Characterization of Aspergillus species on Brazil nut from the Brazilian Amazonian region and development of a PCR assay for identification at the genus level.

BACKGROUND: Brazil nut is a protein-rich extractivist tree crop in the Amazon region. Fungal contamination of shells and kernel material frequently includes the presence of aflatoxigenic Aspergillus species from the section Flavi. Aflatoxins are polyketide secondary metabolites, which are hepatotoxic carcinogens in mammals. The objectives of this study were to identify Aspergillus species occurring on Brazil nut grown in different states in the Brazilian Amazon region and develop a specific PCR method for collective identification of member species of the genus Aspergillus. RESULTS: Polyphasic identification of 137 Aspergillus strains isolated from Brazil nut shell material from cooperatives across the Brazilian Amazon states of Acre, Amapá and Amazonas revealed five species, with Aspergillus section Flavi species A. nomius and A. flavus the most abundant. PCR primers ASP_GEN_MTSSU_F1 and ASP_GEN_MTSSU_R1 were designed for the genus Aspergillus, targeting a portion of the mitochondrial small subunit ribosomal RNA gene. Primer specificity was validated through both electronic PCR against target gene sequences at Genbank and in PCR reactions against DNA from Aspergillus species and other fungal genera common on Brazil nut. Collective differentiation of the observed section Flavi species A. flavus, A. nomius and A. tamarii from other Aspergillus species was possible on the basis of RFLP polymorphism. CONCLUSIONS: Given the abundance of Aspergillus section Flavi species A. nomius and A. flavus observed on Brazil nut, and associated risk of mycotoxin accumulation, simple identification methods for such mycotoxigenic species are of importance for Hazard Analysis Critical Control Point system implementation. The assay for the genus Aspergillus represents progress towards specific PCR identification and detection of mycotoxigenic species.

Monitoring and Optimization of Cupuaçu Seed Fermentation, Drying and Storage Processes.

Cupuaçu [Theobroma grandiflorum (Wild ex Spreng.) K. Schum] seeds have been employed for a long time in the Amazon region for food purposes. Similar to cocoa, processed cupuaçu pulp and seeds can be used to produce juices, ice creams, confectionary products and cupulate®, which is a similar product to chocolate. However, its market penetration requires the mastery of all processing stages to improve the food quality and safety and to make possible an efficient technology transfer to the local small farmers and communities. Based on the above, the current research work aimed at monitoring and optimizing the consecutive fermentation and drying processes of cupuaçu seeds over 7 days each, as well as storage for 90 days. A greenhouse structure incorporating the fermenter and solar drying terrace was designed to be inexpensive, versatile, easily scalable, and easy to maintain and operate by the local small farmers after a short period of training. This research effort also aimed at giving a vision for the future creation of an integrative and sustainable cupuaçu system covering the economic, social, cultural and environmental vectors. The experimental design comprised 5 batches of 100 kg of seeds each. Several microbiological and physicochemical parameters were performed and correlated with processing variables. Microbiological parameters encompassed viable counts of mesophilic microorganisms, coliforms, yeasts, and molds, whereas physicochemical measures included fermentation and drying temperature, pH, acidity, dry matter, ashes, water activity, color, total proteins, lipids and carbohydrates, and energy. The average seed fermentation temperature varied from ca. 28 to 44 °C, reaching the maximum on day 3 and a final value of ca. 31 °C. Regarding solar drying, the average seed temperatures ranged from ca. 24 °C (at the end) to 39 °C on day 3, and an initial value of ca. 29 °C. The average final seed pH value of drying was 5.34 and was kept during storage. During storage, results demonstrated the existence of significant correlations among several experimental parameters under scrutiny. Finally, bean viable counts obtained during storage unfolded acceptable values of total mesophilic bacteria well below the maximum limit. Viable counts of yeast and molds were generally found between 3 and 4 log(CFU/gsample), and total coliforms were also detected, although both were at acceptable levels and well beneath the established maximum limits for food safety.