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.

Hematological and histopathological changes in silver catfish Rhamdia quelen (Siluriformes) exposed to clomazone herbicide in the Madre River, Santa Catarina State, Southern Brazil.

This study evaluated the influence of the clomazone herbicide (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone) contamination on the hematological parameters and histological changes in gills and liver of silver catfish (Rhamdia quelen) from Madre River, Santa Catarina State, Southern Brazil. Fish were collected between March 2010 and January 2012 at two different sites of the Madre River, one site receiving residual water (contaminated site) from rice culture (n=49) and another that do not receive residual water (reference site) (n=48). The herbicide clomazone analysis detected 3.40±1.70 µg/L in the contaminated site and 1.1±0.33 µg/L in the reference site. Fish from contaminated site showed increased (P<0.05) number of monocytes suggesting the possible defense response as a result of chronic exposure to clomazone. On the other hand, no difference was found in the hematocrit percentage, red blood cell count, total thrombocyte number, white blood cell count, lymphocytes, and neutrophils number. Fish from both sites showed histopathological changes in gills and liver, possibly caused by chronic exposure to contamination. The influence of herbicide sub doses on fish health is also discussed.

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.

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.

Selenium and aflatoxin levels in raw Brazil nuts from the Amazon basin.

Whereas selenium (Se) is an important antioxidant in human metabolism to prevent cancer, aflatoxins are highly carcinogenic. Brazil nuts from Eastern and Western Amazon regions were evaluated to find any relationship between Se and aflatoxins levels. A total of 80 (in-shell and shelled) nuts samples were collected directly from different forest sites and analyzed for Se by atomic emission spectrometry and aflatoxins by liquid chromatography tandem mass spectrometry. The limit of quantitation (LOQ) for Se was 2.0 mg/kg, and LOQ for total aflatoxins was 0.390 microg/kg. Nut Se levels from the Eastern region were higher than the Western, in addition to the aflatoxins. The moisture content (mc) and water activity (aw) of the raw nuts from the two regions did not present a significant difference, for either in-shell or shelled. The mc was 24.5% (minimum of 20.1% and maximum of 30.4%) and 22.1% (minimum of 14.6% and maximum of 28.9%) and a w of 0.85 for both regions. Further studies need to be carried out to discover the role of Se on fungi growth stress and aflatoxin production mechanisms.

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.

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.

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.

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.

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.

Fumonisin contamination and fusarium incidence in corn from Santa Catarina, Brazil.

In Brazil, the southern region has the highest incidence of esophageal cancer and also the highest production and consumption of corn (Zea mays) products. Corn samples intended for human consumption from the western, northern, and southern regions of the state of Santa Catarina, southern Brazil, had mean total fumonisin B (B(1), B(2), and B(3)) levels of 3.2, 3.4, and 1.7 mg/kg, respectively. Fusarium verticillioides, the predominant fungus in the corn samples, had mean incidences (percent of kernels infected) of 14, 11, and 18% for the three regions, respectively. Additional corn samples intended for animal feed from the southern region had a mean total fumonisin level of 1.5 mg/kg and a mean F. verticillioides incidence of 10%. The fumonisin levels in corn from the state of Santa Catarina, Brazil, were similar to the high levels determined in other high esophageal cancer incidence regions of the world.

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.

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).

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)).

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.

Polycyclic aromatic hydrocarbons in milk powders marketed in Argentina and Brazil.

The aim of this study was to quantify polycyclic aromatic hydrocarbon (PAH) levels in milk powder samples commercialised in Argentina and Brazil during 2012. Thirty-one samples were available from the retail market. An HPLC method for the determination of PAHs was applied involving a clean-up step with silica cartridges. Recoveries were greater than 79% for all PAHs analysed. Reproducible determination with adequate detection and quantification limits (LOD and LOQ) were attained by HPLC with fluorescence detection for 14 PAHs. Acenaphthylene was determined with a UV-VIS detector. There is no significant difference in any PAHs or in the sum of them between the Argentinean and Brazilian samples. Therefore, the samples were evaluated together. The highest concentration of benzo(a)pyrene (BaP) detected was 0.57 µg kg⁻¹ in milk powder. Contamination of samples expressed as the sum of 15 analysed PAHs varied between 11.8 and 78.4 µg kg⁻¹ and as PAH4 (BaP, chrysene, benzo(a)anthracene and benzo(b)fluoranthene) was between 0.02 and 10.16 µg kg⁻¹. The correlation coefficient for PAH2 (BaP and chrysene) and PAH4 groups was 0.95, for PAH2 and PAH8 it was 0.71, and for PAH4 and PAH8 it was 0.83. All the samples were below the regulatory limit for BaP, but 65% of commercial milk powders do not comply with the European Union limit for PAH4. This is the first report of PAH contamination in powder milk from Argentina and Brazil.