The general objective of the macro-area is to improve the safety of agri-food products, with particular reference to mycotoxins and other contaminants. The research activity in this macro-area are aimed at developing and optimizing traditional and innovative analytical methods for determining mycotoxins, pesticides and their biomarkers, at monitoring, controlling, preventing and reducing mycotoxin contamination in pre- and post-harvest, and their toxicological characterisation. The specific objectives involved in the “Development and optimisation of traditional and innovative analytical methods” for determining mycotoxins and pesticides in matrices of agri-.food interest and their corresponding biomarkers in biological fluids are as follows:

• Synthesising synthetic mediators (molecularly-imprinted polymers) for mycotoxins and pesticides and identifying biological mediators (antibodies, enzymes) to integrate into innovative technologies (biosensors) based on optical and electro-chemical systems for the specific detection of mycotoxins and pesticides to be used in the various agri-food sectors.
• Developing innovative analytical methods for determining mycotoxins and pesticides using the FPIA technique (fluorescence polarization immunoassay);
• Development of analytical methods using gas chromatography and HPLC with a fluorescence detector and/or mass spectrometry for the determination of mycotoxins (trichothecenes, aflatoxins, ochratoxins, zearalenone) and pesticides in agri-food products and the corresponding biomarkers in biological fluids.

The specific objectives of “Monitoring, controlling, preventing and reducing mycotoxin contamination in pre- and post-harvest” are as follows:

• Monitoring of mycotoxins in agri-food products
• Pre- and post-harvest studies on the production of organic wheat and derivative products which are resistant to colonisation by toxigenic fungi and to the accumulation of mycotoxins.
• Assessing the ability of fungicides, natural antagonists and compounds with antimicrobial and/or antioxidant properties to inhibit the growth of toxigenic fungi and the production of mycotoxins.
• Studies of the toxicokinetics of mycotoxins using a computerised gastrointestinal model (TIM) as an alternative to experiments on animals.
• Measuring the capacity of inert adsorbents to trap mycotoxins so as to reduce gastrointestinal absorption using TIM.
• Studying the effects of various production/transformation processes on reducing mycotoxins in foods.

The specific objectives of “Toxicity and risk assessment” are as follows:

• Evaluation of the immunotoxic and estrogenic effects of mycotoxins (individually or in combination) and of xeno-estrogens on human immune system and reproductive cell lines.
• In vivo trials on rainbow trout models and embryo eggs to measure respectively the sub-acute and chronic toxicity of mycotoxins.

The activities carried out in this macroarea as part of our ordinary activities and under the following projects:

• European Project QLK1-1999-00996: “Prevention of Fusarium mycotoxins entering the human and animal food chain”
• European Project QLK1-1999-00986: “Reduction of contamination by fungi and mycotoxins during the organic vegetable production chain: Alternaria-carrot model”;
• European Project QLK5-CT2000-01248: European network for promoting knowledge of mycotoxins”
• MIUR strategic project 2.5.2000: “Innovative systems for the safety of typical milk and cheese production (SISPROLAT)”
• MIPAF project: “Prevention of chemical and mycotoxin contamination in conventional and organic dairy farming”
• MIUR project: “Creating a template for an integrated system for managing food safety in the durum wheat sector (SINSIAF)”
• Syngenta Crop Protection contract: “Traceability of mycotoxins in durum and bread wheat and in the corresponding processed products, and analysis of ochratoxin A in samples of grapes and must”;
• Bayer Crop Science contract on “Traceability of mycotoxins in wheat”;
• contract with VICAM Ltd, USA: “Development and validation of mycotoxin analysis methods”.

