Volatile compounds for biotechnological applications produced during competitive interactions between yeasts and fungi.

Fungi, yeasts and bacteria produce volatile compounds during their metabolism. In this study, the volatile compounds produced by yeast strains (Saccharomyces cerevisiae and Rhodotorula mucilaginosa) and fungal strains (Aspergillus carbonarius and Aspergillus ochraceus) during competitive interactions were investigated by solid-phase microextraction coupled with gas chromatography-mass spectrometry. Fifty-six volatile compounds were identified representing alcohols, aldehydes, esters, ketones, aromatic compounds, acids, furans, phenols, and nitrogen compounds, being the largest amount in the class of esters and alcohols. Eight compounds were identified only in interactive culture conditions such as 2-amino-1-propanol, isopropylamine, dimethylamine, pentyl propanoate, ethyl-2-aminopropanoate, acetone, oxalic acid, and ß-elemene and five of these were produced in cocultures including A. carbonarius. These will be developed for future biotechnological applications such as in the pharmaceutical and biological industry to produce drugs. Antimicrobial and antifungal activities; Solvent and herbicide; flavoring ingredient; solvent, plastic synthesis, nail polish remover and thinner, pesticide and herbicide; important in the complexation of minerals in the soil; and plant-environment interactions, defending predators, pathogens, and competitors.

Active packaging of poly(lactic acid) nanofibers and essential oils with antifungal action on table grapes.

The table grape is a non-climateric fruit that is very susceptible to fungal contamination, in addition to suffering an accelerated loss of quality during storage. The in vitro and in grape antifungal and antiocratoxigenic effects of the essential oils from Alpinia speciosa and Cymbopogon flexuosus against Aspergillus carbonarius and Aspergillus niger were studied. The oils were encapsulated in poly(lactic acid) (PLA) nanofibers as a potential active packaging to be applied to control the degradation of grapes stored during the post-harvest period. Fungal proliferation and ochratoxin A synthesis in A. carbonarius and A. niger decreased in the presence of the active packaging. However, the nanofiber containing the essential oil from C. flexuosus was more efficient in providing a fungicidal effect against A. carbonarius (10% and 20%) and A. niger (20%). In addition, weight loss and color changes were controlled and the parameters of acidity, °Brix, softening and the texture of the grape were maintained. A very small mass loss of the essential oils encapsulated in nanofibers was observed by thermogravimetric analysis, showing that the nanofiber was efficient in enabling the controlled release. The quality and safety of table grapes were maintained for longer periods of storage in the presence of active packaging, so the incorporation of these oils in nanofibers can be a promising way to increase the shelf life of grapes.

Antifungal and physicochemical properties of Ocimum essential oil loaded in poly(lactic acid) nanofibers.

Poly(lactic acid) (PLA) nanofibres containing different proportions of the essential oils from Ocimum basilicum L. and Ocimum gratissimum L. were prepared by solution blow spinning method. The essential oils were extracted by hydrodistillation and characterized by gas chromatography. MEV, contact angle, DSC and FTIR were used to characterize the nanofibres. The effect of bioative nanofibres on the growth of the fungus and on the production of ochratoxin A were evaluated using the fumigation test. Linalool, 1·8-cineole and camphor were the principal components of the essential oil from O. basilicum, and eugenol was the principal constituent in the oil from O. gratissimum. An increase in the average diameter of the nanofibres was observed with the addition of the essential oils. The essential oils acted as a plasticizer, resulting in a reduction in the crystallinity of the PLA. The encapsulation of essential oils in PLA nanofibres was verified by FTIR. An effective antifungal and antimicotoxygenic activity against Aspergillus ochraceus and Aspergillus westerdjikiae was observed for the bioative nanofibres. These results confirm the potential of PLA nanofibres containing the essential oils for the control of toxigenic fungi that cause the deterioration of food and are harmful to human health.

Effect of the essential oils of Satureja montana L., Myristica fragrans H. and Cymbopogon flexuosus S. on mycotoxin-producing Aspergillus flavus and Aspergillus ochraceus antifungal properties of essential oils.

