Fusarium Mycotoxins Stability during the Malting and Brewing Processes.

Mycotoxins are widely studied by many research groups in all aspects, but the stability of these compounds needs further research for clarification. The objective of this study is to evaluate deoxynivalenol and zearalenone stability during all steps of the malting and brewing processes. The levels of these compounds decreased significantly during the production process (barley to beer). During the malting process, the DON levels decreased significantly in the steeping, germination, and malting steps (62%, 51.5%, and 68%, respectively). Considering ZEN, when the levels were compared between barley and the last step of the process, a significant decrease was observed. Most of the mycotoxins produced were transferred to the rootlets and spent grains, which is advantageous considering the final product. Furthermore, the mycotoxin dietary intake estimation was included in this study. The results proved that if the concentrations of target mycotoxins in raw material are under the limits established by the regulations, the levels decrease during the malting and brewing processes and make the beer secure for consumers. The quality of the five commodities involved in the beer process plays a decisive role in the creation of a safe final product.

Assessment of Toxigenic Fusarium Species and Their Mycotoxins in Brewing Barley Grains.

Fusarium species threaten yield and quality of cereals worldwide due to their ability to produce mycotoxins and cause plant diseases. Trichothecenes and zearalenone are the most economically significant mycotoxins and are of particular concern in barley, maize and wheat. For this reason, the aim of this study was to characterize the Fusarium isolates from brewing barley and to assess deoxynivalenol and zearalenone contamination in grains. Characterization of the Fusarium strains was carried out by the phylogeny based on two loci (EF-1α and RPB2). Mycotoxin detection and quantification were performed by LC-MS. The results show that Fusarium was the predominant genus. Phylogenetic study demonstrated that the majority of the strains clustered within the Fusarium sambucinum species complex followed by the Fusarium tricinctum species complex. The results revealed high incidence of deoxynivalenol (DON) and zearalenone (ZEA) contamination (90.6% and 87.5%, respectively). It was observed that 86% of the samples contaminated with ZEA were above the limits set by the EU and Brazilian regulations. These results may highlight the importance of controlling Fusarium toxins in barley, mainly because of its use in the brewing industry and the resistance of various mycotoxins to food processing treatments.

Application of a LC-MS/MS method for multi-mycotoxin analysis in infant formula and milk-based products for young children commercialized in Southern Brazil.

An analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was validated and applied for the analysis of aflatoxin M1 (AFM1), ochratoxin A (OTA) and deoxynivalenol (DON) in infant formula and milk-based products for young children commercialized in Brazil. A total of 38 samples were evaluated, including 12 infant formula, 14 follow-on formula and 12 samples of milk-based products. AFM1 was detected in 12 (32%) samples, and seven (18%) samples contained AFM1 levels above the method limit of quantification in a concentration range between 0.013 and 0.067 ng mL-1 (0.026 ± 0.019). Two samples of milk-based products exceeded the maximum level (ML) fixed by the European Union for AFM1 in baby foods, however, all samples were in agreement with the levels established by the Brazilian regulation. OTA and DON were not detected in any of the analyzed samples.

Incidence of toxigenic fungi and zearalenone in rice grains from Brazil.

Rice (Oryza sativa L.) is one of the most important food crops worldwide. In Brazil, the southern region is the area with the highest production of rice in the country and also has a high average daily intake of rice by the population. The mycoflora, mainly toxigenic Aspergillus and Fusarium species, the presence of AFB1, DON and ZEA in rice grains, as well as daily intake estimates for the Southern Brazilian population were evaluated. The rice grain samples were collected during the 2017 crop from different harvest periods. According to the mycological tests, the samples presented a high count of fungal colonies in the pre and post-harvest, where the incidence of the F. graminearum species complex (52%) was significantly predominant. This group can be responsible for ZEA production, as found in this study in parboiled rice, mainly because most of the isolated strains were producers of high ZEA levels in the pre-harvest (77%) and post-harvest after natural (79%) and artificial (75%) drying of the rice. Only ZEA showed significant results in the rice grain analyzed (60%) at levels of 90.56 to 126.31 µg/kg, where 36% of the samples were significantly higher than the current maximum limit stipulated in Brazilian regulations and by the European Commission. Despite this, the dietary exposure of ZEA estimated for the southern Brazilian population was below the provisional maximum tolerable daily intake level of 0.5 µg/kg body weight/day set at international regulations.

Antifungal activities against toxigenic Fusarium specie and deoxynivalenol adsorption capacity of ion-exchanged zeolites.

