Aspergilli and Penicillia related to the surface of ripening italian grana type cheese and its ripening environment

Information available on the mycoflora associated to ripening Italian “grana type” cheese is very poor. Recently, ochratoxin A (OTA) was detected in samples of packed grated cheese [1]; therefore, the need of information to perform a risk management was highlighted. Moreover, sterigmatocystin (STC) has been reported in cheese and it is considered an emerging problem. Despite the fact that both of them are mycotoxins included in group 2B by IARC [2,3], no European regulation exists. So, the main goal of this work is to give for the first time a general overview about Penicillia and Aspergilli growing on the surface of ripening “grana type” cheese, with particular attention on mycotoxigenic species. To perform this, in 2013 and 2014 crust samples were scratched from ripening grana cheese wheels and also Potato Dextrose Agar plates were exposed to monitor ripening house air. Then, 140 fungal isolates were randomly chosen, purified and monosporic colonies were obtained for their identification at specie level. A polyphasic approach is followed, based on morphological characterisation, toxic extrolites profiling and gene sequencing. The identification is still in progress, but the first results based on the morphological approach showed the presence of mycotoxigenic Aspergilli (Aspergillus flavus and A. versicolor) and various Penicillium species; among them Penicillium chrysogenum, P. implicatum and P. solitum were identified. Only P. chrysogenum was reported to produce the mycotoxins cyclopiazonic acid (CPA) and roquefortine-C (ROQ-C) [4]. These results will be presented and discussed. [1] A. Biancardi, R. Piro, G. Galaverna, C. Dall’Asta, "A simple and reliable liquid chromatography–tandem mass spectrometry method for determination of ochratoxin A in hard cheese" International Journal of Food Sciences and Nutrition 64 (5), 2013, 632 – 640. [2] International Agency for Research on Cancer (IARC) “IARC Monographs on the Evaluation of Carcinogenic Risks to Humans” 31, 1983, 191 – 199. [3] International Agency for Research on Cancer (IARC) “IARC Monographs on the Evaluation of carcinogenic Risks to Humans”, suppl. 7, 1987, 72. [4] J. I. Pitt, D. A. Hocking, “Fungi and Food Spoilage” 1997, 291.

Filamentous fungi in free water and biofilms from Brazilian drinking water systems

In some regions of Brazil, especially where the water is scarce, drinking water is stored in water storage tanks. This practice gives the consumer the guarantee of available water. The water storage conditions such as the exposure to hot weather when the tanks are on rooftops allow the development of microorganisms and microbial biofilms which can deteriorate the water quality and increase the risk to human health [1,2]. This study describes the filamentous fungi (FF) detected in free water and biofilms in drinking water storage tanks in Recife - Pernambuco, Brazil. Five sampling times in triplicate were performed at two distinct points. Colony-forming units (CFU) of FF fungi were determined with 0.45 µm filtration membranes using peptone glucose rose Bengal agar (PGRBA). From the 30 samples analysed a total of 1136 CFU were obtained. The water biofilms were collected from samplers consisting of polyethylene coupons, previously installed in the reservoirs. These coupons were transferred to PGRBA plates and incubated using with the same conditions described for free FF. For the in situ detection of FF in biofilms the Calcofluor White staining technique was used. This procedure demonstrated FF forming biofilms on the surfaces of the coupons. Brazilian legislation does not define limits for FF in drinking water. However considering the potential risk of fungal contamination, the data obtained in this study will contribute to developing future quantitative and qualitative parameters for the presence of fungi in drinking water distribution systems in Brazil. [1] HageskaL, G, Lima, N, Skaar, I. The study of fungi in drinking water. Mycological Research, 113, 2009, 165-172. [2] Skaar I, Hageskal G. Fungi in Drinking Water. In.: Paterson RRM, Lima N. (Eds.) Molecular Biology of Food and Water Borne Mycotoxigenic and Mycotic Fungi. CRC Press, Taylor & Francis Group, Boca Raton, 2015, 597-606.

