Comprehensive identification and risk assessment of regulated and emerging mycotoxins in infant foods and related raw materials and risk management advice: A case study of an infant food company in China.

This study evaluated muti-mycotoxins in 199 samples including processed infant foods and raw materials collected randomly from an infant food company and assessed their role in dietary exposure in infants and young children via probabilistic risk assessment. Approximately 79.6 % (74/93) of the processed infant foods and 65.1 % (69/106) of the raw materials were contaminated by mycotoxins, with a mean occurrence level of 3.66-321.8 µg/kg. Deoxynivalenol (DON) and tenuazonic acid (TeA) were the more prevalent mycotoxins detected, based on their higher frequencies and levels across samples. Co-occurrence of more than two mycotoxins was detected in 61.3 % (57/93) of the processed infant foods and 53.8 % (57/106) of the raw materials. Wheat flour and derived products (e.g., infant noodles and infant biscuits) were contaminated with higher contamination levels and a greater variety of mycotoxins than other samples (e.g., infant cereal and rice grains). The estimated daily exposure to OTA, DON, ZEN, and TEN was lower than the corresponding reference health-based guidance values, indicating acceptable health risks. However, the estimated dietary exposure to alternariol monomethyl ether (AME), alternariol (AOH), and tenuazonic acid (TeA) exceeded the corresponding thresholds of toxicological concern values, indicating potential dietary intake risks. Among the various samples, cereals and cereal-based infant foods emerged as the primary contributors to mycotoxin exposure. Further research is advised to address the uncertainties surrounding the toxicity associated with emerging Alternaria mycotoxins and to conduct cumulative risk assessments concerning multiple mycotoxin exposure in infants and young children.

Infant exposure to ochratoxin A, zearalenone, and deoxynivalenol from the consumption of milk formula and baby cereal in Chile.

Ochratoxin A (OTA), zearalenone (ZEN), and deoxynivalenol (DON) are mycotoxins whose exposure is associated with various adverse health effects, including cancer and renal disorders, estrogenic effects, and immunosuppressive and gastrointestinal disorders, respectively. Infants (<2 years) are the most vulnerable group to mycotoxins, representing a unique combination of restricted food consumption types, low body weight, lower ability to eliminate toxins, and more future years to accumulate toxins. This study aimed to estimate the infant́s exposure to OTA, DON, and ZEN due to the consumption of milk formula and baby cereals in Chile. Milk formula samples (n = 41) and baby cereals (n = 30) were collected and analyzed using commercial ELISA kits for OTA, DON, and ZEA determination. Exposure was assessed by the Estimated Daily Intake (EDI) approach (mean and worst-case scenario, WCS) with the levels found in a modified Lower Bound (mLB) and Upper Bound (UB); ideal consumption (<6m, 7-12 m, and 13-24 m); adjusted by the weight of each group. The risk was estimated by comparing the EDI with a reference tolerable daily intake or by the margin of exposure (MOE) in the case of OTA. DON and OTA occurrence in infant formula were 34 % and 41 %, respectively. The co-occurrence between these mycotoxins was 22 %. Mycotoxin contents were below LOQ values except for OTA determined in one sample (0.29 ng/ml). No milk formulae were contaminated with ZEN. In the case of baby cereals, the occurrences were 17 % for OTA, 30 % for DON, and 7 % for ZEN, all below LOQ. Co-occurrence was seen in two samples between ZEN and OTA. According to exposure calculations, the MOE for OTA was less than 10,000 in all models for milk formula between 0 to 12 months of age and in the UB and WCS for cereal consumption. Health concerns were observed for DON in the WCS and UB for milk consumption in all ages and only in the UB WCS for cereal consumption. Considering the high consumption of milk formula in these age groups, regulation of OTA and other co-occurring mycotoxins in infant milk and food is strongly suggested.

Rapid, on-site quantitative determination of mycotoxins in grains using a multiple time-resolved fluorescent microsphere immunochromatographic test strip.

