Introduction. Fungi (yeasts and moulds) are recognized as one of the main contaminants of dairy products including yogurt and sour milk. These microorganisms can also cause spoilage in a wide range of processed, preserved and refrigerated food products. During the past years, several molecular methods based on immunological and genotypic techniques have been developed for revealing the presence of undesirable microorganisms, including fungi, in different food matrices. However, no commercial kit are already available to detect viable yeasts and moulds in dairy products. Materials and methods. Five antibodies against yeasts and molds were selected from commercially available antibodies and used to produce functionalized magnetic beads to be used to capture and separate microrganisms associated to dairy products. Four yeast type species (Debaryomyces hansenii, Kluyveromyces marxianus, Geotrichum candidum and Pichia anomala) and four mold species (Alternaria alternata, Aspergillus niger, Penicillium italicum and Rhizopus stolonifer) were used. Milk, yogurt and soft cheese were tested as matrices. A RT-PCR protocol was developed for detection of yeast and molds mRNA extracted from contaminated foods. Results. A new method for yeast and molds enrichment from different food dairy products (milk, yogurt and soft cheese) based on the use of antibody coated magnetic beads was developed. A new RT-PCR assay based on a nested amplification was optimized for the detection of yeast and molds in artificially contaminated dairy products.Discussion. The correlation between the amplification signal and the microbial count will allow to use this method for viable contaminants quantification in dairy products. This method can avoid the labor expensive food matrices treatments, often cause of loss of sensitivity. This approach will be transferred also in other food matrices. This procedure can be implemented by the use of automated enrichment systems already available for pathogen microorganisms.

The apiculate yeasts are the species predominating the first stage of grape must alcoholic fermentation and it is important for the production of desired volatile compounds. The aim of the present investigation was to establish a protocol for the enological selection of non-Saccharomyces strains directly isolated from a natural must fermentation during the tumultuous phase. At this scope, fifty H. uvarum isolates were characterized at strain level by employing a new combined PCR-based approach. One isolate representative of each identified strain was used in fermentation assays to assess strain-specific enological properties. The chemical analysis indicated that all the analyzed strains were low producers of acetic acid and hydrogen sulphide, whereas they showed fructophilic character and high glycerol production. Analysis of volatile compounds indicated that one strain could positively affect, during the alcoholic fermentation process, the taste and flavour of alcoholic beverages. The statistical evaluation of obtained results indicated that the selected autochthonous H. uvarum strain showed to possess physiological and technological properties, which satisfy the criteria indicated for non-Saccharomyces wine yeasts selection. Our data suggest that the described protocol could be advantageously applied for the selection of non-Saccharomyces strains suitable for the formulation of mixed or sequential starters together with S. cerevisiae.

Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. Recently, the biosynthetic mechanism of this mycotoxin has been mainly elucidated by experiments of knocking out of the key biosynthetic genes. The mutant strains of A. carbonarius, in which the AcOTAnrps gene had been disrupted, was unable to produce OTA but retained its ability to degrade OTA into OT? when it was grown in presence of exogenous OTA. Microbial degradation of OTA is due to the enzymatic cleavage of the amide bond between L-?-phenylalanine and OT? by proteolytic proteins. Then, an in silico screening has been made on the available genome sequence of A. carbonarius ITEM 5010 to identify genes encoding proteases and to investigate their involvement in the OTA degrading activity of A. carbonarius. Preliminary transcriptomic analysis allowed selecting eight protease encoding genes that were expressed at increased level during OTA production. From the analysis of functional domains of the deduced protein sequences, four identified genes encode for aspartic proteases, three of them encode for serine proteases and one for a metalloprotease. Wild type and three mutant strains of A. carbonarius ITEM 5010 (?AcOTAnrps, ?AcOTApks, ?AcOTAhal) previously obtained and resulted to be unable to produce OTA, have been incubated in presence of OTA under different conditions and time of growth. Expression levels during growth and activation rate of the selected protease genes are under investigation in order to establish their involvement in the degradation activity of A. carbonarius strains.

