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.

A review of mycotoxins in food and feed products in Portugal and estimation of probable daily intakes

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that occur naturally in agricultural commodities worldwide. Aflatoxins, ochratoxin A, patulin, fumonisins, zearalenone, trichothecenes and ergot alkaloids are presently the most important for food and feed safety. These compounds are produced by several species that belong to the Aspergillus, Penicillium, Fusarium and Claviceps genera and can be carcinogenic, mutagenic, teratogenic, cytotoxic, neurotoxic, nephrotoxic, estrogenic and immunosuppressant. Human and animal exposure to mycotoxins is generally assessed by taking into account data on the occurrence of mycotoxins in food and feed as well as data on the consumption patterns of the concerned population. This evaluation is crucial to support measures to reduce consumer exposure to mycotoxins. This work reviews the occurrence and levels of mycotoxins in Portuguese food and feed to provide a global overview of this issue in Portugal. With the information collected, the exposure of the Portuguese population to those mycotoxins is assessed, and the estimated dietary intakes are presented.

Indoor biological agents: evaluation of primary schools environments

Dissertação de mestrado em Human Engineering

Biosynthesis, detection and toxicology of ochratoxins - optimisation of production

Ochratoxin A (OTA) is a very well known mycotoxin found in several food commodities for which maximum limits are being discussed in EC in other to produce appropriate regulations. OTA is one of several ochratoxins produced by Aspergillus and Penicillium species. All the compounds in this group have a molecular structure very similar to OTA and some were already isolated from natural substrates. Several of these compounds such as ochratoxin , methyl and ethyl ester of ochratoxin A, 4-R and S-hydroxyochratoxin A, 10-hydroxyochratoxin A and ochratoxin A open lactone are commercially unavailable. However, they can be easily synthesized through OTA modification. With the main objective of its application on further research works, OTA production, isolation and purification has been optimised from an A. alliaceus strain grown on wheat medium. Synthesis and purification of some OTA derivatives has been achieved and an HPLC method for their detection was optimised. Data about their production by several species of Aspergillus will be presented. The toxicological properties of ochratoxins are still not very clear and a future EC safety limit for OTA will depend on e.g., a better clarification of its carcinogenity. Could OTA derivatives play a role here?

New bio-strategies for ochratoxin A detoxification using lactic acid bacteria

The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Novas bio-estratégias para a destoxificação de ocratoxina A utilizando bactérias do ácido láctico

A ocorrência de bolores micotoxigénicos pertencentes aos géneros Aspergillus, Penicillium e Fusarium em alimentos para consumo Humano e animal, tem um impacto importante sobre a saúde pública e constitui também um importante problema económico. Isto é devido à síntese por este tipo de fungos filamentosos de metabolitos altamente tóxicos conhecidos como micotoxinas. A maioria das micotoxinas são substâncias cancerígenas, mutagénicas, neurotóxicas e imunossupressoras, sendo a ocratoxina A (OTA) uma das mais importantes. A OTA é uma micotoxina, tóxica para os animais e Humanos principalmente devido às suas propriedades nefrotóxicas. Alguns grupos de bactérias gram positivas nomeadamente as bactérias do ácido láctico (BAL) são capazes de controlar o crescimento de fungos, melhorando e aumentando a vida útil de muitos produtos fermentados e, assim, reduzir os riscos para a saúde provocados pela exposição às micotoxinas. Algumas BAL são, também, capazes de destoxificar certas micotoxinas. Em trabalhos anteriores do nosso grupo foi observada a biodegradação da OTA por estirpes de Pediococcus parvulus isoladas de vinhos do Douro. Assim, com este trabalho, pretendeu-se compreender com maior detalhe o processo de biodegradação da OTA pelas referidas estirpes e identificar quais as enzimas que estão associadas à sua biodegradação. Para atingir este objetivo utilizaram-se algumas ferramentas ioinformáticas (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV), desenharam-se primers específicos e realizaram-se PCR específicos para os genes envolvidos. Através da utilização de ferramentas de bioinformática, foi possível identificar várias proteínas que pertencem à família das carboxipeptidases e que podem eventualmente participar no processo da degradação da OTA, tais como D-Ala-D-Ala carboxipeptidase serínica e carboxipeptidase membranar. Estas BAL podem desempenhar um papel importante na destoxificação da OTA, sendo as carboxipeptidases uma das enzimas envolvidas na sua biodegradação.

Estudo de estirpes probióticas como inoculantes de silagens para o controlo de micotoxinas

Dissertação de mestrado em Bioengenharia