The label probe plays a crucial role in enhancing the sensitivity of lateral flow immunoassays. However, conventional fluorescent microspheres (FMs) have limitations due to their short fluorescence lifetime, susceptibility to background fluorescence interference, and inability to facilitate multi-component detection. In this study, carboxylate-modified Eu(III)-chelate-doped polystyrene nanobeads were employed as label probes to construct a multiple time-resolved fluorescent microsphere-based immunochromatographic test strip (TRFM-ICTS). This novel TRFM-ICTS facilitated rapid on-site quantitative detection of three mycotoxins in grains: Aflatoxin B1 (AFB1), Zearalenone (ZEN), and Deoxynivalenol (DON). The limit of detection (LOD) for AFB1, ZEN, and DON were found to be 0.03 ng/g, 0.11 ng/g, and 0.81 ng/g, respectively. Furthermore, the TRFM-ICTS demonstrated a wide detection range for AFB1 (0.05-8.1 ng/g), ZEN (0.125-25 ng/g), and DON (1.0-234 ng/g), while maintaining excellent selectivity. Notably, the test strip exhibited remarkable stability, retaining its detection capability even after storage at 4 °C for over one year. Importantly, the detection of these mycotoxins relied solely on simple manual operations, and with a portable reader, on-site detection could be accomplished within 20 min. This TRFM-ICTS presents a promising solution for sensitive on-site mycotoxin detection, suitable for practical application in various settings due to its sensitivity, accuracy, simplicity, and portability.

Monoclonal-Antibody-Based Immunoassays for the Mycotoxins NX-2 and NX-3 in Wheat.

The fungal infestation of crops can cause major economic losses. Toxins produced by the causative fungi (mycotoxins) represent a potential safety hazard to people and livestock consuming them. One such mycotoxin is deoxynivalenol (DON, also known as vomitoxin), a trichothecene associated with Fusarium Head Blight of wheat. DON is commonly found in cereal crops worldwide. A group of trichothecene mycotoxins closely related to DON, the NX toxins, have been reported to occur in the northeastern United States and southern Canada. While many commercial immunoassays are available to detect DON, there are no rapid screening assays for the NX toxins. We describe the development and isolation of three monoclonal antibodies (mAbs) specific towards two NX toxins: NX-2 and NX-3. The mAbs did not recognize DON or several other closely related trichothecenes. One of the mAbs was selected for development of an enzyme-linked immunosorbent assay (ELISA) for NX-2 and NX-3 in wheat. The dynamic ranges for the assay were 7.7 to 127 µg/kg for NX-2 and 59 µg/kg to 1540 µg/kg for NX-3 in wheat. Recoveries from spiked wheat averaged 84.4% for NX-2 and 99.3% for NX-3, with RSDs of 10.4% and 11.3%, respectively (n = 24). The results suggest that this assay can be used to screen for NX toxins in wheat at levels relevant to human food and animal feed safety.

Impact of Environmental Conditions on the Concentrations of Trichothecenes, Their Glucosides, and Emerging Fusarium Toxins in Naturally Contaminated, Irradiated, and Fusarium langsethiae Inoculated Oats.

Trichothecenes produced by Fusarium species are commonly detected in oats. However, the ratios of the concentrations of free trichothecenes and their conjugates and how they are impacted by different interacting environmental conditions are not well documented. This study aims to examine the effect of water activity (0.95 and 0.98 aw) and temperature (20 and 25 °C) stress on the production of T-2 and HT-2 toxins, deoxynivalenol and their conjugates, as well as diacetoxyscirpenol (DAS). Multiple mycotoxins were detected using liquid chromatography-tandem mass spectrometry from 64 contaminated oat samples. The highest concentrations of HT-2-glucoside (HT-2-Glc) were observed at 0.98 aw and 20 °C, and were higher than other type A trichothecenes in the natural oats' treatments. However, no statistical differences were found between the mean concentrations of HT-2-Glc and HT-2 toxins in all storage conditions analysed. DAS concentrations were generally low and highest at 0.95 aw and 20 °C, while deoxynivalenol-3-glucoside levels were highest at 0.98 aw and 20 °C in the naturally contaminated oats. Emerging mycotoxins such as beauvericin, moniliformin, and enniatins mostly increased with a rise in water activity and temperature in the naturally contaminated oats treatment. This study reinforces the importance of storage aw and temperature conditions in the high risk of free and modified toxin contamination of small cereal grains.

Mycobiome mediates the interaction between environmental factors and mycotoxin contamination in wheat grains.

