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L'approccio biotecnologico prevede l'utilizzo di nuovi microrganismi isolati da differenti fonti con capacità di metabolizzare i composti tossici contenuti nelle AV e ridurne quindi il carico inquinante delle. I microrganismi possono essere prodotti come starter per la fermentazione e il trattamento delle AV in forma di cellule libere o immobilizzate. In tal modo si può ottenere una riduzione sensibile, dal 30 al 50% della domanda chimica di ossigeno (COD) delle acque, della concentrazione di molecole fenoliche presenti, della carica antimicrobica e fitotossica. L'approccio biotecnologico proposto potrebbe garantire nel medio periodo di ottenere delle acque con ridotta o nulla carica inquinante ed umificate, da impiegare senza limitazioni per la ferti-irrigazione dei terreni agricoli. Le AV trattate con la tecnologia proposta, rappresentano inoltre un substrato più facilmente utilizzabile da specifici ceppi batterici per la produzione di biogas.

The understanding of the yeast population dynamics during spontaneous alcoholic fermentation allows to preserve the microbial biodiversity, to use as indigenous fermentation starters so and to improve the organoleptic and sensory properties of the produced wines. However, it is similarly important to investigate the safety aspects of microbial biodiversity, in particular, on the undesired production of biogenic amines (BAs), low-molecular-weight organic bases produced in wine by the activity of microbial-specific amino acid decarboxylases. This study is the first large-scale investigation on vineyard-associated yeast strains from Apulia (Southern Italy). Eight natural must fermentations were carried out by sampling grape (Vitis vinifera) in the most significant production areas for Negroamaro and Primitivo cultivars: Torchiarolo, Copertino, Cutrofiano and Melissano for the former, Galatina, Torchiarolo, Manduria and Gioia del Colle areas for the latter. Yeasts isolates were identified by PCR ribotyping and bioinformatic analysis of the rRNA Internal region denoted as Transcribed Spacer (ITS). The Saccharomyces cerevisiae strains were further identified and differentiate as strain level by evaluating the polymorphism of their interdelta elements. The results of the molecular analyses revealed the presence of twenty different species belonging to 9 genera. In particular, Hanseniaspora uvarum, Metschnikowia pulcherrima and Aureobasidium pullulans were the dominant strains on the grape surface, whereas M. pulcherrima and H. uvarum were predominant during the early fermentation stage. We identified 692 S. cerevisiae isolates and a number of different strain in each of the 8 fermentations strains, ranging from 26 to 55, The strains were assayed for BAs production, either in synthetic media or grape must. Two Pichia manshurica, an Issatchenkia terricola and a M. pulcherrima strains were capable to produce in wine histamine and cadaverine. The production of BAs in the synthetic medium was dissimilar than that detected in wine, thus enhancing significance to assess the yeast BAs production by an "in grape must" assay, in order to reducethe potential health risk for consumer represented by these spoilage yeasts. To the best of our knowledge, this is the primary study regarding the biodiversity and safety aspects of grape-associated yeast strains in this important wine-producing area of Southern Italy.

Table olives are one of the most important traditional fermented vegetables in Southern European countries and their consumption is constantly increasing throughout the world. Today, the industrial production of black table olives is carried out by spontaneous fermentation processes which are not predictable and are strongly influenced by the autochthonous microflora, the physical-chemical conditions, the availability of fermentable substrates and salt content. Evolution of sugars, organic acids, alcohols, mono and polyphenol compounds and volatile compounds associated with the fermentative metabolism of yeasts and bacteria throughout the natural fermentation process of the two Italian olive cultivars Cellina di Nardò and Leccino were determined. A new protocol was developed and applied aimed at the selection of LAB and yeast strains as candidate autochthonous starters for table olive fermentation from Cellina di Nardò and Leccino cultivars.

Valutazione della bioaccessibilità dei principali carotenoidi presenti negli estratti di zucca dopo un processo di digestione in vitro.