Zeolites are often used as adsorbents materials and their loaded cations can be exchanged with metal ions in order to add antimicrobial properties. The aim of this study was to use the 4A zeolite and its derived ion-exchanged forms with Zn2+, Li+, Cu2+ and Co2+ in order to evaluate their antifungal properties against Fusarium graminearum, including their capacity in terms of metal ions release, conidia germination and the deoxynivalenol (DON) adsorption. The zeolites ion-exchanged with Li+, Cu2+, and Co2+ showed an excellent antifungal activity against F. graminearum, using an agar diffusion method, with a zone of inhibition observed around the samples of 45.3 ± 0.6 mm, 25.7 ± 1.5 mm, and 24.7 ± 0.6 mm, respectively. Similar results using agar dilution method were found showing significant growth inhibition of F. graminearum for ion-exchanged zeolites with Zn2+, Li+, Cu2+, and Co2+. The fungi growth inhibition decreased as zeolite-Cu2+>zeolite-Li+>zeolite-Co2+>zeolite-Zn2+. In addition, the conidia germination was strongly affected by ion-exchanged zeolites. With regard to adsorption capacity, results indicate that only zeolite-Li+ were capable of DON adsorption significantly (P < 0.001) with 37% at 2 mg mL-1 concentration. The antifungal effects of the ion-exchanged zeolites can be ascribed to the interactions of the metal ions released from the zeolite structure, especially for zeolite-Li+, which showed to be a promising agent against F. graminearum and its toxin.

Quality and safety of maize (Zea mays L.) from Rondônia state storage units, Northern Brazil.

The quality and safety of maize (Zea mays L.) from different grain storage units (GSUs), located in the main producing region of Rondônia State, Northern Brazil, were evaluated. Maize grains (n = 76) stored in four GSUs were collected from July to November 2014 and evaluated for grain damages, humidity, fungi and fumonisins (FBs) content. The climate conditions data were also obtained from plant growing to storage periods. Regarding the moisture content and water activity (aw), these varied from 10.0% to 16.1% and 0.5 to 0.8, respectively. As expected, fungi spores were present in 94.8% of the samples, prevailing Fusarium genera, with a fungi colony maximum of 2.2 × 104 CFU g-1. Regarding FBs, 60.5% of the samples were contaminated, below Brazilian and United States maximum limits, but 9.2% had levels higher than the European legislation.

Natural occurrence of fumonisins and characteristics of pamonhas sweet type from Southern Brazil.

Fresh and frozen pamonhas, a native Brazilian food made with sweet corn (Zea mays L.), similar to the Mexican tamal, which has been incorporated into the country's diet, were investigated for fumonisins (FBs: FB1 and FB2) contamination and product characteristics. The laboratory conditions of the applied fumonisins method with liquid chromatography and fluorescence detection were validated. Five out of the total samples showed FBs contamination above the national maximum level of 1000-1500 µg/kg. Despite this, in 59.6% (31) of the samples FB1 and in 23.1% (12) FB2 could be detected. The pamonhas characteristics did not show uniformity among the brands surveyed. The moisture content of 59.4% and water activity of 0.97 for both (fresh and frozen) samples showed to be suitable conditions for fungal growth. One sample had a quite high pH. It is essential to establish guidance criteria for pamonha production by the regulatory agencies.

Polycyclic aromatic hydrocarbons in commercial brands of dry whole soybeans for direct human consumption.

A total of 13 polycyclic aromatic hydrocarbons (PAHs) were surveyed in dry whole soybeans (Glycine max L.) sold for human consumption. The analysed samples were commercialised in vegetarian shops and food stores in Southern Brazil regions. The determination of PAHs levels was carried out by high performance liquid chromatography (HPLC)with fluorescence detection. PAHs were present in 89.7% of the samples. Chrysene (Chy) and 5 methyl chrysene (5MeChy) were the main PAHs detected (76.9% and 71.8%, respectively) followed by dibenzo(ai)pyrene (DaiP), however in a much less percentage of samples (23.1%). Apart from those PAHs, also benzo(a)antracene (BaA), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF) and benzo(a)pyrene(BaP) were found. The ΣPAH levels in the positive samples ranged from 0.80 to 38.78 µg kg-1, with exception of one sample that reached 204.46 µg kg-1 (ΣPAHs of six compounds). Despite the present contamination there are no maximum limits set for PAHs in soybeans.

Degradation of bifenthrin and pirimiphos-methyl residues in stored wheat grains (Triticum aestivum L.) by ozonation.