Environmental factors significantly impact grain mycobiome assembly and mycotoxin contamination. However, there is still a lack of understanding regarding the wheat mycobiome and the role of fungal communities in the interaction between environmental factors and mycotoxins. In this study, we collected wheat grain samples from 12 major wheat-producing provinces in China during both the harvest and storage periods. Our aim was to evaluate the mycobiomes in wheat samples with varying deoxynivalenol (DON) contamination levels and to confirm the correlation between environmental factors, the wheat mycobiome, and mycotoxins. The results revealed significant differences in the wheat mycobiome and co-occurrence network between contaminated and uncontaminated wheat samples. Fusarium was identified as the main differential taxon responsible for inducing DON contamination in wheat. Correlation analysis identified key factors affecting mycotoxin contamination. The results indicate that both environmental factors and the wheat mycobiome play significant roles in the production and accumulation of DON. Environmental factors can affect the wheat mycobiome assembly, and wheat mycobiome mediates the interaction between environmental factors and mycotoxin contamination. Furthermore, a random forest (RF) model was developed using key biological indicators and environmental features to predict DON contamination in wheat with accuracies exceeding 90 %. The findings provide data support for the accurate prediction of mycotoxin contamination and lay the foundation for the research on biological control technologies of mycotoxin through the assembly of synthetic microbial communities.

The potential of soil microbial communities to transform deoxynivalenol in agricultural soils-a soil microcosm study.

Infestation of cereal fields with toxigenic Fusarium species is identified as an environmental source for the mycotoxin deoxynivalenol (DON). During rain events, DON may be washed off from infested plants and enter the soil, where microbial transformation may occur. Although some studies showed DON transformation potential of soil microbial communities in liquid soil extracts, these findings can not be transferred to environmental conditions. Accordingly, microbial transformation of DON in soil has to be investigated under realistic conditions, e.g., microcosms mimicking field situations. In this study, we investigated the potential of soil microbial communities to transform DON in six different agricultural soils at two levels (0.5 and 5 µg g-1). The dissipation and the formation of transformation products were investigated in a period of 35 days and compared to a sterilized control. In addition, we measured soil respiration and applied the phospholipid-derived fatty acid (PLFA) analysis to assess whether soil microbial community characteristics are related to the microbial transformation potential. Dissipation of DON in non-sterilized soils was fast (50% dissipation within 0.6-3.7 days) compared to the sterile control where almost no dissipation was observed. Thus, dissipation was mainly attributed to microbial transformation. We verified that small amounts of DON are transformed to 3-keto-deoxynivalenol (3-keto-DON) and 3-epi-deoxynivalenol (3-epi-DON), which were not detectable after 16-day incubation, indicating further transformation processes. There was a trend towards faster transformation in soils with active and large microbial communities and low fungi-to-bacteria ratio.

Development of alkaline phosphatase-linked single-chain variable fragment fusion proteins for one-step immunodetection of deoxynivalenol in cereals.

Deoxynivalenol (DON) is a mycotoxin that widely distributes in various foods and seriously threatens food safety. To minimize the consumers' dietary exposure to DON, there is an urgent demand for developing rapid and sensitive detection methods for DON in food. In this study, a bifunctional single-chain variable fragment (scFv) linked alkaline phosphatase (ALP) fusion protein was developed for rapid and sensitive detection of deoxynivalenol (DON). The scFv gene was chemically synthesized and cloned into the expression vector pET25b containing the ALP gene by homologous recombination. The prokaryotic expression, purification, and activity analysis of fusion proteins (scFv-ALP and ALP-scFv) were well characterized and performed. The interactions between scFv and DON were investigated by computer-assisted simulation, which included hydrogen bonds, hydrophobic interactions, and van der Waals forces. The scFv-ALP which showed better bifunctional activity was selected for developing a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for DON in cereals. The dc-ELISA takes 90 min for one test and exhibits a half inhibitory concentration (IC50) of 11.72 ng/mL, of which the IC50 was 3.08-fold lower than that of the scFv-based dc-ELISA. The developed method showed high selectivity for DON, and good accuracy was obtained from the spike experiments. Furthermore, the detection results of actual cereal samples analyzed by the method correlated well with that determined by high-performance liquid chromatography (R2=0.97165). These results indicated that the scFv-ALP is a promising bifunctional probe for developing the one-step colorimetric immunoassay, providing a new strategy for rapid and sensitive detection of DON in cereals.

Advancing Global Health Surveillance of Mycotoxin Exposures using Minimally Invasive Sampling Techniques: A State-of-the-Science Review.

