Molecular characterization of mycotoxin-producing Aspergillus parasiticus and sensitivity pattern to different disinfectants

Aims@#The study was aimed to isolate and characterize the mycotoxin-producing filamentous Aspergillus parasiticus from the feed samples. The sensitivity pattern of the isolates was assessed against different disinfectants.@*Methodology and results@#Fifty different feed samples were screened for A. parasiticus isolation. Isolates were subjected to macroscopic and microscopic characterization. Polymerase chain reaction was performed to confirm the isolates at the genomic level. Mycotoxin producing potential of the isolates was assessed by thin-layer chromatography (TLC). To quantify the toxins, high performance liquid (HPLC) was employed. The antifungal potential of disinfectants was determined by the well diffusion method followed by minimum inhibitory concentration (MIC) calculation. Out of twenty isolates of A. parasiticus, 11(55%) isolates were observed positive for toxin production. Three toxigenic isolates (AspP2, AspP4 and AspP8) were selected to evaluate their susceptibility against disinfectants by well diffusion method. AspP2 produced maximum (5.90 ng/mL) toxin, followed by AspP4 (3.11 ng/mL) and AspP8 (18.47 ng/mL). Terralin showed maximum fungicidal activity with 29.66 ± 8.08 mm zone of inhibition at 0.42 μg/mL MIC. Hypochlorite and Instru Star showed 99% disinfection with 30, 60 and 90 min contact time (6 mean log reduction) for all A. parasiticus isolates. Alpha Guard inhibited growth after 15 min contact time for all the isolates.@*Conclusion, significance and impact of study@#This study provides data indicating the contamination of feed samples with mycotoxin-producing A. parasiticus isolates and their sensitivity against commercially available disinfectants. Use of these disinfectants in appropriate concentrations and time could help prevent the contamination of food, feed and healthcare settings with the fungal species.

Investigation of aflatoxins and toxigenic fungi contamination during post-harvest processing of Polygalae Radix / 中草药

Objective: To investigate aflatoxins contamination B1, B2, G1, G2 (AFB1, ATB2, AFG1, AFG2), toxigenic fungi species and potential contamination sources of Polygalae Radix during post-harvest processing, and analyze the main ways of aflatoxins contamination. Methods: Twenty-one Polygalae Radix samples were collected from multiple steps during the post-harvest processing in this study. Aflatoxin levels in these samples were determined by immunoaffinity column and HPLC coupled with post-column photochemical derivatization. Dilution-plate method was applied for the fungi isolation followed by strain identification based on morphological characterization and molecular approaches. Results: Aflatoxins were detected in 15 samples, but none of them exceeded the limit set by Chinese Pharmacopoeia. The fungal counts increased significantly from newly harvested samples to post-sweating, and the counts further increased to the maximum (2 × 108 CFU/g) after xylem-removing, then decreased after drying. In contrast, fungal counts of samples dried directly after harvesting did not change much throughout the processing. There was a significant positive correlation between fungal counts and water activity (Aw). A total of 209 fungal belonged to five genera were identified from the samples, and Penicillium was the predominant genus. Cladosporium and Fusarium were increased after sweating, and then Aspergillus increased after xylem-removing and drying. One A. parasiticus strain was confirmed to be able to produce AFB1, AFB2, AFG1, and AFG2. Conclusion: Aflatoxins contamination happened in both field production and post-harvest processing of Polygalae Radix. Especially, the contamination of Penicillium spp. should be paid more attention.

Hongos productores de micotoxinas asociados a hierbas medicinales en la Ciudad de São Paulo, Brasil / Mould and mycotoxins associated in medicinal plants in São Paulo, Brazil