Essential oils can be a useful alternative to the use of synthetic fungicides because they have biological potential and are relatively safe for food and agricultural products. The objectives of the present study were to evaluate the antifungal and antimycotoxigenic activities of the essential oils from Satureja montana L., Myristica fragrans H. and Cymbopogon flexuosus S. against Aspergillus flavus and Aspergillus ochraceus, as well as their effects on ergosterol synthesis and membrane morphology. The antifungal potential was evaluated by mycelial growth analysis and scanning electron microscopy. Fungicidal effects against A. flavus, with MFC of 0.98, 15.62 and 0.98 µL/mL, respectively, were observed for the essential oils from S. montana, M. fragrans and C. flexuosus. Aspergillus ochraceus did not grow in the presence of concentrations of 3.91, 15.62 and 0.98 µL/mL of the essential oils from S. montana, M. fragrans and C. flexuosus, respectively. The essential oils significantly inhibited the production of ochratoxin A by the fungus A. ochraceus. The essential oils also inhibited the production of aflatoxin B1 and aflatoxin B2. The biosynthesis of ergosterol was inhibited by the applied treatments. Biological activity in the fungal cell membrane was observed in the presence of essential oils, given that deleterious effects on the morphologies of the fungi were detected. The essential oils under study are promising as food preservatives because they significantly inhibit toxigenic fungi that contaminate food. In addition, the essential oils hindered the biosynthesis of mycotoxins.

Fungal diversity and occurrence of mycotoxin producing fungi in tropical vineyards.

Grapevine cultivars are distributed worldwide, nevertheless the fermentation of its grape berries renders distinct wine products that are highly associated to the local fungal community. Despite the symbiotic association between wine and the fungal metabolism, impacting both the terroir and mycotoxin production, few studies have explored the vineyard ecosystem fungal community using both molecular marker sequencing and mycotoxin production assessment. In this study, we investigated the fungal community of three grapevine cultivars (Vitis vinifera L.) in two tropical vineyards. Illumina MiSeq sequencing was performed on two biocompartments: grape berries (GB) and grapevine soil (GS); yielding a total of 578,495 fungal internal transcribed spacer 1 reads, which were used for taxonomic classification. GB and GS fungal communities were mainly constituted by Ascomycota phylum. GS harbors a significant richer and more diverse fungal community than GB. Among GB samples, Syrah grape berries exclusively shared fungal community included wine-associated yeasts (e.g. Saccharomycopsis vini) that may play key roles in wine terroir. Mycotoxin production assessment revealed the high potential of Aspergillus section Flavi and Penicillium section Citrina isolates to produce aflatoxin B1-B2 and citrinin, respectively. This is the first study to employ next-generation sequencing to investigate vineyard associated fungal community in Brazil. Our findings provide valuable insights on the available tools for fungal ecology assessment applied to food products emphasizing the coexistence between classical and molecular tools.

Mechanism of action of various terpenes and phenylpropanoids against Escherichia coli and Staphylococcus aureus.

Foodborne, disease-causing microorganisms are increasingly resistant to commercial antibiotics. Thus, there is a need for the development of new agents capable of acting efficiently in the control of these pathogens. Terpenoids and phenylpropanoids stand out for having high biological activity and a broad spectrum of action. The objectives of this study were to evaluate the antibacterial potential of limonene, ß-citronellol, carvone, carvacrol, eugenol and trans-cinnamaldehyde and to investigate the mechanism of activity of these compounds against the bacteria Escherichia coli and Staphylococcus aureus. The terpene and phenylpropanoid compoundswere purchased and their antibacterial potential was assessed by macrodilution. The mechanism of action was verified by tests of potassium ion efflux, salt tolerance, extravasation of cellular contents, absorption of crystal violet and morphological changes analyzed by electron microscopy. Bacteriostatic and bactericidal effects caused by the compounds carvone, carvacrol, eugenol and transcinemaldehyde were observed in both species; antibacterial activity against only S. aureus was observed for ß-citronelol and limonene. Reduced salt tolerance was found for strains of E. coli treated with carvacrol and S. aureus treated with ß-citronelol. There was extravasation of cellular materials induced by treatments with carvone, carvacrol, eugenol and trans-cinnamaldehyde in both microorganisms. The absorption of crystal violet increased for E. coli after incubation with each treatment. Deleterious effects and morphological changes were observed. Therefore, the monoterpenes and phenylpropanoids under study are potentially applicable for antimicrobial use against E. coli and S. aureus, and the mechanism of action involves changes in membrane permeability without cell lysis.

