Occurrence of fumonisins (B<Subscript>1</Subscript>, B<Subscript>2</Subscript>, B<Subscript>3</Subscript>) in maize-based food and feed samples from Indonesia

A survey to evaluate the contamination level of total fumonisins in maize-based foodstuffs, maize and feed from Indonesia is described. The analyses were carried out by enzyme-linked immunosorbent assay (ELISA). Samples were collected from local retail stores around Yogyakarta, Indonesia between February and May 2001. The 101 samples were classified into six categories, i.e. industrially-produced food (n=24), products of small food manufacturers (n=17), maize flour (n=4), maize for food (n=9), maize for feed (n17), and formulated feed (n30). Control of the method showed that the detection limit was 8.7 μg/kg and repeatability is shown by relative standard deviation (RSD) of analyses of contaminated maize (n=5) of 10 %. Results of analyses indicate that 80 samples analysed were contaminated over a large range from 10.0-3307 pg/kg, and the concentration of fumonisins depended on the type of sample. Of four samples of maize flour, none were contaminated (below detection limit). Of 24 samples of industrially produced food, 14 were contaminated in the range 22.8 - 105 μg/kg and 18 of 19 food samples from small manufacturers were contaminated ranging from 12.9 to 234 μg/kg. The highest contamination was observed in maize samples: six of ten samples of maize for food were contaminated between 68.0 - 2471 μg/kg and 16 of 17 samples for feed contained fumonisins over a large range from 17.6 to 3306 μg/kg.     

An antibody-based microarray assay for the simultaneous detection of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript>

Advances in microsystem technology have enabled protein and nucleic acid-based microarrays to be used in various applications, including the study of diseases, drug discovery, genetic screening, and clinical and food diagnostics. Analytical methods for the detection of mycotoxins, however, remain largely based on thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), or enzyme-linked Immunosorbent assay (ELISA) . The aim of our work, therefore, was to transfer an immunological assay from microtitrr plates into microarray format, in order to develop a multiparametric, rapid, sensitive and inexpensive method for the detection of mycotoxins for use in food safety applications. Microarray technology enables the fast and parallel analysis of a multitude of biologically relevant parameters. Not only nucleic acid-based tests but also peptide, antigen, and antibody assays, using different formats of microarrays, have evolved within the last decade. Antibody-based microarrays provide a powerful tool that can be used to generate rapid and detailed expression profiles of a defined set of analytes in complex samples and are potentially useful for generating rapid immunological assays of food contaminants. In this paper, we report a feasibility study of the application of antibody microarrays for the simultaneous (or independent) detection of two common mycotoxins, Aflatoxin B₁ and Fumonisin B₁. We present the development of microarray detection of aflatoxin B1 and fumonisin B1 in standard solutions with detection limits of 3 ng/ml of AFB1 and 43 ng/ml for FB1, and have developed a competitive immunoassay in microarray format for simultaneous analyses. The quality of the microarray data is comparable to data generated by microplate-based immunoassay (ELISA), but further investigations are needed in order to characterise our method more fully. We hope that these preliminary results might suggest that further research is warranted in order to develop hapten microarrays for the immunochemical simultaneous analysis of mycotoxins, as well as for other small molecules (e.g. bacterial toxins or biological warfare agents).     

Production of fumonisins B<Subscript>2</Subscript> and B<Subscript>4</Subscript> in <Emphasis Type="Italic">Tolypocladium</Emphasis> species

Tolypocladium inflatum is known primarily for its production of the cyclosporines that are used as an immunosuppressive drug. However, we report here the production of the carcinogenic fumonisins B₂ and B₄ by this biotechnologically relevant fungal genus. These mycotoxins were detected in 11 strains tested from three species: Tolypocladium inflatum, T. cylindrosporum, and T. geodes. Production of fumonisins by Fusarium spp. and Aspergillus niger is highly medium- and temperature-dependent, so the effect of these parameters on fumonisin production by three T. inflatum strains was studied. Maximum production was achieved on media with high sugar content incubated at 25–30°C. Since these results demonstrate that fumonisin production could be widespread within the genus Tolypocladium, the potential contamination of commercial cyclosporine preparations with fumonisins needs to be investigated.     