Antecedentes: la fitoterapia es una de las más antiguas prácticas utilizadas por la humanidad. Hasta mediados del siglo XIX, cuando se introdujeron los medicamentos, la formulación de estos generalmente era basada en plantas medicinales. Objetivos: Determinar la micobiota y los niveles de aflatoxinas originadas de Aspergillus sección Flavi aislados de las 50 muestras de medicamentos fitoterápicos comercializados actualmente en la ciudad de São Paulo, Brasil. Métodos: Cincuenta (50) muestras de medicamentos fitoterápicos en la forma de hojas (té-25) y cápsulas (25) fueron colectadas de agosto de 2000 a julio de 2001. Los hongos filamentosos aislados fueron identificados al nivel de género de acuerdo con las características morfológicas y criterios taxonómicos. El análisis de aflatoxinas fue realizada por cromatografía de capa fina (TLC). Resultados: El análisis microbiológico mostró que 41 (82 por ciento) de los medicamentos fitoterápicos presentaron un crecimiento fúngico sobre las 100 UFC/g. Un total de 106 especies de seis diferentes géneros fueron aislados (Aspergillus, Penicillium, Mucor, Rhizopus y Alternaria). El género Aspergillus fue el predominante (60.5 por ciento) seguido por Penicillium (20,0 por ciento). Aspergillus niger (30 por ciento) A. flavus (22 por ciento), A. fumigatus (6,5 por ciento) y A. parasiticus fueron las especies de Aspergillus identificadas. Se observó que 13 (56,5 por ciento), de los 23 A. flavus aislados y dos aislados de A. parasiticus produjeron aflatoxinas. Conclusiones: La contaminación observada en la mayoría de los productos y el alto nivel de cepas productoras de aflatoxinas justifica un análisis más cuidadoso de los medicamentos fitoterápicos comercializados y la aplicación de leyes más rigurosas son necesarias para garantizar la calidad de los productos.

Background: phytotherapy is one of the most ancient practices used by humanity. In Antiquity until the middle of the XIX century, when chemotherapeutic drugs were introduced, formulation of medicines was usually based on medicinal plants. Objective: To determine mycobiota and levels of Aspergillus section Flavi aflatoxins isolated from 50 samples of phytotherapeutic remedies currently commercialized in São Paulo, Brazil. Methods: Fifty (50) samples of phytotherapeutic remedies in the form of leaves (teas-25) and powders (capsules-25) were collected from August 2000 to July 2001. Filamentous fungi isolates were identified at the genera level in accordance with morphological characteristics and taxonomic criteria. Aflatoxins were performed by Thin-layer chromatography (TLC). Results: The microbiological analysis showed that 41 (82 percent) of phytotherapeutic remedies presented a fungal growth over 100 CFU/g. A total of 106 species of six different genera were isolated (Aspergillus, Penicillium, Mucor, Rhizopus and Alternaria). The genus Aspergillus was the predominant (60.5 percent) followed by Penicillium genus (20.0 percent). Aspergillus niger (30 percent) A. flavus (22 percent), A. fumigatus (6.5 percent) and A. parasiticus were the species of Aspergillus identified. It was observed that 13 (56.5 percent) of 23 A. flavus isolates and two A. parasiticus isolates produced aflatoxins. Conclusions: The contamination observed in most products and the high level of aflatoxigenic strains justify the concern regarding the execution of more careful analyzes of the commercialized phytotherapeutic remedies and the application of more rigorous laws that may warrant the quality of these products.

Validation of PCR based detection system for aflatoxin producing molds.

Aflatoxins are polyketide secondary metabolites that are produced by certain fungal species in the Aspergillus section Flavi, particularly Aspergillus flavus and Aspergillus parasiticus which contaminate human food as well as animal feed. These are among the most carcinogenic substances known. Due to the toxic and carcinogenic properties of aflatoxins, there is a need to develop reliable methods to detect the presence of aflatoxigenic Aspergilli in contaminated food and feed. Not all Aspergillus strains are able to produce aflatoxins. It requires a detection methodology which can specifically distinguish between the aflatoxin producing and non-producing strains of Aspergillus. Present communication reports validation of a PCR based detection system based on three genes viz., nor-1, apa-2 and omt-1 involved in aflatoxin biosynthesis, that can specifically distinguish the two aflatoxin producing species viz. Aspergillus flavus and Aspergillus parasiticus from non-producers i.e., A. niger, A. fumigates and A. oryzae.