Plant sensitive factor attachment protein receptors (SNAREs) encoded by genes of the same sub-family are generally considered as redundant in promoting vesicle-associated membrane fusion events. Nonetheless, the application of innovative experimental approaches highlighted that members of the same gene sub-family often have different functional specificities. In this work, two closely related Qc-SNAREs-the AtSYP51 and the AtSYP52-are compared in their ability to influence different secretory pathways. Their role in the vesicle sorting to the central vacuole has been revised and they were found to have a novel inhibitory function. When transiently overexpressed, the SYP51 and the SYP52 distributed between the TGN and the tonoplast. Our data demonstrate that these SYPs (syntaxin of plants) act as t-SNARE when present on the membrane of TGN/PVC, whereas they behave as inhibitory or interfering SNAREs (i-SNAREs) when they accumulate on the tonoplast. Moreover, the performed functional analysis indicated that the AtSYP51 and the AtSYP52 roles differ in the traffic to the vacuole. The findings are a novel contribution to the functional characterization of plant SNAREs that reveals additional non-fusogenic roles.

The aim of the present study was to establish a new procedure for the oenological selection of Saccharomyces cerevisiae strains isolated from natural must fermentations of an important Italian grape cultivar, denoted as "Negroamaro". For this purpose, 108 S. cerevisiae strains were selected as they did not produce H2S and then assayed by micro fermentation tests. The adopted procedure made it possible to identify 10 strains that were low producers of acetic acid and hydrogen sulphide and showed that they completed sugar consumption during fermentation. These strains were characterized for their specific oenological and technological properties and, two of them, denoted as 6993 and 6920 strains, revealed themselves to be good candidates as industrial starter cultures. A novel protocol was set up for their biomass production and they were employed for industrial-scale fermentation in two industrial cellars. The two strains successfully dominated the fermentation process and contributed to increasing the wine organoleptic quality. The proposed procedure could be very effective for selecting "company-specific" yeast strains, ideal for the production of typical regional wines. "Winery" starter cultures could be produced on request in a small plant just before or during the vintage season and distributed as a fresh liquid concentrate culture

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. Thepolymorphism of interdelta elements was used to differentiate Saccharomyces cerevisiae strains. Twentydifferent species belonging to 9 genera were identified. Predominant on the grape surface wereMetschnikowia pulcherrima, Hanseniaspora uvarum and Aureobasidium pullulans, whereas M. pulcherrimaand H. uvarum were dominant in the early fermentation stage. A total of 692 S. cerevisiae isolates wereidentified and a number of S. cerevisiae strains, ranging from 26 to 55, was detected in each of the eightfermentations. The strains were tested for biogenic amines (BAs) production, either in synthetic media orgrape must. Two Pichia manshurica, an Issatchenkia terricola and a M. pulcherrima strains were able toproduce histamine and cadaverine, during must fermentation. The production of BAs in wine must wasdifferent than that observed in the synthetic medium. This feature indicate the importance of an "ingrape 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 bythese spoilage yeasts.

The ERY4 laccase gene from Pleurotus eryngii was expressed in Saccharomyces cerevisiae and the recombinant laccase resulted to be not biologically active. This gene was thus modified to obtain chimerical enzymes derived from the substitution of N-, C- and both N- and C-terminal regions with the corresponding regions of Ery3 laccase, another laccase isoform of P. eryngii. The chimerical isoform named 4NC3, derived from the substitution of both N- and C-terminal regions, showed the best performances in terms of enzymatic activities, affinities for different substrates and stability at a broad range of temperatures and pHs. The chimerical 4NC3 laccase isoform was displayed on the cell surface of S. cerevisiae using the N-terminal fusion with either the Pir2 and the Flo1 S. cerevisiae proteins as anchor attachment sequence. Immunofluorescence microscopy and Western blot analyses confirmed the localization of 4NC3 on the yeast cell surface. The enzyme activity on specific laccase substrates revealed that 4NC3 laccase was immobilized in active form on the cell surface. To our knowledge, this is the first example of expression of a chimerical fungal laccase by yeast cell display.