The increased consumption of whole wheat grains and whole wheat products has been associated with reduced risk of developing chronic diseases, such as cardiovascular disease, type 2 diabetes and colon cancer. These beneficial effects have been ascribed to the presence in whole wheat kernels of bioactive compounds which may vary for total content and composition among different wheat species and wheat varieties. In this work we present the profile of hydrophilic and lipophilic bioactive compounds of whole wheat semolina from five durum wheat elite cultivars. Whole semolina samples were analyzed to evaluate the total content and composition of phenolic acids (hydrophilic extract) and the total content and composition of carotenoids, tocopherols and tocotrienols (lipophilic extract). The total phenolic acid content was variable among the cultivars and ranged from 488 ?g/g to 1490 ?g/g whole flour. Among the detected compounds, ferulic acid was the most abundant, followed by sinapic acid and p-coumaric acid. Total carotenoid content varied from 2.64 µg/g whole flour and 4.75 µg/g whole flour and were mostly represented by lutein and zeaxanthin, while ?- e ?-carotene were present in trace amounts. Three different homologues of tocotrienols were detected (?, ? and ?), varying in a range between 18.3 and 28.6 µg/g whole flour, while tocopherols were detected in trace amounts. Duilio and Svevo cultivars, exhibited the highest content of hydrophilic and lipophilic bioactive compounds, respectively, and were selected to test the anti-inflammatory activity of extracts on human intestinal HT-29 cells. Preliminary experiments were carried out in order to assess the highest not cytotoxic concentration of lipophilic and hydrophilic extracts by using MTT test. Both extracts will be used to assess the anti-inflammatory activity on HT-29 cells stimulated by LPS mitogen, by quantification of IL-8. This research shows that whole wheat semolina flours of these five cultivars varied significantly in their contents of bioactive compounds and differences in their anti-inflammatory potential might suggest the possibility that durum wheat varieties could be selected based on potential health benefits.

This work is the first large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy). Yeasts were identified by Internal Transcribed Spacer (ITS) ribotyping and bioinformatic analysis. The polymorphism of interdelta elements was used to differentiate Saccharomyces cerevisiae strains. Twenty different species belonging to 9 genera were identified. Predominant on the grape surface were Metschnikowia pulcherrima, Hanseniaspora uvarum and Aureobasidium pullulans, whereas M. pulcherrima and H. uvarum were dominant in the early fermentation stage. A total of 692 S. cerevisiae isolates were identified and a number of S. cerevisiae strains, ranging from 26 to 55, was detected in each of the eight fermentations. The strains were tested for biogenic amines (BAs) production, either in synthetic media or grape must. Two Pichia manshurica, an Issatchenkia terricola and a M. pulcherrima strains were able to produce histamine and cadaverine, during must fermentation. The production of BAs in wine must was different than that observed in the synthetic medium. This feature indicate the importance of an "in grape must" assessment of BAs producing yeast. Overall, our results suggest the importance of microbiologicalcontrol during wine-making to reduce the potential health risk for consumer represented by these spoilage yeasts.

Strain SPC-1T was isolated from the phyllosphere of Cynara cardunculus L. var. sylvestris (Lamk) Fiori (wild cardoon), a Mediterranean native plant considered the wild ancestor of the globe artichoke and cultivated cardoon. This Gram-negative, catalase-positive, oxidase-negative, non-spore-forming, rod-shaped and non-motile strain secreted copious amounts of an exopolysaccharide and formed slimy, viscous, orange-pigmented colonies and grew optimally at around pH 6.0-6.5 and 26-30°C in the presence of 0-0.5% NaCl. Phylogenetic analysis based on comparisons of 16S rRNA gene sequences demonstrated that SPC-1T clustered together with species of the genus Sphingomonas sensu strictu. The G+C content of the DNA (66.1 mol%), the presence of Q-10 as the predominant ubiquinone, sym-homospermidine as the predominant polyamine, and 2-hydroxymiristic acid (14:0 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the presence of sphingoglycolipid supported this taxonomical position. 16S rRNA gene sequence analysis showed that SPC-1 was most closely related to Sphingomonas hankookensis ODN7T, Sphingomonas insulae DS-28T and Sphingomonas panni C52T (98.19%, 97.91% and 97.11% similarity, respectively). However, DNA-DNA hybridization analysis did not reveal any relatedness at the species level. Further differences were apparent in biochemical traits, and fatty acid, quinone and polyamine profiles leading us to conclude that strain SPC-1T (JCM 17498; ITEM 13494) represents a new species of Sphingomonas, for which the name Sphingomonas cynarae sp. nov. is proposed. A component analysis of the exopolysaccharide (named SPC-1T EPS) suggested that it represents a novel type of sphingan containing glucose, rhamnose, mannose and galactose, while glucuronic acid, which is commonly found in sphingans, was not detected.

