Influence of Limosilactobacillus fermentum IAL 4541 and Wickerhamomyces anomalus IAL 4533 on the growth of spoilage fungi in bakery products.

Fungi are the main microorganisms responsible for the spoilage of bakery products, and their control and subsequent reduction of food waste are significant concerns in the agri-food industry. Synthetic preservatives are still the most used compounds to reduce bakery product spoilage. On the other hand, studies have shown that biopreservation can be an attractive approach to overcoming food and feed spoilage and increasing their shelf-life. However, limited studies show the preservation effects on real food matrices. Therefore, this study aimed to investigate the influence of microorganisms such as lactic acid bacteria (LAB) and yeasts on the growth of spoilage filamentous fungi (molds) on bread and panettones. In general, on conventional and multigrain bread, treatments containing Limosilactobacillus fermentum IAL 4541 and Wickerhamomyces anomalus IAL 4533 showed similar results when compared to the negative control (calcium propionate) in delaying the fungal growth of the tested species (Aspergillus chevalieri, Aspergillus montevidensis, and Penicillium roqueforti). Different from bread, treatments with W. anomallus in panettones delayed the A. chevalieri growth up to 30 days, 13 days longer than observed on negative control (without preservatives). This study showed that biopreservation is a promising method that can extend bakery products' shelf-life and be used as an alternative to synthetic preservatives.

Fungal growth, patulin accumulation and volatile profile in 'Fuji Mishima' apples under controlled atmosphere and dynamic controlled atmosphere.

The objective of this study was to evaluate fungal and patulin contamination, together with its correlation with the volatile compounds (VCs), in 'Fuji Mishima' apples (up to 25% decayed) under controlled atmosphere (CA) and dynamic controlled atmosphere with respiratory quotient (DCA-RQ) of 1.3 combined with different partial pressures of carbon dioxide (0.8, 1.2, 1.6 and 2.0 pCO2). Fruits were stored under the above conditions for 8 months at 0.5 °C plus 7 days shelf life at 20 °C. Toxigenic fungi and patulin accumulation were found in apples from all treatments. Penicillium expansum was the most prevalent species. For all storage conditions, patulin concentrations were above the maximum level allowed in Brazil (50  µg  kg-1) with an exception of DCA-RQ1.3 + 0.8 kPa CO2. This condition, with lower pCO2, showed the lowest patulin accumulation, below the legal limit. The CA provided the highest patulin concentration (166 µg  kg-1). It was observed that fungal growth could also contribute to changes in the volatile composition. Styrene and 3-methyl-1-butanol are considered P. expansum markers in some apple cultivars and were detected in the samples. However, it was not possible to identify volatile organic compounds (VOCs) that are biomarkers from P. expansum, because there were other fungi species present in all samples. In this study, styrene, n-decanoic acid, toluene, phenol and alpha-farnesene were the compounds that showed the most positive correlation with patulin accumulation. On the other hand, a negative correlation of patulin with acids has been shown, indicating that in treatments with a higher patulin concentration there were less acidic compounds.

Understanding Mycotoxin Contamination Across the Food Chain in Brazil: Challenges and Opportunities.

Brazil is one of the largest food producers and exporters in the world. In the late 20th century, the European Union program for the harmonization of regulations for contaminants in food, including mycotoxins, led to the examination of mycotoxin contamination in foods at a global level. The problem of the rejection of food by the European Union and other countries became a Brazilian national priority because of economic and food safety aspects. Ochratoxin A in coffee and cocoa and aflatoxins in Brazil nuts are examples of the impact of technical trade barriers on Brazilian foods. To overcome these threats, several strategies were undertaken by Brazilian and international organizations. In this context, the Codex Commission on Food Contaminants (CCCF) has emerged as a forum to discuss with more transparency issues related to mycotoxins, focusing on establishing maximum levels and codes of practices for some commodities and mycotoxins to ensure fair trade and food safety. Our experience in investigating and understanding mycotoxin contamination across the food chains in Brazil has contributed nationally and internationally to providing some answers to these issues.

Fungi in cake production chain: Occurrence and evaluation of growth potential in different cake formulations during storage.

