CHARACTERIZATION OF THE MYCOFLORA ASSOCIATED TO DRY CURED PORK MEAT PRODUCTS WITH FOCUS ON PENICILLIUM NORDICUM AND ITS BIOCONTROL

Penicillium nordicum è un importante contaminante di salumi, rappresentanando il 10 % e il 26 % della popolazione di Penicillium spp . isolati , rispettivamente dall'aria e dai prodotti carnei stagionati in un'indagine gestita in Italia ( Battilani et al. , 2007). Diverse colonie di P. nordicum isolate dai salumi hanno dimostrato di essere importanti produttori di ocratossina A , OTA ( Sansom e Frisvad , 2004 . Pietri et al, 2006 ; . Battilani et al , 2010). Attualmente, l'impostazione appropriata delle condizioni ambientali (temperatura, umidità relativa e circolazione dell'aria ), è l'unico strumento accettato per impedire la crescita incontrollata di P. nordicum all'interno degli impianti di stagionatura attraverso una accurata analisi dei punti critici di controllo e l’ideazione di un relativo piano HACCP (Hazard Analysis and Critical Control Points) ben struttutato ( Asefa et al , 2011; Virgili et al , 2012). Anche se il sistema HACCP è stato applicato con successo nel settore alimentare ci sono rischi per la sicurezza alimentare non attentamente considerati. Questo è particolarmente vero per quanto riguarda i rischi micotossigeni associati ai prodotti alimentari di origine animale. Il termine "rischi micotossigeni" è utilizzato da Asefa et al. ( 2011) per descrivere lieviti patogeni e metaboliti secondari tossici prodotti da specie fungine tossigene che contaminano i prodotti alimentari e incidono sulla sicurezza alimentare. La maggior parte dei piani HACCP nelle attività di trasformazione alimentare, come ad esempio la produzione di formaggi e di prodotti carnei stagionati, tiene in considerazione principalmente il rischio derivante da agenti batterici (Arvanitoyannis e Mavropoulos, 2000; Barbuti e Parolari, 2002) anche se tali prodotti alimentari vengono spesso contaminati da funghi micotossigeni e dai loro metaboliti (Spotti et al 1989; Spotti et al , 2001a; Battilani et al 2007). Pertanto, dovrebbe essere cruciale definire un piano HACCP specificamente incentrato sui rischi micotossigeni. L'identificazione, il controllo e la standardizzazione della micoflora superficie dei salumi è fondamentale per preservare la sicurezza delle produzioni e la salute dei consumatori . Questo è il contesto in cui deve essere valutata l’efficacia e l’affidabilità per l’identificazione delle popolazioni di Penicillium spp di interessante per la produzione alimentare. In questo contesto , il progetto di ricerca di questa tesi di dottorato ha cercato di approfondire le conoscenze su tali tematiche con l'intento di limitare il rischio micotossigeno nella catena di produzione dei prodotti carnei stagionati. Sono stati affrontati i seguenti argomenti: 1 . studio della composizione e dinamica della microflora fungina presente sulla superficie dei salumi (prodotto testato, salame) e l'aria di ambienti di stagionatura tenendo conto dell'influenza di alcuni parametri di processo (inoculo starter, temperatura, fase produttiva). 2 . sviluppo di un metodo MALDI TOF MS per l'identificazione di Penicilium a livello di specie per le prospettive future di screening diretti della microflora presente sui salumi. 3 . confronto e integrazione di diverse tecniche, come l'analisi morfologica, l’analisi molecolare e l’analisi tramite spettrometria di massa, per l'identificazione delle specie di Penicillium presenti nei salumi. 4 . valutazione dei lieviti selezionati, isolati dalla superficie di prosciutto crudo, per competere con P. nordicum ed inibire l'accumulo di OTA nella prospettiva del loro uso come starter superficiali con funzione di agenti di biocontrollo.Penicillium nordicum is an important contaminant of cured meat products, representing 10% and 26% of the Penicillium spp. isolated, respectively, from the air or the products in a survey managed in Italy (Battilani et al., 2007). Several P. nordicum cured meat isolates proved to be important producers of ochratoxin A, OTA (Sansom and Frisvad, 2004; Pietri et al., 2006; Battilani et al., 2010). Currently, the appropriate setting of environmental conditions (temperature, relative humidity and air circulation), is the only accepted tool to prevent the uncontrolled growth of P. nordicum inside dry-curing plants through a carefully structured Hazard Analysis Critical Control Point (HACCP) plan (Asefa et al., 2011; Virgili et al., 2012). Even if the HACCP system has been successfully applied in the food industry, there are food safety hazards not carefully considered. This is especially true with regard to mycotoxigenic hazards associated with animal food products. The term “mycotoxigenic hazards” is used by Asefa et al. (2011) to describe pathogenic yeasts and toxic secondary metabolites of toxigenic moulds that contaminate food products and affect food safety. Most HACCP plans in food processing activities, such as the production of cheese and dry-cured meat products, considered mainly bacterial agents (Arvanitoyannis and Mavropoulos, 2000; Barbuti and Parolari, 2002), even if such food products get often contaminated with mycotoxigenic fungi and their metabolites (Spotti et al 1989; Spotti et al., 2001a; Battilani et al 2007). Therefore, it should be crucial to define a HACCP plan specifically focused on the mycotoxigenic hazards. The identification, control and standardization of the surface mycoflora of cured meat products is mandatory to preserve the productions safety and the consumers health. This is the context of the effectiveness and reliability evaluation for the Penicillium spp. identification methods of interesting species for food production. In this context, the research project of this PHD thesis tried to fill some gaps of knowledge with the attempt to limit the mycotoxigenic risk in the cured meat products chain. The following topics were faced: 1. study of the composition and dynamic of fungal microflora present on the surface of cured meat products (salami) and the air of seasoning environments taking into account the influence of some process parameters (starter inoculum, curing temperature, stage of seasoning). 2. development of a MALDI TOF MS method for the identification of Penicilium at species level for future direct screening perspectives of the microflora present on cured meat products. 3. comparison and integration of different techniques, as morphological, molecular and mass spectral analysis, for the identification of Penicillium species in cured meat products. 4. evaluation of selected yeasts, isolated from dry-cured ham surface, to compete with P. nordicum and to inhibit OTA accumulation in the perspective of their use as surface starter biocontrol agents

