Fungal Hydrophobins in the Barley‐to‐Beer Chain

Fungal hydrophobins have been shown to induce gushing of beer. In order to study the occurrence and fate of hydrophobins at different stages of the production chain of beer, barley samples artificially infected in the field with Fusarium culmorum, F. graminearum and F. poae were collected during the growing period as well as during various stages of the malting process. In addition, naturally infected malt was brewed in pilot scale and samples were collected throughout the process. The samples were assayed for hydrophobin content using an ELISA method. The results showed that fungi produced hydrophobins that accumulated during barley grain development in the field, but that production was more pronounced during malting. Prolonged storage of barley tended to reduce the ability of fungi to produce hydrophobins in malting. Studies on the fate of hydrophobins during the brewing process revealed that mashing released hydrophobins from the malt into the wort. Some loss of hydrophobins occurred throughout the brewing process with spent grains, cold break (wort boiling) and surplus yeast. In addition, the beer filtration step reduced hydrophobin levels. Despite the substantial loss of hydrophobins during brewing, the level was high enough to induce the gushing detected in the final beer.     

Species-specific PCR-based assays for the detection of Fusarium species and a comparison with the whole seed agar plate method and trichothecene analysis

Species-specific PCR was used for the identification of nine Fusarium species in pure mycelial culture. A PCR-based method was compared with the whole seed agar plate method and trichothecene analysis for three toxin-producing Fusarium species using 85 grain samples of wheat, barley, oat, corn and rye. A simple SDS-based DNA extraction system followed by potassium acetate precipitation resulted in consistent PCR amplification of DNA fragments from cultures and grain samples. The species-specific PCR assays correctly identified pure cultures of Fusarium avenaceum ssp. avenaceum (9 isolates), Fusarium acuminatum ssp. acuminatum (12 isolates), Fusarium crookwellense (7 isolates), Fusarium culmorum (12 isolates), Fusarium equiseti (11 isolates), Fusarium graminearum (77 isolates), Fusarium poae (10 isolates), Fusarium pseudograminearum (23 isolates), and Fusarium sporotrichioides (10 isolates). Multiplex PCR was developed for the simultaneous detection of F. culmorum, F. graminearum and F. sporotrichioides, the three most important trichothecene producing species in Canada. In grain samples, results of PCR assays for these same three species related well with whole seed agar plate method results and determination of Fusarium trichothecenes. The PCR assay described in this study can be used for routine detection and identification of Fusarium spp. in Canada.     

TRI12 based quantitative real-time PCR assays reveal the distribution of trichothecene genotypes of F. graminearum and F. culmorum isolates in Danish small grain cereals

Quantitative real-time PCR assays, based on polymorphisms in the TRI12 gene of the trichothecene pathway, were developed to identify and quantify the trichothecene genotypes producing 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) or nivalenol (NIV) in the Fusarium graminearum species complex, Fusarium culmorum, Fusarium cerealis and Fusarium pseudograminearum. These assays were applied on a total of 378 field samples of cereal grain of wheat, barley, triticale, rye and oats collected from 2003 to 2007 to study the trichothecene genotype composition in Danish cereals. The three genotypes, 3ADON, 15ADON and NIV were found in all five cereal species, great annual variation in the occurrence of the trichothecene genotypes was evident with considerable variation between the samples. 3ADON was the dominant genotype in barley, triticale, rye and oats while 15ADON was most dominant in wheat. The NIV genotype was found at low levels in most samples. Study of genotype composition within the Danish F. graminearum and F. culmorum population was based on principal component analysis (PCA). PCA revealed that the dominating genotype of F. graminearum in wheat is 15ADON. For barley, the PCA analysis indicated that the F. graminearum population consisted of all three genotypes, and in triticale, the F. graminearum population consisted mainly of 15ADON genotype. F. culmorum/F. cerealis showed correlation to the NIV genotype in wheat and triticale but not in barley. F. culmorum/F. cerealis also showed some correlation to 3ADON especially in wheat and triticale. Selected wheat and barley samples from 1957 to 2000 showed low amounts of F. graminearum and F. culmorum in general but with a dominance of the 3ADON genotype. 15ADON was not detected in these samples, except for very low amounts in the sample representing the years from 1997 to 2000. Detection of low amounts of the 15ADON genotype in these historical samples and the relatively high amounts of 15ADON genotype in 2003 and following years correspond well with the occurrence of F. graminearum and indicates that the 15ADON genotype was introduced along with F. graminearum around 2000. The amounts of the 3ADON and 15ADON genotypes correlated well with the total amount of DON whereas the amounts of NIV genotype correlated well with the amount of NIV in wheat and triticale but not in barley where the results indicate that Fusarium poae may also contribute to the NIV content.     