This study aimed to determine the prevalence and populations of fungi in cake production chain. Besides, the growth potential of twelve fungal strains in different cake formulations was evaluated. Raw materials from two different batches (n=143), chocolate cakes (n=30), orange cakes (n=20), and processing environment air samples (n=147) were analyzed. Among the raw materials, wheat flour (3.2±0.3 log CFU per g) and corn meal (3.8±0.8 log CFU per g) belonging to batch #1 showed significant higher fungal counts (p<0.05). The fungal counts in the processing environment air reached up to 2.56 log CFU per m3 (p<0.05). The predominant fungi species in the industrialized cakes were Aspergillus flavus (28.15%), Penicillium citrinum (18.45%), Penicillium paxilli (14.56%), and Aspergillus niger (6.8%), which were also detected in the raw materials and processing environment air. Only Penicillium glabrum and Penicillium citrinum showed visible mycelium (>3mm) in the free of preservative cake formulation at 19th and 44th days of storage at 25°C, respectively. Revealing the biodiversity of fungi in ingredients, air and final products, as well as challenging final products with representative fungal strains may assist to implement effective controlling measures as well as to gather data for the development of more robust cake formulations.

Fungi and mycotoxins in cocoa: from farm to chocolate.

Cocoa is an important crop, as it is the raw material from which chocolate is manufactured. It is grown mainly in West Africa although significant quantities also come from Asia and Central and South America. Primary processing is carried out on the farm, and the flavour of chocolate starts to develop at that time. Freshly harvested pods are opened, the beans, piled in heaps or wooden boxes, are fermented naturally by yeasts and bacteria, then dried in the sun on wooden platforms or sometimes on cement or on the ground, where a gradual reduction in moisture content inhibits microbial growth. Beans are then bagged and marketed. In processing plants, the dried fermented beans are roasted, shelled and ground, then two distinct processes are used, to produce powdered cocoa or chocolate. Filamentous fungi may contaminate many stages in cocoa processing, and poor practices may have a strong influence on the quality of the beans. Apart from causing spoilage, filamentous fungi may also produce aflatoxins and ochratoxin A. This review deals with the growth of fungal species and formation of mycotoxins during the various steps in cocoa processing, as well as reduction of these contaminants by good processing practices. Methodologies for fungal and mycotoxin detection and quantification are discussed while current data about dietary exposure and regulation are also presented.

Occurrence of ochratoxin A in cocoa by-products and determination of its reduction during chocolate manufacture.

This work reports an investigation carried out to assess the natural occurrence of ochratoxin A in 168 samples from different fractions obtained during the technological processing of cocoa (shell, nibs, liquor, butter, cake and cocoa powder) and the reduction of ochratoxin A during chocolate manufacture. Ochratoxin A analyses were performed with immunoaffinity columns and detection by high performance liquid chromatography. Concerning the natural ochratoxin A contamination in cocoa by-products, the highest levels of ochratoxin A were found in the shell, cocoa powder and cocoa cake. The cocoa butter was the least contaminated, showing that ochratoxin A seems to remain in the defatted cocoa solids. Under the technological conditions applied during the manufacture of chocolate in this study and the level of contamination present in the cocoa beans, this experiment demonstrated that 93.6% of ochratoxin A present in the beans was reduced during the chocolate producing.

Aspergillus bertholletius sp. nov. from Brazil nuts.

During a study on the mycobiota of brazil nuts (Bertholletia excelsa) in Brazil, a new Aspergillus species, A. bertholletius, was found, and is described here. A polyphasic approach was applied using morphological characters, extrolite data as well as partial ß-tubulin, calmodulin and ITS sequences to characterize this taxon. A. bertholletius is represented by nineteen isolates from samples of brazil nuts at various stages of production and soil close to Bertholletia excelsa trees. The following extrolites were produced by this species: aflavinin, cyclopiazonic acid, kojic acid, tenuazonic acid and ustilaginoidin C. Phylogenetic analysis using partial ß-tubulin and camodulin gene sequences showed that A. bertholletius represents a new phylogenetic clade in Aspergillus section Flavi. The type strain of A. bertholletius is CCT 7615 ( = ITAL 270/06 = IBT 29228).

Determination of aflatoxins in by-products of industrial processing of cocoa beans.

