Browsing by Author "Kozakiewicz, Zofia"
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- Characterization and differentiation of aflatoxigenic species of Aspergillus section Flavi by MALDI-TOF MSPublication . Rodrigues, Paula; Kallow, Wibke; Erhard, Marcel; Welker, Martin; Kozakiewicz, Zofia; Lima, Nelson; Venâncio, ArmandoAspergillus is a large genus, with a complex taxonomy. The genus is easily identifiedd by its characteristic conidiophore, but species identification and differentiation is complex, mainly because it is traditionally based on a range of morphological features. Aspergillus subgenus Circumdati section Flavi, also refered to as the A.dflavus group, has attracted worldwide attention for its industrial use and toxigenic potential. Sectiond Flavi is divided in two groups of species. One includes the aflatoxigenic species A. flavus, A. parasiticus and A. nomius, which cause serious problems in agricultural commodities, and the other one includes the non-aflatoxigenic species A. oryzae, A. sojae and A.dtamarii, traditionally used for production of fermented foods. Species from A. flavus group are morphologically and genetically very similar, and are therefore difficult to differentiate by both cultural and molecular methods. Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Massd Spectrometry has already shown high potentialities in discriminating very closely related taxa. This work intended to discriminate 30 strains, which were previously classifieddas A. flavus group by morphological methods, using MALDI-TOF MS. These results are compared with those previously obtained by conventional methods.
- Characterization of Aspergillus flavus isolated from winemaking grapesPublication . Soares, C.; Rodrigues, Paula; Kozakiewicz, Zofia; Lima, Nelson; Venâncio, ArmandoConcern about filamentous fungi in vineyards has been traditionally linked to the spoilage of grapes. However, the recent detection of ochratoxin A in wines has increased the interest in mycotoxins. Although not very common in grapes, Aspergillus flavus is occasionally isolated from grapes. A. flavus is the main producer of the well known carcinogenic mycotoxins, the aflatoxins, and the presence of this fungus and aflatoxins is of major food safety concern. The identification of A. flavus is not straightforward due mainly to similarities with its closely related species (A. parasiticus, A. nomius). Various morphological and biochemical parameters may be used for identification: Color of colonies on Czapek Medium (CZ), seriation of conidial heads, conidia ornamentation, production of aflatoxins B1, B2, G1 and G2, and production of cyclopiazonic acid.
- Contributions of Micoteca da Universidade do Minho to food mycologyPublication . Rodrigues, Paula; Soares, Célia; Freitas-Silva, Otniel; Santos, Cledir; Paterson, Russell; Kozakiewicz, Zofia; Venâncio, Armando; Lima, NelsonMicoteca da Universidade do Minho (MUM) is a collection of fungi established in 1996. Research involving secondary metabolites in the form of mycotoxins is paramount. An objective is to provide authentic strains for research in biotechnology, life sciences and challenge testing. Knowledge, information and training in mycology are promulgated by the biological resource centre. MUM is involved in projects which address the health and quality risks from fungal contamination of grape products (e.g. wine), apples, cheese, chilies, nuts and corn: Fungi in drinking water is of particular concern. Aspergillus ochraceous, Aspergillus flavus, Penicillium expansum and Fusarium graminearum especially are relevant to our work: Associated mycotoxins are ochratoxin A, aflatoxins, patulin, citrinin and zearalenone. We study the earthy off-flavours in wine from Botrytis cinearum and P. expansum. The analytical techniques employed are PCR, HPLC, GC-MS and MALDITOF MS for strain characterization. MUM participates in several European and national projects. Post-graduate studies on mycotoxigenic fungi and food security particularly are important. A key objective is international collaboration. MUM strains are well characterized for toxigenicity, making the collection relevant to its mission and industry. This presentation will describe the progress made in this innovative Portuguese laboratory over 10 years.
- Identification and characterization of Aspergillus flavus and aflatoxinsPublication . Rodrigues, Paula; Soares, Célia; Kozakiewicz, Zofia; Paterson, Russell; Lima, Nelson; Venâncio, ArmandoAspergillus flavus is the main producer of the well known carcinogenic aflatoxins. The presence of this fungus and aflatoxins is of huge concern in terms of food safety. The identification of A. flavus is not straightforward due to similarities with closely related species (e.g. A. parasiticus and A. nomius). Also, from the biochemical point of view the closely-related species are able to produce different mycotoxins. In order to clarify the differentiation between species the identification schemes is revisited. Selective media, data from mycotoxins production and molecular biology tools are discussed in order to clarify the concept of A. flavus species.
- Identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus Section Flavi isolated from Portuguese almondsPublication . Rodrigues, Paula; Venâncio, Armando; Kozakiewicz, Zofia; Lima, NelsonAspergillus subgenus Circumdati section Flavi, also referred to as the A. flavus group, has attracted worldwide attention for its industrial use and toxigenic potential. Section Flavi is divided in two groups of species. One includes the aflatoxigenic species A. flavus, A. parasiticus and A. nomius, which cause serious problems in agricultural commodities, and the other one includes the non‐aflatoxigenic species A. oryzae, A. sojae and A. tamarii, traditionally used for production of fermented foods. Differentiating aflatoxigenic from non‐aflatoxigenic species and strains in food commodities is of major importance in food quality control. A polyphasic approach consisting of morphological, chemical and molecular characterization was applied to 31 isolates of Aspergillus Section Flavi originating from Portuguese almonds, with the aim of characterizing and identifying aflatoxigenic and non‐aflatoxigenic strains. On the basis of morphological characters, we found two distinct groups among the population under study: 58% were classified as A. parasiticus and the remaining 42% were classified as A. flavus. Chemical characterization involved the screening of the isolates for aflatoxins B (AFB) and G (AFG), and also for cyclopiazonic acid (CPA), by HPLC. All A. parasiticus isolates were strong AFB and AFG producers, but no CPA production was detected. The A. flavus isolates showed to be more diversified, with 77% being atoxigenic, whereas 15% produced CPA and low levels of AFB and 8% produced the 3 groups of mycotoxins. Molecularly, two genes of the aflatoxin biosynthetic pathway, aflD (=nor1) and aflQ (=ord1= ordA) were tested for presence and expression (by PCR and RT‐PCR, respectively). The presence of both genes did not correlate with aflatoxigenicity. aflD expression was not considered a good marker for differentiating aflatoxigenic from non‐aflatoxigenic isolates, but aflQ showed a good correlation between expression and aflatoxin‐production ability.
- MALDI-TOF MS potentialities and limits to characterise aflatoxigenic species of Aspergillus Section FlaviPublication . Rodrigues, Paula; Santos, Cledir; Kallow, Wibke; Erhard, Marcel; Welker, Martin; Kozakiewicz, Zofia; Lima, Nelson; Venâncio, Armando
- Micotoxinas: contributos da micoteca da Universidade do Minho (mum) para a segurança alimentarPublication . Rodrigues, Paula; Soares, Célia; Freitas-Silva, Otniel; Santos, Cledir; Paterson, Russell; Kozakiewicz, Zofia; Venâncio, Armando; Lima, NelsonA Micoteca da Universidade do Minho (MUM), fundada em 1996, tem como missão ser uma colecção de fungos filamentosos com o objectivo principal de manter e fornecer estirpes com qualidade e autenticidade para a investigação em biotecnologia e ciências da vida, e laboratórios de ensaio, actuando também como um centro de conhecimento, informação e formação na área da micologia. Dentro desta missão, a MUM tem estado envolvida em projectos que procuram dar resposta aos riscos alimentares derivados da contaminação fúngica.
- Polyphasic approach including maldi-tof mass spectrometry to characterise aflatoxigenic species of aspergillus section flaviPublication . Rodrigues, Paula; Santos, Cledir; Kozakiewicz, Zofia; Venâncio, Armando; Lima, NelsonAflatoxins are toxic compounds which are produced as secondary metabolites by the fungi Aspergillus flavus, A. parasiticus and A. nomius growing on a variety of food products and are known to be carcinogenic, mutagenic, teratogenic and immunosuppressive1,2. Aspergillus is a large genus, with a complex taxonomy. The genus is easily identified by its characteristic conidiophore, but species identification and differentiation is complex, mainly because it is traditionally based on a range of morphological features. One includes the aflatoxigenic species referred above A. flavus, A. parasiticus and A. nomius, which cause serious problems in agricultural commodities, and the other one includes the non-aflatoxigenic species A. oryzae, A. sojae and A. tamarii, traditionally used for production of fermented foods. Species from A. flavus group are morphologically and genetically very similar, and are therefore difficult to differentiate by both cultural and molecular methods. Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry has already shown high potentialities in discriminating very closely related taxa. In this work is presented a polyphasic approach including MALDI-TOF MS to discriminate A. flavus group strains.
- A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section flavi isolated from portuguese almondsPublication . Rodrigues, Paula; Venâncio, Armando; Kozakiewicz, Zofia; Lima, NelsonA polyphasic approach consisting of morphological, chemical and molecular characterization was applied to 31 isolates of Aspergillus Section Flavi originating from Portuguese almonds, with the aim of characterizing and identifying aflatoxigenic and non-aflatoxigenic strains. On the basis of morphological characters (mainly colony color on Czapek-Dox agar and conidia morphology), we found two distinct groups among the population under study: 18 isolates (58%) had dark-green colonies and rough conidia, and were classified as A. parasiticus; the remaining 13 isolates (42%) had yellow-green colonies and smooth to finely rough globose conidia, and were classified as A. flavus. Chemical characterization involved the screening of the isolates for aflatoxins B (AFB) and G (AFG), and also for cyclopiazonic acid (CPA), by HPLC with fluorescence and UV detection, respectively. All A. parasiticus isolates were strong AFB and AFG producers, but no CPA production was detected, showing a consistent mycotoxigenic pattern. The A. flavus isolates showed to be more diversified, with 77% being atoxigenic, whereas 15% produced CPA and low levels of AFB and 8% produced the 3 groups of mycotoxins. Aflatoxin production was also screened on Coconut Agar Medium (CAM), and the results were consistent with the HPLC analysis. Sclerotia production showed no correlation to aflatoxigenicity. Molecularly, two genes of the aflatoxin biosynthetic pathway, aflD (= nor1) and aflQ (= ord1= ordA) were tested for presence and expression (by PCR and RT-PCR, respectively). The presence of both genes did not correlate with aflatoxigenicity. aflD expression was not considered a good marker for differentiating aflatoxigenic from non-aflatoxigenic isolates, but aflQ showed a good correlation between expression and aflatoxin-production ability.