Deoxynivalenol in the wheat milling process and wheat-based products and daily intake estimates for the Southern Brazilian population

Fusarium head blight of wheat is caused by the Fusarium species that produces mycotoxins, such as deoxynivalenol (DON). The distribution of DON in wheat products can lead to high economic and health impacts. The objective of this study was to evaluate the natural distribution of DON in the wheat milling process and wheat-based products, as well as the daily intake estimates for the Southern Brazilian population. The fractions of wheat grains (milled wheat, finished flour and bran) were produced in a mill. Additionally, wheat-derived products, such as pasta, bread and crackers were analyzed. The bran fraction had the highest mean concentration of DON (2278 μg kg⁻¹), followed by milled wheat and finished flour (1895 μg kg⁻¹ and 1305 μg kg⁻¹). The distribution factor in the finished flour (69%) fraction demonstrates that DON was reduced when compared to milled wheat, by contrast of bran fraction that presents higher DON levels (120%). A percentage of 35% bran, 35% finished flour and 30% milled wheat samples would not be in compliance with future Brazilian regulations for DON levels. From the wheat-based products analyzed, 17% of whole bread and 10% of salted cracker products were contaminated with DON, with a median of 437 μg kg⁻¹ and 624 μg kg⁻¹, respectively. The finished flour was the fraction that most contributes to the daily intake of DON in Southern Brazil, representing 89.6% of the provisional maximum tolerable daily intake.     

Effect of milling on the content of deoxynivalenol,nivalenol, and zearalenone in Japanese wheat

The effect of milling on the content of zearalenone (ZEA), a Fusarium mycotoxin, in a semi-processed wheat product was investigated and compared with that of deoxynivalenol (DON) or nivalenol (NIV). Two grain samples of Japanese soft wheat varieties, norin 61 and chikugoizumi, were milled to obtain three breaking flours (1B, 2B, 3B), three middling flours (1M, 2M, 3M) and two outer-layer fractions (bran and shorts). Patent flour for human consumption was made from 1B, 1M, 2B and 2M flours, while low-grade flour for animal feed was made from 3B to 3M flours. The contents of ZEA in patent flour, low-grade flour, bran and shorts were analyzed by an in-house validated analytical method using solvent extraction, multifunctional cartridge cleanup, and HPLC-fluorescence detection. A greater than 50% reduction in ZEA was observed in the patent flour of both samples, while 4–74% reduction in DON and NIV in the patent flour of the same two samples was observed. The results of this study revealed that the transfer ratios of ZEA had different features from those of deoxynivalenol and nivalenol.     

Mycotoxin glucosylation in commercial wheat varieties: Impact on resistance to Fusarium graminearum under laboratory and field conditions

Fusarium head blight (FHB) is a devastating disease on small cereal crops and is primarily caused by Fusarium graminearum. FHB typically is associated with a contamination of the infected grains with mycotoxins, which can act as virulence factors for the advancing fungus. Some wheat plants have evolved a typical resistance designated Fhb1 resistance, which is associated with a glucosyltransferase function, capable of detoxifying the deposited deoxynivalenol (DON), a mycotoxin abundantly produced by F. graminearum. However, the resulting ‘masked mycotoxin’, deoxynivalenol-3-glucoside (DON-3G), may be metabolized in the digestive tract of animals, releasing the native toxin anew. The present study aims to assess the glucosylation capacity in the Belgian commercial wheat pool and to determine the importance of DON glucosylation in the complex background of natural field circumstances. Clear indications were found that several Belgian commercial wheat cultivars, to some extent, do possess a glucosyltransferase function capable of detoxifying significant amounts of DON. However, the level of glucosylation in the field did not correlate well with disease severity. The present research provides the first large-scale assessment of this glucosylation capacity in Belgian commercial wheat cultivars.     