This study has examined the occurrence of aflatoxins in 168 samples of different fractions obtained during the processing of cocoa in manufacturing plants (shell, nibs, mass, butter, cake and powder) using an optimised methodology for cocoa by-products. The method validation was based on selectivity, linearity, limit of detection and recovery. The method was shown to be adequate for use in quantifying the contamination of cocoa by aflatoxins B(1), B(2), G(1) and G(2). Furthermore, the method was easier to use than other methods available in the literature. For aflatoxin extraction from cocoa samples, a methanol-water solution was used, and then immunoaffinity columns were employed for clean-up before the determination by high-performance liquid chromatography. A survey demonstrated a widespread occurrence of aflatoxins in cocoa by-products, although in general the levels of aflatoxins present in the fractions from industrial processing of cocoa were low. A maximum aflatoxin contamination of 13.3 ng g(-1) was found in a nib sample. The lowest contamination levels were found in cocoa butter. Continued monitoring of aflatoxins in cocoa by-products is nevertheless necessary because these toxins have a high toxicity to humans and cocoa is widely consumed by children through cocoa-containing products, like candies.

The effect of cocoa fermentation and weak organic acids on growth and ochratoxin A production by Aspergillus species.

The acidic characteristics of cocoa beans have influence on flavor development in chocolate. Cocoa cotyledons are not naturally acidic, the acidity comes from organic acids produced by the fermentative microorganisms which grow during the processing of cocoa. Different concentrations of these metabolites can be produced according to the fermentation practices adopted in the farms, which could affect the growth and ochratoxin A production by fungi. This work presents two independent experiments carried out to investigate the effect of some fermentation practices on ochratoxin A production by Aspergillus carbonarius in cocoa, and the effect of weak organic acids such as acetic, lactic and citric at different pH values on growth and ochratoxin A production by A. carbonarius and Aspergillus niger in culture media. A statistical difference (ρ<0.05) in the ochratoxin A level in the cured cocoa beans was observed in some fermentation practices adopted. The laboratorial studies demonstrate the influence of organic acids on fungal growth and ochratoxin A production, with differences according to the media pH and the organic acid present. Acetic acid was the most inhibitory acid against A. carbonarius and A. niger. From the point of view of food safety, considering the amount of ochratoxin A produced, fermentation practices should be conducted towards the enhancement of acetic acid, although lactic and citric acids also have an important role in lowering the pH to improve the toxicity of acetic acid.

Mycobiota of cocoa: from farm to chocolate.

The present work was carried out to study the mycobiota of cocoa beans from farm to chocolate. Four hundred and ninety-four samples were analyzed at various stages of cocoa processing: (i) primary stage at the farm (fermentation, drying, and storage), (ii) secondary stage at processing (testa, nibs, liquor, butter, cake and powder) and (iii) the final chocolate product (dark, milk, white and powdered) collected from retail outlets. Direct plating or dilution plating on Dichloran 18% Glycerol agar were used for cocoa beans and processed product analyses, respectively. Fungi were isolated and identified using different keys of identification. The largest numbers and diversity of fungi were observed in the samples collected at the farm, especially during drying and storage. The species with the highest occurrence among samples were: Absidia corymbifera, Aspergillus sp. nov., A. flavus, Penicillium paneum and yeasts. A total of 1132 potentially toxigenic fungi were isolated from the following species or species groups: A. flavus, Aspergillus parasiticus, Aspergillus nomius, Aspergillus niger group, Aspergillus carbonarius and Aspergillus ochraceus group. The highest percentage of toxigenic fungi was found at the drying and storage stages. The industrial processing reduced the fungal contamination in all fractions and no fungi were found in the final chocolate products. The knowledge of which fungi are dominant at each processing stage of cocoa provides important data about their ecology. This understanding leads to a reduction in fungal spoilage and mycotoxin production in this product.

Aflatoxigenic fungi and aflatoxin in cocoa.