Mycotoxins are a heterogeneous group of toxins produced by fungi that can grow in staple crops (e.g., maize, cereals), resulting in health risks due to widespread exposure from human consumption and inhalation. Dried blood spot (DBS), dried serum spot (DSS), and volumetric tip microsampling (VTS) assays were developed and validated for several important mycotoxins. This review summarizes studies that have developed these assays to monitor mycotoxin exposures in human biological samples and highlights future directions to facilitate minimally invasive sampling techniques as global public health tools. A systematic search of PubMed (MEDLINE), Embase (Elsevier), and CINAHL (EBSCO) was conducted. Key assay performance metrics were extracted to provide a critical review of the available methods. This search identified 11 published reports related to measuring mycotoxins (ochratoxins, aflatoxins, and fumonisins) using DBS/DSS and VTS assays. Multimycotoxin assays adapted for DBS/DSS and VTS have undergone sufficient laboratory validation for applications in large-scale population health and human biomonitoring studies. Future work should expand the number of mycotoxins that can be measured in multimycotoxin assays, continue to improve multimycotoxin assay sensitivities of several biomarkers with low detection rates, and validate multimycotoxin assays across diverse populations with varying exposure levels. Validated low-cost and ultrasensitive minimally invasive sampling methods should be deployed in human biomonitoring and public health surveillance studies to guide policy interventions to reduce inequities in global mycotoxin exposures.

Near infrared hyperspectral imaging as a sorting tool for deoxynivalenol reduction in wheat batches.

The present study aimed to evaluate the feasibility of using near-infrared hyperspectral imaging (NIR-HSI) and chemometrics for classification of individual wheat kernels according to their deoxynivalenol (DON) level. In total, 600 wheat kernels from samples naturally contaminated over the maximum EU level were collected, and the DON content in each individual wheat kernel was analyzed by UHPLC. Linear discriminant analysis (LDA) was employed for building classification models of DON using the EU maximum level as cut off level, and they were tested on balanced and imbalanced test sets. The results showed that the models presented a balanced accuracy of 0.71, that would allow to obtain safe batches from contaminated batches once the unsafe kernels had been rejected, but often more than 30% of the batch would be rejected. The work confirmed that NIR-HSI could be a feasible method for monitoring DON in individual kernels and removing highly contaminated kernels prior to food chain entry.

Inhibition mechanism of fusarium graminearum growth by g-C3N4 homojunction and its application in barley malting.

The increase of deoxynivalenol (DON) caused by Fusarium graminearum (F. graminearum) during the malting process is a serious safety problem. In our work, the inhibition mechanism of F. graminearum growth by g-C3N4 homojunction and its application in barley malting were studied. The reason why the growth activity of F. graminearum decreased after photocatalysis by g-C3N4 homojunction was that under visible light irradiation, a large amount of •O2- elicited by g-C3N4 homojunction destroyed the cell structure of F. graminearum, leading to the deficiency of cell membrane selective permeability and serious disorder of intracellular metabolism. The application of photocatalysis technology in malting can effectively inhibit the growth of F. graminearum and the accumulation of ergosterol was reduced by 30.55 %, thus reducing the DON content in finished malt by 31.82 %. Meanwhile, the physicochemical indexes of barley malt after photocatalytic treatment still met the requirements of second class barley malt in Chinese light industry standard QB/T 1686-2008. Our work provides a new idea for the control of fungal contamination in barley malt.

Mycotoxin occurrence in kernels and straws of wheat, barley, and tritordeum.

Thirty-two varieties of common and durum wheat, hordeum, barley, and tritordeum collected over two harvesting years (2020 and 2021) were investigated for the presence of multiple Fusarium-related mycotoxins in asymptomatic plants. DON, 3-AcDON, 15-AcDON, T-2, HT-2, and ZEN together with the emerging mycotoxin ENN B and the major modified form of DON, namely DON3Glc, were quantified by means of UHPLC-MS/MS. Overall, DON and ENN B were the most frequently detected mycotoxins, albeit large inter-year variability was observed and related to different climate and weather conditions. Straws had higher mycotoxin contents than kernels and regarding DON occurrence tritordeum was found to be the most contaminated group on average for both harvesting years, while barley was the less contaminated one. Emerging mycotoxin ENN B showed comparable contents in kernels compared to straw, with a ratio close to 1 for tritordeum and barley. Regarding the occurrence of the other evaluated mycotoxins, T-2 and HT-2 toxins have been spotted in a few tritordeum samples, while ZEN has been frequently found only in straw from the harvesting year 2020. The data collected confirms the occurrence of multiple Fusarium mycotoxins in straws also from asymptomatic plants, highlighting concerns related to feed safety and animal health. The susceptibility of Tritordeum, hereby reported for the first time, suggests that careful measures in terms of monitoring, breeding, and cultural choices should be applied when dealing with his emerging crop.

