PENICILLIA AND ASPERGILLI ASSOCIATED TO GRANA CHEESE MULTIPHASIC IDENTIFICATION APPROACH, ECOLOGICAL NEEDS AND TOXINS PRODUCTION

Recentemente, l’ocratossina A (OTA) è stata segnalata in formaggio grattugiato confezionato: si ritiene sia stata prodotta dallo sviluppo di alcuni funghi durante il periodo di maturazione. Gli studi presentati in questa tesi hanno avuto lo scopo di migliorare la conoscenza dei funghi, Aspergilli e Penicilli, associati al formaggio di tipo grana. Si è dunque provveduto alla loro identificazione tramite approccio multifasico, eseguendo anche prove ecologiche per definire i fabbisogni delle specie presumibilmente dominanti. Obiettivo finale sarà quello di predire il rischio di contaminazione da micotossine alle condizioni ambientali di stagionatura del prodotto. Aspergillus puulaauensis e alcune specie del genere Penicillium (P.crustosum and P. solitum) sono state rilevate con maggior frequenza, mentre non sono state rilevate nei campioni di crosta le specie P. nordicum e P. verrucosum. In ogni caso, le condizioni ambientali dei locali di maturazione (15-22°C; 72-88% UR) sono risultate favorevoli allo sviluppo di queste ultime, dando adito a preoccupazione circa una possibile contaminazione da OTA; tuttavia, il ruolo di altre specie micotossigene, ad esempio P. crustosum, non va sottovalutato. Le azioni di contrasto verso questi funghi micotossigeni dovrebbero, innanzitutto, ridurre l’inoculo presente nell’aria: l’ozono sembra efficace in tal senso; inoltre, l’utilizzo della luce blu potrebbe ridurre notevolmente la crescita fungina sulla superficie dei formaggi e delle scaffalature. Le informazioni fornite in questa tesi saranno utili ai produttori e agli altri operatori della filiera di questo prodotto, aiutandoli ad attuare una miglior gestione del rischio e a garantire un prodotto sicuro e di elevata qualità ai consumatori.Ochratoxin A (OTA) was detected a few years ago in Italian packed grated cheese, and supposed to be produced by fungal growth during cheese ripening. The works managed and presented in this thesis aimed to improve knowledge about mycotoxigenic Aspergilli and Penicillia associated to Italian grana cheese. A multiphasic approach was applied for their identification and ecological trials were organised to define fungal needs of the expected dominant species. The objective was to predict the risk of mycotoxin contamination during the ripening condition of grana cheese. Aspergillus puulaauensis and some Penicillium spp (P.crustosum and P. solitum) were the prevalent species associated to grana cheese, while neither P. nordicum, nor P. verrucosum were detected in cheese rind. However, the environmental conditions of the ripening rooms (15-22°C; 72-88% RH) are favourable to P. nordicum and P. verrucosum, reinforcing concern about possible OTA contamination, but other mycotoxigenic species such as P. crustosum must not be neglected. Actions against those mycotoxigenic fungi should mainly reduce total inoculum in the environmental air: ozone is reported to be effective in this sense; moreover, blue light may significantly reduce fungal growth on cheese and shelves surfaces. Information provided therein will be useful for producers and stakeholders to perform a better risk management and guarantee a safe, high-quality product to consumers

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

Caracterização de linhagens de Aspergillus e Penicillium isoladas da superfície de queijo curado italiano do tipo “grana”

O queijo curado Italiano do tipo “grana” é um produto de alto valor comercial. Apesar de se observar o crescimento de linhagens pertencentes aos géneros Aspergillus e Penicillium, que têm espécies potencialmente produtoras de micotoxinas, não existe muita informação acerca da micobiota associada a este produto. As micotoxinas, produzidos por algumas espécies fúngicas, podem ter efeitos nocivos graves na saúde humana e animal. Assim, é essencial estudar o micobiota deste queijo, de modo a se poder fazer uma análise do risco associado às espécies envolvidas. A ocorrência de micotoxinas no queijo tem vindo a ser gradualmente reportada, como a ocratoxina A (OTA) e a esterigmatocistina (STC). Apesar destas micotoxinas estarem incluídas no grupo 2B pela IARC/International Agency for Research on Cancer, i.e., serem potencialmente cancerígenas para os humanos, neste momento, não existe legislação Europeia que as regule no queijo. O objectivo deste trabalho foi obter pela primeira um levantamento sobre linhagens de Penicillium e Aspergillus que crescem na superfície do queijo curado Italiano do tipo “grana”, dando especial atenção às espécies micotoxigénicas. Para o efeito, entre os anos de 2013 e 2014, foram obtidas amostras raspadas da crosta do aro da cura e foram expostas ao ar placas com meio de batata e dextrose agarizado para monitoramento das câmaras de cura. Dos fungos isolados foram escolhidos aleatoriamente 140 linhagens de colónias monoespóricas (89% Penicillium e 11% Aspergillus) para a identificação ao nível de espécie usando a abordagem polifásica com a caracterização morfológica, perfil de extrólitos e molecular. As espécies mais representativas foram: Penicillium solitum (54), P. crustosum (20), P. commune (12), P. nordicum (11), Aspergillus puulaaeunsis (6), A. flavus (5), P. charlesii (3), P. chrysogenum (3), P. paneum (3), P. citrinum (2), P. jugoslavicum (2). Em conclusão, este trabalho permitiu fazer pela primeira vez um levantamento das espécies contaminantes deste tipo de queijo bem como verificar a presença de linhagens produtoras de aflatoxinas (A. flavus), de STC (A. puulaaeunsis) e de OTA (P. nordicum). Mediante estes resultados, pode-se inferir que o risco de ocorrência de micotoxinas neste produto é real sendo essencial implementar medidas que impeçam o desenvolvimento desta micobiota durante a cura do queijo