Fate of deoxynivalenol and deoxynivalenol-3-glucoside during wheat milling and Chinese steamed bread processing

To investigate the fate of deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) during wheat milling and Chinese steamed bread (CSB) processing, Fusarium-contaminated wheat samples containing DON and D3G at five different levels were milled and processed to CSB. Mycotoxin levels in milling fractions, doughs and CSB were determined with UPLC-MS/MS. Concentrations of DON and D3G were 1.2–2.2 times and 2.9–4.4 times higher in bran than that in wheat grain and they were slightly lower in shorts compared to bran. Reductions of 79–90% for DON and 23–39% for D3G in flour were observed, respectively, compared to wheat grain. With respect to wheat grain, the distribution of DON was 35% in bran, 27% in shorts, and 9% in flour, and the distribution of D3G was 77% in bran, 58% in shorts, and 37% in flour. Milling decreased the total amount of DON but increased the amount of D3G, which may result from the binding of DON to starch during the milling process. DON levels approximately doubled when the mixed and fermented dough was processed into CSB. D3G concentrations in mixed and fermented doughs and in CSB did not differ significantly, but were almost 50% lower than in flour. Dough making decreased the amount of D3G and steaming increased the amount of DON. CSB processing may thus release bound DON in flour.     

Mycotoxin occurrence in grains and the role of postharvest management as a mitigation strategies. A review

Mycotoxins are poisonous compounds produced by certain species of fungi found in contaminated grain. There are five major groups of mycotoxins which can occur in grains: Aflatoxin, fumonisin, deoxynivalenol (DON), ochratoxin (OT), and zearalenone (ZEN). Their occurrence may start in the field, harvesting, handling, storage, and processing. DON, ZEN, and fumonisins may start to cause the grains at the field/or pre-harvest while aflatoxin and OT are mostly occurring during storage due to improper postharvest handling. Most of the grains susceptible to mycotoxins such as maize, peanut/groundnut, sorghum, millet, wheat, and rice were reviewed. The main postharvest factors for the cause of grain mycotoxin contamination are mechanical injury, insect infestation, time of harvesting, drying method, types of storage structure and conditions, handling and processing. Temperature, moisture and humidity are the main factors for the growth and development of mycotoxins. Developing countries especially African are more vulnerable for the causes due to lack of well-established infrastructures, regulations, and standards. Postharvest mitigation strategies are an important and cost-effective method to control the cause. The core grain postharvest interventions used as mitigating strategies of mycotoxin includes rapid and proper drying, postharvest insect control, proper transportation and packaging, good storage conditions, use of natural and chemical agents and irradiation. Grain processing such as sorting, cleaning, milling, fermentation, baking, roasting, flaking, nixtamalization and extrusion cooking are also reported to reduce mycotoxin concentration. In general, system approach to good manufacturing practice and HACCP based implementation are important to mitigate mycotoxins in grains.     

Fusarium mycotoxins in wheat samples harvested in Serbia: A preliminary survey

The objective of this study was to determine the occurrence of trichothecenes of both the A-type and B-type, masked mycotoxin derived from DON - deoxynivalenol-3-glucoside (DON-3-Glc), 3- and 15-acetyldeoxynivalenol (ADONs), fusarenon-X (FUS-X) and nivalenol (NIV)) as well as zearalenone (ZON) in winter wheat. Total of 54 samples were collected during the harvest of 2007 representing the most important Serbian wheat-growing regions. The samples were prepared by one-step simple method and analyzed by high performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The obtained recoveries proved that the used method could be successfully applied for multi-component analysis of Fusarium mycotoxins. DON, DON-3-Glc and HT-2 contents were detected approximately in 28%, 13% and 6% of the total number of samples, respectively. The amount of these toxins ranged from 17 μg/kg for DON-3-Glc to 309 μg/kg for DON. ADONs, FUS-X, NIV, T-2 toxin as well as ZON were below the limit of detection. Different susceptibility of wheat cultivars towards detected mycotoxins was observed. The results were compared to the EC Regulative and with available the literature data concerning the neighboring countries. This is first report on the simultaneous presence of 8 Fusarium mycotoxins in the wheat cultivated in the Balkan Countries region.     