Pesticide insecticides are used on wheat grains in storage units but their efficiency is hindered by persistent residues in the grains. Therefore, this study aims to evaluate the effectiveness of ozone (O3) gas treatment on the degradation of residual bifenthrin and pirimiphos-methyl insecticides commonly used in storage wheat grains, as well as to evaluate degradation of their by-products. The residues of bifenthrin decreased after 180 min of exposure in a concentration of 60 µmol/mol (a 37.5 ± 7.4% reduction) with 20% moisture content and 0.9 water activity. On the other hand, under the same experimental conditions, the pirimiphos-methyl residues significantly decreased in the wheat grains (71.1 ± 8.6%) after 30 min of exposure. After O3 gas treatment, three by-products of pirimiphos-methyl (m/z=306.1) containing different molecular mass to charge ratios (m/z=278.1, 301.1 and 319.2) were identified by LC-MS. O3 is a strong oxidizer that has shown the potential to reduce pesticide residues in stored grain in order to ensure food quality and safety.

Fumonisins and fungi in dry soybeans (Glycine Max L.) for human consumption.

This survey reports the occurrence of fumonisins (FBs) and fungi in dry soybeans sold for human consumption. The variation levels were 138-1495 µg kg(-1) and 178-552 µg kg(-1) for FB1 and FB2, respectively. In addition, potentially toxigenic fungi as Fusarium, Aspergillus and Penicillium genera were isolated in the samples. These can be considered as indicator-toxin and can produce considerable amounts of mycotoxins. Despite FB presence in the soybeans for human consumption, there is no legal regulation. Therefore, it is important to emphasise the need for frequent monitoring of these contaminants in soybeans.

Fumonisins B1 and B2 in the corn-milling process and corn-based products, and evaluation of estimated daily intake.

The distribution of fumonisins (FBs: FB1 and FB2) in the corn-milling process and in corn-based products, as well as daily intake estimates for the Brazilian population were evaluated. Among corn fractions samples, corn meal had the highest mean concentration of FB1 (1305 µg kg(-1)) and FB2 (651 µg kg(-1)) and a distribution factors of 452% and 256% in relation to corn grain, respectively. On the other hand, the distribution factor of FB1 and FB2 in corn flour was found to be 144% and 88% respectively, which demonstrates that fumonisins in this fraction were reduced compared with corn grain. As a result, almost half the corn meal samples (47%) would be non-compliant with future Brazilian regulation (2017) for fumonisins. However, corn-based products, such as corn flakes and popcorn, were in compliance with the regulation. The average probable daily intake and maximum probable daily intake of fumonisins estimated for the Santa Catarina state (Brazil) population were below the provisional maximum tolerable daily intake of 2 µg kg(-1) body weight day(-1) for all corn samples. Despite this, the adoption of practices to control the occurrence of fumonisins should be applied to the corn-milling fractions that may contain a higher concentration of this toxin, such as corn meal, often used for animal feed in Brazil.

Fungi and the natural occurrence of deoxynivalenol and fumonisins in malting barley (Hordeum vulgare L.).

The industrial use of barley grain has experienced continuous growth, mainly due to its economic importance for malt production. From a technological perspective, fungal persistence can reduce product marketability and cause economic losses. In this sense, the aim of the present study was to determine the presence and identification of mycoflora and the occurrence of deoxynivalenol (DON) and fumonisins (FBs) in malting barley. The samples presented a low count of fungal colonies, with values ranging from 10.5 to 0.5 × 10(1)CFU g(-1) and the species most found were Fusarium graminearum and Fusarium verticillioides with 26% and 12% of incidence, respectively. In the samples analyzed for mycotoxins occurrence, DON and FBs were present in 18% and 12%, respectively. The high concentrations of toxins found in the malting samples may be strongly influenced by agricultural practices and the weather conditions during critical phases of plant growth.

Efficacy of zinc compounds in controlling Fusarium head blight and deoxynivalenol formation in wheat (Triticum aestivum L.).

The efficiency of zinc compounds (zinc sulfate, ZnSO4 and zinc oxide, ZnO in regular and nanosize, respectively) on wheat plants was evaluated against growth of Fusarium graminearum and DON formation. In addition, any possible effects on the grain microstructures were observed by scanning electron microscopy (SEM), and the remaining residue of Zn on wheat plants was analyzed. The plants were inoculated with F. graminearum and treated with Zn compounds (100mM) onto spikelets at the anthesis stage. When wheat plants reached maturation, grains were harvested and evaluated for Fusarium (number of colonies, CFU/g), DON formation, and SEM observation, followed by determination of possible remaining Zn residue. The groups treated with ZnSO4 and ZnO-NP showed a reduction in number of CFU of F. graminearum when compared to the control. Similarly for DON formation, i.e. the toxin was reduced to non-detected levels in the treated group. ZnO-NP efficiently reduced F. graminearum and DON formation in the grains at low concentration. Zn remained within the international recommended level for consumption and the treatment did not cause any damage to wheat grains. New strategies of control using Zn compounds in addition to conventional treatments could increase the efficiency against FBH and DON formation.