Fermentação em estado sólido de Aspergillus parasiticus e produção de aflatoxinas / Solid state fermentation of Aspergillus parasiticus and aflatoxin production

Aflatoxinas são metabólitos secundários de fungos com grande potencial carcinogênico, produzidos principalmente por Apergillus flavus e Aspergillus parasiticus. Em vista da ampla variedade de alimentos em que se encontram essas micotoxinas, o presente estudo teve como objetivo determinar condições de cultivo para o Aspergillus parasiticus e produção das quatro principais aflatoxinas (AFB1, AFB2, AFG1 e AFG2) considerando diferentes substratos conhecidos pela contaminação por estas micotoxinas, entre eles arroz branco, arroz cateto, amendoim, milho e farinha de trigo integral, e diferentes valores de umidade e pH. Ao analisar por cromatrografia em camada delgada os extratos dos diferentes substratos, verificou-se a produção de aflatoxinas em todos os alimentos, porém na cromatografia líquida de alta eficiência foi possível perceber a maior produção de aflatoxinas no arroz cateto e na farinha de trigo integral. Para a continuidade do trabalho, utilizou-se o arroz cateto e então preparou- se diferentes meios sólidos com valores de pH entre 3,5 e 7,5 e umidade entre 42 % e 62 %. Ao analisar por CLAE, todas as amostras apresentaram produção de AF, porém as amostras com o maior valor de água agregada (62%) apresentaram maior produção enquanto a variação de pH não apresentou influência nesta produção.

Aflatoxins are secondary metabolites of fungi with great carcinogenic potencial, mainly produced by Aspergillus flavus and Aspergillus parasiticus. In order of the wide variety of foods where mycotoxins are found in, this study aimed to determine growth conditions for Aspergillus parasiticus and production of four major aflatoxins (AFB1, AFB2, AFG1 and AFG2) considering different substrates known for contamination by these aflatoxins, including white rice, cathetus rice, peanuts, maize and whole wheat flour, and different pH and humidity values. When extracts of different substrates were analyzed by thin layer chromatography, it has been verified the production of aflatoxins in all foods; however, when high performance liquid chromatography (HPLC) analysis was performed, the greater production of aflatoxins was observed in cathetus rice and whole-wheat flour. Cathetus rice was then selected to continue the study and different solid medium with pH values between 3.5 and 7.5 and humidity percentages between 42 % and 62 % were prepared. When analyzed by HPLC, all samples showed production of aflatoxins, but the samples with higher humidity value (62%) showed greatest production while the pH changes had no effect on this production.

Modulation of antimicrobial metabolites production by the fungus Aspergillus parasiticus

Biosynthesis of active secondary metabolites by fungi occurs as a specific response to the different growing environments. Changes in this environment alter the chemical and biological profiles leading to metabolites diversification and consequently to novel pharmacological applications. In this work, it was studied the influence of three parameters (fermentation length, medium composition and aeration) in the biosyntheses of antimicrobial metabolites by the fungus Aspergillus parasiticus in 10 distinct fermentation periods. Metabolism modulation in two culturing media, CYA and YES was evaluated by a 2² full factorial planning (ANOVA) and on a 2³ factorial planning, role of aeration, medium composition and carbohydrate concentration were also evaluated. In overall, 120 different extracts were prepared, their HPLC profiles were obtained and the antimicrobial activity against A. flavus, C. albicans, E. coli and S. aureus of all extracts was evaluated by microdilution bioassay. Yield of kojic acid, a fine chemical produced by the fungus A. parasiticus was determined in all extracts. Statistical analyses pointed thirteen conditions able to modulate the production of bioactive metabolites by A. parasiticus. Effect of carbon source in metabolites diversification was significant as shown by the changes in the HPLC profiles of the extracts. Most of the extracts presented inhibition rates higher than that of kojic acid as for the extract obtained after 6 days of fermentation in YES medium under stirring. Kojic acid was not the only metabolite responsible for the activity since some highly active extracts showed to possess low amounts of this compound, as determined by HPLC.

Effects of carbon, nitrogen and pH on the growth of Aspergillus parasiticus and aflatoxins production in water.

