Elisabete Yurie Sataque Ono

Effect of climatic conditions on natural mycoflora and fumonisins in freshly harvested corn of the State of Paraná, Brazil

Natural mycoflora associated with fumonisins were analyzed in 150 samples of freshly harvested corn from Central-Southern, Central-Western and Northern regions of the State of Paraná, Brazil and correlated to climatic conditions. The corn samples were frequently contaminated with Fusarium sp.(98.7 to 100%) and Penicillium sp. (93 to 100%), when compared to Aspergillus sp. (not detected to 27.7%). The highest contamination with potentially mycotoxigenic fungi occurred in corn harvested in the Central-Western region, where total mould and yeast counts ranged from 5.5 × 103 to 5.2 × 106 CFU/g, with 98.7% contaminated byFusarium sp. and 93% by Penicillium sp. In this region F. moniliforme (F. verticillioides) was the predominant Fusariumsp., and was isolated in 85.9% of the samples. Aspergillus sp. was isolated from 27.7% samples. FB1 was detected in 100% of the samples (mean of 2.39 μg/g) and FB2 in 97.7% (mean of 1.09 μg/g). Fumonisins were also detected in all samples from Northern region, with mean of 4.56 μg/g (FB1) and 2.20 μg/g (FB2).Considering 1.0 μg/g as the threshold, 72% of the corn samples from the Central-West and 92% from the North were contaminated with concentrations above this value, in contrast to a 18.5% contamination rate from Central-Southern samples. Between corn planting to harvesting season, the average maximum temperature and relative humidity were 26 °C and 77.1%(Central-Southern), 27 °C and 69% (Northern)and 29.9 °C and 89.1% (Central-Western).Therefore, the higher fumonisins contamination of corn from Northern region when compared to the Central-South were due to the differences in rainfall levels (92.8 mm in Central-Southern, 202 mm in Northern) during the month preceding harvest.

Evaluation of fumonisin-aflatoxin co-occurrence in Brazilian corn hybrids by ELISA

The natural co-occurrence of fumonisins and aflatoxins was investigated in freshly harvested corn kernels (150 samples, 62 hybrids), acquired from the Central-Southern (27 samples, 21 hybrids), Central-Western (86 samples, 51 hybrids) and Northern (37 samples, 18 hybrids) regions of the State of Paraná

Post-harvest storage of corn: effect of beginning moisture content on mycoflora and fumonisin contamination.

The effect of storage on mycoflora profile was monitored bimonthly in 36 corn (Zea mays L.) samples, dividing the same sample into groups dried to 11 and 14% moisture content (1008 analysis). These groups were further subdivided based on the initial total count (moulds and yeasts) up to 104 CFU g-1 (12 samples, range 1.6 × 104 to 9.0 × 104, mean 3.8 × 104 CFU g-1) and up to 105 CFU g-1 (24 samples, range 1.0 × 105 to 5.0 × 105, mean 2.7 × 105 CFU g-1). In the corn group dried to 11%, the fumonisin content was analysed at the initial stage (freshly harvested) and at the end of 12-month storage. Fusarium spp. and Penicillium spp. prevailed at the freshly harvested stage (100%), maintaining this profile throughout 12 months, in corn dried to both 11 and 14%. Cladosporium spp., Aspergillus spp. and Phoma spp. were also detected at lower frequencies during the storage. Fusarium spp. and the total fungal colony count during 12-month storage carried out with samples dried to 11 or 14% moisture content were statistically evaluated using ANOVA for randomized complete block design. The correlation between storage time and Fusarium spp. and total fungal colony count data was analysed by Pearsons correlation test. There was no difference in Fusarium spp. and total counts in the 104 CFU g-1 initial total count group throughout the storage time (p < 0.05). There was a negative correlation between fungal population and storage time (p < 0.05) in the 105 CFU g-1 initial total count group. Fumonisins were detected in all freshly harvested corn, at a mean concentration of 9.9 ± 6.0 µg g-1 (range 0.74-22.6 μg g-1). These values did not change in the 12-month stored corn (mean of 9.9 ± 5.8 μg g-1, range 0.81-23.7 μg g-1). These post harvest data indicated the importance of moisture content at the crop harvesting/predrying stage to control fungal growth and further fumonisin production.

Natural occurrence of deoxynivalenol in wheat from Paraná State, Brazil and estimated daily intake by wheat products.