Research activities in the “Quality of agri-food products” macro-area aim to reach their various objectives using methods such as plant biotechnologies, proteomics and nutritional physiology, taking into account both their sustainability and applicability on the one hand, and on the other the research carried out in the sector by the rest of the national and international scientific community. The overall objective of improving “Food Quality: molecular and functional characterization of animal and plant proteins using a proteomic approach” was attained by achieving the following specific objectives:

• Improving diagnostic testing for food allergies, selecting naturally hypoallergenic plant varieties and using technology to produce anergic foods
• Developing an objective evaluation method based on comparing the total protein in OGM foods with the corresponding non-GM foods, including their potential for allergenicity
• Identifying and evaluating the biological function of proteins associated with milk-fat globule membranes from humans, cows and equine species (horse and donkey). Identifying and characterising the main allergens in cow’s milk. Looking for a valid alternative foodstuff for children who cannot be breast-fed and/or cannot drink cow’s milk.

The overall objective of “Plant Biotechnologies: improving the quality and defences of agriculturally important plants” was to improve the quality of plant-based foods by studying the genes and enzymes which help to determine the organoleptic qualities, healthiness and disease-resistance of the plant species involved (tomato, strawberry, almond and hazelnut). This objective was attained by achieving the following specific objectives:

• Producing “functional foods” aimed at preventing food-related diseases. In particular, this activity aimed to induce the synthesis of new antioxidant molecules from the stilbene family in tomatoes, by intervening on the flavonoid metabolism.
• Biochemical and molecular characterization of plant lipoxygenases and other enzymes from the same metabolic pathway (hydroperoxide lyase) involved in producing volatile molecules, aromatic components produced during the ripening process of strawberries, almonds and hazelnuts
• Producing recombinant lipoxygenase and hydroperoxide lyase to find out whether natural aromas can be produced using these recombinant enzymes.
• Identifying proteins expressed differently during the ripening process of fruits (proteomic analysis)
• Defining the role played by oxylipins in plant response to pathogens and whether plant resistance can be increased by altering the expression of some genes in the lipoxygenase biosynthesis pathway.
• Molecular characterisation of the ripening process in Wild Strawberry (Fragaria vesca L.) and commercial strawberries (Fragaria x ananassa Duch.) by AFLP analysis of cDNA obtained from fruits harvested at various stages of ripening. Identification and characterisation of molecular targets characteristic of stages of ripening and of the varieties analysed.

The overall objective of “Bio-factory: production of plant molecules of agro-industrial, nutritional and pharmacological interest” concentrated in particular on the antioxidant biomolecules useful in food. This objective was attained by achieving the following specific objectives:

• developing an innovative process for producing lycopene from tomatoes using an inert gas (carbon dioxide at supercritical conditions) instead of organic solvents which are toxic for the human organism
• selecting Italian almond cultivars with a high tocopherol content in order to acquire useful knowledge to enable the production of almond oil containing tocopherol
• optimising an in vitro production system of alpha-tocopherol (vitamin E) from sunflower (Helianthus annuus L.) suspension cell cultures
• studying the key steps in the biosynthetic pathway of tocopherols through molecular analysis of sunflower cells in active synthesis
• development of an in vitro production system of anthocyanins from calluses and cell cultures of sour cherry. Identification of the various factors (nutrients, phytoregulators, light) which induce the biosynthesis of anthocyanins in vitro in calluses and cell cultures of sour cherry.

The overall objective of improving the “Quality of foodstuffs for livestock and by-products” was attained by achieving the following specific objectives:

• Identifying forage species and storage methods which would allow a farm access to protein with high nutritional value, to use for feeding ruminants, as an alternative to using proteins of animal origin
• characterising the fatty acid composition of meadows/pastures in the Alpine valleys compared with that in silage stacks characterising the lipid fraction and components of the flavoured compounds in Alpine milk and cheeses identification of “typical geographical markers” (branched fatty acids, monoterpenes and sesquiterpenes) capable of certifying the geographical origin of DOP cheese products. Applying the NIRS technique to characterise the lipid component of cheeses produced in winter and in summer in Alpine environments
• Evaluating the possible use of by-products from the agri-food industry for feeding horses
• Evaluating the chemical composition of nutrient sources for a potential use in formulations for commercially-bred fish Comparing the effectiveness of diets based on plant raw material for freshwater fish (warm or cool) and for marine fish.

