Elisabete Hiromi Hashimoto

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.

Further Investigation of Microbial Degradation of Microcystin Using the Advanced Marfey Method

It is known that microcystin (MC) is subject to microbial degradation to provide three types of products, linearized MCLR (Adda-Glu-Mdha-Ala-Leu-MeAsp-Arg), tetrapeptide Adda-Glu-Mdha-Ala, and Adda. They can be readily detected by the usual HPLC, because they commonly have an Adda moiety with a diene and an absorption maximum at 238 nm as the chromophore. However, no other degradation products without such a chromophore have been isolated to date. In this study, cell preparation of a bacterium B-9 that can degrade MC and detection of the degradation products were devised. First, we regulated the B-9 hydrolytic activity by washing with sodium chloride solution to obtain a desired cell preparation, which permitted an additional intermediate and the final products of MCLR to be obtained. Second, the resulting products could be firmly identified using the advanced Marfey method with the aid of log D. As a result of these experiments, the following degradation products were further identified: a tetrapeptide, Adda-Glu-Mdha-Ala, tripeptides Adda-Glu-Mdha, Glu-Mdha-Ala, and Arg-MeAsp-Leu, a dipeptide, Glu-Mdha, and amino acids Adda, Arg, and methylamine derived from Mdha. The present study expands the hydrolytic activity of the B-9 strain, which can hydrolyze not only cyanobacterial cyclic peptides but also MC to the intermediates and final products. The established characterization method composed of the advanced Marfey method and log D would be a standard technique for the structural characterization of a mixture of amino acids and peptides.

Mycotoxicological quality evaluation of corn samples used by processing industries in the Northern region of Paraná State, Brazil.

Based on fungal and fumonisin contamination of 870 freshly harvested samples, the quality of corn used by processing industries in the Northern region of Paraná State, Brazil (2003 and 2004 crop-year) was evaluated. Sampling was carried out for each crop at two points in the production chain, i.e. at reception by the processors and at the pre-drying step. Corn samples were more frequently contaminated with Fusarium sp. (100%) and Penicillium sp. (84.1–95.3%) than Aspergillus sp. (5.6–19.8%). Fumonisin B1 (FB1) was detected in all samples from the two points in both crop-years. FB1 levels ranged 0.02–11.83 µg g−1 in the reception and 0.02–10.98 µg g−1 in the pre-drying samples of the 2003 crop. Samples from the 2004 crop showed FB1 levels ranging 0.03–12.04 µg g−1 in the reception and 0.06–7.74 µg g−1 in the pre-drying samples. FB2 levels ranged 0.02–5.25 µg g−1 in the reception and 0.01–7.89 µg g−1 in the pre-drying samples (2003 crop-year). In samples from the 2004 crop, FB2 levels ranged 0.02–6.12 µg g−1 in the reception and 0.05–3.47 µg g−1 in the pre-drying samples. Low fumonisin levels were detected in most corn samples used by processors in the Northern region of Paraná State, showing a decreasing trend in fumonisin contamination over the years.

Biomonitoring of microcystin and aflatoxin co-occurrence in aquaculture using immunohistochemistry and genotoxicity assays

This work investigated the effects of co-occurring aflatoxin B1 (AFB1) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB1 by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB1 was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB1 showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

Microbial Degradation of Amino Acid-Containing Compounds Using the Microcystin-Degrading Bacterial Strain B-9

Strain B-9, which has a 99% similarity to Sphingosinicella microcystinivorans strain Y2, is a Gram-negative bacterium with potential for use in the degradation of microcystin-related compounds and nodularin. We attempted to extend the application area of strain B-9 and applied it to mycotoxins produced by fungi. Among the tested mycotoxins, only ochratoxin A was completely hydrolyzed to provide the constituents ochratoxin α and l-phenylalanine, and levels of fumonisin B1 gradually decreased after 96 h. However, although drugs including antibiotics released into the aquatic environment were applied for microbial degradation using strain B-9, no degradation occurred. These results suggest that strain B-9 can only degrade amino acid-containing compounds. As expected, the tested compounds with amide and ester bonds, such as 3,4-dimethyl hippuric acid and 4-benzyl aspartate, were readily hydrolyzed by strain B-9, although the sulfonamides remained unchanged. The ester compounds were characteristically and rapidly hydrolyzed as soon as they came into contact with strain B-9. Furthermore, the degradation of amide and ester compounds with amino acids was not inhibited by the addition of ethylenediaminetetraacetic acid (EDTA), indicating that the responsible enzyme was not MlrC. These results suggest that strain B-9 possesses an additional hydrolytic enzyme that should be designated as MlrE, as well as an esterase.

Safety and Quality in the Agricultural Product Chain in Brazil

An agriculture-intensive country should be aware of natural toxins, including both mycotoxins and cyanotoxins, which are closely associated with the quality of raw materials, for food safety and industry. The major production chains – corn, wheat, beef, and broiler chicken – are the top components of agribusiness, and they should be tracked by reliable and practical tools. The corn chain is of particular concern in food production; intensive controls, multi-year mycotoxin monitoring, and improved harmless/sustainable management methods for uninterrupted farming in the tropicsubtropics are needed to achieve a long-lasting trend. The rapid control of natural toxins (mycotoxin and cyanotoxin) has focused on immunochemical methods developed with highly specific monoclonal antibodies (mAb) matched with chroma‐ tographic methods. In parallel, the promising widespread application of nondestructive analytical methods based on NIR (Near Infrared Reflectance)