Luís Roberto Batista

Toxigenic fungi associated with processed (green) coffee beans (Coffea arabica L.)

Processed (green) coffee beans from Coffea arabica in Brazil were assessed for the presence of Aspergillus and Penicillium species both before and after surface sterilisation, the aflatoxigenic and ochratoxigenic potential of the isolates and ochratoxin A levels. Contamination by Aspergillus and Penicillium species was found on 96% and 42%, respectively, of 45 samples from 11 localities. After disinfection with 1% sodium hypochlorite, the levels fell to 47% and 24%, respectively. One hundred and eighty isolates were identified to species level and comprised Aspergillus sections Circumdati (10 species), Flavi (3), Nigri (3), Versicolores (4), while two were teleomorphic species. Eight species of Penicillium were isolated. Within section Circumdati, 75% of the isolates produced ochratoxin A and all except Aspergillus elegans and Aspergillus insulicola have previously been reported to produce ochratoxin A. One-third of the 18 isolates of Aspergillus flavus produced aflatoxin B1 and B2. None of the isolates belonging to Aspergillus section Nigri or Penicillium produced ochratoxin A. Of the 40 bean samples analysed, 58% were infected with potentially ochratoxigenic fungi but only 22% of these were contaminated with ochratoxin A at levels that varied from 0.47 to 4.82 ng/g, with an average contamination level of 2.45 ng/g.

Succession of bacterial and fungal communities during natural coffee (Coffea arabica) fermentation

Bacteria, yeasts and filamentous fungi were isolated during natural coffee processing. Bacteria were isolated in greater numbers at the beginning of the fermentation, when the moisture of the coffee beans was around 68%. Gram-positive bacteria represented 85.5% of all bacteria isolated, and Bacillus was the predominant genus (51%). Gram-negative species of the genera Serratia, Enterobacter and Acinetobacter were also found. Approximately 22% of 940 randomly chosen isolates of microorganisms were yeasts. Debaryomyces (27%), Pichia (18.9%) and Candida (8.0%) were the most commonly found genera, and these three genera tended to appear more often as the fruit was fermented and dried. Aspergillus was the most abundant genus besides Penicillium, Fusarium and Cladosporium, with 42.6% of the total fungi isolates. The genera and species identified included members known to have pectinase and cellulase activities. Of the 10 organic acids analyzed and quantified in coffee beans, acetic and lactic acids may have been generated by microbial activity. Butyric acid was not detected in any sample.

Identification of fungi of the genus Aspergillus section nigri using polyphasic taxonomy

In spite of the taxonomy of the Aspergillus species of the Nigri Section being regarded as troublesome, a number of methods have been proposed to aid in the classification of this Section. This work aimed to distinguish Aspergillus species of the Nigri Section from foods, grains and caves on the basis in Polyphasic Taxonomy by utilizing morphologic and physiologic characters, and sequencing of s-tubulin and calmodulin genes. The morphologic identification proved useful for some species, such as A. carbonarius and Aspergillus sp UFLA DCA 01, despite not having been totally effective in elucidating species related to A. niger. The isolation of the species of the Nigri Section on Creatine Sucrose Agar (CREA) enabled to distinguish the Aspergillus sp species, which was characterized by the lack of sporulation and by the production of sclerotia. Scanning Electron microscopy (SEM) allowed distinguishing the species into two distinct groups. The production of Ochratoxin A (OTA) was only found in the A. carbonarius and A. niger species. The sequencing of β-tubulin gene was efficient in differing most of the Aspergillus species from the Nigri Section with the exception of Aspergillus UFLA DCA 01, which could not be distinguished from A. costaricaensis. This species is morphologically similar to A. costaricaencis for its low sporulation capacity and high sclerotia production, but it differs morphologically from A. costaricaensis for its conidial ornamentation and size of vesicles. Equally, based on partial calmodulin gene sequence data Aspergillus UFLA DCA 01 differs from A. costaricaensis.