Solvent-dependent transformation of aflatoxin B<Subscript>1</Subscript> in soil

To date, all studies of aflatoxin B₁ (AFB₁) transformation in soil or in purified mineral systems have identified aflatoxins B₂ (AFB₂) and G₂ (AFG₂) as the primary transformation products. However, identification in these studies was made using thin layer chromatography which has relatively low resolution, and these studies did not identify a viable mechanism by which such transformations would occur. Further, the use of methanol as the solvent delivery vehicle in these studies may have contributed to formation of artifactual transformation products. In this study, we investigated the role of the solvent vehicle in the transformation of AFB₁ in soil. To do this, we spiked soils with AFB₁ dissolved in water (93:7, water/methanol) or methanol and used HPLC-UV and HPLC-MS to identify the transformation products. Contrasting previous published reports, we did not detect AFB₂ or AFG₂. In an aqueous-soil environment, we identified aflatoxin B_2a (AFB_2a) as the single major transformation product. We propose that AFB_2a is formed from hydrolysis of AFB₁ with the soil acting as an acid catalyst. Alternatively, when methanol was used, we identified methoxy aflatoxin species likely formed via acid-catalyzed addition of methanol to AFB₁. These results suggest that where soil moisture is adequate, AFB₁ is hydrolyzed to AFB_2a and that reactive organic solvents should be avoided when replicating natural conditions to study the fate of AFB₁ in soil.     

Aflatoxins B<Subscript>1</Subscript> and M<Subscript>1</Subscript>: risks related to milk produced in Brazil

This work shows data on the occurrence of aflatoxins in milk produced in Brazil. A review of the literature on this contamination. Several studies carried out in Brazil show that levels of aflatoxin M₁ in milk are higher than the ones established by the legislation, an evidence of the lack of control and inspection of these mycotoxins. Taking into account that milk has been widely consumed as an important source of nutrients, mainly by children, it is fundamental to carry out a thorough study of the occurrence of aflatoxins and take measures to mitigate milk contamination.     

Methylenecyclopropene: local vision of the first <Superscript>1</Superscript>B<Subscript>2</Subscript> excited state

The ¹A₁ ground and the first ¹B₂ excited states of the methylenecyclopropene (triafulvene) are described by localized wave functions, based on 20 structures valence bond structures. The results are compared to CASSCF(4,4) calculations for both the energetics and the dipole moment. Additional calculations with partial electronic delocalization are presented, and it is shown that the dipole moment modification does not correspond to a situation where the antiaromatic situation prevails (with 4n electrons in the cycle). Part of the analysis uses a “trust factor” that helps to decide if a wave function is appropriate to describe a given state. The trust factor compares the VB wave function to the CASSCF’s with their overlap. Finally, the valence bond density is used to produce density maps that illustrate the electron transfer upon excitation.

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Graphical Abstract A projector-based method compares CASSCF wave functions to local wave functions, including Lewis structures as shown in the picture. A “trust factor” (τ) is obtained. Both the ground state and the first excited state of the methylenecyclopropene are discussed

     

Effect of chemical and microbial vitamin B<Subscript>12</Subscript> analogues on production of vitamin B<Subscript>12</Subscript>

Strain improvement by genetic manipulation or optimization of fermentation conditions for overproduction of vitamin B₁₂ has a drawback due to feed back inhibition. To resist the feed back inhibition by analogues of vitamin B₁₂ in Propionibacterium freudenrechii subsps. shermanii (OLP-5), we have tested with microbially separated B₁₂ analogues from three different strains. Microbial analogues were differentiated from commercially available vitamin B₁₂ by high pressure liquid chromatography and spectrophotometric method. An analogue isolated from NRRL-B-4327 was shown to increase vitamin B₁₂ concentration from 18.53 ± 0.15 to 31.67 ± 0.58 mg/l in OLP-5 strain. The presence of chemical analogue (ICH₂ Co(DH)₂ (H₂Py)₄) increased vitamin B₁₂ production from 16.13 ± 0.15 to 18.53 ± 0.15 mg/l in OLP-5. These findings revealed that addition of B₁₂ analogues in fermentation media have developed strain resistance to feed back inhibition by vitamin B₁₂.     

