Efecto de extractos de plantas sobre el crecimiento y producción de aflatoxinas por Aspergillus flavus Link ex Fries y A. parasiticus Speare

Tesis (Maestría en Ciencias con Especialidad en Microbiología) UANL

Inhibición del crecimiento y la producción de toxinas de Aspergillus flavus y A. parasiticus por extractos de plantas del género Agave

Tesis (Maestría en Ciencias con Especialidad en Microbiología) UANL

Influencia de extractos de agavaceas sobre el crecimiento de Aspergillus flavus Link ex. Fries y A. parasiticus Speare

Tesis (Maestría en Ciencias con Especialidad en Microbiología) UANL

Use of PCR for detecting Aspergillus flavus in maize treated by gamma radiation process

The aim of this study was to verify the effects of gamma radiation process on the fungal DNA and the application of PCR in the detection of Aspergillus flavus in irradiated maize grains. The samples were inoculated with a toxigenic strain and incubated under controlled conditions of relative humidity, water activity, and temperature for 15 days. After incubation, the samples were treated with gamma radiation with doses of 5 and 10 kGy and individually analyzed. The use of PCR technique showed the presence of DNA bands of Aspergillus flavus in all irradiated samples that showed no fungal growth in agar medium.

Characterization of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from corn grains of different geographic origins in Brazil

Aflatoxins can cause great economic losses and serious risks to humans and animals health. The largest aflatoxin producers belong to Aspergillus section Flavi and can occur naturally in food commodities. Studies showed that molecular tools as well as the type of sclerotia produced by the strains could be helpful for identification of Aspergillus species and could be correlated with levels of toxin production. The purpose of this work was to characterize the genetic diversity using AFLP technique, the type of sclerotia and the ability of aflatoxin production by isolated strains from corn of different origins in Brazil, and to verify whether qPCR based on aflR and aflP genes is appropriate for estimating the level of aflatoxin production. All the 75 strains were classified as A. flavus and the AFLP technique showed a wide intraspecific variability within them. Regarding sclerotia production, 34% were classified as S and 66% as L type. Among the aflatoxin-producers, 52.8% produced aflatoxin B-1, while 47.2% aflatoxins B-1 and B-2. Statistical analysis showed no correlation between sclerotia production and aflatoxigenicty, and no correlation between the phylogenetic clusters and aflatoxin production. Concerning the relative expression of aflR and aflP, Pearson's correlation test demonstrated low positive correlation between the expression of the aflR and aflP genes and the production of AFB(1) and AFB(2), but showed high positive correlation between aflR and aflP expression. In contrast to the other reference strains, A. oryzae ATCC 7282 showed no amplification of aflR and aflP. The results highlight the need for detection of reliable and reproducible markers with a high positive correlation with aflatoxin production.

Molecular phylogeny and phenotypic variability of clinical and environmental strains of Aspergillus flavus

Aspergillus flavus is the second most common cause of aspergillosis infection in immunocompromised patients and is responsible for the production of aflatoxins. Little is known about the population structure of A. flavus, although recent molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. The aim of this study was to carry out a morphological, physiological, and molecular analysis of a set of clinical and environmental isolates to determine whether this variability is due to species divergence or intraspecific diversity, and to assess whether the clinical isolates form a separate group. The amdS and omtA genes were more phylogenetically informative than the other tested genes and their combined analysis inferred three main clades, with no clear distinction between clinical and environmental isolates. No important morphological and physiological differences were found between the members of the different clades, with the exception of the assimilation of D-glucosamine, which differentiates the members of the clade II from the others. (C) 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

A survey on prevalence rate of aflatoxigenic species of Aspergillus and residues of aflatoxins by ELISA method in cold-water cultural fish (rainbow trout) feeds in Tehran and West Azarbayjan provinces-Iran