Table olives are a typical food of the Mediterranean diet and an important source of phenolic compoundswith high biological potential for human health. Their concentrations (?1-2% of FW) confer to table olivesantioxidant, anti-inflammatory, and antitumoral properties. The polyphenols content and composition in table olives can be affected by several factors, such as cultivars, climate, fruits ripeness, and, mainly, theprocessing methods. Among the de-bittering processes, the Greek method represents a spontaneous fermentation procedure that is driven by a mixed population of microorganisms, mainly consisting of yeastsand lactic acid bacteria (LAB). In this work, the effects of fermentation by autochthonous yeast and LAB starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto(TDB) and Cellina di Nardò (CEL) were studied in comparison with the commercial products. The samples were characterized by HPLC-DAD for their polyphenols composition; 18 compounds were identified andthe cultivar related effect was highlighted. The main identified phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucosideand quercetin in CEL. Further, the fermentation using selected LAB and yeasts influenced differently the composition and amount of polyphenols of the three cultivars, in particular the BDC amount was doubledcompared with the commercial sample. Instead, for TDB and CEL, the treatment did not influence the polyphenols composition. It is noteworthy that the use autochthonous microbial starter to drive table olivesfermentation process allows to maintain stable or increases polyphenols concentration in fermented table olives, significantly reducing the time necessary for de-bittering and improving organoleptic and sensorycharacteristics of the final product. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7 mg Trolox eq/g FW; 8.5 ?mol hydroxytyrosol eq/100g FW).Moreover, the polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted.Finally, fermented table olives are excellent source of health promoting compounds. Indeed, hydroxytyrosol and tyrosol are almost 8 times more than in olive oil for which a nutritional EU claim (No433/2012 of 23 May 2012) that attributes the protective effect from oxidative stress by polyphenols on blood lipids, has been established.

Table olives fermentation is a process unpredictable and frequently performed using traditional practices often inadequate to obtain products with acceptable quality and safety standards. In the present study, the efficacy of selected yeast strains as starters to drive fermentations of green and black table olives by the Greek method was investigated. Pilot-scale production by spontaneous fermentation as a control, olives started with previously selected Saccharomyces cerevisiae strains; fermentation driven by commercial S. cerevisiae baker"s yeast strain were carried out for each one of Manzanilla, Picual, and Kalamàta table olives cultivars.RESULTS: Time of fermentation was significantly shortened to 40 days to complete the transformation process for all the three tested cultivars. Inoculated table olives were enhanced in their organoleptic and nutritional properties in comparison with corresponding samples obtained by spontaneous fermentation. The use of starters was also able to improve safety traits of table olives in terms of biogenic amines reduction as well as of absence of undesired microorganisms at the end of the process.CONCLUSIONS: Autochthonous, but also non-authochthonous yeasts can be used to start and control le olives fermentations and can significantly improve quality and safety aspects of table olives produced by many smallholder farmers.

Tableoliveshavebeena component of theMediterranean diet for centuries,with the trendfor their consumptioncurrently increasing worldwide. They are rich in bioactive molecules with nutritional, antioxidant, anti-inflammatory orhormone-like properties. In the present study, the concentrations of phenolics, triterpenic acids, carotenoids and vitamins, aswell as fatty acid profiles and antioxidant activity,were analyzed in the edible portion of black table olives (Olea europea L.) fromItalian (Cellina di Nardò and Leccino) and Greek (Kalamàta and Conservolea) cultivars fermented with selected autochthonousstarters and in the corresponding monovarietal olive oils.RESULTS: On a fresh weight basis, Cellina di Nardò and Leccino table olives showed the highest total phenolic content. Nosignificant differences were found with respect to the levels of total triterpenic (maslinic and oleanolic) acids and vitaminE among cultivars. All table olives were characterized by high amounts of oleic, linoleic and palmitic acids. Oils were richerin lipophilic antioxidants (carotenoids and tocochromanols) than table olives, which, instead, showed a higher content ofpolyphenols and triterpenic acids than oils.CONCLUSION: The present study demonstrates that fermented table olives are an excellent natural source of unsaturated fattyacids, as well as being nutritionally important health-promoting bioactive compounds.