Ergosterol as a rapid measurement of Ganoderma rot of oil palm

Palm oil (PO) is a very important commodity for many countries and especially Indonesia and Malaysia who are the predominant producers. PO is used in ca. 30% of supermarket foods, cosmetics, cooking and as biodiesel. The growth of oil palms in plantations is controversial as the production methods contribute to climate change and cause environmental damage [1]. The plant is subjected to a devastating disease in these two countries caused by the white rot fungus Ganoderma. There are no satisfactory methods to diagnose the disease in the plant as they are too slow and/or inaccurate. The lipid compound ergosterol is unique to fungi and is used to measure growth especially in solid substrates. We report here on the use of ergosterol to measure the growth of Ganoderma in oil palms using HPLC and TLC methods [2]. The method is rapid and correlates well with other methods and is capable of being used on-site, hence improving the speed of analysis and allowing remedial action. Climate change will affect the health of OP [1] and rapid detection methods will be increasingly required to control the disease. [1] Paterson, RRM, Kumar, L, Taylor, S, Lima N. Future climate effects on suitability for growth of oil palms in Malaysia and Indonesia. Scientific Reports, 5, 2015, 14457. [2] Muniroh, MS, Sariah M, Zainal Abidin, MA, Lima, N, Paterson, RRM. Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC. Journal of Microbiological Methods, 100, 2014, 143–147.

Polyphasic identification of a Rhizopus species and a putative novel Gongronella which both degrade the fungicide metalaxyl

Apresentação efetuada na MicroBiotec’15

Biosynthetic production of curcuminoids

Curcuminoids are natural phenylpropanoids from plants that have been reported as potential cancer-fighting drugs. Nevertheless, these compounds present a poor bioavailability. Cellular uptake is low and curcuminoids are quickly metabolized once inside the cell, requiring repetitive oral doses to achieve an effective concentration for therapeutic activity [1]. Herein, we report an engineered artificial pathway for the production of curcuminoids in Escherichia coli. Arabidopsis thaliana 4-coumaroyl-CoA ligase and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1) were used and 188 µM (70 mg/L) of curcumin was obtained from ferulic acid [2]. Bisdemethoxycurcumin and demethoxycurcumin were also produced, but in lower concentrations, by feeding p-coumaric acid or a mixture of p-coumaric acid and ferulic acid, respectively. Additionally, curcuminoids were produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase from Rhodotorula glutinis and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis were used [3]. Caffeoyl-CoA 3-O-methyl-transferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic acid, p-coumaric acid or tyrosine as a substrate, 3.9, 0.3, and 0.2 µM of curcumin were produced, respectively. This is the first report on the use of DCS and CURS1 in vivo to produce curcuminoids. In addition, curcumin, the most studied curcuminoid for therapeutic purposes and considered in many studies as the most potent and active, was produced by feeding tyrosine using a pathway involving caffeic acid. We anticipate that by using a tyrosine overproducing strain, curcumin can be produced in E. coli without the need of adding expensive precursors to the medium, thus decreasing the production cost. Therefore, this alternative pathway represents a step forward in the heterologous production of curcumin using E. coli. Aiming at greater production titers and yields, the construction of this pathway in another model organism such as Saccharomyces cerevisiae is being considered.

Parceria de cuidados, da investigação à prática

Apresentação efetuada no "(Novos) cenários em pediatria, 6.º Encontro Nacional de Enfermagem em pediatria", em Évora, Portugal, 2015

Da idiossincrasia da variante brasileira do português e do seu papel para o estatuto de importância crescente desta língua

[Excerto] 0. Introdução 0.1. O português do Brasil, sendo um exemplo, é também e sobretudo um caso exemplar1 de uma língua transplantada: é que o Brasil, aquele país-continente, tem sido e – não há razões para pensar o contrário – continuará a ser um cadinho rácico [europeu (vários) + índio (vários) + africano (vários)], cultural (a mentalidade brasileira) e linguístico (a variante brasileira, bem tropical e afectiva, do português). Isto quer, pois, significar que a realidade brasileira (que se caracteriza, para além da unidade territorial, pela unidade linguístico-cultural e por um enorme sentido de tolerância) só pode ter resultado, resultar e continuar a resultar da interacção mútua, livre e sem preconceitos, isto é, da miscigenação lato sensu étnico-linguístico-cultural.2 Isto, por um lado. (...)