Fungal communities in Brazilian cassava tubers and food products.

Cassava (Manihot esculenta Crantz) is one of the most widely cultivated foods in the world and is of great socio-economic importance, especially in developing countries. It is predominantly consumed in boiled form, but also is used to produce a number of products, including cassava starch, sour starch, cassava flour and tapioca flour (hydrated cassava starch). Fungal spoilage can occur throughout the production chain, impairing both productivity and quality, as well as posing a potential risk of contamination by mycotoxins. We used multidisciplinary approaches based on phenotypic and molecular data (ITS/BenA/TEF-1a/RPB2 loci) to investigate the mycobiota of 101 samples (including roots, soil and products) collected in the state of São Paulo, Brazil. A total of 20 fungal groups/genera were morphologically characterized, and 37 different species were molecularly identified. The predominant groups in cassava tubers were Fusarium spp., Penicillium spp. and Trichoderma spp. In cassava products, the most frequent groups were Penicillium spp. and Paecilomyces spp. Potentially toxigenic species were also found, including Paecilomyces saturatus, Penicillium citrinum, P. paneum, P. brevicompactum, P. chrysogenum, Fusarium foetens and Fusarium mundagurra. In soil-cultivated cassava samples, the groups found most frequently were Penicillium spp., Cladosporium spp. and Fusarium spp. Some of the species found in cassava tubers and/or product samples were also present in the soil, including F. mundagurra, Neocosmospora solani, P. citrinum and P. brevicompactum. In general, there was a higher occurrence of Penicillium spp., Fusarium spp. and Trichoderma spp., and the predominant species were F. fabacearum and P. citrinum. The mycobiota of Brazilian cassava proved to be extremely diverse, and the occurrence of several species in cassava tubers and/or products are reported herein for the first time. Potentially toxigenic species were found in cassava tubers, cassava products and soil, showing how important it is to constantly monitor these substrates.

Aspergillus section Flavi and aflatoxins in Brazilian cassava (Manihot esculenta Crantz) and products.

Aflatoxins are carcinogenic compounds produced by some species of Aspergillus, especially those belonging to Aspergillus section Flavi. Their occurrence in food may start in the field, in the post-harvest, or during storage due to inadequate handling and storage. Because cassava is a staple food for a high percentage of the Brazilian population, we evaluated the presence of aflatoxin-producing species in cassava tubers, cassava products (cassava flour, cassava starch, sour starch, and tapioca flour), and in soil samples collected from cassava fields. In addition, the levels of aflatoxin contamination in cassava products were quantified. A total of 101 samples were analyzed, and 45 strains of Aspergillus section Flavi were isolated. Among the identified species, Aspergillus flavus, Aspergillus arachidicola, Aspergillus novoparasiticus, and Aspergillus parasiticus were found. The majority of strains (73.3%) tested for their aflatoxin-producing ability in synthetic media was positive. Despite that, cassava and cassava products were essentially free of aflatoxins, and only one sample of cassava flour contained traces of AFB1 (0.35 µg/kg).

Potential of volatile compounds produced by fungi to influence sensory quality of coffee beverage.

Fungi are known producers of a large number of volatile compounds (VCs). Several VCs such as 2,4,6 trichloroanisole (TCA), geosmin and terpenes have been found in coffee beverages, and these compounds can be responsible for off-flavor development. However, few studies have related the fungal contamination of coffee with the sensory characteristics of the beverage. The aim of this research was to investigate the production of VCs by fungi isolated from coffee and their potential as modifiers of the sensory coffee beverage quality. Three species were isolated from coffee from the southwest of São Paulo state and selected for the study: Penicillium brevicompactum, Aspergillus luchuensis (belonging to section Nigri) and Penicillium sp. nov. (related to Penicillium crustosum). VCs produced by the fungal inoculated in raw coffee beans were extracted and tentatively identified by SPME-GC-MS. Different VCs that may interfere in the coffee beverage quality were detected in the raw coffee beans inoculated with these fungal species (mainly A. luchuensis). Oct-1-en-3-ol was detected in the raw coffee inoculated with A. luchuensis. This compound, which is characterized by earthy and moldy/mushroom aroma, can be related to negative characteristics of coffee beverage in sensory analysis. On the other hand, the presence of some fungal species in the coffee, even at a high percentage of infection, did not necessarily result in loss of the sensorial quality of the beverage, since the samples with a high infection of P. brevicompactum showed positive sensory evaluation.

Growth and mycotoxin production by fungi in atmospheres containing 80% carbon dioxide and 20% oxygen.