Fundamental study on the influence of Fusarium infection on quality and ultrastructure of barley malt

Barley infection with Fusarium species has been a long standing problem for the malting and brewing industries. In this study, we evaluate the impact of Fusarium culmorum infected raw barley on the final malt quality. Barley grains were infected for 5 days at optimum fungal growth conditions. Grains were fully characterized and compared to standard barley grains. Due to fungal infection, germinative energy of infected barley grains decreased by 45%; its water sensitivity increased dramatically, and grains accumulated 199 μg/kg of deoxynivalenol (DON). Barley grains were subsequently malted for 8 days, fully characterized and compared to standard malt grains. Fungal growth behavior was evaluated during malting using a PCR-based assay and mycotoxins were measured using HPLC. Fungal biomass increased in grains, during all stages of malting. Infected malt accumulated 8-times its DON concentration during malting. Kernel ultrastructure was evaluated using scanning electron and confocal laser scanning microscopy. Infected malt grains were characterized by extreme structural proteolytic, (hemi)-cellulolytic and starch deterioration with increased friability and fragmentation. Infected grains had higher protease and β-glucanase activities, lower amylase activity, a greater proportion of free amino and soluble nitrogen, and a lower β-glucan content. Malt loss was over 27% higher in infected malt in comparison to the control. The results of this study revealed that 20% F. culmorum infected barley kernels lead to a significant reduction in malt quality as well as mycotoxin formation.     

Multiplex real-time PCR detection of fumonisin-producing and trichothecene-producing groups of Fusarium species

Some species of Fusarium can produce mycotoxins during food processing procedures that facilitate fungal growth, such as the malting of barley. The objectives of this study were to develop a 5' fluorogenic (Taqman) real-time PCR assay for group-specific detection of trichothecene- and fumonisin-producing Fusarium spp. and to identify Fusarium graminearum and Fusarium verticillioides in field-collected barley and corn samples. Primers and probes were designed from genes involved in mycotoxin biosynthesis (TRI6 and FUM1), and for a genus-specific internal positive control, primers and a probe were designed from Fusarium rDNA sequences. Real-time PCR conditions were optimized for amplification of the three products in a single reaction format. The specificity of the assay was confirmed by testing 9 Fusarium spp. and 33 non-Fusarium fungal species. With serial dilutions of purified genomic DNA from F. verticillioides, F. graminearum, or both as the template, the detection limit of the assay was 5 pg of genomic DNA per reaction. The three products were detectable over four orders of magnitude of template concentration (5 pg to 5 ng of genomic DNA per reaction); at 50 ng template per reaction, only the TRI6 and FUM1 PCR products were detected. Barley and corn samples were evaluated for the presence of Fusarium spp. with traditional microbiological methods and with the real-time PCR assay. The 20 barley samples and 1 corn sample that contained F. graminearum by traditional methods of analysis tested positive for the TRI6 and internal transcribed spacer (ITS) PCR products. The five corn samples that tested positive for F. verticillioides by traditional methods also were positive for the FUMI and ITS PCR products. These results indicate that the described multiplex real-time PCR assay provides sensitive and accurate differential detection of fumonisin- and trichothecene-producing groups of Fusarium spp. in complex matrices.     

ATP citrate lyase 1 (acl1) gene-based loop-mediated amplification assay for the detection of the Fusarium tricinctum species complex in pure cultures and in cereal samples