Antibacterial and Antifungal Activities and Toxicity of the Essential Oil from Callistemon viminalis Complexed with ß-Cyclodextrin.

The essential oil from Callistemon viminalis (EOC) is rich in monoterpenes, with a variety of biological properties: antibacterial, antifungal, insecticide, and antioxidant. Inclusion complexes (ICs) with cyclodextrins (CDs) is an alternative to prevent toxicity, improve the activity, and reduce the concentration to be used. Thus, the objective of this work was to prepare an IC (EOC/ß-CD) and evaluate the antibacterial, antifungal and phospholipase activities, as well as the toxicity. Antimicrobial activity used the agar diffusion test and antifungal activity the disc diffusion test. Toxicity tests were carried out using Lactuca sativa L. The inhibition of phospholipase activity using the venom of Bothrops atrox as an inducer was performed. Antibacterial and antifungal tests demonstrated a decrease in the minimum inhibitory concentration (MIC) of the IC. It was most significantly observed for the bacterium Listeria monocytogenes, for which there was a decrease in the MIC from 250 µg mL-1 to 62.5 µg mL-1 after complexation, and for the fungus Aspergillus flavus, with a decrease in MIC from 125 µg mL-1 to 62.5 µg mL-1 after complexation. Toxicity tests with Lactuca sativa showed a decrease in toxicity after complexation in all parameters analyzed, with no statistical difference from the negative control. Inhibition of phospholipase activity induced by Bothrops atrox venom was more expressive in the highest proportion studied (1:10 m:m), exerting 23% inhibition. The assays demonstrated that the complexation between the EOC and ß-CD is a promising alternative for use in different branches, especially in food industry, to fully exploit its application potential.

Effect of the essential oils from Melaleuca alternifolia, Melaleuca quinquenervia and Backhousia citriodora on the synthesis of ochratoxin A by Aspergillus niger and Aspergillus carbonarius isolated from tropical wine grapes.

The influence of essential oils (EOs) extracted from the leaves of Melaleuca alternifolia, Melaleuca quinquenervia and Backhousia citriodora on ochratoxin A (OTA) synthesis by fungi was studied. The extraction of EOs was performed by hydrodistillation (Clevenger apparatus) over a 2-h period and subsequently analyzed by GC-MS and GC-FID. The toxigenic activity of the essential oils (31.25; 15.62 and 7.81 µg mL-1) was evaluated by inhibiting the production of OTA by Aspergillus niger and Aspergillus carbonarius in Czapek agar medium culture. The quantification of the toxin was performed by HPLC. The production of OTA was dependent on the fungal species, incubation temperature (15 and 25 °C) and the presence of the essential oils. In tests carried out at 15 °C, the oils caused a reduction in OTA synthesis that ranged from 57.60 to 76.93% and from 54.78 to 98.68% for the fungal species A. carbonarius and A. niger, respectively. At 25 °C, reductions ranged from the 38.66 to 75.93% and from 17.94 to 71.79% for the respective fungi. The study concluded that natural products could be potential biocontrol agents against OTA contamination in food.

Influence of physical and chemical characteristics of wine grapes on the incidence of Penicillium and Aspergillus fungi in grapes and ochratoxin A in wines.

The incidence of filamentous fungi and toxin levels in grapes and wines varies depending on the variety of grapes, the wine region, agricultural practices, weather conditions, the harvest and the winemaking process. In this sense, the objective of this study was to evaluate the diversity of Aspergillus and Penicillium fungi isolated from wine grapes of the semi-arid tropical region of Brazil, evaluate the presence of ochratoxin A (OTA) in the experimental wine and verify if there is a correlation between occurrence of these fungi and the physicochemical characteristics of the wine grapes grown in the region. For the isolation of fungi we used the direct plating technique. The presence of OTA in the experimental wine was detected by high-performance liquid chromatography. The species found were Aspergillus niger, A. carbonarius, A. aculeatus, A. niger Aggregate, A. flavus, A. sojae, Penicillium sclerotiorum, P. citrinum, P. glabrum, P. decumbens, P. solitum and P. implicatum. All isolates of A. carbonarius were OTA producers and all P. citrinum were citrinin producers. The highest concentration of OTA was found in red wine (0.29µg/L). All species identified in this study, except A. flavus, showed a positive correlation with at least one physicochemical parameter assessed, highlighting the pectin content, total sugar, total acidity and phenolic compounds.