Mycotoxins are considered as the most hazardous fungal metabolites for human, animals and plant health. Recently, more attention has been paid on the occurrence of this group of fungi in different water sources throughout the globe. In this study, Aspergillus parasiticus ATCC strain was used as representative strain producing aflatoxins in drinking water. This study aimed to investigate the activation of fungi in drinking water and their ability to produce aflatoxins (B1, B2, G1, and G2) in water under different ratios of C:N using different concentrations of total organic carbon (TOC) and total nitrogen (TN). Glucose and ammonium sulphate were used for changing the levels of TOC and TN in the selected water media. Similarly, the effects of different water pH levels from 4.5 to 8.2 on the growth of this group of fungi and aflatoxins production were also investigated. The results indicate that the growth of fungi was highest, at C:N ratio of 1:1 as compared to other selected ratios. Furthermore, the findings indicate that the pH levels 5.5-6.5 showed best growth of fungi as compared to other pH levels. Aflatoxin concentrations were measured in the water samples using HPLC technique, but selected fungi were not able to produce aflatoxins in water at applied concentrations of TOC and TN mimicking the ratios and concentrations present in the natural aquatic environment.

The potential effects of Zataria multiflora Boiss essential oil on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes of toxigenic Aspergillus parasiticus

This study aims at evaluating the effects of Zataria multiflora (Z. multiflora) essential oil (EO) on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes. Total RNAs of Aspergillus parasiticus (A.parasiticus) ATCC56775 grown in yeast extract sucrose (YES) broth medium treated with Z. multiflora EO were subjected to reverse transcription-polymerase chain reaction (RT-PCR). Specific primers of nor-1, ver-1, omt-A and aflR genes were used. In parallel mycelial dry weight of samples were measured and all the media were assayed by high-pressure liquid chromatography (HPLC) for aflatoxinB1 (AFB1), aflatoxinB2 (AFB2), aflatoxinG1 (AFG1), aflatoxinG2 (AFG2) and aflatoxin total (AFTotal) production. The results showed that mycelial dry weight and aflatoxin production reduce in the presence of Z. multiflora EO (100 ppm) on day 5 of growth. It was found that the expression of nor-1, ver-1, omt-A and aflR genes was correlated with the ability of fungus to produce aflatoxins on day 5 in YES medium. RT-PCR showed that in the presence of Z.multiflora EO (100 ppm) nor-1, ver-1 and omtA genes expression was reduced. It seems that toxin production inhibitory effects of Z. multiflora EO on day 5 may be at the transcription level and this herb may cause reduction in aflatoxin biosynthesis pathway genes activity.

Qualidade da castanha-do-brasil do comércio de Rio Branco, Acre / Quality of Brazil nuts marketed in Rio Branco, Acre

Este trabalho teve como objetivo avaliar a qualidade de castanhas-do-brasil beneficiadas e comercializadas em Rio Branco, Acre. Foram analisadas amostras das três marcas de castanha encontradas no mercado local quanto às variáveis: atividade de água, teor de umidade, contagem total de fungos filamentosos, quantificação de Aspergillus flavus e de A. parasiticus, bem como quantificação de aflatoxinas B1, B2, G1 e G2. As castanhas do comércio se encontravam com um teor de umidade e atividade de água adequados, o que pode ter sido responsável pela baixa contaminação por fungos e por aflatoxinas. Quanto a estas micotoxinas, as amostras estão de acordo com o recomendado pela Anvisa, podendo ser esta uma consequência da grande divulgação no Estado do uso de Boas Práticas no manejo da castanha.

The goal of this paper was to evaluate the quality of Brazil nuts processed and marketed in the city of Rio Branco, in the state of Acre (Brazil). We analysed three samples for water activity, moisture content, total fungus quantification of Aspergillus flavus and A. parasiticus, as well as quantification of total aflatoxin, Afla B1, Afla B2, Afla G1 and Afla G2. The nut samples from the market showed an appropriate moisture content and water activity, which may have been responsible for the low fungus contamination and aflatoxin production. As to these mycotoxins, the samples were consistent with Anvisa's recommendations, which may be a consequence of good management of the nuts in Acre.