The effect of atmosphere containing 80% CO(2) and 20% O(2) on growth of Mucor plumbeus, Fusarium oxysporum, Byssochlamys fulva, Byssochlamys nivea, Penicillium commune, Penicillium roqueforti, Aspergillus flavus, Eurotium chevalieri and Xeromyces bisporus was investigated. Production of aflatoxin by A. flavus, patulin by B. nivea, roquefortine C by P. roqueforti, and cyclopiazonic acid by P. commune was also studied. Fungal growth was evaluated by three methods: colony diameter, hyphal length or mycelium dry weight and ergosterol content. Among the nine fungal species examined, two E. chevalieri and X. bisporus, did not grow under these conditions. In this study, fungi differed in their response to modified atmospheres in biomass, ergosterol content, mycotoxin production and morphology. Reductions of 57.8-96.9%, 73.7-99.6% and 91.5-99.9% were obtained in colony diameter, hyphal length and ergosterol content, respectively, under this atmosphere compared to air. Ergosterol content was more affected in most species than other measurements. Patulin, cyclopiazonic acid and roquefortine C were produced in this atmosphere, although levels were very low and aflatoxin was not produced at all. Growth was quite extensive as measured by colony diameters, but hyphal lengths were low and ergosterol production was also affected in all species of this study.

Growth and mycotoxin production by food spoilage fungi under high carbon dioxide and low oxygen atmospheres.

The influence of high carbon dioxide and low oxygen concentrations on growth by the foodborne fungal species, Mucor plumbeus, Fusarium oxysporum, Byssochlamys fulva, Byssochlamys nivea, Penicillium commune, Penicillium roqueforti, Aspergillus flavus, Eurotium chevalieri and Xeromyces bisporus was investigated. Production of aflatoxin by A. flavus, patulin by B. nivea and roquefortine C by P. roqueforti was also studied. Fungal growth was evaluated under atmospheres consisting of 20, 40 and 60% CO(2) plus <0.5% O(2), on two media, Czapek Yeast Extract agar and Potato Dextrose agar. Several methods for measuring fungal growth were used: colony diameter, ergosterol content, hyphal length and/or mycelium dry weight. Among the nine species, three groups were distinguished with respect to their growth responses under modified atmospheres: (i) species which did not grow in 20% CO(2) <0.5% O(2) (P. commune, E. chevalieri and X. bisporus); (ii) species which grew in 20% CO(2) <0.5% O(2), but not 40% CO(2) <0.5% O(2) (P. roqueforti and A. flavus); (iii) species which grew in 20%, 40% and 60% CO(2) <0.5% O(2) (M. plumbeus, F. oxysporum, B. fulva and B. nivea). Facultatively anaerobic behaviour was observed in these last four species, which grew under the same conditions as the obligate anaerobe, Clostridium sporogenes. The production of aflatoxin, patulin, and roquefortine C was greatly reduced under all of the atmospheres tested.

Incidence of toxigenic fungi and ochratoxin A in dried fruits sold in Brazil.

A total of 117 dried fruit samples (black sultanas, white sultanas, dates, dried plums, dried figs and apricots) from different origins were analysed both for toxigenic fungi and for the presence of ochratoxin A. Amongst the fungi found, Aspergillus niger was predominant, with 406 isolates, of which 15% were ochratoxin A producers. They were followed by A. ochraceus, with 15 isolates and 87% ochratoxigenics, and A. carbonarius, with only five isolates of which 60% were ochratoxin A producers. The average infection rates for A. niger in black sultanas, plums, figs, dates and white sultanas were 22.0, 8.0, 4.0, 1.5 and 0.5%, respectively. The apricot samples were not contaminated by any fungi or ochratoxin A. Black sultana and dried figs contained the highest contamination with ochratoxin A, with 33 and 26.3% of the samples containing more than 5 microg kg(-1) respectively, while all the white sultanas, dates and plums had no sample that exceeded this limit.

Determination of ochratoxin A in green coffee by immunoaffinity column cleanup and liquid chomatography: collaborative study.