Spontaneous grapes must fermentations are promoted by the indigenous yeasts, that are able to confer a distinctive style and quality to the produced wine. The spontaneous fermentations of grape must are at first dominated by non-Saccharomyces yeasts and, in a final stage the alcoholic fermentation process is completed by dominant S. cerevisiae strains (Bauer and Pretorius, 2000).The autochthonous yeast strains are associated to a specific vineyard niche habitat and their role in natural fermentation allows the production of wines with particular features in each microclimatic area (Pérez-Coello et al., 1999). However, in order to avoid the unpredictability of must spontaneous fermentation, the winemakers employ commercial dry active yeast culture for wine industrial productions. Increasing interest in the application of locally selected yeasts for fermentation management has been reported (Tristezza et al., 2012). The employment of autochthonous strains of S. cerevisiae as starters seems to be preferable since they are adapted to all the constraints related to a specific wine-production area (Lopes et al., 2007) and are thus capable to dominate more efficiently the indigenous microflora during the fermentation process. Moreover, autochthonous yeast strains can assure the preservation and/or the enhancement of the typical oenological and sensory features which could be considered representative of an oenological region (Rodríguez et al., 2010). In the present study, we developed and applied a strategy to select S. cerevisiae strains from a larger number of yeast isolates. This was achieved adopting a number of key parameters indicative of the strains technological and enological properties. S. cerevisiae population has been isolated from natural fermentations of grape musts, which derive from grapes sampled from the six most representative Negroamaro and Primitivo producing-areas in Apulia (Southern Italy). The yeast populations were identified by molecular assays (AFLP and sequencing) and some selected representative strains were subjected to physiological, oenological and technological characterization. At the end of the selection procedure, that lasted three years, three indigenous S. cerevisiae strains (one for Primitivo and two for Negroamaro), characterized by interesting technological and oenological properties, were selected. The three selected strains were evaluated by both laboratory tests and semi-industrial scale fermentations to confirm their ability to act as autochthonous fermentation starters. An optimized procedure was worked out for the production of starter biomasses, in order to test them in Negroamaro and Primitivo wine production, at an industrial scale, in six different wineries. The employment of autochthonous starter cultures for the industrial production of typical wines in Apulia will be discussed.

Two approaches were developed in order to select microorganisms suitable to be used in olive mill wastewaters bioremediation. By the first approach, three hundred yeasts were isolated from five industrial mills and identified by molecular analysis. The different strains were selected according to their capacity to grow in OMW (olive mill wastewaters) as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One Geotrichum candidum isolate was used to set up a whole-cell immobilization system in calcium alginate gel and the COD and phenolic reduction obtained using the immobilized cells showed respectively a 2.2- and 2-fold increase compared to the removal obtained using free cells. By the second approach, a new protocol was developed to isolate and select aerobic microorganisms from different industrial samples and environmental niches (soils, OMWs) and able to detoxify olive mill wastewaters.