Essential oil from Chenopodium ambrosioides L: secretory structures, antibacterial and antioxidant activities / Óleo essencial de Chenopodium ambrosioides L: estruturas secretoras, atividade antibacteriana e antioxidante

The aim of this study was to evaluate the antibacterial and antioxidant activities of essential oil from Chenopodium ambrosioides L. and to determine its secretory structures. The essential oil was extracted through the hydrodistillation technique using a modified Clevenger apparatus (2 hours) and chemically characterized by GC/MS and GC-FID. The antioxidant activity was determined by monitoring the reduction of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and by the oxidation of the ß-carotene/linoleic acid system. The evaluation of antibacterial activity was performed by the agar cavity diffusion technique using the microorganisms Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella Cholerasuis. The characterization of trichomes was accomplished by Scanning Electron Microscopy (SEM) and histochemical tests with Nadi and Sudan IV reagents. The antioxidant activity demonstrated by the ß-carotene/acid linoleic test, with IC50 = 455.7 µg mL-1. This oil also presented antibacterial activity for both Gram-negative and Gram-positive bacteria. The minimal inhibitory concentration ranged 62.5 to 250 µL mL-1. The presence of terpenes in the glandular trichomes was observed, suggesting that the essential oil is secreted by these structures.

Objetivou-se neste trabalho avaliar as atividades antibacteriana e antioxidante do óleo essencial de Chenopodium ambrosioides L. e determinar as suas estruturas secretoras. A extração do óleo essencial foi realizada pelo método de hidrodestilação, utilizando o aparelho de Clevenger modificado com duração de 2 horas, sendo, posteriormente, analisado por CG/EM e CG-DIC. A atividade antioxidante foi determinada monitorando-se a redução do radical livre DPPH (2,2-difenil-1-picril-hidrazila) e utilizando o ensaio de oxidação do sistema ß-caroteno/ácido linoleico. A avaliação da atividade antibacteriana foi realizada por meio da técnica difusão cavidade em ágar, utilizando os micro-organismos Staphylococcus aureus, Listeria monocytogenes, Escherichia coli e Salmonella Cholerasuis. A caracterização dos tricomas foi realizada por Microscopia Eletrônica de Varredura (MEV) e pelos testes histoquímicos com os reagentes de Nadi e Sudan IV. A atividade antioxidante foi evidenciada pelo teste ß-caroteno/ácido linoleico, com CI50 455,7 µg mL-1. O óleo também apresentou atividade antibacteriana tanto para bactérias Gram -negativas como para Gram-positivas. A concentração mínima inibitória do mesmo variou entre 62,5 e 250 µL mL-1. Constatou-se a presença de compostos terpênicos nos tricomas glandulares, inferindo-se que o óleo essencial seja secretado nessas estruturas.

Effect of Fungicides on Growth and Ochratoxin A Production by Aspergillus carbonarius from Brazilian Wine Grapes.

Prevention in the field of mycotoxin-producing fungi is the most effective strategy for controlling the presence of mycotoxins in foods. Chemical fungicides are widely used to protect crops, so their implications on mycotoxin production need to be considered. Therefore, the aim of this study was to evaluate the effect in vitro and on grapes of five fungicides commonly used on grape cultures in Brazil on Aspergillus carbonarius growth and ochratoxin A (OTA) production. At the doses recommended by manufacturers, most fungicides significantly reduced A. carbonarius growth and OTA production in vitro, whereas this effect was influenced by the type of fungicide, dose, and temperature. Temperature was the main factor that influenced the effectiveness of fungicides. In general, at 15°C, fungicides showed the greatest reduction on fungal growth and OTA production. Fungicide effect on grapes was different to that on a semisynthetic grape medium. All fungicide doses were not effective at controlling A. carbonarius in grapes. Thus, the direct effect of fungicides on grapes must be studied to obtain a better approximation of field conditions. The results indicate that the use of fungicides at the doses recommended by manufacturers is better than the application at low doses. This study showed that at the lowest doses, where fungal growth is not inhibited, fungicides positively stimulate OTA production.