A minor survey of deoxynivalenol in Fusarium infected wheat from Yangtze–Huaihe river basin region in China

Deoxynivalenol (DON) is frequently present in infected cereals such as wheat, barley and maize. Fusarium asiaticum and Fusarium graminearium are the key fungal species which produces DON, when they infect wheat in China. Our present study is to do a minor survey on the presence of DON in the highly infected wheat regions from Jiang su and An hui province, China, harvested during the year 2010. A total of 56 wheat samples which are naturally infected by Fusarium sp., of which 35 samples from Jiang su province and 21 samples from An hui province were taken up for the study. The samples were analyzed for the presence of DON by using high performance liquid chromatography (HPLC) combined with ultraviolet detection. The recoveries of DON from the spiked samples (500–15,000 μg/kg) were obtained in the range of 85.7–90.2%, with the average relative standard deviation of 2.7%. The average percentage of contamination in all the samples was 89.3%, with the DON concentration ranging between 259 and 4975 μg/kg, and with the mean of 1962 μg/kg. According to the Chinese standard limits for DON, nearly 70% of the samples were above the maximum tolerable level of 1000 μg/kg. However, only 44% of the samples exceeded the European commission limits for unprocessed wheat of 1750 μg/kg. Regions where there was higher level of Fusarium sp. contamination due to climatic changes like heavy rainfall with high humidity, showed the presence of higher levels of DON in the samples. Cropping of Fusarium resistant wheat cultivars like Sumai 3 in the regions where the wheat is highly susceptible for the Fusarium contamination could help in the reduction of contamination in wheat.     

PCR detection of Fusarium fungi with similar profiles of the produced mycotoxins

The real-time PCR-based system has been developed for the detection and quantification of the most common Fusarium species with the similar profiles of mycotoxins they can potentially produce. Corresponding group-specific primers for various Fusarium fungi have been designed based on the translation elongation factor 1α (tef1α) gene sequences. The specificity of the selected primers and TaqMan probes has been tested with 26 single-spore isolates of seven most common Fusarium species and 21 wheat and barley grain samples. PCR results were analyzed relative to the most common mycotoxin deoxynivalenol (DON) content in infected grain samples.     

Near infrared spectroscopy for determination of mycotoxins in cereals

The use of near infrared transmittance instrumentation for determination of the mycotoxin deoxynivalenol (DON) in wheat kernel samples was investigated in a Nordic pilot project. Dilution series of wheat field infected with Fusarium culmorum was used in the calibration trials. Principal component analysis and partial least square regression calculations showed that the number of factors required to predict DON was 11–13 depending on wavelength range used. The best regression model was obtained for the wavelength range 670–1100 nm with a slope of 0.949, a correlation coefficient of 0.984 and a standard error of 381 μg DON per kg. The results indicate that it may be possible in the future to develop a calibration model, which can be used to screen for DON in wheat at concentrations just above the proposed European limits for wheat flour.     

Natural occurrence of zearalenone in Tunisian wheat grains

Fusarium mycotoxins are worldwide occurring in cereals and they are frequently reported in fresh or stored grains. Cereals represent a staple food for the Tunisian population; it therefore has a high social, economic and nutritional relevance. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by a variety of Fusarium fungi in temperate and warm countries. Fungi-producing ZEN contaminates corn, barley, wheat, sorghum and rice. A total of 205 samples of wheat were collected during the harvest year of 2010 from the major cropping areas in Tunisia and they were analyzed for zearalenone contamination. The aim of this study was to investigate for the first time the presence of ZEN in widely-consumed cereals in Tunisia, especially durum and tender wheat, to compare the levels of contamination by ZEN with the European norms and to suggest some factors that can promote the production of ZEN in Tunisia. To perform this study, we developed and validated in our laboratory conditions an HPLC method for quantitative analysis of ZEN in solid cereal samples. Our results showed that the incidence of ZEN contamination was 75%. The levels of contamination determined in the positive samples ranged between 3 and 560 μg/kg with a mean value of 60 μg/kg. These important amounts of ZEN in wheat can be attributed to the Tunisian climate, warm temperature and prolonged wetness witch are favor to Fusarium growth and mycotoxin production during the cultivation and the final ripening period of wheat grains.     