Stereo and scanning electron microscopy of in-shell Brazil nut (Bertholletia excelsa H.B.K.): part two-surface sound nut fungi spoilage susceptibility.

This work reports the in-shell Brazil nut spoilage susceptible morpho-histological characteristics and fungi infection (shell, edible part, and brown skin) through stereo and scanning electron microscopies (SEM). The following characteristics related to shell (a) morphology-that allow fungi and insects' entrance to inner nut, and (b) histology-that allow humidity absorption, improving environment conditions for living organisms development, were identified. (a.1) locule in testae-the nut navel, which is a cavity formed during nut detaching from pods (located at 1.0 to 2.0/4th of the shell B&C nut faces linkage). It allows the nut brown skin (between shell and edible part) first contact to the external environment, through the (a.2) nut channel-the locule prolongation path, which has the water/nutrients cambium function for their transport and distribution to the inner seed (while still on the tree/pod). Both, locule followed by the channel, are the main natural entrance of living organisms (fungi and insects), including moisture to the inner seed structures. In addition, the (a.3) nut shell surface-which has a crinkled and uneven surface morphology-allows water absorption, thus adding to the deterioration processes too. The main shell histological characteristic, which also allows water absorption (thus improving environment conditions for fungi proliferation), is the (b.1) cell wall porosity-the multilayered wall and porous rich cells that compose the shell faces double tissue layers and the (b.2) soft tissue-the mix of tissues 2 faces corner/linkage. This work also shows in details the SEM nut spoilage susceptible features highly fungi infected with hyphae and reproductive structures distribution.

Mycoflora and deoxynivalenol in whole wheat grains (Triticum aestivum L.) from Southern Brazil.

The fungal species Fusarium graminearum is related to deoxynivalenol (DON) formation. The aim of this study was to evaluate mycoflora and DON occurrence in 53 whole wheat grain samples collected in Southern Brazil during the 2012 crop. Wheat grains showed adequate values of water activity ranging from 0.48 to 0.72, within the required limits of moisture content, ranging from 9.1% to 13.9%. In addition, low counts of fungal colonies, ranging from 10 to 8.2 × 10(2), were found. For Fusarium genera, there was predominance of Fusarium verticillioides (34%) and F graminearum (30.2%). For Aspergillus species, 37.7% of Aspergillus flavus was determined. Regarding the Penicillium species, Penicillium digitatum (49%) was the most found species. DON was detected in 47.2% (25 out of 53) of the samples analysed, with levels ranging from 243.7 to 2281.3 µg kg(-1) (mean: 641.9 µg kg(-1)).

Stereoscopy and scanning electron microscopy of Brazil nut (Bertholletia excelsa H.B.K.) shell, brown skin, and edible part: part one--healthy nut.

UNLABELLED: In this article, tissue layers and cells characteristics of the Brazil nut (Bertholletia excelsa H.B.K.) shell (tegument), brown skin (testae), and edible part (cotyledons) were identified by stereoscopy (SM) and scanning electron microscopies (SEM). (a) The shell (a lignin rich, protective wall) varies in thickness throughout the nut structure and comprises different tissue types (total 3)/texture (hard/mid-hard/soft), layers (2 to 5), colors (light to dark brown and white to cream), cell shape (amorphous/flattened on both surfaces; polygonal and cylindrical with thick, porous primary and secondary wall in cross-section), and vascular distribution (helically and polyedrical thickened vessels at soft tissue and locule/channel structures). These variations are observed either in the shell faces, face corners, nut tips, or locule in testae. (b) The brown skin (shell nut part linked to both the shell and edible part) is made of flattened irregular-shaped parenchymal cells distributed in several layers with more flexible fibrous, thinner wall tissue than shell. It has both rough and smooth shiny texture on the upper and lower surfaces, respectively. However, the nut (c) edible part, that is the nut storage tissue, shows several different tissue/cell layers starting from epidermis (double/triple cells sequence of round and palisade shapes) layer-the endosperm tissue. The parenchymal tissues show cells of irregular shape with small and larger sizes distributed in regular and randomly layers, respectively, separated by a short meristem tissue layer. The cortex cells increase in size as they approach the cotyledons junction. The Brazil nut part's tissue layers and cells were identified by the SM and SEM microscopy methods applied, which provides knowledge for further understanding of nut alterations that may occur either in the forest or during the factory processing. PRACTICAL APPLICATION: Knowledge about the characteristics and nature of the waste woody tissues from the Brazil nut factories is of interest for potential applications in the industry. Understanding the nut tissues and cells structures helps in judging how much whole nut edible part gets spoiled/deteriorated (either raw or processed), for further development of procedures to prevent and/or control such spoiling/deterioration for achieving nut quality and safety (to be discussed in Part Two).