A collaborative study was conducted to evaluate a method using immunoaffinity column cleanup with liquid chromatography (LC) for the determination of ochratoxin A (OTA) in green coffee at levels that could be included in possible future regulations of the European Union. The test portion was extracted with methanol-3% aqueous sodium hydrogen carbonate solution (50 + 50, v/v). The extract was filtered, and the filtrate was diluted with phosphate-buffered saline and applied to an immunoaffinity column containing antibodies specific for OTA. After washing, the toxin was eluted from the column with methanol and quantified by LC with fluorescence detection. Pairs of 4 homogeneous noncontaminated and naturally contaminated materials (mean levels of < 0.12, 2.44, 5.15, and 13.46 ng/g) and blank samples (< 0.12 ng/g) for spiking were sent to 20 participant laboratories from 8 countries. The materials were analyzed according to the method description and all difficulties encountered in the analysis were reported. Statistical analysis was carried out according to the Harmonized Protocol of the International Union of Pure and Applied Chemistry. The relative standard deviation for repeatability (RSDr) ranged from 7.42 to 20.94%, and the relative standard deviation for reproducibility (RSDR) ranged from 16.34 to 29.17%. The method showed acceptable within-laboratory and between-laboratories precision for green coffee materials, as evidenced by HorRat values of < or = 0.85, at the studied range, for spiked and naturally contaminated materials. The mean recovery was 92.8% for green coffee material spiked with OTA at a level of 4.82 ng/g.

Utilization of AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples.

AIMS: The objective of this work was to test whether ochratoxin A (OTA) production of Aspergillus niger and A. carbonarius is linked to a certain genotype and to identify marker sequences with diagnostic value aiding identification of A. carbonarius, a fungus of major concern regarding OTA production in food and food raw materials. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated mainly from Brazilian coffee sources. The ability of isolates to produce OTA was tested by thin layer chromatography (TLC). Strains were genetically characterized by AFLP fingerprinting and compared with each other and with reference strains. Cluster analysis of fingerprints showed clear separation of A. niger from A. carbonarius strains. To obtain marker sequences, AFLP fragments were isolated from silver stained polyacrylamide gels, cloned and sequenced. Sequences obtained were used to develop species- specific PCR primers for the identification of A. carbonarius in pure culture and in artificially and naturally infected samples of green coffee. CONCLUSIONS: No clear correlation between genetic similarity of the strains studied and their potential to produce OTA was found. The PCR assays designed are a useful and specific tool for identification and highly sensitive detection of A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assays allow specific and sensitive detection and identification of A. carbonarius, a fungus considered to be one of the major causative agents for OTA in coffee and grape-derived products. Assays may provide powerful tools to improve quality control and consumer safety in the food processing industry.

The source of ochratoxin A in Brazilian coffee and its formation in relation to processing methods.

A total of 408 Brazilian coffee samples was examined during the 1999 and 2000 coffee harvest seasons for the presence of ochratoxin A (OA) and fungi with the potential to produce it. Samples came from four regions: Alta Paulista (western area of São Paulo State), Sorocabana (southwest São Paulo State), Alta Mogiana (northeast São Paulo State) and Cerrado Mineiro (western area of Minas Gerais State). Cherries and beans were examined at different stages: immature, mature and overripe cherries from trees, overripe cherries from the ground and beans during drying and storage on the farm. For mycological studies, the cherries and beans were surface disinfected with chlorine, plated on Dichloran 18% Glycerol Agar at 25 degrees C for 5-7 days and analysed for the presence of Aspergillus ochraceus and closely related species, A. carbonarius and A. niger. More than 800 isolates of fungi belonging to these species were identified and studied for the ability to produce OA using the agar plug technique and thin layer chromatography (TLC). A. niger was the species found most commonly (63% of isolates of these three species), but only 3% of them produced OA. A. ochraceus also occurred commonly (31% of isolates), and 75% of those studied were capable of OA production, a much higher percentage than reported elsewhere. A. carbonarius was found (6% of isolates) only in Alta Paulista, the hottest region studied, and only from beans in the drying yard or in storage. However, 77% of the A. carbonarius isolates were capable of producing OA. Average infection rates for cherries taken from trees were very low, but were higher in fruit taken from the ground, from the drying yard and from storage, indicating infection by toxigenic species after harvest. The average OA content in 135 samples of mature cherries from trees, overripe from trees, overripe from the ground, drying yard and storage was 0.1, <0.2, 1.6, 2.1 and 3.3 microg/kg, respectively. Although individual OA levels varied widely, only 9 of the 135 samples analysed exceeded 5 microg/kg OA, with one sample of poor quality dried coffee in excess of 100 microg/kg OA. The causes of high contamination were investigated on the farms concerned and several critical points were found, relating both to local climatic conditions and the drying processes used.

Research on the origin, and on the impact of post-harvest handling and manufacturing on the presence of ochratoxin A in coffee.