Biological activity of the essential oils from Cinnamodendron dinisii and Siparuna guianensis.

This study had analyzed the antibacterial, antifungal and trypanocidal activity of the essential oils from Cinnamodendron dinisii Schwacke (Canellaceae) and Siparuna guianensis Aublet (Siparunaceae). The essential oils were obtained from fresh leaves by hydrodistillation, using a modified Clevenger apparatus. Chemical analysis by gas-liquid chromatography coupled to mass spectrometry (GC-MS) showed that these essential oils are rich in monoterpene and sesquiterpene hydrocarbons. Activity against the pathogenic bacteria Escherichia coli , Listeria monocytogenes , Pseudomonas aeruginosa , Salmonella choleraesuis and Staphylococcus aureus was evaluated with the agar cavity diffusion method, while activity on the filamentous fungi Aspergillus flavus , Aspergillus niger , Aspergillus carbonarius and Penicillium commune was evaluated by the disk diffusion technique. Trypanocidal activity was tested against Trypanosoma cruzi epimastigotes, using the Tetrazolium salt (MTT) colorimetric assay. Both essential oils exhibited low inhibitory effect towards bacteria, showing high MIC values (125-500 µg mL (-1) ), with Gram positive bacteria being more susceptible. Better inhibitory effect was obtained for the evaluated fungi, with lower MIC values (7.81-250 µg mL (-1) ), being A. flavus the most susceptible species. Both essential oils presented low trypanocidal activity, with IC 50 /24 h values of 209.30 µg mL (-1) for S. guianensis and 282.93 µg mL (-1) for C. dinisii . Thus, the high values observed for the MIC of evaluated bacteria and for IC 50 /24 h of T. cruzi , suggest that the essential oils have a low inhibitory activity against these microorganisms. In addition, the low MIC values observed for the tested fungi species indicate good inhibitory activity on these microorganisms's growth.

Biological activity of the essential oils from Cinnamodendron dinisii and Siparuna guianensis

This study had analyzed the antibacterial, antifungal and trypanocidal activity of the essential oils from Cinnamodendron dinisii Schwacke (Canellaceae) and Siparuna guianensis Aublet (Siparunaceae). The essential oils were obtained from fresh leaves by hydrodistillation, using a modified Clevenger apparatus. Chemical analysis by gas-liquid chromatography coupled to mass spectrometry (GC-MS) showed that these essential oils are rich in monoterpene and sesquiterpene hydrocarbons. Activity against the pathogenic bacteria Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella choleraesuis and Staphylococcus aureus was evaluated with the agar cavity diffusion method, while activity on the filamentous fungi Aspergillus flavus, Aspergillus niger, Aspergillus carbonarius and Penicillium commune was evaluated by the disk diffusion technique. Trypanocidal activity was tested against Trypanosoma cruzi epimastigotes, using the Tetrazolium salt (MTT) colorimetric assay. Both essential oils exhibited low inhibitory effect towards bacteria, showing high MIC values (125–500 μg mL−1), with Gram positive bacteria being more susceptible. Better inhibitory effect was obtained for the evaluated fungi, with lower MIC values (7.81–250 μg mL−1), being A. flavus the most susceptible species. Both essential oils presented low trypanocidal activity, with IC50/24 h values of 209.30 μg mL−1 for S. guianensis and 282.93 μg mL−1 for C. dinisii. Thus, the high values observed for the MIC of evaluated bacteria and for IC50/24 h of T. cruzi, suggest that the essential oils have a low inhibitory activity against these microorganisms. In addition, the low MIC values observed for the tested fungi species indicate good inhibitory activity on these microorganisms’s growth.

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.

Methods to preserve potentially toxigenic fungi.

Microorganisms are a source of many high-value compounds which are useful to every living being, such as humans, plants and animals. Since the process of isolating and improving a microorganism can be lengthy and expensive, preserving the obtained characteristic is of paramount importance, so the process does not need to be repeated. Fungi are eukaryotic, achlorophyllous, heterotrophic organisms, usually filamentous, absorb their food, can be either macro or microscopic, propagate themselves by means of spores and store glycogen as a source of storage. Fungi, while infesting food, may produce toxic substances such as mycotoxins. The great genetic diversity of the Kingdom Fungi renders the preservation of fungal cultures for many years relevant. Several international reference mycological culture collections are maintained in many countries. The methodologies that are most fit for preserving microorganisms for extended periods are based on lowering the metabolism until it reaches a stage of artificial dormancy. The goal of this study was to analyze three methods for potentially toxigenic fungal conservation (Castellani's, continuous subculture and lyophilization) and to identify the best among them.

