Stability of the mycotoxin deoxynivalenol (DON) during the production of flour-based foods and wheat flake cereal

Deoxynivalenol (DON) is a mycotoxin found in cereal grains and cereal-based foods. DON concentrations in finished products are reduced under some processing conditions, but not others. DON concentrations in flour, wheat and selected foods made from them under commercially relevant conditions were compared by GC with electron capture detection. Average concentrations (n?=?9/item) in cookies, crackers and pretzels ranged from 61% (cookies) to 111% (pretzels) compared with flour (100%?=?0.46?µg?g^?1). Lesser amounts were found in donuts and bread: their respective DON concentrations were 44% and 30% that of flour. Mass balance estimates for DON (µg?g^?1 flour equivalents) ranged from 50% (bread?=?0.23?µg?g^?1 flour equivalents) to 120% (donuts), indicating that dilution by recipe ingredients contributed to DON reductions in bread and accounted for all of the apparent reduction in donuts. Mass balance estimates averaged 76% (crackers) to 107% (pretzels) for the other flour products. DON concentrations were higher in cereal flakes (0.55?µg?g^?1 in the finished product and 0.58?µg?g^?1 on a mass balance basis) than in wheat (0.40?µg?g^?1), suggesting that DON concentrations might increase during processing of wheat cereals under some conditions. In summary, DON concentrations of finished food products were reduced?≥50% only in bread and donuts. Reduction in bread resulted from a combination of DON ‘loss’ and dilution by recipe ingredients whereas the reduction in donuts was due entirely to dilution. These results are further evidence of DON stability during the preparation of popular flour or wheat-based products.     

粮油食品中呕吐毒素危害及风险分析

该文分析呕吐毒素性质,在自然界和粮油食品中存在情况,及对人类和动物危害,并进行风险评估,同时提出预防呕吐毒素危害建议。     

小麦脱氧雪腐镰刀菌烯醇（DON）快速筛查方法的研究

基于脱氧雪腐镰刀菌烯醇(DON)产毒菌镰刀菌形态特征,以其大型分生孢子为检测目标物,初步建立一种新的小麦DON筛查方法。样品直接用水振荡提取,100目滤布过滤,滤液于显微镜下镜检。检测时间约3 min。对不同感染水平的小麦阳性样品进行镰刀菌大孢子检验(n=6),方法相对标准偏差(RSD)为7.1%~21.6%。对我国小麦主产区的72个小麦样品进行检验,用HPLC法作对照。结果表明,样品中有46个未检出镰刀菌大孢子,占样品总数的63.9%,这些样品DON含量为0~792.4μg/kg,均未超过国家限量标准1 000μg/kg;有26个样品检出大孢子,DON未超标为6个,但均有检出,其余大部分样品镰刀菌生长量与其产生DON的产量呈正相关。由此可见,通过本方法,可快速的对批量小麦DON超标与未超标样品进行筛查区分。     

Changes in available methionine and tryptophan contents during cereal flake production

The changes in available methionine and tryptophan contents during industrial production of wheat, rye, barley and oat flakes have been investigated. The contents of available methionine and tryptophan were lowered significantly (p = 0.05) under conditions of technological processing. The total decrease in available methionine ranged from 13 to 26% and tryptophan from 10 to 20%. Hydrothermal treatment (120 °C, 60 min, 1.99 × 10⁵ Pa) of whole cereal grain had a considerable impact on the damage to available methionine, but the highest decrease in tryptophan was determined after the flaking process (70 °C, 0.3 mm gap). The total decrease in these amino acids correlates significantly (p = 0.001) and positively with values for lipid oxidation products, expressed on a total lipid basis, in cereal flakes as well as in untreated cereal grains. Available methionine and tryptophan contents in proteins of cereal flakes equalled or exceeded requirements for adults recommended by the FAO 1985 reference pattern. © 2000 Society of Chemical Industry     

Cross-reactivity features of deoxynivalenol (DON)-targeted immunoaffinity columns aiming to achieve simultaneous analysis of DON and major conjugates in cereal samples