The effect of <Emphasis Type="Italic">Baccharis glutinosa</Emphasis> extract on the growth of mycotoxigenic fungi and fumonisin B<Subscript>1</Subscript> and aflatoxin B<Subscript>1</Subscript> production

The aim of this study was to evaluate the effect of Baccharis glutinosa isolated extract on the growth of Aspergillus flavus and Aspergillus parasiticus, and their aflatoxin B₁ production; and growth of Fusarium verticillioides, and their fumonisin B₁ production. The three fungi were exposed to an antifungal fraction, designated as fraction F6-1, isolated from B. glutinosa by methanolic extraction followed by silica gel chromatography. The growth of the fungi was evaluated in kinetics of radial extension growth, kinetics of spores germination, length and diameter of hyphae, spores diameter, as well as in aflatoxin B₁ and fumonisin B₁ production. Fraction F6-1 caused radial growth inhibition of the three fungi mainly F. verticillioides. Spores germination of A. flavus and A. parasiticus was delayed in the early stage of the incubation time, although they completely germinated at 27 h. In contrast, spore germination of F. verticillioides was inhibited 87.7% up to 96 h. The lengths and diameters of hyphae, and spore diameters of the three fungi, were significantly smaller in comparison with those of the controls, and several morphological alterations were observed. Concerning aflatoxin B₁ and fumonisin B₁, fraction F6-1 did not show any inhibition effect at the concentration used. Fraction F6-1 was able to significantly inhibit the development of the three fungi, mainly F. verticillioides. The strong inhibitory effect of F6-1 on hyphae and spores suggests that it interacted with the fungi cell walls, which caused severe deformities. Nevertheless, this fraction was unable in inhibiting mycotoxin production from the three fungi at the concentration tested.     

Aflatoxin B<Subscript>1</Subscript> in chilies from the Punjab region,Pakistan

The occurrence of aflatoxin B1 (AFB1) in chilies from Pakistan was determined by using HPLC in work undertaken in Pakistan. Whole (n = 22) and powdered (n = 22) chilies were analyzed. Sixteen (73.0%) and 19 (86.4%) samples of whole and ground chilies, respectively, were contaminated. The mean concentration in powdered chilies (32.20 μg/kg) was higher statistically than in whole chilies (24.69 μg/kg). Concentrations ranged from 0.00 to 89.56 μg/kg for powdered chilies, compared with 0.00–96.3 μg/kg for whole chilies. The limits of detection and quantification were 0.05 μg/kg and 0.53 μg/kg, respectively. The concentrations were high in general and greater than the statutory limit set by the European Union. There is considerable scope for improvements in chili production in Pakistan.     

Exposure assessment and risk characterization of aflatoxin B<Subscript>1</Subscript> in Malaysia

Aflatoxin B₁ (AFB₁) was detected in 57% of the nuts and nut products marketed in Penang, Malaysia using the liquid chromatography tandem mass spectrometry. The contamination levels ranged from 0.40 to 222 μg/kg and 17 out of 128 samples (13.3%) contained AFB₁ above the European Commission permitted level (2 μg/kg). Estimated dietary exposure of AFB₁ in nuts and nut products were 0.36 ng per kg body weight and day and 8.89 ng per kg body weight and day, representing the low and high-level of exposure, respectively. Dose-response modelling resulted in benchmark dose lower confidence limit (BMDL₁₀) values of 0.305 ng per kg body weight and day, with the best fitted from the log-logistic model. The derived margin of exposure (MoE) values ranged from 34 to 847 suggested that AFB₁ would be of public health concern and might reasonably be considered as a high priority for risk management actions.     

Determination of serum aflatoxin B<Subscript>1</Subscript>-lysine to evaluate the efficacy of an aflatoxin-adsorbing feed additive in pigs fed an aflatoxin B<Subscript>1</Subscript>-contaminated diet

