugates.

The fate of Fusarium mycotoxins (deoxynivalenol and zearalenone) through wort fermenting by Saccharomyces yeasts (S. cerevisiae and S. pastorianus).

The aim of this study was to evaluate the effect of 15 commercial yeasts in the mitigation of the Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) during the brewing process. Saccharomyces strains (10 strains of S. cerevisiae and 5 of S. pastorianus) were used to ferment DON and ZEN contaminated wort. Wort samples were taken every 24 h during fermentation, while mycotoxin analysis in yeast was performed at the end of fermentation (96 h); additionally, pH and ethanol content were measured daily. For mycotoxin analysis, after immunoaffinity purification of sample extracts, analysis was performed using an Ultra-High-Pressure Liquid Chromatograph coupled with a diode array or fluorescence detector (UHPLC-DAD/FLD). Mycotoxin presence had no significant effect on the ethanol production during brewing. At the end of fermentation, 10-17% of DON and 30-70% of ZEN had been removed, 6% of the initial concentration of DON and 31% of the ZEN being adsorbed by the yeast. Beermakers must pay careful attention to the raw material since a high percentage of DON could be present at the end of the beer fermentation process. Future studies should focus on the quantification of "masked" mycotoxins that are relevant to food security.

Frequency and levels of mycotoxins in beer from the Mexican market and exposure estimate for deoxynivalenol mycotoxins.

The aim of the present study was to evaluate the occurrence of 23 mycotoxins in beer purchased in Mexico and to assess two exposure scenarios in the Mexican population through beer consumption. Multi-mycotoxin analysis of a total of 61 different beers (132 samples) was carried out using UHPLC-MS/MS equipment. Probability density functions were used to describe mycotoxins contamination. The daily intake of mycotoxins was estimated using a semi-probabilistic approach, applying the Monte Carlo method. Deoxynivalenol (DON) and its metabolites (deoxynivalenol-3-glucoside (DON3G) and 3-acetyl-deoxynivalenol (3ADON)) were the mycotoxins found in higher proportions in contaminated samples. None of the other mycotoxins overpassed the limit of quantification (LOQ) of the method. The combined intake of DON and its analogues ranged from 5.24 to 86.59 ng kg-1 bw day-1, which represent from 1.20 to 19.83% of the DON TDI. The results suggest that depending on the individual consumption of beer and depending on the type of beer, the intake of DON via beer could represent a significant percentage of the tolerable daily intake (TDI).

Transfer of Fusarium mycotoxins from malt to boiled wort.

The fate of deoxynivalenol, deoxynivalenol-3-glucoside, 3- and 15-acetyl-deoxynivalenol, zearalenone, α- and ß-zearalenol and fumonisins (fumonisin B1 and fumonisin B2) through mashing and wort boiling was studied. Three different mycotoxin contamination scenarios were considered. In almost all samples an increase in the level of mycotoxins in wort was observed during mashing followed by a decrease after just 30 min of the process, with levels remaining constant until the end of boiling. Deoxynivalenol and its metabolites were reduced to their initial level contained in the malt before mashing, or even lower, however in none of the samples they were completely eliminated. Zearalenone was not quantitated at the end of boiling, although there was a significant initial level of ZEN. ß-Zearalenol remained unaltered during the process. Fumonisins were reduced by between 50 and 100 per cent during mashing and boiling.

The role of mycotoxins in the human exposome: Application of mycotoxin biomarkers in exposome-health studies.

Mycotoxins are secondary metabolites produced by fungi that may contaminate different foods intended for human consumption, resulting in a widespread exposure worldwide. The novel exposome paradigm has the ambition to decipher the different environmental insults threating human health throughout the entire lifespan. Given the large potential impact of mycotoxins in terms of human exposure and related health effects, the ambition of this review is to present this group of chemical compounds and the high interest to be included in exposome projects. Furthermore, we also attempt to approach the novel exposome paradigm to more traditional disciplines such as mycotoxin exposure assessment and mycotoxicology, introducing the new methodological challenges and translational needs. Hence, we provide an overview of major biomarkers currently developed, biological matrices where these may be found, an overview of internal exposure levels and potential co-occurrence with environmental chemicals and finally an overview of major health effects with the illustrative example of the potent xenoestrogen zearalenol. Conversely, these new approaches may be an excellent opportunity to fill many research gaps on mycotoxins research as the identification of associations with human health, elucidation of join effect with other environmental exposures or the decipher of underlying molecular mechanisms by using advanced OMICs technologies.

Aflatoxin B1, ochratoxin A and zearalenone in sorghum grains marketed in Tunisia.