Validation and Ecological Niche Investigation of a New Fungal Intraspecific Competitor as a Biocontrol Agent for the Sustainable Containment of Aflatoxins on Maize Fields

Crop yield and plant products quality are directly or indirectly affected by climate alterations. Adverse climatic conditions often promote the occurrence of different abiotic stresses, which can reduce or enhance the susceptibility to pests or pathogens. Aflatoxin producing fungi, in particular, whose diffusion and deleterious consequences on cereals commodities have been demonstrated to highly depend on the temperature and humidity conditions that threaten increasingly larger areas. Biological methods using intraspecific competitors to prevent fungal development and/or toxin production at the pre-harvest level are particularly promising, even if their efficacy could be affected by the ecological interaction within the resident microbial population. A previously characterized Aspergillus flavus atoxigenic strain was applied in two maize fields to validate its effectiveness as a biocontrol agent against aflatoxin contamination. At one month post-application, at the harvest stage, its persistence within the A. flavus population colonizing the maize kernels in the treated area was assessed, and its efficacy was compared in vitro with a representation of the isolated atoxigenic population. Results proved that our fungal competitor contained the aflatoxin level on maize grains as successfully as a traditional chemical strategy, even if representing less than 30% of the atoxigenic strains re-isolated, and achieved the best performance (in terms of bio-competitive potential) concerning endogenous atoxigenic isolates

Long-term voice monitoring with smartphone applications and contact microphone

In recent years, the growing interest in the recognition of voice disorders as occupational diseases has required screening methods adaptable to the clinical requirements, capable to extend the collection of baseline data. In this framework, the use of smartphones has gained increasing interest, thanks to advancements in digital technology, which made them suitable for recording and analyzingacoustic signals. Two smartphone applications, based on the Voice Care® technology, have been developed for long-term monitoring of voice activity when combined with a cheap contact microphone embedded in a collar. The applications have been tested in laboratory and used for the monitoring of teachers at kindergarten, primary school, and university. Vocal Holter App allows the selection of short and long term monitoring mode, and three different clusters of vocal parameters related to intensity, intonation, and load, respectively. Most of the results are based on the distributions of occurrences of vocal parameters. A headlight informs the person under monitoring of pathologic voice. Vocal Holter Rec allows data recording and to perform a personalized analysis based on updated parameters. The equipment allows downloading and saving data on a dedicated web site for further processing, comparisons over time, or sharing with physicians or rehabilitators

Development of innovative technologies for the management of aflatoxin contamination