The research was carried out as part of our ordinary activities and under the following projects:

• Food Allergy Risk Evaluation based on improved Diagnosis, Allergens and test methods” (EU contract QLK4 2001-00301, FAREDAT)
• Developing a methodological model for producing and assessing hypoallergenic food products (MIUR – Strategic Project Law 449/97)
• Assessment of hypoallergenicity and nutritional value of foods based on horse milk (Convention with the Turin Foundation for Biotechnologies)
• Development of methods for the characterisation of the protein fraction in plants of agri-food interest (Biennial convention with Parma University)
• Bioactive amines in wines (COFIN project Turin University, Dept of Animal and Human Biology)
• Structural characterisation of some members of the lipocalin family (COFIN project Turin University, Dept of Animal and Human Biology)
• Aerobic deterioration of silage, with particular reference to maize silage. Survey into the situation of local farms, including the effects on the quality of silage, milk and cheese products, and methods of prevention (Piedmont Regional Government Project)
• Creation of hay silage to boost alpine pasture resources: use in the production of organic milk and DOP and quality cheese in mountain environments (Piedmont Regional Government project)
• Biological and production evaluation in optimising the profitability of sturgeon farming (MiPAF Project)
• Effect of plant lipid sources on production performance, body characteristics and quality of meat in acipenseridae and salmonids. (MiPAF Project)
• Development of innovative technologies and biotechnological processes in controlled conditions in the plant culture sector”-( Cluster 5-MURST);
• Establishing a universal system for detection of Kunitz-type proteinase inhibitor genes in potato (Nato funds-Russia collaborative linkage grants JSTC.RCLG.980102);
• Study of genes of biomedical and agri-food interest” – (Cluster MURST)
• Recognition of Italian ecotype of wood strawberry (Fragaria vesca L.) by genetic and molecular analysis of the fragrance (SCRIGNO Programme – FISR fund)
• Production of Lycopene with innovative systems (MIUR funds).

Coordinator: Antonio Logrieco
Unit: Bari

Unit: Bari

Our research in the “Toxigenic fungi and biopesticides” macroarea aimed to acquire products and systems to improve the healthiness and quality of plant products, with particular reference to integrated and microbiological control of agricultural plant diseases and reducing the risk both of toxigenic fungi being present and of passing on their toxic metabolites down the food chain. The research is highly innovative, and includes the use of tools and techniques from microbial biotechnology, such as sequencing and the functional characterisation of the genes of phytopathogenic, toxigenic and antagonistic micro-organisms and the molecular interactions that are established with plants. To this end, it appears to be vital to maintain and increase a mycological collection documenting microbial biodiversity and metabolic variability. The production of biologically active metabolites by fungi and bacteria capable of acting either as antagonists against harmful micro-organisms, plants and insects (biopesticides), or as plant growth promoters (biofertilisers and bio-phytohormones) is another main objective for low environmental impact farming and eco-compatible products. The overall objective of the “Agri-food mycology” research line is to identify, prevent and control toxigenic fungi (producers of mycotoxins) in the most important agri-food sectors for Southern Italy (cereals, vine crops and horticultural products), and has the following specific objectives:

• Assessment of toxicological risk in the main food matrices. This objective is achieved by monitoring the presence of toxigenic fungi on the main cereal (maize, wheat, rice) and other crops of agri-food interest in order to identify the potential contamination by mycotoxins in the respective food products.
• Developing specific and simple methods for the early diagnosis of infective and toxigenic agents of agrarian crops and food supplies.
• Molecular characterisation of toxigenic species through sequencing techniques on the variable regions of ribosomal DNA, and analysis of the polymorphisms of randomised restriction and amplification of total DNA.
• Isolation and characterisation of specific DNA sequences from toxigenic fungi for the characterisation and identification of Fusarium, Penicillium and Aspergillus isolates, whether or not they produce toxic metabolites;
• Development of a DNA optical sensor and the construction of DNA arrays for the identification of toxigenic fungi in the various forms of durum wheat.
• Development of various PCR tests for the early recognition of toxigenic fungi in food matrices and the use of TaqMan probes for real time PCR experiments on food matrices, such as: wheat, maize, coffee, grapes, must.
• Increasing the ITEM fungus collection and developing a data base.