Fusarium-inoculated wheat: deoxynivalenol contents and baking quality in relation to infection time

Cereals contaminated by Fusarium spp. and their mycotoxins, e.g. deoxynivalenol (DON), are not only a risk to human and animal health but also show reduced technological properties. This study examines the influence of high Fusarium infection levels of wheat on its baking quality in relation to infection time. Nearly uninfected wheat, wheat inoculated with a spore-mixture of different Fusarium spp. at the time of flowering, and wheat inoculated with spores of Fusarium graminearum at different growth stages were investigated. Extension and baking tests, HPLC analyses of gluten proteins, and DON determination were performed as well as X-ray absorption spectroscopy to analyse the sulfur speciation of gluten proteins on a molecular level. Our results indicate that high Fusarium infection levels accompanied with high DON contents did not necessarily deteriorate baking quality. This finding was independent of infection time.     

Limited survey of deoxynivalenol in wheat from different crop rotation fields in Yangtze-Huaihe river basin region of China

The food toxin, deoxynivalenol (DON) is frequently present in cereals such as wheat, barley and maize, which are infected by Fusarium asiaticum and Fusarium graminearium. Crop rotation and climatic conditions play a major role in the Fusarium infection in wheat. In this present study, a minor survey was conducted to find out the impact of crop rotation affecting the Fusarium infection leading to DON contamination in wheat samples from selected infected regions of Jiang su and An hui provinces, China, especially during harvest period in 2012. A total of 84 wheat samples from the highly Fusarium infected region were collected, of which 30, 8, 39 and 7 samples were from the fields where cotton, corn, rice, and soya bean was a rotation crop, respectively. DON concentration in wheat sample was analyzed by high performance liquid chromatography (HPLC) combined with ultraviolet detection. DON contamination in the wheat samples was 95% with the mean DON concentration of 3881.2 μg/kg from both the regions. Average DON contamination in wheat samples from cotton, soya beans, corn, and rice rotation fields were 2067.5, 2853.6, 3517.5, and 4899.3 μg/kg, respectively. Nearly 34% of the wheat samples from the cotton rotation fields were below the DON maximum tolerable limits of EU (1750.0 μg/kg) and only 3% samples were above 4000.0 μg/kg DON concentration. However, the average DON contamination level was the highest in wheat samples (4899.3 μg/kg) where rice was a rotation crop, with 64% of the samples were above 4000.0 μg/kg. The present study shows cotton could be a promising rotation crop in the regions where wheat is more prone to the infection of Fusarium sp, which may minimize the economic loss to the farmers in wheat.     

Occurrence of deoxynivalenol in durum wheat in Tunisia

Wheat is frequently contaminated by the deoxynivalenol (DON) which is a member of the trichotecene family, the most important group of mycotoxins produced by the Fusarium moulds. As Tunisian population is a big consumer of cereals mainly durum wheat, human exposure to DON can be, consequently high. This survey was performed to study the occurrence of DON in Tunisian durum wheat area during the crop of 2007. A total of 65 samples of durum wheat from five cultivating locations in the North of Tunisia, the major cropping area, were analysed. To detect and to quantify the mycotoxin DON, an efficient HPLC/UV method was developed, including immunoaffinity step for DON extraction from durum wheat followed by liquid chromatography (LC) for quantification. As DON is a water soluble toxin, the extraction procedure from wheat samples was performed using water. Samples were centrifuged then passed through the immunoaffinity columns. After column’s washing, the toxin was slowly eluted by methanol. Wheat sample extracts were injected to the LC system set at a wavelength of 220 nm. From 65 samples, 83% showed DON contamination with averages ranging from 12.8 ± 5% to 30.5 ± 13.3% μg/g exceeding the maximum permitted limit of 1.75 μg/g set by the European Commission in wheat.     

Effect of deoxynivalenol detoxification by ozone treatment in wheat grains

Considering the wide existence of mycotoxin deoxynivalenol (DON) in wheat and derived products, finding ways to detoxify DON in wheat grains as original resources for food-chain contamination is very important to protect human health and reduce economic losses. This study aimed to evaluate the effectiveness of destroying DON in wheat via ozonation under different conditions, such as moisture content (MC), ozone concentration, exposure time, and form of raw-material. The achieved data showed that DON reduction significantly improved with increased ozone concentration and exposure time. The whole wheat flour (WWF) was easier to degrade than the wheat kernels. Higher MC decreased larger amounts of DON. The maximum reduction of DON was 20.10% for the MC group, compared with the 11.79% and 16.29% for the other MC groups. DON concentration decreased from 3.89 mg/kg to 0.83 mg/kg under the generally recognized Maximum Residue Limit (MRL) level of 1 mg/kg, when WWF was treated with 100 mg/L of ozone treatment for 60 min. The first-order kinetic model established in this work showed a good R-squared value (R² > 0.877) and was consistent with the results. Therefore, ozonation is an effective and rapid way to degrade DON in wheat, especially in whole wheat flour.     