Immunoaf?nity columns (IACs) are a well-established tool in the determination of regulated mycotoxins in food and feed commodities. However, they also have the potential to become attractive pre-concentration and clean-up materials for the determination of masked (also called modified) mycotoxins, which have been recognised as important contributors to the toxicological hazard deriving from fungal spoilage of goods. However, the information available in the literature concerning the cross-reactivity of DON-IACs against the major conjugates (DON-3-G, 15-AcDON and 3-AcDON) is incomplete and often contradictory. We have carried out a detailed characterisation of the cross-reactivity of the four main IACs brands against DON and its conjugates as well as an assessment of the competition among the analytes. Only one IAC enabled the simultaneous analysis of all relevant DON forms while two missed 15-AcDON and the fourth one missed DON-3-G and 3-AcDON. In the case of the multivalent IAC, the analytes modified at the C-3 position compete for the antibody binding with preference for 3-AcDON (less spatially hindered) while DON-3-G has the more-hindered access to the active sites. Taking into consideration the levels of DON conjugates existing in real samples, the cross-reactivity of one DON-IAC allows a quantitative analysis of all of these analytes. Important but rather neglected aspects such as the continuous supply of IACs with identical characteristics, and of columns which are strictly blank, are also addressed in this paper.     

室温下麦粒中脱氧雪腐镰刀菌烯醇(DON)及其隐蔽型(D3G)毒素累积分析

为了研究麦粒中脱氧雪腐镰刀菌烯醇（DON）及其隐蔽型脱氧雪腐镰刀菌烯醇-3-葡萄糖苷（D3G）的累积情况,将禾谷镰刀菌菌株F-1和5035分别人工接种至经高温灭菌、8 k Gy和15 k Gy辐照的麦粒中25℃培养7、14、21、28、35 d后,通过LC-MS/MS方法测定DON和D3G毒素在麦粒中的含量。不同处理的麦粒中DON含量均随培养时间延长逐渐增加;禾谷镰刀菌接种灭菌麦粒及15 k Gy辐照麦粒均未检出D3G毒素;禾谷镰刀菌接种8 k Gy辐照小麦籽粒后检测到隐蔽型毒素D3G,培养7 d时D3G最高且其含量随着培养时间延长逐渐降低;本文使用指数模型拟合25℃下F-1与5035接种麦粒（8 k Gy辐照）中D3G/DON的比值与时间关系的R2分别为0.97、0.93,R2均在0.9以上,说明指数模型能够较好地拟合本实验条件下时间与D3G/DON的比值的变化,为进一步后续研究提供依据。      

Analysis and occurrence of deoxynivalenol (DON) in cocoa

The primary objective of this study was to establish a current situation assessment of the possible occurrence of deoxynivalenol in cocoa and cocoa products. Since there was no analytic method for determining DON in cocoa and cocoa products, a special method was developed. The applicability and consistency of the method was confirmed by performing recovery assays on various cocoa products. A special post-column derivatisation procedure was used to increase selectivity and raise sensitivity by a factor of 80. The method’s limit of detection (LOD) was thereby reduced to 7 μg/kg; the limit of quantification (LOQ) was 14 μg/kg. The method was used to test 230 samples for possible DON content, ranging from cocoa beans to cocoa bean shells, nibs, cocoa liquor and cocoa powders through to finished cocoa-based products. In the case of cocoa beans and cocoa bean shells, DON content close to the detection limit was only determined in isolated cases. No DON content was detected in nibs, cocoa liquor, cocoa powders and finished cocoa-based products. Analogous to ochratoxin A and aflatoxins, the results show DON is more likely to be found in cocoa bean shells. Separation of shells during cocoa processing can reduce potential DON contents. Since no DON was determined in the fractions relevant for chocolate production, these assays show it does not represent a considerable issue for the cocoa and chocolate industry.     

Stability of deoxynivalenol in heat-treated foods.

The effects of high-temperature and -pressure processing of foods spiked with deoxynivalenol (DON) were examined. In extruded corn grits, extruded dry dog food, and autoclaved moist dog food, there were no significant reductions (P < 0.05) in DON after processing. Autoclaved cream-style corn showed a reduction in DON of only 12%. Overall, DON was stabile to the high temperature and pressure processes tested. The use of an alpha-amylase in the extraction method for analysis by an enzyme-linked immunosorbant assay (ELISA) improved the recovery of DON from the spiked extruded and autoclaved products by as much as 26% over the standard ELISA method.     