The occurrence of deoxynivalenol (DON) was evaluated in 113 wheat samples from the northern and central/southwestern regions of Paraná State, Brazil during the 2008 and 2009 growing seasons, and this rate of occurrence was used to estimate the DON dietary exposure. The DON determination was carried out by an indirect competitive enzyme-linked immunosorbent assay. DON was detected in 66.4% samples at levels ranging from 206.3 to 4732.3 μg/kg (mean 1894.9 μg/kg). The estimated daily intake (EDI) of DON through bread and pasta was evaluated in the inhabitants of Londrina City in northern Paraná State, Brazil. The average intake of these inhabitants was 0.79 μg/kg body weight (b.w.) for bread and 0.35 μg/kg b.w. for pasta. The total EDI was 1.13 μg/kg, which is above the Provisional Tolerable Daily Maximum Intake (PTDMI) of 1 μg/kg b.w. To our knowledge, this is the first report on natural DON occurrence in wheat and DON dietary exposure estimation from Paraná, Brazil.

Penicillium expansum versus antagonist yeasts and patulin degradation in vitro

Taking into account the preliminary antagonistic/biodegradation property showed by Pichia membranifaciens and Sporobolomyces roseus, which decreased the initial patulin concentration of 588.4 to 290.0 µg/mL, ability of P. ohmeri 158 in biocontrol against Penicillium expansum and patulin decrease in vitro was performed. The culture supernatant of P. ohmeri 158 was effective against 66.17% micelial growth, indicating antibiosis related with the killer phenomenon. The initial patulin concentration of 223 µg in the presence of P. ohmeri 158 cells was decreased over 83% of the original concentration, when incubated at 25oC/2 days and > 99% after 5 days incubation time, with undetectable patulin level after 15 days. The initial pH 4.0 decreased to pH 3.3 along 15 days experiment, suggesting that patulin decrease was an active process and a consequence of yeast metabolism. The results suggested that P. ohmeri 158 could be a promising alternative for the inhibition of P. expansum growth and patulin degradation.

A comparison between enzyme immunoassay and HPLC for ochratoxin A detection in green, roasted and instant coffee

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for ochratoxin A (OTA) detection in green, roasted and instant coffees was developed using anti-OTA monoclonal antibody. Immunological reagents prepared were OTA-BSA (4.76 mg/mL), anti-OTA.7 MAb (2x103-fold dilution) and HRP-anti IgG (103-fold dilution). The detection limit was 3.73 ng OTA/g and correlation coefficients (r) between this immunoassay and high performance liquid chromatography were 0.98 for green coffee, 0.98 for roasted and 0.86 for instant. OTA levels detected by ic-ELISA were higher than by HPLC, with ELISA/HPLC ratio of 0.66 - 1.46 (green coffee), 0.96 - 1.11 (roasted) and 0.93 - 1.82 (instant). ELISA recoveries for OTA added to coffee (5 - 70 ng/g) were 81.53 % for green coffee, 46.73 % for roasted and 64.35 % for instant, while recoveries by HPLC were 80.54 %, 45.91 % and 55.15 %, respectively. Matrices interferences were minimized by samples dilution before carrying out the ELISA assay. The results indicate that MAb-based ic-ELISA could be a simple, sensitive and specific screening tool for OTA detection, contributing to quality and safety of coffee products.

A Comparative Study of Indirect Competitive ELISA and HPLC for Fumonisin Detection in Corn of the State of Paraná, Brazil

The performance of a monoclonal antibody (MAb)-based indirect competitive ELISA (ICELISA) for fumonisin detection in corn was evaluated using 150 post-harvest corn samples from the April 1995 and March-April 1996 crop, in the State of Paraná, Brazil. Compared to high performance liquid chromatography (HPLC), the correlation coefficient (r) of the results from the two methods was 0.94. The IC-ELISA detected total fumonisin levels of 1.1 to 1.5-fold higher than HPLC in 85 samples (56.6%). The concentration range of 1.0 to 5.0 μg g−1 was observed in 26% of samples analysed by ELISA and in 29% by HPLC. In addition, four samples (2.7%) showed an ELISA/HPLC ratio of 0.7 to 0.99 and seven samples (4.7%) a 1.0 ratio. In 29 (13.3%) samples, IC-ELISA detected 1.6 to 2.0-fold higher values than HPLC. These data showed high correlation (r = 0.91) of IC-ELISA and HPLC for fumonisin concentration in corn contaminated with ≤ 10 μg g−1, with a detection limit of 93 ng g−1. Matrix interference was minimized by diluting the sample before the ELISA assay. MAb-based IC-ELISA may be effectively applied for fumonisin screening in corn, with advantages of simplicity, ease of sample preparation and sensitivity.