Principal mycotoxins in wheat flour from the Serbian market: Levels and assessment of the exposure by wheat-based products

The presence of eleven principal mycotoxins from the wheat flour bought in supermarkets in Novi Sad, the capitol of the northern Serbian province of Vojvodina, was determined. The samples were prepared by simple one-step method and analyzed by ultra-high performance liquid chromatography with heated-electrospray ionization triple quadrupole mass spectrometry (UHPLC/HESI-MS/MS). Deoxynivalenol (DON) was the predominant mycotoxin for all analyzed samples followed by zearalenone (ZON) and T-2 toxin, with frequency of occurrence: 88.7%, 33.3% and 26.7%, respectively. Aflatoxins (AFs), ochratoxin A (OTA), HT-2 toxin, fumonisins B1 (FB1) as well as B2 (FB2) were below the limit of detection. All the samples complied with current European/Serbian legislation, except one sample that exceeded the DON maximum level of 750 μg/kg. In addition, mycotoxin intakes through consumption of wheat-based products were estimated for average adult consumers based on Serbian market basket and then compared with the tolerable daily intake (TDI) proposed by Scientific Committee on Food of the European Union. The calculated intakes of ZON and T-2 were lower than the respective TDIs. However, intakes of DON were assessed to be close to the level of TDI for adults. This is the first study on the intake assessment for mycotoxins present in the wheat flour through the consumption of wheat-based products on the Serbian market.     

Occurrence and risk assessment of mycotoxins in subsistence farmed maize from Zimbabwe

Maize is the staple food of Zimbabweans and is consumed daily in the majority of households, particularly subsistence farming households. The objectives of this study were first, to determine the occurrence of mycotoxins in maize produced and consumed by subsistence farmers in Zimbabwe and second, to determine mycotoxin exposure through maize consumption and subsequently the human health risk. A total of 95 maize meal samples were collected from the household stores of randomly selected subsistence farming households. Maize intake data and agronomic practices of these households were investigated A multi-mycotoxin LC-MS/MS method was used to analyze and quantify mycotoxin contamination in the maize samples. Mycotoxin contamination was compared across agro-ecological zones in order to determine differences in mycotoxin contamination levels and presented. Of the toxicologically relevant mycotoxins, aflatoxin B1 (AFB1), fumonisin B1 (FB1), FB2, deoxynivalenol (DON) and zearalenone (ZEN) were detected in 1, 95, 31, 24 and 15 % of the samples at mean levels of 11, 242, 120, 217 and 110 μg/kg respectively. Other mycotoxins detected in the maize were 15-acetyl-deoxynivalenol (15-ADON), nivalenol (NIV), FB3, alternariol-methylether (AME), AFB2, AFG1 and diacetoxyscirpenol (DAS) and the percentage contamination ranged between 1 and 4 % in the maize samples. Contamination of the maize by the mycotoxins was observed at minimum levels below limit of detection for each mycotoxin and maximum levels of 105, 530, 67, 108, 3, 4 and 14 μg/kg for 15-ADON, NIV, FB3, AME, AFB2, AFG1 and DAS respectively. The median levels of each mycotoxin were reported below the limit of detection, with the exception of FB1 (median, 146 μg/kg), which was further considered in the exposure and risk assessment. Dietary exposure was derived from combining mean maize intake data and median FB1 contamination. Mean maize intake was estimated to be 26.8, 37.2, 30.1, 15.8 and 15.0 g/kg bodyweight (bw)/day for under 5s, children, adolescents, adults and the elderly respectively. Subsequently FB1 exposure from maize was calculated, to be 3.91, 5.40, 4.40, 2.30 and 2.20 μg/kg bw/day for these populations. Exposure to FB1 through maize intake was observed to equate to 196, 272, 220, 115 and 110 % of the provisional maximum tolerable daily intake (2 μg/kg bw/day) for under 5s, children, adolescents, adults and the elderly respectively. Subsistence farming communities in Zimbabwe are at risk of high exposure to FB1 and the risk was highest for under 5s, children and adolescents respectively.     