Ozone treatment to reduce deoxynivalenol (DON) and zearalenone (ZEN) contamination in wheat bran and its impact on nutritional quality

Wheat bran is an important source for human and animal feed. Its nutritional aspects include a high content of fibre and minerals, as well as phenolic compounds that help prevent chronic diseases. However, wheat can be susceptible to contamination by fungus, which can produce mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEN), causing adverse health effects. Therefore, methods should be developed to reduce possible contamination. Ozone can be used for this purpose as it is considered safe and environmental friendly. The aim of this study was to evaluate the reduction of DON and ZEN concentrations in wheat bran using the ozonation process as well as to evaluate the effect of ozonation on the nutritional quality of bran. Considering this, wheat bran naturally contaminated with both DON and ZEN was processed using ozone at different conditions. The nutritional quality of the bran was evaluated after processing considering the following aspects: the total phenolic content and the bran antioxidant capacity (by using both DPPH and ABTS radicals). The results showed that the degradation of ZEN was higher and faster than the degradation of DON, which could be explained by their molecular structures. The total phenolic content and antioxidant capacity of the bran were not affected by the ozonation process, which is preferable from a nutritional point of view. Therefore, ozonation was demonstrated to be a possible method for reducing mycotoxins in wheat bran, although more studies are needed in order to better understand and optimise processing and product quality.     

Effects of oral exposure of pigs to deoxynivalenol (DON) sulfonate (DONS) as the non-toxic derivative of DON on tissue residues of DON and de-epoxy-DON and on DONS blood levels

The Fusarium toxin deoxynivalenol (DON) is of outstanding importance in pig nutrition because of its frequent occurrence in cereal grains at levels high enough to cause adverse effects such as a decrease in feed intake and impairment of the immune system. Thus, simple decontamination procedures would be useful. The present study aimed to examine the effects of wet preservation of triticale contaminated with DON and zearalenone (ZON) with sodium metabisulphite (SBS) on the treatment-related non-toxic derivative of DON (DON-sulfonate, DONS), and on ZON and its metabolites in blood and various physiological specimens of piglets. The uncontaminated control triticale (CON) and the DON-contaminated triticale (FUS) were included in the diets either untreated or SBS treated (CON-SBS, FUS-SBS) and fed to piglets for 28 days starting from weaning. The diet concentrations for DON were 0.156, 0.084, 2.312 and 0.275 mg kg^?1, for DONS were <0.05, <0.05, <0.05 and 1.841 mg kg^?1, and for ZON were <0.001, 0.006, 0.017, and 0.016 mg kg^?1 for each of CON, CON-SBS, FUS and FUS-SBS, respectively. DONS was present in the blood of piglets fed the FUS-SBS at a median concentration of 15.5 ng ml^?1 (3–67 ng ml^?1), while the median DON concentration amounted to 2 ng ml^?1 (0–5 ng ml^?1) at the same time. The median DON concentration in the blood of piglets fed the FUS diet reached a median concentration of 10.5 ng ml^?1 (5–17 ng ml^?1). Moreover, the relative differences between the DON concentrations in other physiological specimens (muscle, liver, kidney, bile and urine) in piglets fed the FUS-SBS and the FUS diet were comparable with the blood DON concentration differences. Although these differences can be taken as an indication for DONS stability after absorption and distribution further studies examining DONS in these other physiological specimens directly are necessary to substantiate this conclusion. Moreover, ZON and α-zearalenol could only be detected in bile and urine where their levels were not influenced by the SBS treatment.     

Quantitative dietary exposure assessment of the Catalonian population (Spain) to the mycotoxin deoxynivalenol

The mycotoxin deoxynivalenol (DON) is one of the most common contaminants of cereals worldwide, and its occurrence has been widely reported in raw foods and foodstuffs, around the European region, including Catalonia. In the present work, a stochastic methodology has been applied to accurately assess the exposure of the Catalonian population (Spain) to DON through food consumption. Raw contamination data was provided by a large survey conducted in this region, in addition to the raw consumption data from a nutritional study specifically designed to assess the dietary intake of the main foodstuffs related to DON contamination for all population age groups. Contamination and consumption data were combined by simulation using an essentially parametric (P-P) method. The P-P method draws sampling values from distribution functions fitted to consumption and contamination data sets. Moreover, to quantify the accuracy and reliability of the statistics estimates, we built the related confidence intervals using a pseudo-parametric bootstrap method. Considering the results drawn from the P-P simulation method, the Catalonian population should be expected to be exposed at moderated levels of deoxynivalenol, the infants and individuals with ethnic dietary patterns being the most exposed population groups.     