Equine Mycotoxins.

The main mycotoxins involved in adverse equine health issues are aflatoxins, fumonisins, trichothecenes, and probably ergovaline (fescue grass endophyte toxicosis). Most exposures are through contaminated grains and grain byproducts, although grasses and hays can contain mycotoxins. Clinical signs are often nonspecific and include feed refusal, colic, diarrhea, and liver damage but can be dramatic with neurologic signs associated with equine leukoencephalomalacia and tremorgens. Specific antidotes for mycotoxicosis are rare, and treatment involves stopping the use of contaminated feed, switching to a "clean" feed source, and providing supportive care.

Role of ferroptosis in food-borne mycotoxin-induced toxicities.

Contamination by toxic substances is a major global food safety issue, which poses a serious threat to human health. Mycotoxins are major class of food contaminants, mainly including aflatoxins (AFs), zearalenone (ZON), deoxynivalenol (DON), ochratoxin A (OTA), fumonisins (FBs) and patulin (PAT). Ferroptosis is a newly identified iron-dependent form of programmed or regulated cell death, which has been found to be involved in diverse pathological conditions. Recently, a growing body of evidence has shown that ferroptosis is implicated in the toxicities induced by certain types of food-borne mycotoxins, which provides novel mechanistic insights into mycotoxin-induced toxicities and paves the way for developing ferroptosis-based strategy to combat against toxicities of mycotoxins. In this review article, we summarize the key findings on the involvement of ferroptosis in mycotoxin-induced toxicities and propose issues that need to be addressed in future studies for better utilization of ferroptosis-based approach to manage the toxic effects of mycotoxin contamination.

Phylogenetic Variation of Tri1 Gene and Development of PCR-RFLP Analysis for the Identification of NX Genotypes in Fusarium graminearum Species Complex.

NX toxins have been described as a novel group of type A trichothecenes produced by members of the Fusarium graminearum species complex (FGSC). Differences in structure between NX toxins and the common type B trichothecenes arise from functional variation in the trichothecene biosynthetic enzyme Tri1 in the FGSC. The identified highly conserved changes in the Tri1 gene can be used to develop specific PCR-based assays to identify the NX-producing strains. In this study, the sequences of the Tri1 gene from type B trichothecene- and NX-producing strains were analyzed to identify DNA polymorphisms between the two different kinds of trichothecene producers. Four sets of Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were successfully developed to distinguish the common type B trichothecene producers and NX producers within FGSC. These promising diagnostic methods can be used for high-throughput genotype detection of Fusarium strains as a step forward for crop disease management and mycotoxin control in agriculture. Additionally, it was found that the Tri1 gene phylogeny differs from the species phylogeny, which is consistent with the previous studies.

Exposure Assessment to Deoxynivalenol of Children over 3 Years Deriving from the Consumption of Processed Wheat-Based Products Produced from a Dedicated Flour.

Wheat-based products are largely consumed by children worldwide. Deoxynivalenol (DON) is known for its acute and chronic toxicity and is the most common contaminant of cereal grains. Since no legal limits are set for DON in wheat-based products and specific foods intended for children over 3 years on the market, a high risk of overexposure to this contaminant may emerge. The main objective of the study, conducted in 2018-2019, was to produce a wheat flour intended for children over three years, characterized by a high level of safety in terms of DON content, to be used to produce wheat-derived products. The dedicated flour was produced by adopting tailored procedures like the selection of wheat suppliers, the predetermination of the safe contamination of DON in the final products, and the evaluation of the transfer rate from the wheat flour to derived products (bread, breadsticks, biscuits, plumcake, and focaccia). The results showed that the daily exposure of children was considered to be safe, in a range between 7% (biscuits) and 67% (bread) of DON tolerable daily intake (TDI) and that only by producing a flour characterized by DON levels much lower than those in force, can "safe" products be marketed.