Incidence and distribution of filamentous fungi during fermentation, drying and storage of coffee (Coffea arabica L.) beans

O objetivo deste estudo foi isolar e caracterizar fungos filamentosos presentes em diferentes estagios de beneficiamento de cafe processado pelo metodo natural, incluindo: colheita, fermentacao, secagem e armazenamento. O cafe cereja foi colhido manualmente e entao colocado em uma plataforma de cimento, onde permaneceu ate atingir 11% de umidade. A contagem microbiana foi realizada em todas as amostras durante a fermentacao e secagem do cafe. A populacao de fungos filamentosos no cafe cereja ainda nos pes (tempo 0) foi em torno de 1,5 x 103 UFC/g. Este numero aumentou vagarosamente durante a fermentacao e secagem, alcancando valores de 2 x 105 UFC/g em 22 dias do processamento. Duzentos e sessenta e tres isolados de fungos filamentosos foram identificados. A distribuicao das especies durante fermentacao e secagem foi bastante variada, mas no armazenamento dos graos ocorreu o predominio de especies de Aspergillus. Foram encontradas 38 especies de fungos distribuidas nos seguintes generos: Pestalotia (4), Paecelomyces (4), Cladosporium (26), Fusarium (34), Penicillium (81) e Aspergillus (112).

Detection of ochratoxin A in tropical wine and grape juice from Brazil

BACKGROUND Ochratoxin A (OTA) is the main mycotoxin found in grapes, wines and grape juices and is considered one of the most harmful contaminants to human health. In this study, samples of tropical wines and grape juices from different grape varieties grown in Brazil were analysed for their OTA content by high-performance liquid chromatography. RESULTS The detection and quantification limits for OTA were 0.01 and 0.03 µg L(-1) respectively. OTA was detected in 13 (38.24%) of the samples analysed, with concentrations ranging from < 0.03 to 0.62 µg L(-1). OTA was not detected in any of the grape juice samples. Most of the red wine samples proved to be contaminated with OTA (75%), while only one white wine sample was contaminated. However, the OTA levels detected in all samples were well below the maximum tolerable limit (2 µg L(-1)) in wine and grape juice established by the European Community and Brazilian legislature. CONCLUSION The results of this study indicate a low risk of exposure to OTA by consumption of tropical wines and grape juices from Brazil.

Biological control of mycotoxin-producing molds

Mycotoxins are produced by the secondary metabolism of many fungi and can be found in almost 25% of the worlds agricultural commodities. These compounds are toxic to humans, animals, and plants and therefore, efforts should be made to avoid mycotoxin contamination in food and feed. Besides, up to 25% of all harvested fruits and vegetables are lost due to storage molds and/or mycotoxin contamination and many methods have been applied to mitigate these issues, but most of them rely on the use of fungicides. Although chemicals are often the first defensive line against mycotoxigenic fungi, the indiscriminate use of fungicides are awakening the public perception due to their noxious effects on the environment and human/animal health. Thus, there is an increasing public pressure for a safer and eco-friendly alternative to control these organisms. In this background, biological control using microbial antagonists such as bacteria, fungi and yeasts have been shown to be a feasible substitute to reduce the use of chemical compounds. Despite of the positive findings using the biocontrol agents only a few products have been registered and are commercially available to control mycotoxin-producing fungi. This review brings about the up-to-date biological control strategies to prevent or reduce harvested commodity damages caused by storage fungi and the contamination of food and feed by mycotoxins.

Effect of temperature, water activity, and pH on growth and production of ochratoxin A by Aspergillus niger and Aspergillus carbonarius from Brazilian grapes.

The growth of ochratoxigenic fungus and the presence of ochratoxin A (OTA) in grapes and their derivatives can be caused by a wide range of physical, chemical, and biological factors. The determination of interactions between these factors and fungal species from different climatic regions is important in designing models for minimizing the risk of OTA in wine and grape juice. This study evaluated the influence of temperature, water activity (aw), and pH on the development and production of OTA in a semisynthetic grape culture medium by Aspergillus carbonarius and Aspergillus niger strains. To analyze the growth conditions and production of OTA, an experimental design was conducted using response surface methodology as a tool to assess the effects of these abiotic variables on fungal behavior. A. carbonarius showed the highest growth at temperatures from 20 to 33°C, aw between 0.95 and 0.98, and pH levels between 5 and 6.5. Similarly, for A. niger, temperatures between 24 and 37°C, aw greater than 0.95, and pH levels between 4 and 6.5 were optimal. The greatest toxin concentrations for A. carbonarius and A. niger (10 μg/g and 7.0 μg/g, respectively) were found at 15°C, aw 0.99, and pH 5.35. The lowest pH was found to contribute to greater OTA production. These results show that the evaluated fungi are able to grow and produce OTA in a wide range of temperature, aw, and pH. However, the optimal conditions for toxin production are generally different from those optimal for fungal growth. The knowledge of optimal conditions for fungal growth and production of OTA, and of the stages of cultivation in which these conditions are optimal, allows a more precise assessment of the potential risk to health from consumption of products derived from grapes.