Quantitative dietary exposure assessment of the Catalonian population (Spain) to the mycotoxin deoxynivalenol

The mycotoxin deoxynivalenol (DON) is one of the most common contaminants of cereals worldwide, and its occurrence has been widely reported in raw foods and foodstuffs, around the European region, including Catalonia. In the present work, a stochastic methodology has been applied to accurately assess the exposure of the Catalonian population (Spain) to DON through food consumption. Raw contamination data was provided by a large survey conducted in this region, in addition to the raw consumption data from a nutritional study specifically designed to assess the dietary intake of the main foodstuffs related to DON contamination for all population age groups. Contamination and consumption data were combined by simulation using an essentially parametric (P-P) method. The P-P method draws sampling values from distribution functions fitted to consumption and contamination data sets. Moreover, to quantify the accuracy and reliability of the statistics estimates, we built the related confidence intervals using a pseudo-parametric bootstrap method. Considering the results drawn from the P-P simulation method, the Catalonian population should be expected to be exposed at moderated levels of deoxynivalenol, the infants and individuals with ethnic dietary patterns being the most exposed population groups     

Influence of temperature and pH on the degradation of deoxynivalenol (DON) in aqueous medium: comparative cytotoxicity of DON and degraded product

Deoxynivalenol (DON), a toxic fungal metabolite, is stable under different processing conditions; however, its stability in aqueous medium at different temperatures and low pH (1–2) (present in the gastrointestinal tract) has not been investigated. In the present study, DON standard was used to study the influence of temperature and pH on DON stability in aqueous medium, the characterisation of the degraded product, and the comparative toxicity profile of the degraded and the parent compound. The results suggest that standard DON was unstable at 125–250°C showing 16–100% degradation whereas DON at pH 1–3 had 30–66% degradation, with a concomitant increase in the formation of a degraded product. Further ESI-MS characterisation of the dominant precursor ion of the HPLC eluate of the DON-degraded product was found to be m/z 279, resembling the known metabolite DOM-1. The degraded product of DON was reconfirmed as DOM-1 by comparison with standard DOM-1 and both gave a similar λ_max at 208 nm. Comparative studies of both standard DOM-1 and the degraded product of DON showed no cytotoxicity up to 6400 ng ml^–1 while significant cytotoxicity was observed for DON (400 ng ml^–1). The results suggest that a highly acidic environment (pH 1–2) could be responsible for the de-epoxydation of DON leading to the formation of DOM-1.     

酸面团和谷物发酵对面制食品营养品质的影响

酸面团发酵可改善焙烤及蒸煮食品的风味、质构,并延长食品的货架寿命,谷物发酵也可提高食品的营养品质及功能特性。酸面团可以提高全麦粉、富含纤维或无面筋产品的食用品质,有效抑制淀粉消化率从而降低血糖、调节生物活性成分的含量和生物可利用性、提高矿物质的生物利用效率,谷物发酵过程中面筋蛋白的降解可以降低或减少小麦中过敏原的含量。该文从营养价值提高及功能特性改善方面综述了酸面团及谷物发酵对面制食品的作用及机理。     

Effects of the mycotoxin deoxynivalenol on human primary hepatocytes

Toxic effects of the mycotoxin deoxynivalenol (DON) observed in animals range from diarrhea, vomiting, gastro-intestinal inflammation to necrosis of several tissues. In the last years, DON has been tested in hepatocytes of several animal species for its cytotoxicity. However, these tests are limited to the use of animal cells. No studies using human hepatocytes are available. Further investigations with the human hepatocellular liver carcinoma cell line HepG2 might be limited due to the disadvantages of cell lines (e. g. immortalization, tumor derivation, longtime cultivation) and do not necessarily reflect the response of normal human cells. In order to overcome this problem and to be closer to the human situation, we studied the effect of DON in human primary hepatocytes and compared these data to the effects in the HepG2 cell line. Cell viability, apoptotic and necrotic cell death, albumin secretion and metabolic activity were determined. It could be demonstrated that DON has a distinct cytotoxic effect on human primary hepatocytes. Viability, protein content and albumin secretion were reduced in a dose-dependent manner. The apoptotic key enzyme caspase-3 was activated, while LDH release occurred only after long incubation time due to a secondary necrosis. Furthermore, we studied the metabolism of DON using LC-MS/MS. DON was neither metabolized by primary hepatocytes cells nor by the HepG2 cell line.     