The availability of a collection of microfungi which produce bioactive metabolites, as well as being an important source of microbial biodiversity, also provides a number of potential biotechnology solutions. This objective is achieved by isolating fungal strains from agriculturally important plants and food products, and registering the most representative strains of the isolated populations with the most interesting toxigenic and biological characteristics. The overall objective of the “Microbial bio-products for environmentally friendly agriculture” research line is to develop scientific knowledge, materials and technology related to the use of micro-organisms for improving the health and quality of agri-food production, in order to obtain high value-added products such as organic ones, and to reduce the environmental impact of farming practices and the risks attached to them. This is to be attained through the following objectives: Development of biopesticides (antagonists, mycoherbicides, bioinsecticides) and biofertilisers for environmentally friendly farming. This objective is split into various specific objectives, such as:

• isolation and characterisation of fungi and bacteria that are potentially useful in biological and integrated control against biotic agents of plant disease and agricultural products in pre- or post-harvest or as biofertilisers;
• development of methods for their formulation and application;
• use of natural substances of microbial origin in the fight against phytopathogenic and mycotoxigenic agents in pre- and post-harvest and against weeds.

Study of the mechanisms of micro-organisms used as biopesticides and/or biofertilisers and of their molecular determinants (secondary and primary metabolites and their genes). This objective is split into various specific objectives, such as:

• Identification, characterisation and study of the expression of genes involved in the synthesis of primary or secondary microbial metabolites with a role in the action of biopesticides and biofertilisers;
• study of the useful/pathogenic physiological and molecular interactions between plant and micro-organism: analysis of gene expression and production of enzymes by the plant in response to bio-inoculants; c) measurement of the IGR induced by phyto-hormone producing bacteria (auxins and cytokinins), both individually and when combined with micro-organisms used as biological control agents in order to evaluate any synergies arising.

Risk assessment of releasing fungi and bacteria used as biopesticides and/or biofertilisers into the environment. This objective is split into the following specific objectives:

• Toxicological characterisation of the secondary metabolites produced by useful micro-organisms;
• Monitoring of useful micro-organisms and the dynamics of their toxic metabolites (production, release and persistence) on treated products, in the environment and in the food chain;
• Transfer of pathogenicity factors and/or virulence to useful fungi and bacteria;
• Transfer of genes coding for resistance to substances responsible for the antagonistic properties of biological control agents;
• Evaluation of the quality of products obtained from useful micro-organisms.

The research was carried out as part of our ordinary activities and under the following projects:

• Project on “Antagonistic fungi and their metabolites for biological control of phytopathogenic fungi” (MIUR – Piano MAIA Cluster 6+7, Prog.1.1, Workpackage 1, 2, 3, 4).
• Project on “Study of the molecular mechanisms in various plant-pathogen systems” (MURST Project – LAG Plan).
• Project on “Creating a template for an integrated system for managing food safety in the durum wheat sector” (SINSIAF) (MIUR – PON Project)” Activity 3.1/3.2
• Project on “Toxigenic Fusariums and biological control” (Bilateral project CNR/SAV, Slovakia)
• Project on “Early diagnosis of toxigenic Fusarium species and ochratoxigenic fungi in plant products” (EU Project: Quality of Life-QLK1-1999-01380).
• Project on “Risk assessment and integrated management of ochratoxin A (OTA) in grapes and wine” (EU project QLK1-CT-2001-01761).
• Project on “Sustainability, product safety and quality in cereals: development of new quantitative models for identifying the risk of mycotoxigenic Fusarium species” (EU Project: QLk5-CT-2000-01517)
• Project on “Determining the risk of using fungal micro-organisms in biological control” (EU QLK1-CT2001-01391).