In this study, serum aflatoxin B₁ (AFB₁)-lysine was determined in order to evaluate the in vivo efficacy of a hydrated sodium calcium aluminosilicate (HSCAS) in pigs fed AFB₁. Twenty-four 49-day-old crossbred barrows were maintained in individual cages and allowed ad libitum access to feed and water. A completely randomized design was used with six animals assigned to each of four dietary treatments for 21 days as follows: (A) basal diet (BD), (B) BD supplemented with 0.5 % HSCAS, (C) BD supplemented with 1.1 mg/kg AFB₁, and (D) BD supplemented with 0.5 % HSCAS and 1.1 mg/kg AFB₁. HSCAS was able to alleviate the toxic effects of AFB₁ on pigs and reduce (P < 0.05) the levels of serum AFB₁-lysine. Cumulative reductions of adduct yield values, calculated through the equation [(pg AFB₁-lysine/mg albumin) / (μg AFB₁/kg body weight)], were 53.0, 62.8, and 72.1 after 7, 14, and 21 days of oral exposure, respectively. AFB₁-lysine has potential as an AFB₁-specific biomarker for diagnostic purposes and for evaluating the efficacy of chemoprotective interventions in pigs.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Exposure to aflatoxin B<Subscript>1</Subscript> in Thailand by consumption of brown and color rice

This study assessed the aflatoxin B₁ (AFB₁) intake of the Thai population through consumption of contaminated brown and color rice. A total of 240 rice samples from two harvesting periods were collected in June/July 2012 (period I) and in December 2012/January 2013 (period II) and analyzed for AFB₁ by HPLC with fluorescence detection (limit of detection (LOD)?=?0.093 ng/g). Exposure assessment was based on AFB₁ levels in rice and food intake data for rice according to Thai National Consumption. Frequency and levels of AFB₁ were higher in period I (59 %, <LOD?=?26.61 μg kg^?1) than in period II (10 %, <LOD?=?3.51 μg kg^?1). Only one sample exceeded the Thai standard limit for total aflatoxin of 20 μg kg^?1, but 12 out of 240 rice samples exceeded the European Union maximum level for AFB₁ of 2 μg kg^?1. The data showed that the quality and safety of Thai rice largely comply with the requirement for both exports and domestic consumption. According to the Thai National Consumption data, the estimated AFB₁ intake via rice consumption in period I and period II was 0.80 and 0.12 μg kg^?1 bw day^?1, respectively. The potential risk for cancer, based on the recommendation of the JECFA, was estimated to be 0.011 person/year/100,000 people at a mean consumption. Although the risk via consumption of Thai rice seems to be low, the maximum levels of AFB₁ in this staple food suggest that careful monitoring and surveillance of AFB₁ contamination in rice is essential to ensure the safety of rice.     

Aflatoxin B<Subscript>1</Subscript> levels in groundnut products from local markets in Zambia

In Zambia, groundnut products (milled groundnut powder, groundnut kernels) are mostly sold in under-regulated markets. Coupled with the lack of quality enforcement in such markets, consumers may be at risk to aflatoxin exposure. However, the level of aflatoxin contamination in these products is not known. Compared to groundnut kernels, milled groundnut powder obscures visual indicators of aflatoxin contamination in groundnuts such as moldiness, discoloration, insect damage or kernel damage. A survey was therefore conducted from 2012 to 2014, to estimate and compare aflatoxin levels in these products (n = 202), purchased from markets in important groundnut growing districts and in urban areas. Samples of whole groundnut kernels (n = 163) and milled groundnut powder (n = 39) were analysed for aflatoxin B₁ (AFB₁) by competitive enzyme-linked immunosorbent assay (cELISA). Results showed substantial AFB₁ contamination levels in both types of groundnut products with maximum AFB₁ levels of 11,100 μg/kg (groundnut kernels) and 3000 μg/kg (milled groundnut powder). However, paired t test analysis showed that AFB₁ contamination levels in milled groundnut powder were not always significantly higher (P > 0.05) than those in groundnut kernels. Even for products from the same vendor, AFB₁ levels were not consistently higher in milled groundnut powder than in whole groundnut kernels. This suggests that vendors do not systematically sort out whole groundnut kernels of visually poor quality for milling. However, the overall contamination levels of groundnut products with AFB₁ were found to be alarmingly high in all years and locations. Therefore, solutions are needed to reduce aflatoxin levels in such under-regulated markets.     