Table olives have been a component of the Mediterranean diet for centuries and their consumption is increasing worldwide. They contain several nutritional components such as unsaturated fatty acids, vitamin E, triterpenic acids, sterols, polyphenols and carotenoids [1]. The content of these bioactive molecules depends on the olive variety, the ripening stage of the olive fruit, the cultivation conditions and the processing method [2]. There are numerous commercial preparations of table olives and in a recent work we have developed and validated a new procedure for table olive production based on the use of selected autochthonous microbial starters and a productive process optimized to mimic the microbial evolution observed during spontaneous fermentations. [3]. In this work, we characterized some bioactive compounds and coloring pigments of fermented black table olives, belonging to two Italian (Cellina di Nardò and Leccino) and two Greek (Kalamàta and Conservolea) cultivars. Greek table olives resulted richer in carotenoids, vitamin E and triterpenic acids (maslinic and oleanoic acids) than Italian cv, contents ranged from 2.28 to 6.91 µg/g fw (edible portion), 53.64 to 59.15 µg/g fw and 2.19 to 3.28 mg/g respectively. Polyphenol compounds are known to be responsible for the bitter taste of the olive fruit and most of them are involved in table olives colour changes. In all tested black table olive cultivars, polyphenols content ranged from 0.87 mg/g fw (in Conservolea) to 23.49 mg/g fw (Cellina di Nardò). Moreover, we analysed the content of tyrosol, hydroxytyrosol, oleuropein aglycone, verbascoside, isoverbascoside, rutin, pyrocathecol, luteolin glucoside, quercetin, cyanidine rutinoside and cyniadine glucoside. In particular, Cellina di Nardò is characterized by the highest levels of anthocyanin pigments such as cyanidin 3-O-glucoside (14.77 mg/g fw) and cyanidin-3-O-rutinoside (3.17 mg/g fw) responsible for the characteristic black color. The results indicated that fermented black table olives are an excellent natural source of bioactive compounds and can represent a potential important functional food.

A pressing necessity of the Apulian wine industry is to being able to pilot and to control the wine production to obtain wines with peculiar characteristics and with respect of the typicality guaranteed by the denominations of origin. The employment of selected autochthonous yeast strains would be a potent instrument to improve the organoleptic and sensory characteristics of typical regional wines. In fact, indigenous yeasts are better adapted to a specific must and therefore they are able to exalt the peculiarities of the derived wine. The present work described the genetic diversity of autochthonous Saccharomyces cerevisiae strains derived from natural must fermentations of an important Apulian grape cultivar, denoted as “Primitivo”. The yeast strains showing the best technological and oenological properties were selected and their fermentative performances were assayed by either laboratory tests and industrial scale fermentations. Two autochthonous yeast strains showed to be good candidates as industrial starter cultures, since they dominated the fermentation process and produced wines characterized by peculiar oenological and organoleptic features, that were judged very pleasant by a panel of winemakers.

The effects of fermentation by autochthonous microbial starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto (TDB) and Cellina di Nardò (CEL) were studied, highlighting also the cultivars influence. In BDC with starter, polyphenols amount doubled compared with commercial sample, while in TDB and CEL, phenolics remain almost unchanged. The main phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucoside and quercetin in CEL. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7 mg Trolox eq/g FW; 8.5 ?mol hydroxytyrosol eq/100g FW). The polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted. Finally, fermented table olives are excellent source of health promoting compounds, since hydroxytyrosol and tyrosol are almost 8 times more than in olive oil.

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.

This study, for the first time describes the volatiles profile of sparkling wine produced by the traditional method, using a base wine obtained from the "Maresco" cultivar . Maresco cultivar, is one of the minor autochthonous vines in Apulia region, with the largest cultivation area located in Valle d'Itria (Central Apulia, Southern Italy). The grapes are characterized by a good balance of acidity and structure intense, a floral aroma and great a potential for the development of aromatic sparkling wine. Volatile fraction was investigated using an optimized HS-SPME method combined with the gas chromatography/mass spectrometry (GC-MS). The procedure was optimized for the following parameters: i) SPME fiber selection, ii) sample amount; iii) NaCl addition, iv) pre-incubation and extraction time, v) incubation and extraction temperature. Two-factors three-level designs were used in the optimization of pre-incubation/extraction time and temperature. As resulting by the optimization analyses, the following conditions were selected for volatiles extraction required the use of DVB/CAR/PDMS 50/30 ?m as fiber, 5 ml of wine, 2 gr of NaCl, stirring, incubation at 40°C for 30 min and extraction at 40°C for 10 min. Twenty-two compounds were identified, including alcohols, esters, volatiles acids and terpens. The results obtained by the GC-MS analyses for the characterization of volatile compounds in Maresco sparkling wine will be discussed.