The major risk factors and processing steps that can lead to contamination of green coffee with ochratoxin A (OTA) have been identified. Surveys of the green coffee production chain indicate that Aspergillus ochraceus and A. carbonarius are the most potent OTA producers on coffee. Both have been successfully grown in vitro on green coffee and coffee cherries, respectively, producing high amounts of OTA (5-13 mg kg(-1)). The so-called dry processing of coffee, which is cherry drying, was identified as one of the steps during which OTA formation can take place, particularly under humid tropical conditions. Cherries contain sufficient amounts of water to support mould growth and OTA formation during the initial 3-5 days of drying on the outer part of the cherries. Not surprisingly, after dehulling, husks can be highly contaminated with OTA, as also indicated by its enhanced concentration in soluble coffees adulterated with husks and parchment. A minimum water activity of 0.80 (about 14% MC) is required for in vitro OTA production on green coffee, a fact that does not rule out the possibility of OTA contamination due to improper transportation and storage of green coffee. However, this appears not to be a major route for OTA contamination of coffee. OTA contamination can clearly be minimized by following good agricultural practice and a subsequent post-harvest handling consisting of appropriate techniques for drying, grading, transportation and storage of green coffee; these procedures are well established.

Comparison of culture media, simplate, and petrifilm for enumeration of yeasts and molds in food.

The efficacy of three culture media, dichloran rose bengal chloramphenicol (DRBC), dichloran 18% glycerol agar (DG18), and potato dextrose agar (PDA) supplemented with two antibiotics, were compared with the Simplate and Petrifilm techniques for mold and yeast enumeration. The following foods were analyzed: corn meal, wheat flour, cassava flour, bread crumbs, whole meal, sliced bread, ground peanuts, mozzarella cheese, grated parmesan cheese, cheese rolls, orange juice, pineapple pulp, pineapple cake, and mushroom in conserve. Correlation coefficients of DRBC versus PDA and DG18 for recovering total mold and yeast counts from the composite of 14 foods indicated that the three media were generally equivalent. Correlation coefficients for Petrifilm versus culture media were acceptable, although not as good as between culture media. Correlation coefficients of Simplate versus DRBC, DG18, PDA, and Petrifilm for recovering total yeasts and molds from a composite of 11 foods demonstrated that there was no equivalence between the counts obtained by Simplate and other culture media and Petrifilm, with significant differences observed for the most foods analyzed.

Growth of fungi and mycotoxin production on cheese under modified atmospheres.

The use of modified atmospheres to prevent fungal growth and mycotoxin production in cheese was evaluated. Eight fungal species: Mucor plumbeus, Fusarium oxysporum, Byssochlamys fulva, B. nivea, Penicillium commune, P. roqueforti, Aspergillus flatus and Eurotium chevalieri were inoculated onto cheese and incubated under conditions of decreasing concentrations of O2 (5% to < 0.5%) and increasing concentrations of CO2 (20-40%). Fungal growth was measured by colony diameter and ergosterol content. All fungi examined grew in atmospheres containing 20% and 40% CO2 with 1% or 5% O2, but growth was reduced by 20-80%, depending on species, compared with growth in air. The formation of aflatoxins B1 and B2, roquerfortine C and cyclopiazonic acid was greatly decreased but not totally inhibited in these atmospheres. At 20% or 40% CO2 with < 0.5% O2, only B. nivea exhibited growth, which was very slow. Growth of F. oxysporum, B. fulca, P. commune and A. flavus showed good correlations between colony diameter and ergosterol content. However, for the other species correlations were inconsistent.

Occurrence of ochratoxin A-producing fungi in raw Brazilian coffee.

Ochratoxin A (OA)-producing fungi were identified in coffee at different stages of maturation. The toxin was quantified in coffee during terrace drying and in coffee stored in barns. By direct plating, a high level of contamination (100%) was found in the coffee beans studied, with the genus Aspergillus representing 33.2%, of which Aspergillus ochraceus and Aspergillus niger represented 10.3 and 22.9%, respectively, of the strains isolated from the coffee beans. The capacity to produce ochratoxin was determined in 155 strains of A. ochraceus and A. niger using both the agar plug method and extraction with chloroform, giving positive results for 88.1% of the A. ochraceus strains and 11.5% of the A. niger strains. Analysis for OA in the terrace and barn coffee samples showed that, independent of cultivar, year harvested, or production region, all except one of the samples analyzed showed mycotoxin levels below the limit suggested by the European Common Market (8 microg/kg), thus indicating that the problem is restricted and due to severe faults in harvesting and storage practices.