The combined data set of the acl1 and tef-1α gene sequences of 61 fungal strains assigned to Fusarium tricinctum, Fusarium avenaceum, Fusarium acuminatum, Fusarium arthrosporioides, Fusarium flocciferum and Fusarium torulosum were used to study the phylogenetic relations between taxa. F. tricinctum, F. acuminatum and F. avenaceum formed distinct clades. Members of the F. tricinctum/F. acuminatum clade fall into three well supported lineages, of which the largest includes the epitype of F. tricinctum. Loop-mediated isothermal amplification (LAMP) was used to amplify a 167bp portion of the acl1 gene in F. tricinctum (Corda) Saccardo. DNA amplification was detected in-tube by indirect calcein fluorescence under black light after 60min of incubation at 65.5°C. The assay had a detection limit of 0.95pg of purified genomic DNA of F. tricinctum CBS 410.86 per reaction, corresponding to ca. 18 genomic copies of the acl1 gene. Specificity of the assay was tested using purified DNA from 67 species and subspecies of Fusarium as well as 50 species comprising 22 genera of other filamentous fungi and yeasts. The assay detected 21 of the 23 F. tricinctum strains tested. Cross reactivity was observed with eight out of 13 strains in F. acuminatum but with none of 17 F. avenaceum strains tested. Specificity was further confirmed by conventional PCR with primers designed from the same gene. Detection of F. tricinctum from culture scrapings directly added to the reaction master mix, in DNA extracts from wheat, in single barley grains or in washings of bulk grain samples are proposed as possible applications showing the suitability of the method for food analysis. Finally it was demonstrated that the LAMP reaction can be run using simple lab equipment such as a heating block, water bath, hybridization oven or household equipment, e.g. a microwave oven.     

Fusarium avenaceum -- the North European situation

The Fusarium species complex found on small-grain cereals in Northern Europe is largely dominated by F. avenaceum, while other important species include F. tricinctum, F. poae, F. culmorum and F. graminearum. The dominance of F. avenaceum has in recent years initiated extensive analytical activity in Norway and Finland in order to gain insight into the contamination of grain with secondary metabolites related to the fungus. Of these, moniliformin is the most studied compound with regard to toxicity. However, the data from analytical surveys indicate that field conditions in Northern Europe do not favour production of the metabolite. Instead, enniatins are regularly found in ppm-concentrations in grain, especially wheat and barley, while the bio-production of a range of other F. avenaceum related metabolites has so far barely been investigated. This paper summarises the results from mycological and chemical analyses of grain samples from Norway and Finland for major Fusarium species and F. avenaceum-related secondary metabolites.     

Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize

Zearalenone (ZEA) is a mycotoxin produced by some species of Fusarium, especially by Fusarium graminearum and F. culmorum. ZEA induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals. In the present study, a real-time PCR (qPCR) assay has been successfully developed for the detection and quantification of Fusarium graminearum based on primers targeting the gene PKS13 involved in ZEA biosynthesis. A standard curve was developed by plotting the logarithm of known concentrations of F. graminearum DNA against the cycle threshold (Ct) value. The developed real time PCR system was also used to analyze the occurrence of zearalenone producing F. graminearum strains on maize. In this context, DNA extractions were performed from thirty-two maize samples, and subjected to real time PCR. Maize samples also were analyzed for zearalenone content by HPLC. F. graminearum DNA content (pg DNA/ mg of maize) was then plotted against ZEA content (ppb) in maize samples. The regression curve showed a positive and good correlation (R2=0.760) allowing for the estimation of the potential risk from ZEA contamination. Consequently, this work offers a quick alternative to conventional methods of ZEA quantification and mycological detection and quantification of F. graminearum in maize.     

Trichothecene and moniliformin production by Fusarium species from western Canadian wheat

Fusarium graminearum, Fusarium culmorum, and Fusarium avenaceum, isolated from Fusarium-damaged wheat harvested in western Canada, were cultured and evaluated for mycotoxin production. Extracts of the culture media were assayed for trichothecenes by gas chromatography-mass spectrometry and for moniliformin by liquid chromatography. Deoxynivalenol (DON) was found in 28 of 42 isolates of F. graminearum and 42 of 42 isolates of F. culmorum at levels ranging from 0.5 to 25.0 microg/g. 15-AcetylDON was found in 28 of 42 isolates of F. graminearum at levels ranging from 1.0 to 7.1 microg/g. 3-AcetylDON was found in 41 of 42 isolates of F. culmorum at levels ranging from 0.8 to 13.0 microg/g. Several other trichothecenes were assayed but not detected in the culture medium. Moniliformin was present in 40 of 42 isolates of F. avenaceum at levels ranging from 1.3 to 138.1 microg/g, but was not present in any of the isolates of F. graminearum or F. culmorum.     