This paper reports the occurrence of aflatoxigenic fungi and the presence of aflatoxins in 226 cocoa samples collected on Brazilian farms. The samples were taken at various stages of fermentation, drying and storage. A total of 819 potentially aflatoxigenic fungi were isolated using Dichloran 18% Glycerol agar after surface disinfection, and identified by standard techniques. The ability of the fungi to produce aflatoxins was determined using the agar plug technique and TLC. The presence of aflatoxins in cocoa samples was determined by HPLC using post-column derivatization with bromide after immunoaffinity column clean up. The aflatoxigenic fungi isolated were Aspergillus flavus, A. parasiticus and A. nomius. A considerable increase in numbers of these species was observed during drying and storage. In spite of the high prevalence of aflatoxigenic fungi, only low levels of aflatoxin were found in the cocoa samples, suggesting the existence of limiting factors to the accumulation of aflatoxins in the beans.

Ochratoxigenic fungi and ochratoxin A in cocoa during farm processing.

This study investigated the occurrence of fungi with the potential to produce ochratoxin A (OTA), and the occurrence of OTA, in Brazilian cocoa beans. Two hundred and twenty two samples of cocoa were evaluated, taken at various stages of fermentation, drying and storage. Samples were collected from Bahia, the main cocoa producing region in Brazil. Fungi with the potential to produce OTA were isolated by direct plating of cocoa beans on Dichloran 18% Glycerol agar after surface disinfection, and identified by standard techniques. The ability of the fungi to produce OTA was estimated using the agar plug technique and TLC. The presence of OTA in cocoa samples was determined by HPLC after immunoaffinity column clean up. The most common ochratoxigenic species found were Aspergillus carbonarius and A. niger aggregate, with lower numbers of A. melleus, A. westerdijkiae and Av. ochraceus. A considerable increase in the numbers of these species was observed during drying and storage. OTA was found at all stages of cocoa processing, with the major incidence during drying and storage. The OTA levels found were in general low and there was a strong positive correlation between the presence of A. carbonarius and OTA contamination in the beans.

Pulmonary aspergillosis outbreak in Rhea americana in southern Brazil.

Commercial raising of rheas is currently in expansion in the south of Brazil, and many diseases previously restricted to other avian species are currently emerging on rhea farms, especially as a result of careless management of these animals. The objective of the present article is to report a pulmonary aspergillosis outbreak that occurred in great rhea (Rhea americana) in the south of Brazil. About 50 birds aged 30 to 60 days died suddenly and one of them was submitted to autopsy which revealed the presence of white caseous nodules 0.5 mm in diameter occupying 95% of the lung area. One lung was sent to the Federal University of Santa Maria for histopathological and mycological analyses. Histopathological analysis revealed multifocal areas with necrosis and inflammatory infiltrates and the presence of fungal hyphae, giant cells and fibrous tissue proliferation at the periphery. Aspergillus fumigatus was recovered as pure culture from all culture media. This appears to be the first report of aspergillosis among great rhea in Brazil and the second in the world.

Pneumonia intersticial em bovinos associada à ingestão de batata-doce (Ipomoea batatas) mofada / Interstitial pneumonia in cattle fed moldy sweet potatoes (Ipomoea batatas)

Uma doença respiratória foi diagnosticada em cinco dentre 23 bovinos (21,7 por cento) após terem sido alimentados com batata-doce (Ipomoea batatas) mofada em uma pequena propriedade rural em São Vicente do Sul, Rio Grande do Sul, Brasil. Três dos cinco bovinos afetados morreram espontaneamente, e o quarto foi sacrificado para necropsia quando mostrava sinais clínicos respiratórios avançados. A manifestação clínica iniciara cerca de 24 horas após a ingestão das batatas-doces e a evolução clínica foi de 1 a 4 dias. Os sinais clínicos incluíam dispnéia (respiração laboriosa e abdominal), taquipnéia, pescoço estendido com cabeça baixa e dilatação ritmada das narinas. Dois bovinos foram necropsiados. Os achados de necropsia incluíam pulmões distendidos, pálidos e de consistência borrachenta, que não colapsavam quando o tórax era aberto; enfisema e edema acentuados eram evidentes no pulmão. Os linfonodos e o baço apresentavam alterações características de hiperplasia linfóide. Histologica-mente, as lesões eram típicas de pneumonia intersticial. Os septos alveolares estavam espessados por fibroblastos e células inflamatórias, havia hipertrofia e hiperplasia de pneumócitos tipo II; os septos interlobulares estavam distendidos por edema e enfisema. A cultura de amostras das batatas-doces mofadas produziu Fusarium solani e F. oxysporum