Aspergillus flavus, a phytopathogenic species that naturally colonize economically relevant crops such as maize, peanuts and spices, is considered the main producer of highly toxic and cancerogenic compounds known as aflatoxin B1 and B2. Aflatoxins, that possess teratogenic and mutagenic properties, can be frequently found at high concentration in food and feed commodities, since such molecules often are transferred along the food chain reaching the consumers through contaminated matrices. For this reason, the concentration of aflatoxin B1 in food and feed is strictly regulated by national and international Food Security Authorities. The control is thus a complex problem that requires the development of wide range of contamination management strategies, from the field (pre- and post- harvest) to the fork. This Ph.D. project proposes a green approach, focused on the development of integrated strategies for reduce aflatoxin pre-harvest contamination, coping innovative technologies of plant defense, with lower environmental impact and application costs to increase the sustainability of corn cultivation in Emilia Romagna Region. Part of this work is addressed to characterize the antifungal and antimycotoxigenic properties of newly synthesized bioinorganic compounds, safe for the environment and the human health, with the final goal to obtain a Q-SAR database. The second part is focused on the definition of an aflatoxin containment strategy based on the use of intraspecific bio-competitor, investigated through a two-year in field experimentation. Additional topics are also included, aimed at a deeper and accurate molecular characterization of A. flavus isolates in terms of genetics, biodiversity and new developing tools of detection

Sabotage at the Powerhouse? Unraveling the Molecular Target of 2-Isopropylbenzaldehyde Thiosemicarbazone, a Specific Inhibitor of Aflatoxin Biosynthesis and Sclerotia Development in Aspergillus flavus, Using Yeast as a Model System

Amongst the various approaches to contain aflatoxin contamination of feed and food commodities, the use of inhibitors of fungal growth and/or toxin biosynthesis is showing great promise for the implementation or the replacement of conventional pesticide-based strategies. Several inhibition mechanisms were found taking place at different levels in the biology of the aflatoxin-producing fungal species such as Aspergillus flavus: compounds that influence aflatoxin production may block the biosynthetic pathway through the direct control of genes belonging to the aflatoxin gene cluster, or interfere with one or more of the several steps involved in the aflatoxin metabolism upstream. Recent findings pointed to mitochondrial functionality as one of the potential targets of some aflatoxin inhibitors. Additionally, we have recently reported that the effect of a compound belonging to the class of thiosemicarbazones might be related to the energy generation/carbon flow and redox homeostasis control by the fungal cell. Here, we report our investigation about a putative molecular target of the 3-isopropylbenzaldehyde thiosemicarbazone (mHtcum), using the yeast Saccharomyces cerevisiae as model system, to demonstrate how the compound can actually interfere with the mitochondrial respiratory chain

Aspergillus Goes Viral: Ecological Insights from the Geographical Distribution of the Mycovirome within an Aspergillus flavus Population and Its Possible Correlation with Aflatoxin Biosynthesis

Microbial multi-level interactions are essential to control the success of spreading and survival of most microbes in natural environments. Phytopathogenic mycotoxigenic fungal species, such as Aspergillus flavus, represent an important issue in food safety. Usually, non-toxigenic strains are exploited for biocontrol strategies to mitigate infections by toxigenic strains. To comprehend all the biological variables involved in the aflatoxin biosynthesis, and to possibly evaluate the interplay between A. flavus toxigenic and non-toxigenic strains during intraspecific biocompetition, the “virological” perspective should be considered. For these reasons, investigations on mycoviruses associated to A. flavus populations inhabiting specific agroecosystems are highly desirable. Here, we provide the first accurate characterization of the novel mycovirome identified within an A. flavus wild population colonizing the maize fields of northern Italy: a selection of A. flavus strains was biologically characterized and subjected to RNAseq analysis, revealing new mycoviruses and a peculiar geographic pattern distribution in addition to a 20% rate of infection. More interestingly, a negative correlation between viral infection and aflatoxin production was found. Results significantly expanded the limited existent data about mycoviruses in wild A. flavus, opening new and intriguing hypotheses about the ecological significance of mycoviruses