Comprehensive evaluation of malt volatile compounds contaminated by Fusarium graminearum during malting

Fusarium graminearum is the most important filamentous fungi in the malting process, and greatly affects malting performance and malt quality. Analysis of the emitted volatiles from F. graminearum mycelium was used to evaluate the malt flavour contaminated by F. graminearum during malting based on solid‐phase microextraction combined with gas chromatography–mass spectrometry. GC–MS analysis of different mycelium samples revealed a broad spectrum of volatile compounds including aldehydes, ketones, alcohols, organic acids and aromatics, which were highly similar to those of barley and malt. Statistical assessment of the data via principal component analysis demonstrated that the major contributors to the time‐dependent dynamic changes in the volatiles were the volatile aldehyde, alcohol and ketone fractions in the contaminated malting process. The results showed that volatile concentration was modulated by the ‐metabolism of the green malt and F. graminearum. Volatile compounds from green malt were influenced by F. graminearum fungi contamination during malting, although some were probably generated by this fungus. Copyright © 2017 The Institute of Brewing & Distilling     

Expected shifts in Fusarium species' composition on cereal grain in Northern Europe due to climatic change

In Northern Europe, changes in climate may result in better growing conditions for many crops. However, the expected warmer and more humid conditions are favourable for Fusarium head blight infections on cereals. The Fusarium species prevalent in Nordic areas to date are the same as in Central Europe: F. avenaceum, F. culmorum, F. graminearum and F. poae. The prevalence of F. graminearum in cereal grain has already increased in Central Europe and is likely to increase in the North due to the expected changes in weather conditions, reduced tillage and the predicted increase in maize cultivation in Nordic countries. The possible weather extremes predispose cereals to Fusarium infections by increasing the populations of insect pests injuring plants. Adverse conditions may even create conditions suitable for F. subglutinans or F. verticilloides to infect maize and possibly other cereals in rotation in southern parts of Scandinavia. The importance of the species that infect in relatively dry conditions, F. langsethiae and F. poae, may also increase on winter cereals which are predicted to be more prevalent in future farming. If the number of crop species cultivated will increase and non-cereal crops are included in rotations effects of reduced tillage on Fusarium infections in grain could be limited. The predicted changes in climate towards 2050 are expected to slightly change Fusarium species composition in Northern Europe. An increase in F. graminearum and possibly the invasion of northern parts of Central Europe and Denmark by fumonisin producers is expected.     

Effect of ozone treatment on the safety and quality of malting barley

Molds and their mycotoxins are an expensive problem for the malting and brewing industries. Deoxynivalenol (DON) is a mycotoxin that is associated with Fusarium spp. These fungi frequently cause Fusarium head blight in wheat and barley in the midwestern region of the United States; Manitoba, Canada; Europe; and China. Barley growers and malt producers would benefit from a postharvest control method for mold growth and DON production. We evaluated the use of gaseous ozone (O(3)) for preventing Fusarium growth and mycotoxin production while maintaining malt quality characteristics. Micromalting was performed in three replications under standard conditions. Ozone treatment was applied to malting barley during steeping via a submerged gas sparger. Ozone treatment conditions were 26 mg/cm(3) for 120 min after 2 and 6 h of steeping. The effects of gaseous ozone on DON, aerobic plate counts, Fusarium infection, and mold and yeast counts of barley throughout the malting process were measured. Various quality parameters of the malt were measured after kilning. Statistical tools were used to determine the significance of all results. Ozonation of malting barley during steeping did not lead to significant reductions in aerobic plate counts but did lead to a 1.5-log reduction in mold and yeast counts in the final malt. The influence of gaseous ozone on DON concentration was inconclusive because of the low initial concentrations of DON in the barley. Ozone significantly reduced Fusarium infection in germinated barley. Gaseous ozone did not negatively influence any aspect of malt quality and may have subtle beneficial effects on diastatic power and β-glucan concentrations.     

Epidemiology of Fusarium head blight on small-grain cereals

Fusarium head blight (FHB) is one of the most serious diseases affecting wheat and barley worldwide. It is caused by Fusarium graminearum along with F. culmorum, F. avenaceum and other related fungi. These fungi also produce several mycotoxins. Though the disease results in reduced seed quality and yield, the toxins which may accompany the disease are often a more serious problem. Pathogen inoculum is usually very abundant, however production and dispersal of inoculum are weather-sensitive processes. An abundance of colonized substrate (i.e. maize or cereal debris) in a region contributes to airborne inoculum throughout the area. Local residues beneath the cereal crop (i.e. from previous crop) may have a less obvious effect, particularly in regions where long-distance dispersal is likely due to wind conditions. The host is most susceptible to infection at anthesis and shortly thereafter. A warm, moist environment characterized by frequent precipitation or heavy dew is highly favorable to fungal growth, infection and development of disease in head tissues. As the fungus grows, it produces mycotoxins which are water-soluble and may be translocated between tissues or leeched from source tissues. Important epidemiological issues have arisen recently and include an apparent shift in prevalence of Fusarium species on infected heads in Europe toward F. graminearum; and the presence of multiple chemotypes and aggressiveness variants within a species in a region.     