Senescence is an integrated response of plants to various internal (developmental) and external (environmental) signals. It is a highly regulated process leading eventually to the death of cells, single organs such as leaves, or even whole plants. In cereals, which are monocarpic plants, senescence represents the final stage of development. In order to study senescence in durum wheat (Triticum turgidum subsp. durum), a cDNA-AFLP analysis was performed. The transcription profiles of plants at different developmental stages (flowering and senescent) were compared. About 2000 cDNA fragments, ranging in size from 160 to 1900 bp, were reproducibly detected. This allowed the identification of 57 differentially expressed cDNAs corresponding to genes belonging to different functional categories related to cellular metabolism, transcription, maintenance of DNA structure, transport and signal transduction. This paper reports the identification of novel durum wheat candidate genes involved in the senescence process, and provides new information about the senescence programme of this important crop species.

Artemisinin is a sesquiterpene antimalarial compound produced, though at low levels (0.1-1% dry weight), in Artemisia annua in which it accumulates in the glandular trichomes of the plant. Due to its antimalarial properties and short supply, efforts are being made to improve our understanding of artemisinin biosynthesis and its production. Native ?-cyclodextrins, as well as the chemically modified heptakis(2,6-di-O-methyl)-?-cyclodextrin (DIMEB) and 2-hydroxypropyl-?-cyclodextrins, were added to the culture medium of A. annua suspension cultures, and their effects on artemisinin production were analysed. The effects of a joint cyclodextrin and methyl jasmonate treatment were also investigated. Fifty millimolar DIMEB, as well as a combination of 50 mM DIMEB and 100 ?M methyl jasmonate, was highly effective in increasing the artemisinin levels in the culture medium. The observed artemisinin level (27 ?mol g-1 dry weight) was about 300-fold higher than that observed in untreated suspensions. The influence of ?-cyclodextrins and methyl jasmonate on the expression

Artemisia annua L., is a herbal plant traditionally used for the treatment of fever. The glandular trichomes of this plant accumulate, although at low levels, artemisinin which is highly effective against malaria. Due to the great importance of this compound many efforts have been made to improve the knowledge on artemisinin production both in plants and in cell cultures. In this study A. annua suspension cultures were established in order to investigate the effects of methyl jasmonate (MeJA) and miconazole treatments on artemisinin biosynthesis. Twenty-two mM MeJA induced a three-fold increase of artemisinin production soon after 30 min. Two hundred mM miconazole induced a 2.5 fold increase of artemisinin production after 24 h treatment but had severe effects on cell viability. The influence of these treatments on the expression of biosynthetic genes was also investigated. MeJA induced an up-regulation of CYP71AV1 while miconazole induced an up-regulation of CPR and DBR2.

Plants respond to environmental stimuli, such as heat shock, by re-programming cellular activity through differential gene expression, mainly controlled at the transcription level. The current study refers to two sunflower small heat shock protein (sHSP) genes arranged in tandem in head-to-head orientation and linked by a 3809 bp region. These genes exhibit only slight structural differences in the coding portion. They code for cytosolic class I sHSPs and are named HaHSP17.6a and HaHSP17.6b according to the molecular weight of the putative proteins. The genomic organization of these genes is consistent with the idea that many HSP genes originate from duplication events; in this case, probably an inversion and duplication occurred. The HaHSP17.6a and HaHSP17.6b genes are characterized by different expression levels under various heat stress conditions; moreover, their expression is differently induced by various elicitors. The differential regulation observed for HaHSP17.6a and HaHSP17.6b genes differs from previous observations on duplicated sHSP genes in plants.