“DIFESAMAIS”: AN ITALIAN PROJECT FOR THE MANAGEMENT OF AFLATOXIN CROPS CONTAMINATION

Corn aflatoxins contamination is one of the main issue that farmers of large areas of Europe have to cope with. Different strategies have been implemented to try to reduce, if not eliminate, toxin contamination at the pre-harvest level; however, a careful analysis of the costs/efficacy of each of the various inputs must be performed in order to provide an acceptable income for the farmers. On the other hand, there is an increasing market demand for food and feed commodities obtained with environmentally low-impact practices possibly through the experimentation of innovative and organic defense techniques. Minimization of mycotoxin risk requires a multifactorial approach since climatic, agronomic and phytoiatric factors are found to interact each other in complex, local ecological realities. The “DIFESAMAIS” project intends to design and validate an innovative combined approach addressed to the development and the valorization of organic farming/integrated production of corn, with particular attention to the maintenance of biodiversity. Both academic and industrial partners (PROGEO S.C.A. and AGRITES s.r.l.) were involved in the present project. The main stakeholders are farmers which will have the opportunity to acquire the economic expertise and the technical tools to implement an ecologically sustainable maize cultivation by a reduction of chemical inputs. Among the various activities performed in the framework of the Project, we will report the validation of a sustainable procedure to reduce aflatoxin contamination by bio-competition approaches, based on: 1) the use of non-aflatoxigenic strains of Aspergillus flavus, and 2) the validation of technologies for Ostrinia nubilalis control. Aflatoxin containment, biocontrol, maize protection Funding have been provided by the Emilia Romagna Rural Developing Program (PSR 2014-2020), Project n. 5004325 “DIFESAMAIS: Development of innovative defense technologies to increase environmental sustainability of corn production”

Double Gamers—Can Modified Natural Regulators of Higher Plants Act as Antagonists against Phytopathogens? The Case of Jasmonic Acid Derivatives

As key players in biotic stress response of plants, jasmonic acid (JA) and its derivatives cover a specific and prominent role in pathogens-mediated signaling and hence are promising candidates for a sustainable management of phytopathogenic fungi. Recently, JA directed antimicrobial effects on plant pathogens has been suggested, supporting the theory of oxylipins as double gamers in plant-pathogen interaction. Based on these premises, six derivatives (dihydrojasmone and cis-jasmone, two thiosemicarbazonic derivatives and their corresponding complexes with copper) have been evaluated against 13 fungal species affecting various economically important herbaceous and woody crops, such as cereals, grapes and horticultural crops: Phaeoacremonium minimum, Neofusicoccum parvum, Phaeomoniella chlamydospora, Fomitiporia mediterranea, Fusarium poae, F. culmorum, F. graminearum, F. oxysporum f. sp. lactucae,F. sporotrichioides, Aspergillus flavus, Rhizoctonia solani,Sclerotinia spp. and Verticillium dahliae. The biological activity of these compounds was assessed in terms of growth inhibition and, for the two mycotoxigenic species A. flavus and F. sporotrichioides, also in terms of toxin containment. As expected, the inhibitory effect of molecules greatly varied amongst both genera and species; cis-jasmone thiosemicarbazone in particular has shown the wider range of effectiveness. However, our results show that thiosemicarbazones derivatives are more effective than the parent ketones in limiting fungal growth and mycotoxins production, supporting possible applications for the control of pathogenic fungi

PROJECT AFLATOX®: A NEW APPROACH FOR THE DEVELOPMENT OF ANTIFUNGAL AND ANTIMYCOTOXIGENIC COMPOUNDS

Aflatoxins represent a major issue for a food and feed economy based on cereal cultivations, and aflatoxins contamination constitutes a health emergency because these mycotoxins, besides being toxic, are among the most carcinogenic substances known. Even if Aspergillus species were dominant in tropical regions, due to the climatic change they recently became a serious concern also in Europe. In Italy, this problem is particularly relevant in the Po Valley, in which maize market is strictly linked to dairy products such as the well-known Parmigiano Reggiano cheese. Despite of countless efforts, to date the problem of food and feed contamination remains unsolved, since the essential factors that affect aflatoxins production are various and hardly to handle together. In this scenario, the exploitation of bioactive natural sources to obtain new agents with novel mechanisms of action may represent a successful strategy to minimize at the same time mycotoxin contamination and the use of harmful pesticides. In 2015 Aflatox® Project was granted from Cariplo Fundation: purpose of our project was the development of new-generation inhibitors of aflatoxigenic Aspergillus spp proliferation and toxin production, through the modification of naturally occurring molecules; a panel of at least 180 compounds, based on a class of molecules named thiosemicarbazones, have been analyzed for their antifungal and antiaflatoxigenic ability. Cytotoxicity, genotoxicity and epi-genotoxicity of new-synthesized compounds was assessed on both human cell lines and in vitro model systems, and finally data were assembled in a Quantitative Structure-Activity Relationship (QSAR) database correlating chemical structures with biological/toxicological activities. This study was supported by a grant from “Fondazione Cariplo” (Project N. 2014-0555)