Evaluation of antifungal activity of essential oils against potentially mycotoxigenic Aspergillus flavus and Aspergillus parasiticus

The antifungal activity of essential oils of fennel (Foeniculum vulgare Mill., Apiaceae), ginger (Zingiber officinale Roscoe, Zingiberaceae), mint (Mentha piperita L., Lamiaceae) and thyme (Thymus vulgaris L., Lamiaceae) was evaluated against mycotoxin producers Aspergillus flavus and A. parasiticus. High Resolution Gas Chromatography was applied to analyze chemical constituents of essential oils. The effect of different concentrations of essential oils was determined by solid medium diffusion assay. Mycelial growth and sporulation were determined for each essential oil at the concentrations established by solid medium diffusion assay. At the fifth, seventh and ninth days the mycelial diameter (O mm) and spore production were also determined. FUN-1 staining was performed to assess cell viability after broth macrodilution assay. Trans-anethole, zingiberene, menthol and thymol are the major component of essential oils of fennel, ginger, mint and thyme, respectively. The effective concentrations for fennel, ginger, mint and thyme were 50, 80, 50 and 50% (oil/DMSO; v/v), respectively. The four essential oils analysed in this study showed antifungal effect. Additionally, FUN-1 staining showed to be a suitable method to evaluate cell viability of potential mycotoxigenic fungi A. flavus and A. parasiticus after treatment with essential oils.

Ochratoxigenic fungi associated with green coffee beans (Coffea arabica L.) in conventional and organic cultivation in Brazil.

The genera Aspergillus comprises species that produce mycotoxins such as aflatoxins, ochratoxins and patulin. These are cosmopolitan species, natural contaminants of agricultural products. In coffee grains, the most important Aspergillus species in terms of the risk of presenting mycotoxins belong to the genera Aspergillus Section Circumdati and Section Nigri. The purpose of this study was to assess the occurrence of isolated ochratoxigenic fungi of coffee grains from organic and conventional cultivation from the South of Minas Gerais, Brazil, as well as to evaluate which farming system presents higher contamination risk by ochratoxin A (OTA) produced by fungi. Thirty samples of coffee grains (Coffea arabica L.) were analysed, being 20 of them of conventional coffee grains and 10 of them organic. The microbiological analysis was done with the Direct Plating Technique in a Dichloran Rose Bengal Chloramphenicol Agar (DRBC) media. The identification was done based on the macro and micro morphological characteristics and on the toxigenic potential with the Plug Agar technique. From the 30 samples analysed, 480 filamentous fungi of the genera Aspergillus of the Circumdati and Nigri Sections were isolated. The ochratoxigenic species identified were: Aspergillus auricoumus, A. ochraceus, A. ostianus, A. niger and A. niger Aggregate. The most frequent species which produces ochratoxin A among the isolated ones was A. ochraceus, corresponding to 89.55%. There was no significant difference regarding the presence of ochratoxigenic A. ochreceus between the conventional and organic cultivation systems, which suggests that the contamination risk is similar for both cultivation systems.

Essential oil of Origanum majorana L., Illicium verum Hook. f. and Cinnamomum zeylanicum Blume: chemical and antimicrobial characterization

Essential oils of Origanum majorana L. (marjoram), Illicium verum Hook. f. (star- anise) and Cinnamomum zeylanicum Blume (cinnamon) were obtained by steam distillation using a modified Clevenger device. The antimicrobial activity of each oil was evaluated against the bacteria Staphylococcus aureus, Escherichia coli and the fungi Aspergillus flavus and Aspergillus parasiticus by observing their growth and/or mycelial inhibition through comparison with the standard dish (without oil). The essential oils were analyzed using a gas chromatograph coupled to a mass spectrometer for identification and coupled to a flame ionization detector for quantification. The major constituents of marjoram, star-anise and cinnamon essential oils were 4-terpineol, trans- anetole and cinnamic aldehyde, respectively. In in vitro tests, essential oils of marjoram and cinnamon promoted an inhibitory effect on the bacteria S. aureus and E. coli, while the essential oil of star-anise presented activity only against E. coli. Marjoram, star-anise and cinnamon oils were effective against the studied fungi, presenting an inhibitory effect. The minimal inhibitory concentration for the mycelial growth of A. parasiticus was 1 and 0.01 L mL -1 for star-anise and cinnamon oils, respectively. The minimal inhibitory concentration for A. parasiticus was 0.25, 2 and 2 L mL -1 for cinnamon, star-anise and marjoram oils, respectively.