Artemisinin is an effective antimalarial compound obtained by thè aerial parts of thè herbal plant Artemisia annua L. Unfortunately, thè production of artemisinin in plants is very low (0.1 -1% on a dry weight basis) and its chemical synthesis is very difficult. In recent years biotechnological attempts to improve artemisinin production either in plant or genetically engineered microrganisms were made. A. annua celi and tissue cultures were also explored for thè production of artemisinin, although thè yieids obtained have been so far quite low. We have recently established A. annua celi cultures able to biosynthesize artemisinin and to respond to thè elicitation by methyl jasmonate (MeJA). Interestingly, amounts of artemisinin produced by these cultures were also found in thè spent culture medium. Cyclodextrins (CDs) are known to increase thè water solubility of various compounds, by forming host-guest inclusion complexes. Chemically modified CDs, such as 2,6 dimethyl-p-cyclodextrins (DIMEB) have been successfully used to improve thè production of health-promoting plant metabolites such as resveratrol in grape celi cultures. The aim of this work was to evaluate thè ability of DIMEB to enhance artemisinin production in A. annua suspension celi cultures. HPLC analysis of artemisinin and Real Time PCR gene expression analysis were carried out in DIMEB treated and untreated cultures. DIMEB induced a 300-fold increase of artemisinin levels in thè spent culture medium after a three-day-treatment. The addition of MeJA induced a further increase of thè artemisinin amounts. The expression levels of artemisinin biosynthetic genes (cytochrome P450 monoxygenase, cytochrome P450 reductase and artemisinic aldehyde A11 reductase) were not altered by thè DIMEB treatment. These results suggest that DIMEB improved artemisinin yieids of A. annua celi cultures, probably not affecting thè biosynthetic pathway but enhancing thè solubility and stability of artemisinin molecules.

Apulia is the second Italian wine-producing region and the most important producer of red-rosé wine. One peculiar characteristic of Apulian wines is that the podologic characteristics and the climatic conditions of this region contribute to enrich the wine of aromatic essences and to give, therefore, a characteristic and intense taste to the finished product. The Apulian wine industry is living a moment of great qualitative transformation and the challenge of the market had therefore addressed the productive row towards the use of innovative systems to guarantee and exalt the qualitative characteristics of regional wines. A heartfelt requirement is to being able to pilot and to control the productive activity to obtain wines with peculiar characteristics, with respect of the typicality, that is guaranteed by the denominations of origin. The selection and the employment of new combinations of microorganisms, obtained from the native micro flora would be a powerful instrument to improve the organoleptic and sensory characteristics of the product. Autochthonous yeasts are the micro-organisms better adapted to a specific must, which detain characteristics determined by the variety of the grapes and the terroir and, therefore they are able to exalt the peculiarities (aromas, structure and colour) of a wine. In these last year the research activity of the Institute of Science of Food Production of C.N.R. has been directed to the exploitation of autochthonous microbiota to enhance the quality of regional wines. This research line has been granted by a number of National and Regional Projects and it has been conducted in collaboration between the ISPA and a large number of wine companies. The research activity has generated a virtuous circle, thus allowing the standardization of protocols for wine yeast enological selection and biomass production, the constitution of a Yeast Collection but, most of all, the transfer of technology to a large number of SMEs belonging of the wine production chain. Natural fermentations of Negroamaro, Primitivo and Susumaniello musts have been performed and the Saccharomyces cerevisiae population has been analyzed and characterized by molecular, physiological, enological and technological tests allowing the identification of four indigenous S. cerevisiae strains candidate as autochthonous fermentation starters. The enological properties of the above strains have been evaluated during the vintages 2006-2009, by performing 37 large scale vinification trials in 21 different industrial cellars in Apulia. The obtained results and their implications for the valorisation and the improvement of Apulian typical wines will be discussed.