Comparison of extraction and clean-up procedures for analysis of zearalenone in corn, rice and wheat grains by high-performance liquid chromatography with photodiode array and fluorescence detection.

The aim of this work was the optimization of some procedures usually used in the analysis of zearalenone (ZEA) in corn and other cereals by high-performance liquid chromatography (HPLC) with photodiode array and/or fluorescence detection. The comparison of five extraction solvents is presented. Three solid-phase extraction cartridges (C-18, silica, Florisil) and immuno-affinity columns were also compared to obtain the best recovery of the mycotoxin with the minimal presence of co-extractives in the chromatograms. Mixtures of methanol-1% aqueous NaCl (80.20 or 60:40 v/v) were the best extraction solvents. Florisil provided higher recovery of ZEA than C-18, and silica proved unsuitable. The immuno-affinity column was very efficient in cleaning the extracts, but its sample capacity was lower than that of SPE columns due to saturation. The mobile phase (methanol-water 80:20 v/v) gave a low retention time for ZEA (approximately 5 min), high sensitivity and acceptable separation between this mycotoxin and alpha-zearalenol. The optimized protocol is straightforward, provides high ZEA recoveries in spiked corn (mean 102.4%), has an acceptable sensitivity and has a lack of interference with fluorescence detection (detection limit 4 ng ZEA g(-1) corn). The photodiode array detector was useful, except at very low ZEA levels, to confirm the identity of the mycotoxin. The method was applied to search for ZEA accumulation in corn, wheat and rice grains inoculated with selected strains of Fusarium graminearum, F. oxysporum and method was applied to search for ZEA accumulation in corn, wheat and rice grains inoculated with selected strains of Fusarium graminearum, F. oxysporum and F. culmorum.     

The use of tri5 gene sequences for PCR detection and taxonomy of trichothecene-producing species in the Fusarium section Sporotrichiella

Purified DNA from isolates of Fusarium poae, Fusarium sporotrichioides, Fusarium kyushuense and Fusarium langsethiae was used as a template to amplify a 658-bp fragment from the trichodiene synthase (tri5) gene of these fungi with the gene-specific PCR primer pair Tox5-1/Tox5-2. Fragments obtained were isolated and sequenced. DNA sequence alignments revealed high similarity between the sequences derived from F. sporotrichioides and F. langsethiae (98.7%) and less similarity between the latter species and F. poae (90.9%). Phylogenetic analysis of the aligned sequences using the tri5 sequence of Fusarium pseudograminearum as an outgroup revealed clear separation between one group consisting of F. poae and F. kyushuense and another consisting of F. sporotrichioides and F. langsethiae. The two latter species could not be distinguished phylogenetically on the basis of their tri5 sequences. Taxon-specific reverse primers were designed from the aligned sequences and combined with the tri5 gene-specific forward primer Tox5-1. The new reverse primers enabled specific amplification of a fragment of approximately 400 bp from DNA isolated from F. sporotrichioides, F. poae, F. langsethiae and F. kyushuense, respectively. All primers were tested for cross-reactivity with DNA from 26 fungal species potentially capable of producing trichothecenes. Only the primer designed for F. langsethiae cross-reacted with F. sporotrichioides. PCR assays were applied in analysis of artificially and naturally infected samples of barley and oats. On artificially infected barley, species were selectively detected by the corresponding primers. In naturally infected oats, F. langsethiae was identified by the combination of two PCR assays designed for detection of F. sporotrichioides and F. langsethiae, respectively.     

Mycotoxins in cereal grain. Part 14. Histochemical examination of Fusarium-damaged wheat kernels

Wheat kernels with visible Fusarium-damage, naturally infected, have been examined with histochemical techniques to observe mycelium growth inside kernels and change in kernels cells. Kernels infected with F. culmorum were found to be damaged more extensively, comparatively to those infected with F. graminearum, F. avenaceum and F. nivale/(Microdochium nivale). Growth of Fusarium mycelium was most abundant between pericarp and aleurone layer and alongside of scutellum. Mycelium colonized also endosperm and embryo--both plumula and radicula.     