A total of 64 samples of sorghum (37 Tunisian sorghum samples and 27 Egyptian sorghum samples) were collected during 2011-2012 from markets in Tunisia. Samples were analysed for contamination with aflatoxin B1, ochratoxin A and zearalenone by High-Performance Liquid Chromatography Coupled with Fluorescence Detection (HPLC-FLD). Aflatoxin B1 was found in 38 samples in the range 0.03-31.7 µg kg-1. Ochratoxin A was detected in 24 samples with concentrations ranging from 1.04 to 27.8 µg kg-1. Zearalenone was detected in 21 samples and the concentration varied between 3.7 and 64.5 µg kg-1. ANOVA analysis of the influence of the country of origin on the incidence and concentration of mycotoxins in the samples studied showed no significant difference (P > 0.05) between the two batches of samples for each of the three mycotoxins studied. The studied mycotoxins contaminate sorghum and may also co-exist because of the diversity of the mycobiota in this cereal.

Mycotoxins and beer. Impact of beer production process on mycotoxin contamination. A review.

Beer is the most consumed alcoholic beverage in the world. Its contamination with mycotoxins is of public health concern, especially for heavy drinkers. Beer production implies a variety of operations which might impact the initial level of mycotoxins in a positive or negative way. The complexity of these operations do not give to the brewer a complete control on chemical and biochemical reactions that take place in the batch, but the knowledge about mycotoxin properties can help in identifying the operations decreasing their level in foodstuffs and in the development of mitigation strategies. This review discusses available data about mycotoxin evolution during malting and brewing process. The operations that may lead to a decrease in mycotoxin load are found to be steeping, kilning, roasting, fermentation and stabilization operations applied over the process (e.g. clarification). Also, other general decontamination strategies usually employed in food industry, such as hot water treatment of barley, ozonation or even the use of lactic acid bacteria starter cultures during malting or fermentation are considered.

Assessment of intraspecies variability in fungal growth initiation of Aspergillus flavus and aflatoxin B1 production under static and changing temperature levels using different initial conidial inoculum levels.

Intraspecies variability in fungal growth and mycotoxin production has important implications for food safety. Using the Bioscreen C we have examined spectrophotometrically intraspecies variability of A. flavus using 10 isolates under different environments, including temperature shifts, in terms of growth and aflatoxin B1 (AFB1) production. Five high and five low AFB1 producers were examined. The study was conducted at 5 isothermal conditions (from 15 to 37 °C) and 4 dynamic scenarios (between 15 and 30 °C). The experiments were carried out in a semisolid YES medium at 0.92 aw and two inoculum levels, 102 and 103 spores/mL. The Time to Detection (TTD) of growth initiation was determined and modelled as a function of temperature through a polynomial equation and the model was used to predict TTD under temperature upshifts conditions using a novel approach. The results obtained in this study have shown that a model can be developed to describe the effect of temperature upshifts on the TTD for all the studied isolates and inoculum levels. Isolate variability increased as the growth conditions became more stressful and with a lower inoculum level. Inoculum level affected the intraspecies variability but not the repeatability of the experiments. In dynamic conditions, isolate responses depended both on the temperature shift and, predominantly, the final temperature level. AFB1 production was highly variable among the isolates and greatly depended on temperature (optimum temperature at 30-35 °C) and inoculum levels, with often higher production with lower inoculum. This suggests that, from an ecological point of view, the potential isolate variability and interaction with dynamic conditions should be taken into account in developing strategies to control growth and predicting mycotoxin risks by mycotoxigenic fungi.

Hydrolysers of modified mycotoxins in maize: α-Amylase and cellulase induce an underestimation of the total aflatoxin content.

Aflatoxins are the most potent genotoxic and carcinogenic mycotoxins. To date, research has only focused on the presence of free aflatoxins in agricultural commodities. Therefore, the main objective of this study was to investigate the occurrence of possible modified aflatoxins in maize. Different hydrolysis methods were applied to convert modified mycotoxins into their free aflatoxins. Eighteen aflatoxin-contaminated maize samples were incubated with potassium hydroxide, trifluoromethanesulfonic acid and several enzymes to induce hydrolysis. Potassium hydroxide caused a total reduction of aflatoxins, while trifluoromethanesulfonic acid did not lead to an increase in free aflatoxins, neither did treatment with a protease. However, α-amylase and cellulase incubation caused significant increases in the total free aflatoxin content, 15 ±â€¯8% and 13 ±â€¯5%, respectively. These results show that a small proportion of aflatoxins could be associated to matrix substances in plants. Consequently, hydrolysis could occur during food processing and during mammalian digestion, leading to an underestimation of the total aflatoxin content.