Polyphasic identification of Penicillia and Aspergilli isolated from Italian grana cheese

"Available online 18 January 2018"Penicillium and Aspergillus genera, both including mycotoxin producing species, were reported as associated to cheese and cheese working environment, but never studied in an extensive way in Italian grana cheese (Grana Padano and Parmigiano Reggiano). The aim of this work was to address the identification of Aspergilli and Penicillia associated to grana cheese in order to lay down the basis for risk assessment and safe processing for a high quality production. One hundred and four strains belonging to Aspergillus and Penicillium genera were obtained from cheese crust and from ripening room air (with the latter largely dominant), and identified following a polyphasic approach, strongly required for the identification at the species level. Morphological observation was used along with molecular techniques, RAPD-PCR fingerprinting and calmodulin gene sequencing (CaM), the former aimed to limit as much as possible the latter sequencing effort. Seventy four percent of the strains were assigned to Penicillium subgenus Penicillium, section Fasciculata. Main mycotoxin producing species identified were A. flavus, P. crustosum and P. verrucosum, while the dominant species in both air and cheese crust was P. solitum, which has never been so far reported as mycotoxigenic. Results obtained in this study confirmed that mycotoxin contamination is a possible issue to face during grana cheese making.The present work was supported by the following institutions: Consorzio del Formaggio Parmigiano Reggiano, Consorzio per la tutela del Formaggio Grana Padano, Fondazione Romeo ed Enrica Invernizzi, the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/ 2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Simone Decontardi carried out this work within the PhD school “Agrisystem” of the Universita Cattolica del Sacro Cuore (Italy).info:eu-repo/semantics/publishedVersio

Modeling Growth and Toxin Production of Toxigenic Fungi Signaled in Cheese under Different Temperature and Water Activity Regimes

The aim of this study was to investigate in vitro and model the effect of temperature (T) and water activity (aw) conditions on growth and toxin production by some toxigenic fungi signaled in cheese. Aspergillus versicolor, Penicillium camemberti, P. citrinum, P. crustosum, P. nalgiovense, P. nordicum, P. roqueforti, P. verrucosum were considered they were grown under different T (0–40 °C) and aw (0.78–0.99) regimes. The highest relative growth occurred around 25 °C; all the fungi were very susceptible to aw and 0.99 was optimal for almost all species (except for A. versicolor, awopt = 0.96). The highest toxin production occurred between 15 and 25 °C and 0.96–0.99 aw. Therefore, during grana cheese ripening, managed between 15 and 22 °C, ochratoxin A (OTA), penitrem A (PA), roquefortine-C (ROQ-C) and mycophenolic acid (MPA) are apparently at the highest production risk. Bete and logistic function described fungal growth under different T and aw regimes well, respectively. Bete function described also STC, PA, ROQ-C and OTA production as well as function of T. These models would be very useful as starting point to develop a mechanistic model to predict fungal growth and toxin production during cheese ripening and to help advising the most proper setting of environmental factors to minimize the contamination risk

Penicillium species identification and new insights on mycotoxins in food commodities (apples, chilli and cheese)

The 10th International Palestinian Conference of Laboratory Medicine and The 15th Arab Conference of Clinical BiologyAmong certain groups of filamentous fungi that produce mycotoxins, relevant contaminates in food, the genus Penicillium is of great importance. Penicillium is ubiquitous in nature and inevitable, although it can be controlled from the field to the fork. Mycotoxins are fungal secondary metabolites that cause sickness or death in people when ingested, inhaled, and/or absorbed. Major mycotoxins associated with common penicillia are: Ochratoxin A (P. verrucosum and P. nordicum), patulin (P. expansum), citrinin (P. expansum), cyclopazonic acid (P. camemberti), penicillic acid (P. radicicola) and secalonic acid D, F (P. griseofulvum). Penicillia identification is time-consuming and sounder polyphasic identification, which includes phenotypic and genotypic approaches, is recommended. However, in many laboratories, the standard character for identification is still morphology. Taking this into account, results from Penicillium species isolated from Tunisian apples, Chilean traditional chilli (Merkén), Italian cheeses and their mycotoxin profiles (patulin and ochratoxin A) will be presented in this work. For morphological analyses, isolates were inoculated in triplicate in different media. Fungi grown in MEA for colony and microscopy analyses were used. Multilocus sequence analysis was performed through comparison of partial -tubulin, calmodulin and ITS with sequences available in GenBank. Specific primers for genes involved in the mycotoxins pathways were used for PCR amplification. After extraction the mycotoxins were quantified using HPLC-FLD (fluorescence detection). From Tunisian apples isolates, a novel species Penicillium tunisiense of section Ramosa is proposed. This is not a patulin producer with the idh gene negative in contrast with the other dominant P. expansum isolates. In addition, ochratoxigenic strains P. verrucosum and P. crustosum were isolated from chilli and cheese samples, respectively, and characterised with genes involved this mycotoxin production. Our findings show that mycotoxigenic Penicillium strains, as food contaminants, remain an important field of study and more knowledge needs to be learned.info:eu-repo/semantics/publishedVersio

Penicillia diversity from food identified polyphasically, including mycotoxin production

Portuguese Foundation for Science and Technology (FCT). It was under the scope of the strategic funding of the UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01–0145-FEDER-006684) and the BioTecNorte operation (NORTE-01–0145-FEDER-000004), funded by the European Regional Development Fund through Norte2020—Programa Operacional Regional do Norte (Portugal