Fumonisins in corn (Zea mays L.) from Southern Brazil.

A total of 232 samples of corn commercialised in Santa Catarina state, Southern Brazil (temperate zone climate), were evaluated from 2007 to 2012 for fumonisins (FBs: FB1 and FB2). Before performing this study, a FBs method with liquid chromatography and fluorescence detection (ex. 335; em. 440 nm) was validated first. FBs were detected in 46.6% (108 samples), with values ranging from 66 to 7832 µg kg(-1) for FB1 and 110 to 1201 µg kg(-1) for FB2. The number of contaminated corn samples for FB1 and FB2 varied and often presented contamination of FB1 only. Per year of analysis, the numbers were: n = 22/8(FB1/FB2), 44/5(FB1/FB2), 25/12(FB1/FB2), 4(FB1), 6(FB1) and 7(FB1) in 2007, 2008, 2009, 2010, 2011 and 2012, respectively. The contamination percentage was 42.3/15.4, 59.5/6.8, 43.8/21.1, 36.4, 35.3 and 33.3%, respectively, during these years. Consumers can be exposed to these mycotoxins and their health can be at risk through the consumption of contaminated corn.

Effect of zinc compounds on Fusarium verticillioides growth, hyphae alterations, conidia, and fumonisin production.

BACKGROUND: Several strategies are used to eliminate toxigenic fungi that produce fumonisins in grains. Fusarium verticillioides can be controlled by the application of synthetic fungicides in the field or during storage. However, there may also be residuals, which may remain in the foods. Inorganic compounds such as zinc are cheap, stable and could present strong antifungal activity. Some Zn compounds can be utilized as dietary supplements and are authorized for the fortification of foods. Knowing the advantages and that low concentrations of Zn can have antimicrobial activity, our objective was to evaluate the effects of Zn compounds on the growth of F. verticillioides and the production of fumonisin and conidia. In addition, we aimed to verify that Zn compounds cause morphological alterations of the hyphae, mortality and production of reactive oxygen species. RESULTS: Zn compounds efficiently reduced fungal growth and fumonisin production. Treatment using zinc perchlorate gave the best results. All treatments inhibited conidia production and caused morphological alterations of the hyphae. It was possible to observe cell death and production of reactive oxygen species. CONCLUSION: Zn compounds have advantages compared to other antifungal compounds. In particular, they are non-toxic for the organism in appropriate amounts. They could be studied further as potential fungicides in agriculture.

Antioxidant treatment prevents cognitive impairment and oxidative damage in pneumococcal meningitis survivor rats.

Pneumococcal meningitis is associated with the highest fatality case ratios in the world. Most of patients that survive present neurologic sequelae at later times as well as biochemicals alterations such as oxidative stress in both earlier and later times after central nervous system infection. In this context, we evaluated the effect of antioxidant treatment on memory and oxidative parameters in the hippocampus of meningitis survivor rats 10 days after infection. To this aim, the animals underwent a magna cistern tap receiving either 10 µL sterile saline as a placebo or an equivalent volume of a Streptococcus pneumoniae suspension at the concentration 5x10(9) cfu/mL. The animals submitted to meningitis were divided into the following groups: 1) treated with antibiotic, 2) treated with basic support plus N-acetylcysteine, 3) treated with basic support plus deferoxamine, 4) treated with basic support plus N-acetylcysteine and deferoxamine, or 5) treated with N-acetylcysteine plus deferoxamine. Ten days after meningitis, the animals underwent inhibitory avoidance and habituation to an open field tasks and, immediately after, were assessed for oxidative damage in the hippocampus and cortex. The meningitis group showed significantly decreased performance in latency retention compared with the sham group in the inhibitory avoidance task. In the open-field task, the meningitis group presented memory impairment after meningitis. All these memory impairments were prevented by N-acetylcysteine plus deferoxamine with or without basic support and its isolate use. In addition, there was an increase of lipid phosphorylation in cortex and hippocampus and all the combined antioxidants attenuated lipid phosphorylation in both structures. On the other hand, there was an increase of protein phosphorylation in cortex and N-acetylcysteine plus deferoxamine with or without basic support prevented it. Thus, we hypothesize that oxidative stress may be related to cognitive impairment in pneumococcal meningitis.