La fermentazione malolattica (FML) è un evento fermentativo successivo alla fermentazione alcolica compiuta dai batteri lattici, in particolare da Oenococcus oeni. La FML influenza la qualità del vino ed è considerata, per questo, un evento importante nel processo di vinificazione. Tale processo avviene sia spontaneamente ad opera della flora indigena o artificialmente mediante inoculo di colture starter. Quest'ultimo processo è una pratica comunemente utilizzata in cantina e le fermentazioni condotte con l'inoculo di ceppi batterici selezionati sono più facili da controllare e garantiscono lo standard qualitativo del prodotto. Scopo di questo studio é stata la caratterizzazione biomolecolare e tecnologica di ceppi isolati da mosto di uve Salentine "Primitivo" tipica cultivar della Puglia. Durante le differenti fasi del processo di FML (inizio, media e fine) è stata isolata una popolazione di 220 isolati di batteri lattici, da cui sono stati identificati 87 ceppi di Oenococcus oeni. L'identificazione è stata effettuata mediante analisi PCR specie-specifica utilizzando oligonucleotidi specifici disegnati per il gene MLE e successivo sequenziamento dei prodotti di PCR relativi all'RNA contenente la regione per il gene 16S rRNA. Questo ha permesso di ottenere delle sequenze discriminanti le diverse specie. L'analisi genotipica è stata eseguita con lo sviluppo della tecnica molecolare AFLP, per la prima volta adottata sul genoma di questo batterio, al fine di sviluppare profili molecolari discriminanti che differenziano e caratterizzano gli Oenococcus oeni a livello di ceppo. In seguito a questa analisi è stato possibile differenziare quattro differenti gruppi di isolati, tre dei quali hanno mostrato significativi livelli di omologia intraspecifica. L'analisi AFLP, si è dimostrata una valida alternativa ad altri metodi molecolari. Un totale di 28 ceppi, scelti dai differenti sottogruppi dei cluster ottenuti dagli AFLP, sono stati testati per la loro capacita di metabolizzare il malato. Significative differenze sono state osservate tra i ceppi relativamente alla a) velocità del consumo di acido malico, b) biomassa prodotta e al c) tasso specifico di consumo di acido malico. I risultati ottenuti e la loro implicazione biotecnologia verranno discusse.

CNR-ISPA develops analyses of food products and compounds with the use of innovative technologies: ?Identification: genes, proteins and metabolites in food products and plants, biosynthesis pathways, biomarkers, DNA arrays and expression profiling, Northern blots, RT-PCR ?Analysis: effects of biocompounds on cell cultures, cell growth and survival, biochemical assays, cell activation /inhibition, Western blots, monitoring changes in cell phenotype, cell-to-cell contact, analysis of gap-junctions using microinjection and confocal microscopy ?Detection: compounds / proteins in food using Protein arrays and antibody chips (enzymes, allergens, protein-protein interaction studies.

During the time frame of COST853, we presented results on DNA arrays for detection of Fusarium species belonging to the Liseola Section. In this work, we focused the attention on the detection on Aspergillus carbonarius, a common contaminant of several food commodities and the main responsible for ochratoxin A (OTA) accumulation in grapes and wine. OTA is a mycotoxin highly toxic for humans. In order to rapidly identify the A. carbonarius species, specific sequences were selected in the ITS1 and ITS2 region of various Aspergillus species. Two generic primers (P1For, P2Rev) were designed in order to amplify the fungal rDNA (including ITS1 and ITS 2 region). In the DNA macroarray experiments, oligonucleotides specific for A. carbonarius, A. japonicus and A. aculeatus, were provided with an NH2-terminal modification for covalent binding to epoxy-slides (Nexterion, Schott). These immobilised sequences were used as probe to test their specificity in hybridization experiments with the cyanine 3-labeled amplified products. Surface plasmon resonance (SPR) is an emerging technology that enables the label-free detection of biomolecular interactions. In the SPR sensor experiments, the probes were prepared biotinylated in their 5' end and immobilized on a dextran-streptavidin pre-coated gold chip by means of the well known biotin-streptavidin interaction. The probes were tested for biospecific interactions with label-free amplified products. Conclusions: we tested two different methods to verify the specificity of various probes. Fungal DNA amplified with P1For and P2Rev generic primers was used as target. The advantage of the DNA macroarray method consists in the possibility to immobilize a high number of different probes on the same chip, on the contrary, only two probes can be immobilized on the chip in the SPR method. Nevertheless the SPR method has the advantage that the amplified target DNA is label-free. The experiments allowed the identification of the working conditions useful for obtaining specific hybridization signals. The obtained results show that the two methods are both specific and sensitive.