An aminotransferase from bacterium ATCC 55552 deaminates hydrolyzed fumonisin B<Subscript>1</Subscript>

Previous research identified several microorganisms and pathways capable of degrading the mycotoxin fumonisin B₁ (FB₁). Degradation of FB₁ by microorganisms seems to comprise two essential steps: hydrolysis to hydrolyzed fumonisin B₁ (HFB₁) and deamination of the hydrolysis product. One of the previously studied microorganisms was the Gram negative bacterium ATCC 55552. The gene corresponding to the first step of FB₁ degradation in this bacterium was identified, but the genetic basis for deamination of the hydrolyzed intermediate remained unexplained (Duvick et al. 2000, PCT patent application WO200004158). Here we report the sequence and HFB₁-deaminating activity of a novel aminotransferase encoded by the bacterium ATCC 55552. The corresponding gene was identified, sequenced, and over-expressed in Escherichia coli. Cell lysates of the recombinant E. coli strain showed distinct HFB₁-deaminating activity in the presence of pyridoxal phosphate and pyruvate, as was demonstrated by liquid chromatography–mass spectrometry. Thus, we suggest the novel enzyme to be part of the fumonisin catabolic pathway of the bacterium ATCC 55552.     

Increased CO<Subscript>2</Subscript> evolution caused by heat treatment in wood-decaying fungi

Wood-decaying fungi are regarded as the main decomposers of woody debris in boreal forests. Given that fungal respiration makes a significant contribution to terrestrial carbon flows, it is important to understand how the wood-decaying fungal metabolism is regulated in relation to different environmental conditions and disturbances. In the present study, we investigated the effect of temperature stress on wood decomposition rate in 18 species of wood-decaying fungi, representing a broad range of species–habitat associations. Heat shock duration and temperature were calibrated to match the conditions of a forest fire. We found a general increase in fungal decay rate after heat shock; the response was more pronounced in species associated with fire-prone forests. The underlying mechanism is unclear, but possibly relates to an up-regulation at the cellular level in response to heat shock. Our results show that the decomposition rate of dead wood can be strongly affected by environmental triggers.     

Bonding,aromaticity, and planar tetracoordinated carbon in Si<Subscript>2</Subscript>CH<Subscript>2</Subscript> and Ge<Subscript>2</Subscript>CH<Subscript>2</Subscript>

Natural bond orbital (NBO) analyses and dissected nucleus-independent chemical shifts (NICS _{π z z} ) were computed to evaluate the bonding (bond type, electron occupation, hybridization) and aromatic character of the three lowest-lying Si₂CH₂ (1-Si, 2-Si, 3-Si) and Ge₂CH₂ (1-Ge, 2-Ge, 3-Ge) isomers. While their carbon C₃H₂ analogs favor classical alkene, allene, and alkyne type bonding, these Si and Ge derivatives are more polarizable and can favor “highly electron delocalized”? and “non-classical”? structures. The lowest energy Si ₂CH₂ and Ge ₂CH₂ isomers, 1-Si and 1-Ge, exhibit two sets of 3–center 2–electron (3c-2e) bonding; a π-3c-2e bond involving the heavy atoms (C–Si–Si and C–Ge–Ge), and a σ-3c-2e bond (Si–H–Si, Ge–H–Ge). Both 3-Si and 3-Ge exhibit π and σ-3c-2e bonding involving a planar tetracoordinated carbon (ptC) center. Despite their highly electron delocalized nature, all of the Si₂CH₂ and Ge₂CH₂ isomers considered display only modest two π electron aromatic character (NICS(0) _{π z z} =--6.2 to –8.9 ppm, computed at the heavy atom ring center) compared to the cyclic-C ₃H₂ (–13.3 ppm).

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Graphical Abstract The three lowest Si2CH2 and Ge2CH2 isomers.

     

Bonding analysis and stability on alternant B<Subscript>16</Subscript>N<Subscript>16</Subscript> cage and its dimers

Bonding analysis is performed on alternant B₁₆N₁₆ cage based on a combined study of DFT with NBO method. The main feature of such analysis is the separation of bonding structure into two components: σ skeleton and π bond system. Each component is further decomposed into contributions from various NBOs, thus we obtain the details of bonding interactions of every BN unit. Based on these results, relative stability of four covalent dimers of B₁₆N₁₆ is predicted and this prediction is verified by DFT calculations. So the possibility of forecasting properties of oligomers just from analysis on monomer is highlighted in this way.