Aflatoxins are one kind of fungal toxins produced by species of toxigenic Aspergillus (A. flavus and A. parasiticus) and in other words they are secondary metabolites which are considered as one of the threatening factors of food consumer's health. In this research 96 samples of cold-water cultural fish feed, rainbow trout, during the seasons of spring and summer of 2007 (every fifteenth of the month) were randomized (by simple and stratified random) to determine: 1. The prevalence rate of aflatoxigenic species of Aspergillus in stored feed of cold-water cultural fish in West Azarbayjan cultural fish farms in both seasons (spring and summer); 2. The residues of total aflatoxin in stored feed of fish in cultural fish farms of West Azarbayjan in both seasons by ELISA method; and 3. The residues of that toxin in feed produced in aquatic feed factories in Tehran and West Azarbyjan provinces with the same method. In order to study prevalence rate of toxigenic species of Aspergillus, pour-plate culture method by general medium such as Malt Extract Agar (M.E.A.) and Sabouraud-Dextrose Agar (S.D.A.) and by standard No.997 of Iranian Standard Institute were used. The produced colonies were examined microscopically. To determine the aflatoxins residues, ELISA method using Agra-Quant kit of Romer Lab company, were applied. The results of this survey indicated that only 8.3% of the samples were infected by A. flavus. A. parasiticus was not observed. There were no significant differences between the prevalence rate of AFT and seasons/months, either (P<0.05). Evaluating mean of aflatoxin rate showed that the rates of this variable are lower than the tolerance levels designated by the joint FAO/WHO expert committee (The mean of AFT in all data was lower than 11 ppb). Furthermore, mean of total AFT residues rates of stored feed of various cultural center of West Azarbayjan and Tehran factories were comparable in spring and summer, and no significant differences were observed (P<0.05). But there were significant differences between the total aflatoxin rates in the feed of West Azarbayjan factory and spring and summer (P<0.05), and AFT residues in spring (8.6 ppb) were higher than summer (6.1 ppb). Prevalence rates of AFT in Tehran feed factories (9.2 ppb) are higher than W. Azarbayjan (7.4 ppb). In other words, location was considered as a decisive factor in total AFT rates of samples. Moreover, the results indicated that there was significant difference between total aflatoxin rates of feed and cultural centers (P<0.05). The mean of AFT rates in embankment dam cultural fish farms (6.75 ppb) and multi-functions cultural fish farms (6.25 ppb) was higher than individual cultural pond (4.67 ppb). In conclusion, the finally results of this survey indicated that the lower rates of Aspergillus is not effective on the presence of total aflatoxin rates in trout feed. Due to low levels of aflatoxin rates (lower than 20 ppb), the produced feed of cold-cultural fishes, Rainbow Trout, in Tehran and West Azarbayjan provinces, in spring and summer of 2007, were safe and healthy both for fish and their consumers.

Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium. © 2010 Springer-Verlag.

Anthranilate Hydroxylase from Aspergillus niger: New Type of NADPH-Linked Nonheme Iron Monooxygenase

Anthranilate hydroxylase from Aspergillus niger catalyzes the oxidative deamination and dihydroxylation of anthranilic acid to 2,3-dihydroxybenzoic acid. This enzyme has been purified to homogeneity and has a molecular weight of 89,000. The enzyme is composed of two subunits of 42,000 with 2 gram-atoms of nonheme iron per mol. Fe2+-chelators like alpha,alpha'-dipyridyl and o-phenanthroline are potent inhibitors of the enzyme activity. Absorption and fluorescence spectra of the enzyme offer no evidence for the presence of other cofactors like flavin. Flavins and flavin-specific inhibitors like atebrin have no effect on the activity of the enzyme. The enzyme incorporates one atom of oxygen each from 18O2 and H218O into the product 2,3-dihydroxybenzoic acid. Based on these studies, it is concluded that anthranilate hydroxylase from A. niger is a new type of NADPH-linked nonheme iron monooxygenase.