Methods to preserve potentially toxigenic fungi

Microorganisms are a source of many high-value compounds which are useful to every living being, such as humans, plants and animals. Since the process of isolating and improving a microorganism can be lengthy and expensive, preserving the obtained characteristic is of paramount importance, so the process does not need to be repeated. Fungi are eukaryotic, achlorophyllous, heterotrophic organisms, usually filamentous, absorb their food, can be either macro or microscopic, propagate themselves by means of spores and store glycogen as a source of storage. Fungi, while infesting food, may produce toxic substances such as mycotoxins. The great genetic diversity of the Kingdom Fungi renders the preservation of fungal cultures for many years relevant. Several international reference mycological culture collections are maintained in many countries. The methodologies that are most fit for preserving microorganisms for extended periods are based on lowering the metabolism until it reaches a stage of artificial dormancy . The goal of this study was to analyze three methods for potentially toxigenic fungal conservation (Castellani's, continuous subculture and lyophilization) and to identify the best among them.

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.

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.

Controle microbiológico de alimentos utilizando óleo essencial de Pimpinella anisum (erva-doce) / Food microbiological control using essential oil of Pimpinella anisum

O consumo de produtos naturais que substituam aditivos químicos tem sido uma opção para aqueles que procuram hábitos saudáveis e segurança alimentar. Os condimentos possuem comprovada atividade biológica sobre fungos e bactérias. Por isso, sempre foram utilizados como conservantes de alimentos. Este trabalho foi realizado com o objetivo de avaliar o efeito “in vitro” do óleo essencial de erva-doce (Pimpinella anisum), sobre as bactérias Staphylococcus aureus e Escherichia coli e sobre os fungos Aspergillus flavus e Aspergillus parasiticus. O óleo essencial foi extraído pela técnica hidrodestilação utilizando aparelho de Clevenger modificado e testado nas concentrações de 50; 40; 30; 20; 10; 5; 1; 0,5 e 0.1% para as bactérias e nas concentrações de 2; 1; 0,5; 0;25 e 0,1 µL/mL para fungos. O óleo apresentou uma elevada atividade bacteriana com uma concentração mínima inibitória (CMI) de 1% para a E.coli, porém não apresentou atividade frente a S. aureus. Os valores de CMI encontrados para o A. flavus e A. parasiticus foram de 0,5 e 2 µL/mL, respectivamente. Verificou-se que o óleo essencial apresentou efeito inibitório sobre os micro-organismos estudados, sendo, portanto, uma alternativa no controle microbiológico de alimentos.

Molecular ecology and polyphasic characterization of the microbiota associated with semi-dry processed coffee (Coffea arabica L.).

This work was aimed at isolating and identifying the microbiota present during the semi-dry method of coffee processing using polyphasic methods and to evaluate microbial diversity with PCR-DGGE. Samples of Coffea arabica L. were collected during different processing stages in southern Minas Gerais, Brazil. The bacterial and fungal isolates were phenotypically characterised and grouped according to the ARDRA technique, in which the 16-23S and ITS1-5.8S regions of the rDNA were sequenced for species identification. The bacterial counts varied from 3.7 to 7 log CFU g(-1). The yeast counts ranged from 3.4 to 6.9 log CFU g(-1), and the filamentous fungal population varied from 2 to 3.7 log CFU g(-1). Bacillus subtilis, Escherichia coli, Enterobacter agglomerans, Bacillus cereus and Klebsiella pneumoniae were the predominant bacteria detected during the processing of the coffee, and Pichia anomala, Torulaspora delbrueckii and Rhodotorula mucilaginosa were the dominant yeasts. All of the yeast and bacterial species detected by PCR-DGGE were isolated using culture-dependent methods, with the exception of one uncultivable bacterial species. Aspergillus was the most common genus among the filamentous fungal isolates. The use of polyphasic methods allowed a better characterization of the microbiota that is naturally present in semi-dry processed coffee.