Assessment of deoxynivalenol (DON) adsorbents and characterisation of their efficacy using complementary in vitro tests

Deoxynivalenol (DON) is a prevalent and resistant mycotoxin found in cereals and related products. Adsorbents appear to provide an opportunity to decrease DON absorption in animals but, due to their specificity, it is very difficult to evaluate their actual efficacy. It is pointless to extrapolate results obtained with one mycotoxin to another and even to extrapolate results obtained in vitro in buffer to an in vivo situation. We carried out experiments to characterize the properties of potential DON adsorbents. Initial tests in buffer pH 7 allowed us to focus on six adsorbents: activated charcoal, cholestyramin, Saccharomyces cerevisiae mannans, algal β-glycan, fungal β-glycan and leguminous plant. The use of equilibrium sorption models suggested a non-saturated phenomenon and involved variable mechanisms according to the specific material. Subsequent tests with a Caco-2 cell model showed a high reduction in DON cytotoxicity on proliferative intestinal cells and DON absorption by differentiated intestinal cells when adsorbent was added (except for cholestyramin). Otherwise, values were not always in accordance with those obtained in buffer. Our work allowed us to identify five potential DON adsorbents and to propose a complementary in vitro test allowing improved determination of adsorbent properties.     

Effect of fermentation on naturally occurring deoxynivalenol (DON) in Argentinean bread processing technology.

The stability of naturally occurring DON was evaluated during the fermentation stage of the bread-making process on a pilot scale. Two different products, French bread and Vienna bread, were prepared with naturally contaminated wheat flour (150 mg kg(-1)) under controlled experimental conditions. Dough was fermented at 30, 40 and 50 degrees C according to standard procedures employed in Argentinean low-technology bakeries. When the dough was fermented at 50 degrees C, the maximum reduction was 56% for the Vienna bread, with French bread being reduced by 41%. DON reduction during bread-making occurs not only in the baker due to thermal decomposition, but also during the fermentation step. The Argentinean traditional bread-making process might reduce DON levels during the fermentation stages if the dough is leavened at temperatures > 30 degrees C.     

Stability and kinetics of leaching of deoxynivalenol,deoxynivalenol-3-glucoside and ochratoxin A during boiling of wheat spaghettis

The stability of deoxynivalenol (DON), deoxynivalenol-3-glucoside (DON-3-glucoside) and ochratoxin A (OTA) during spaghetti production and cooking was investigated. Initial mycotoxin concentration, boiling time and use of egg as ingredient were assayed as factors. DON was stable during kneading and drying, but a consistent reduction of DON (> 40%) was observed in boiled spaghettis. According to our results, DON was transferred to broth, where it was not degraded, and boiling time determined the extent of the transfer. A DON leaching model was fitted to data with a high goodness of fit (r² = 0.99). This model can be used for prediction of final DON concentration in cooked pasta, and a useful tool in risk assessment models. DON-3-glucoside is totally stable through the pasta making process; moreover DON-3-glucoside is slightly released from pasta components and it is leached to broth. Similarly, OTA is also stable during pasta making; however, it is scarcely transferred to broth during boiling. The presence of egg as ingredient did not affect the final mycotoxin concentration in pasta in any case.     

Cross-reactivity of some commercially available deoxynivalenol (DON) and zearalenone (ZEN) immunoaffinity columns to DON- and ZEN-conjugated forms and metabolites

Seven commercially available deoxynivalenol (DON) and zearalenone (ZEN) immunoaffinity columns (IACs) were tested for cross-reactivity to conjugated forms (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, DON-3-glucoside, DON-3-glucuronide, ZEN-glucosides, ZEN-glucuronide) and metabolites (de-epoxydeoxynivalenol, α-zearalenol, β-zearalenol) and nivalenol (NIV), using a semi-quantitative multi-mycotoxin ultra-performance liquid chromatography-tandem mass spectrometry method. The DON IACs showed cross-reactivity for nearly all DON derivatives tested. The ZEN IACs showed limited cross-reactivity to some of the ZEN derivatives. The IACs were evaluated for their potential use as sample clean-up for mycotoxins in serum.