Modelling climatic risks of aflatoxin contamination in maize

Aflatoxins are highly carcinogenic mycotoxins produced by two fungi, Aspergillus flavus and A. parasiticus, under specific moisture and temperature conditions before harvest and/or during storage of a wide range of crops including maize. Modelling of interactions between host plant and environment during the season can enable quantification of preharvest aflatoxin risk and its potential management. A model was developed to quantify climatic risks of aflatoxin contamination in maize using principles previously used for peanuts. The model outputs an aflatoxin risk index in response to seasonal temperature and soil moisture during the maize grain filling period using the APSIM's maize module. The model performed well in simulating climatic risk of aflatoxin contamination in maize as indicated by a significant R2 (P ≤ 0.01) between aflatoxin risk index and the measured aflatoxin B1 in crop samples, which was 0.69 for a range of rainfed Australian locations and 0.62 when irrigated locations were also included in the analysis. The model was further applied to determine probabilities of exceeding a given aflatoxin risk in four non-irrigated maize growing locations of Queensland using 106 years of historical climatic data. Locations with both dry and hot climates had a much higher probability of higher aflatoxin risk compared with locations having either dry or hot conditions alone. Scenario analysis suggested that under non-irrigated conditions the risk of aflatoxin contamination could be minimised by adjusting sowing time or selecting an appropriate hybrid to better match the grain filling period to coincide with lower temperature and water stress conditions.

Anthranilate Hydroxylase from Aspergillus niger: Evidence for the Participation of Iron in the Double Hydroxylation Reaction

Evidence was obtained for the participation of iron in the double hydroxylation reaction catalyzed by anthranilate hydroxylase from Aspergillus niger (UBC 814). Omission of iron from the growth medium gave inactive preparations of anthranilate hydroxylase which could be reactivated by incubating the enzyme preparations with ferric citrate. The enzyme was susceptible to inhibition by metal chelating agents. The Ki for o-phenanthroline, which inhibited the enzyme activity non-competitively with respect to anthranilate, was calculated to be 0.9 mM. The inhibition by o-phenanthroline was counteracted by ferric complexes such as ferric-ethylenediaminetetraacetic acid and ferric citrate. Anthranilate afforded protection against inhibition by o-phenanthroline.

Involvement of protocatechuic acid in the metabolism of phenylacetic acid by Aspergillus niger

Abstract is not available.

Inactivation of Aspergillus flavus spores by curcumin-mediated photosensitization

Minimizing fungal infection is essential to the control of mycotoxin contamination of foods and feeds but many potential control methods are not without their own safety concerns for the consumers. Photodynamic inactivation is a novel light-based approach which offers a promising alternative to conventional methods for the control of mycotoxigenic fungi. This study describes the use of curcumin to inactivate spores of Aspergillus flavus, one of the major aflatoxin producing fungi in foods and feeds. Curcumin is a natural polyphenolic compound from the spice turmeric (Curcuma longa). In this study the plant has shown to be an effective photosensitiser when combined with visible light (420 nm). The experiment was conducted in in vitro and in vivo where A. flavus spores were treated with different photosensitiser concentration and light dose both in buffer solution and on maize kernels. Comparison of fungal load from treated and untreated samples was determined, and reductions of fungal spore counts of up to 3 log CFU ml−1 in suspension and 2 log CFU g−1 in maize kernels were obtained using optimal dye concentrations and light dose combinations. The results in this study indicate that curcumin-mediated photosensitization is a potentially effective method to decontaminate A. flavus spores in foods and feeds.

Studies on Aspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grown Aspergillus niger was increased 3-5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH4 + , and further, the enzyme is repressed by increasing concentrations of NH4 +. In contrast to other micro-organisms, the Aspergillus niger enzyme was neither specifically inactivated by NH4+ or L-glutamine nor regulated by covalent modification.Glutamine synthetase from Aspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn2+ or Mg2+-dependent synthetase and Mn2+-dependent transferase activity.Aspergillus niger glutamine synthetase was completely inactivated by two mol of phenylglyoxal and one mol of N-ethylmaleimide with second order rate constants of 3·8 M–1 min–1 and 760 M–1 min–1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH4+, Mn2+ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.