Study of co-occurrence of mycotoxins in cocoa beans in Brazil by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

In this study, 135 samples of cocoa beans collected in the Amazon and Atlantic Forest regions of Brazil were analysed to evaluate the possible co-occurrence of 34 mycotoxins. The results indicate that 42% of the cocoa samples exhibited quantifiable levels for 11 mycotoxins: aflatoxins (AFs) B1, B2 and G1; ochratoxin A; citrinin; cyclopiazonic acid; tenuazonic acid; paxilline; sterigmatocystin; zearalenone and fumonisin B2. Of the samples, 18% exhibited the co-occurrence of up to six mycotoxins. No toxins belonging to the groups of trichothecenes or ergot alkaloids were detected. Contingency analysis of the incidence of mycotoxins did not show significant differences between the two regions evaluated. Seven samples were contaminated with AFs, while only one contained ochratoxin A above 10 µg kg-1. The accuracy of the method was evaluated by proficiency testing for ochratoxin A, where satisfactory Z-scores were obtained.

The Multidirectional Influence of Feed-Borne Deoxynivalenol and Zearalenone on Animal Health.

Mycotoxins are secondary fungal metabolites which pose a significant threat for global food and feed security [...].

Occurrence of Types A and B Trichothecenes in Cereal Products Sold in Romanian Markets.

In view of the frequent occurrences of mycotoxins in cereals, this study assessed the presence of trichothecenes in 121 samples from Romanian markets. These samples were divided into five groups based on product type: (1) bread and bakery products containing white flour, (2) half-brown bread with whole wheat flour, (3) brown bread containing rye flour, (4) pasta, and (5) raw wheat. Gas Chromatography-Mass Spectrometry was used to detect 13 different mycotoxins, which included the Type A compounds HT-2 toxin and T-2 toxin, as well as the Type B compounds deoxynivalenol and nivalenol. Results indicated trichothecene contamination in 90.08% of our samples, with deoxynivalenol predominating by at least 78% in each examined group. Co-occurrence of three or four trichothecenes were found in 23.85% of our samples. Our study underscores the necessity of consistent monitoring of staple foods to prevent the intake of harmful trichothecenes by consumers.

Inactivation of zearalenone (ZEN) and deoxynivalenol (DON) in complete feed for weaned piglets: Efficacy of ZEN hydrolase ZenA and of sodium metabisulfite (SBS) as feed additives.

Female pigs respond sensitive both to DON and ZEN with anorexia and endocrine disruption, respectively, when critical diet concentrations are exceeded. Therefore, the frequent co-contamination of feed by DON and ZEN requires their parallel inactivation. The additive ZenA hydrolyzes ZEN while SBS inactivates DON through sulfonation. Both supplements were simultaneously added (+, 2.5 g SBS and 100 U ZenA/kg) to a control diet (CON-, 0.04 mg DON and < 0.004 mg ZEN/kg; CON+, 0.03 mg DON and < 0.004 mg ZEN/kg) and a Fusarium toxin contaminated diet (FUS-, 2.57 mg DON and 0.24 mg ZEN/kg; FUS+, 2.04 mg DON and 0.24 mg ZEN/kg). The 4 diets were fed to 20 female weaned piglets each (6 kg initial body weight) for 35 days; the piglets were sacrificed thereafter for collecting samples. Supplements improved performance and modified metabolism and hematology independent of dietary DON contamination. The mechanisms behind these changes could not be clarified and require further consideration. SBS reduced DON concentration in feed by approximately 20% and to the same extent in blood plasma and urine suggesting that no further DON sulfonate formation occurred in the digestive tract before absorbing DON in the upper digestive tract or that additionally formed DON sulfonates escaped absorption. DON sulfonates were detected in feces suggesting that unabsorbed DON sulfonates reached feces and/or that unabsorbed DON was sulfonated in the hindgut. The observed reduction rate of 20% was evaluated to be insufficient for feeding practice. Galenic form of SBS added to dry feed needs to be improved to support the DON sulfonation in the proximal digestive tract.ZenA was active in the digestive tract as demonstrated by the presence of its hydrolyzed none-estrogenic reaction products hydrolyzed ZEN (HZEN) and decarboxylated and hydrolyzed ZEN (DHZEN) both in feces, systemic circulation, and urine of group FUS+ compared to group FUS-. The presence of these hydrolysis products was paralleled by a significant decrease in high-estrogenic ZEN concentrations which, in turn, was related to a decrease in relative weights of uteri and ovaries when compared to group FUS-. Thus, ZenA was proven to be effective; both in terms of biomarkers and biological effects.