Incidencia de fungos toxigênicos e aflatoxinas em arroz / Incidence of toxigenic fungi and aflatoxins in rice

Um dos problemas mais sérios que confrontam a qualidade do arroz é a presença de fungos produtores de micotoxinas, principalmente as espécies pertencentes aos gêneros Aspergillus, Penicillium e Fusarium. Neste estudo, objetivou-se verificar os níveis de aflatoxinas e identificar a população fúngica associada a grãos de arroz beneficiado e comercializado em Belo Horizonte e algumas cidades do sul do estado. Foram analisadas um total de 60 amostras de arroz: orgânico, parboilizado, integral, polido, sendo a incidência de aflatoxinas verificada em uma amostra. Os resultados demonstraram que as espécies aflatoxigênicas identificadas foram A. parasiticus e A. flavus. Utilizando Cromatografia Líquida de Alta Eficiência para avaliar a incidência de aflatoxina em arroz, apenas uma apresentou contaminação de 1,2 ìg Kg-1. Apesar da presença de fungos aflatoxigênicos, as amostras não apresentaram níveis preocupantes de aflatoxinas que pudessem colocar em risco a segurança do produto e a saúde dos consumidores.

One of the most serious problems facing the quality of rice is the presence of mycotoxin-producing fungi, especially the species belonging to the genera Aspergillus, Penicillium and Fusarium. The objective of this study was to check the levels of aflatoxins and identify the fungal population associated with grains of rice received and sold in Belo Horizonte and some cities in the southern state. We analyzed a total of 60 samples of rice: organic, parboiled, full, polished, and the incidence of aflatoxins found in a sample. The results showed that the aflatoxigenic species identified were A. parasiticus and G2 and A. flavus. Using high performance liquid chromatography to assess the incidence of aflatoxin in rice, only one showed a contamination of 1.2 ìg kg-1. Despite the presence of aflatoxigenic fungi, the samples showed no worrying levels of aflatoxin that could endanger the safety of the product and consumer health.

Purification and characterization of mycoferritin from Aspergillusflavus MTCC 873.

The fungus Aspergillus flavus MTCC 873, a non-toxigenic isolate demonstrated its capability to synthesize mycoferritin (MF) upon induction with iron in yeast extract sucrose (YES) medium. The molecular mass, yield, iron and carbohydrate contents of the MF were 440 kDa, 0.015 mg/g of wet mycelia, 0.8 and 30.4%, respectively. Native gel-electrophoresis revealed a band corresponding to dimeric form of equine spleen ferritin (ESF). Subunit analysis by SDS-PAGE revealed a single protein band with an apparent molecular mass of 24 kDa, suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross-reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy (TEM) revealed an apparent particle size of 100 Å. N-terminal amino acid sequence of MF revealed a sequence of SLPLQDYA, which showed identities with other eukaryotic ferritin sequences. The spectral characteristics (UV/VIS, fluorescence and circular dichroic spectra) were similar to ESF. The fungus, unlike A. parasiticus 255 (non-toxigenic) was incapable of producing aflatoxins, when grown in YES media.

Culture conditions for the production of a-galactosidase by Aspergillus parasiticus MTCC-2796: a novel source / Condiciones del cultivo para la producción de a-galactosidasa por Aspergillus parasiticus MTCC-2796: una nueva fuente

Aspergillus parasiticus microbial type culture collection (MTCC)-2796, a new source of a-galactosidase is an efficient producer of enzyme in basic medium under submerged fermentation conditions. Maximum a-galactosidase production (156.25 Uml-1) was obtained when the basic medium is supplemented with galactose (0.5 percent w/v) and raffinose (0.5 percent w/v) as carbon source and yeast extract as nitrogen source. Enzyme production was also enhanced considerably in the presence of wheat bran (1.0 percent w/v). Enzyme secretion was strongly inhibited by the presence of Hg2+, Cu2+, and Co2+ in the medium and to some extent by Zn2+ and Ni2+, while marginal increase in the enzyme production was observed when Mg2+ and Mn2+ were added in the medium. Among amino acids checked (aparagine, cysteine, glutamine, leucine and proline), glutamine (1 mM) was found to be an enhancer for the enzyme production. The temperature and pH range for the production of enzyme were 25ºC to 35ºC and 6.5 to 7.5, respectively with maximum activity (50 Uml-1) at 30ºC and pH 6.5 under static fermentation condition.