小麦赤霉病源呕吐毒素的合成、检测及防控

赤霉病是主要由禾谷镰刀菌侵染小麦、大麦和其他小谷类作物而造成作物减产的世界性病害。小麦赤霉病发病需要高温和高湿的环境条件,感染程度及发病范围主要由寄主自身的抗性决定,对粮食产业以及人类健康的危害主要来源于籽粒中积累的呕吐毒素(deoxynivalenol, DON)。赤霉病需要多层次、全流程的防治,而降低DON毒素的危害是防治赤霉病需要重点突破的环节。本文概述了DON的合成过程、致病机制、检测方法和脱毒策略,并提出防治赤霉病的思路,重点挖掘与DON毒素诱导、运输和转化相关的基因资源,为通过生物育种途径培育DON低积累小麦品种、减少DON危害提供参考。     

Influence of agronomic and climatic factors on Fusarium infestation and mycotoxin contamination of cereals in Norway

A total of 602 samples of organically and conventionally grown barley, oats and wheat was collected at grain harvest during 2002-2004 in Norway. Organic and conventional samples were comparable pairs regarding cereal species, growing site and harvest time, and were analysed for Fusarium mould and mycotoxins. Agronomic and climatic factors explained 10-30% of the variation in Fusarium species and mycotoxins. Significantly lower Fusarium infestation and concentrations of important mycotoxins were found in the organic cereals. The mycotoxins deoxynivalenol (DON) and HT-2 toxin (HT-2) constitute the main risk for human and animal health in Norwegian cereals. The impacts of various agronomic and climatic factors on DON and HT-2 as well as on their main producers F. graminearum and F. langsethiae and on total Fusarium were tested by multivariate statistics. Crop rotation with non-cereals was found to reduce all investigated characteristics significantly--mycotoxin concentrations as well as various Fusarium infestations. No use of mineral fertilisers and herbicides was also found to decrease F. graminearum, whereas lodged fields increased the occurrence of this species. No use of herbicides was also found to decrease F. langsethiae, but for this species the occurrence was lower in lodged fields. Total Fusarium infestation was decreased with no use of fungicides or mineral fertilisers, and with crop rotation, as well as by using herbicides and increased by lodged fields. Clay and to some extent silty soils seemed to reduce F. graminearum in comparison with sandy soils. Concerning climate factors, low temperature before grain harvest was found to increase DON; and high air humidity before harvest to increase HT-2. F. graminearum was negatively correlated with precipitation in July but correlated with air humidity before harvest. F. langsethiae was correlated with temperature in July. Total Fusarium increased with increasing precipitation in July. Organic cereal farmers have fewer cereal intense rotations than conventional farmers. Further, organic farmers do not apply mineral fertiliser or pesticides (fungicides, herbicides or insecticides), and have less problem with lodged fields. The study showed that these agronomic factors were related to the infestation of Fusarium species and the concentration of mycotoxins. Hence, it is reasonable to conclude that farming system (organic versus conventional) impacts Fusarium infestation, and that organic management tends to reduce Fusarium and mycotoxins. However, Fusarium infestation and mycotoxin concentrations may be influenced by a range of factors not studied here, such as local topography and more local climate, as well as cereal species and variety.     

Fate of enniatins and beauvericin during the malting and brewing process determined by stable isotope dilution assays

The fate of enniatins A, A1, B, B1 and beauvericin during the malting and brewing process was investigated. Three batches of barley grains were used as starting material, one was naturally contaminated, two were artificially inoculated with Fusarium fungi. Samples were taken from each key step of the malting and brewing procedure, the levels of the toxins were determined with stable isotope dilution assays using liquid chromatography–tandem mass spectrometry detection. Significant increases of the toxins were found during germination of two batches of barley grains, resulting in green malts contamination up to a factor of 3.5 compared to grains before germination. Quantitative PCR analyses of fungal DNA revealed in all batches growth of Fusarium avenaceum during germination. After kilning, only 41–72% of the total amounts of the toxins in green malts remained in kilned malts. In subsequent mashing stage, the toxins in kilned malts predominantly were removed with spent grains. In the final beer, only one batch still contained 74 and 14 μg/kg of enniatin B and B1, respectively. Therefore, the carryover of these enniatins from the initial barley to final beer was less than 0.2% with the main amounts remaining in the spent grains and the malt rootlets.