Gaseous allyl isothiocyanate to inhibit the production of aflatoxins,beauvericin and enniatins by Aspergillus parasiticus and Fusarium poae in wheat flour

There is a growing concern about the presence of mycotoxins in foods, since up to 25% of cereals and cereal foods are contaminated with these compounds. Moreover, the general public is against the use of synthetic preservatives in foods and the use of natural antimicrobials in foods is a current trend. Allyl isothiocyanate (AITC) is a volatile antimicrobial derived from oriental and black mustard. The objective of this work was to evaluate the capacity of gaseous AITC in inhibiting the production of mycotoxins by Aspergillus parasiticus (aflatoxin producer) and Fusarium poae (beauvercin and enniatin producer) in wheat flour. Petri dish lids filled with 2 g of wheat flour were inoculated with 10⁴ conidia/g of A. parasiticus or F. poae and placed in a 1 L mason jar. AITC was added at 0.1, 1 or 10 µL/L in the gaseous phase. Jars were hermetically closed and kept at 23 °C for 30 d. Mycotoxins were identified and quantified by LC-MS/MS. Even 0.1 µL/L of AITC was able to produce 6.9–23% reduction of mycotoxin production. In general, synthesis of aflatoxins and beauvericin was more affected than enniatins. The use of AITC at 10 µL/L completely inhibited the production of all mycotoxins for 30 d. AITC at low doses could be added to flour packages in order to inhibit the production of potentially dangerous mycotoxins.     

Evaluation of an extraction method and spin column cleanup procedure for Fusarium mycotoxins and their masked derivatives from grain matrix

Fusarium mycotoxins in foodstuffs at levels in violation of allowed legal limits belong to the most frequently occurring type of food contamination. Out of the large variety of known mycotoxins, deoxynivalenol (DON), zearalenone (ZEN), and the T-toxins are the most prevalent and are responsible for extensive and recurring economic damage. For ensuring consumer safety, it is required to continually identify faster, simpler and more reliable analytical methodologies. In the present work, miniaturized centrifugation (spin) columns for the extraction of mycotoxins from grain samples were investigated, with a focus on matrix removal, applicability of different extraction solvents, and recovery of mycotoxin. The method was further extended to the extraction of naturally occurring DON and ZEN conjugates (masked mycotoxins). The spin column method was observed to routinely provide a total matrix reduction of >90% from typical wheat samples in a single centrifugation step. The use of H₂O/ACN (20/80; v/v) as an extraction solvent provided non-masked DON, ZEN and T-toxin recoveries of greater than 80%. For the masked DON and ZEN conjugates (DON-3-glucoside and ZEN-14-sulfate), recoveries were lower. Overall, the evaluated miniaturized spin column procedure enabled rapid loss-free extraction of unconjugated mycotoxins from conventional grain matrix and a significantly reduced matrix load in the extracts.     

Retention of deoxynivalenol and its derivatives during storage of wheat grain and flour

Fate of deoxynivalenol (DON) and its derivatives—deoxynivalenol-3-glucoside (D3G), 15-acetyldeoxynivalenol (15-AcDON) and 3-acetyldeoxynivalenol (3-AcDON) during six-month storage of wheat grain and flour was investigated. Wheat grain and flour polluted by DON, D3G, 15-AcDON and 3-AcDON were packaged in polyethylene bags, kraft paper bags and cloth bags, and stored at room temperature and 4–6 °C, and mycotoxin levels were analyzed by UPLC-MS/MS after storing for 30, 90 and 180 days. Levels of DON, 15-AcDON and 3-AcDON in wheat grain showed a generally decrease during the storage duration, and DON concentrations averagely decreased by 40–50%. An obviously increase of DON (more than 70%) and decrease of 15-AcDON and 3-AcDON were observed after storage in wheat flour. There were no significant differences of D3G levels in most of wheat grain and wheat flour samples after storage, but some increased dramatically with retention level up to 240%. The results suggested conversion of DON and its derivatives may occur during wheat grain and flour storage.