Molecular identification of Aspergillus spp. isolated from coffee beans

Some species belonging to the genus Aspergillus are potential producers of ochratoxin A (OA), a mycotoxin with nephrotoxic, immunosuppressive, teratogenic and carcinogenic effects. The aim of the present study was to identify the species of Aspergillus that contaminate the inside of coffee beans collected in the stage of maturation and drying, from 16 producing areas located in the northern region of the State of Parana, in the South of Brazil. A total of 108 isolates of Aspergillus spp. was identified at the species level, by sequencing the internal transcribed spacer (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). The results revealed the presence of potentially ochratoxigenic species in 82% of the geographic regions studied, among which Aspergillus niger was the species most frequently detected, followed by A. ochraceus and A. carbonarius. The presence of A. carbonarius in immature coffee fruits harvested from trees is reported for the first time.

Fumonisins in corn: correlation with Fusarium sp. count, damaged kernels, protein and lipid content

A contaminacao natural por fungos e fumonisinas foi avaliada em 109 amostras de milho recem-colhido do Estado do Parana e correlacionada com graos ardidos (%). Alem disso, graos sadios e ardidos de 24 amostras de milho foram selecionados a fim de comparar o perfil da microbiota fungica e niveis de fumonisinas. A correlacao entre os teores de proteinas/lipidios e os niveis de fumonisinas tambem foi analisada nos 15 hibridos de milho mais frequentemente cultivados no Estado do Parana. A contagem total de fungos em 109 amostras de milho recem-colhido variou de 1,9x104 a 3,5x106 UFC/g, Fusarium sp. de 1,0x103 a 2,2x106 UFC/g e, niveis de fumonisinas de 0,13 a 20,38 µg/g. A contagem total de fungos/Fusarium spp. e niveis de fumonisinas apresentaram correlacao positiva (p<0,05). Adicionalmente, houve uma correlacao positiva entre graos ardidos (%) e a contagem total de fungos/ Fusarium spp. (p < 0,05). Os niveis de fumonisinas nos graos sadios variaram de 0,57 a 20,38 µg/g, enquanto que nos graos ardidos variaram de 68,96 a 336,38 µg/g. Nao foi observada correlacao significativa entre os niveis de fumonisinas e os teores de proteinas/lipidios. Esses resultados ratificam a importância do monitoramento constante da contaminacao por fungos toxigenicos e fumonisinas em milho e derivados a fim de garantir a qualidade e seguranca dos produtos e minimizar o risco potencial a saude humana e animal.

Reliable indirect competitive ELISA used for a survey of ochratoxin A in green coffee from the North of Paraná State, Brazil.

The performance of an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a monoclonal antibody (mAb) for ochratoxin A (OTA) detection was evaluated in a comparative study with high-performance liquid chromatography (HPLC) analysis using 68 freshly harvested coffee samples from the North of Paraná State, Brazil. The anti-OTA mAb showed high specificity and low cross-reactivity with OTA analogues (OTB and OTα), but cross-reacted with OTC. This ic-ELISA showed a detection limit of 3.75 ngg−1 sample, when compared to 0.80 ngg−1 by HPLC, with an ic-ELISA/HPLC correlation coefficient of 0.90. As regards OTA analysis of these coffee samples, natural contamination was detected in 10 samples (14.7%) by both methods, where the ic-ELISA values (range 3.9–7.3 ngg−1) were 1.1 to 1.6-fold higher than HPLC data (2.7–4.7 ngg−1). Five samples (7.4%) were OTA positive (range 0.84–1.30 ngg−1) only by HPLC assay, probably due to the higher detection limit reached by ic-ELISA. OTA was undetectable in 53 samples (77.9%) by both methods, while all positive samples (range 0.84–7.30 ngg−1) showed OTA levels lower than 8 ngg−1 (maximum limit recommended by the European Union). The matrix interference of green coffee was minimized by dilution of sample extracts before carrying out the ELISA assay. This mAb-based ic-ELISA can be effectively applied for OTA screening in coffee, because it is simple, sensitive and sample preparation is easy.