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.

Olive quick decline syndrome (OQDS) caused by X. fastidiosa is currently causing severe damages to the production and reducing the life span of the plants in the Salento peninsula of Apulia (Italy). No effective means of control of X. fastidiosa is currently available. The objective of this study was to evaluate in vitro antimicrobial activities against X. fastidiosa (strain Salento-1) of different classes of compounds having diverse origins, i.e. traditional antibiotics, plant-derived natural products, and microbial metabolites. A preliminary bioassay, performed by the agar disc diffusion method, revealed that 17 of the 31 antibiotics tested did not affect bacterial growth at a dose of 5 ?g. Olive mill wastewaters (OMWs), which are known to possess a broad range of antimicrobial activity, are able to inhibit X. fastidiosa in vitro. Most interestingly when we analysed different OMWs derived micro, ultra and nano-filtered fractions as well as some of the single phenolic compounds that they contain, we found that the OMWs micro-filtered fraction is the most effective against the bacterium but only few phenolics are active in their pure form. Also some fungal extracts and bacteria toxins showed noteworthy inhibitory effect to strain Salento-1 growth. The possible use of some of these products for curative/preventive treating OQDS-affected or at-risk olive plants will be discussed.

Olive quick decline syndrome (OQDS) is causing severe damages to the olive trees in Salento (Apulia, Italy) and poses a severe threat for the agriculture of Mediterranean countries. DNA-Based Typing Methods have pointed out that OQDS is caused by a single outbreak strain of Xylella fastidiosa subsp. pauca referred to as CoDiRO or ST53. Since no effective control measures are currently available, the objective of this study was to evaluate in vitro antimicrobial activities of different classes of compounds against Salento-1 isolated by a OQDS affected plant and classified as ST53. A bioassay based on agar disc diffusion method, revealed that 17 out of the 32 tested antibiotics did not affect bacterial growth at a dose of 5 g disk-1. When we assayed micro-, ultra- and nano-filtered fractions of olive mill wastewaters (OMWs) we found that the micro-filtered fraction resulted the most effective against the bacterium. Moreover, some phenolics (4-methylcathecol, cathecol, veratric acid, caffeic acid, oleuropein) were active in their pure form. Noteworthy, also some fungal extracts and fungal toxins showed inhibitory effects on bacterial growth. Some of these compounds can be further explored as potential candidate in future applications for curative/preventive treating OQDS-affected or at-risk olive plants.

Table olives are one of the most important fermented food in the Mediterranean countries. Apart from lactic acid bacteria and yeasts that mainly conduct the olive fermentation, molds can develop on the brine surface, and can have either deleterious or useful effects on this process. From the food safety point of view, occurring molds could also produce mycotoxins, so, it is important to monitor and control them. In this respect, identification of molds associated to two Italian and two Greek fermented black table olives cultivars, was carried out. Sixty strains were isolated and molecularly identified as Penicillium crustosum (21), P roqueforti (29), P paneum (1), P expansum (6), P. polonicum (2), P commune (1). A group of 20 selected isolates was subjected to technological (beta-glucosidase, cellulolytic, ligninolytic, pectolytic, and xylanolytic activities; proteolytic enzymes) and safety (biogenic amines and secondary metabolites, including mycotoxins) characterization. Combining both technological (presence of desired and absence of undesired enzymatic activities) and safety aspects (no or low production of biogenic amines and regulated mycotoxins), it was possible to select six strains with biotechnological interest. These are putative candidates for future studies as autochthonous co-starters with yeasts and lactic acid bacteria for black table olive production.

Jellyfish have been considered as food for more than thousand years in Asia, where a multi-phase processing consisting on mixtures of salt and alum are used as traditional procedure. Jellyfish could also become an attractive choice for western food market for many reasons. The need for fishery diversification and for new food resources, the increasing market demand for innovative food products or ingredients, and finally the great availability of a resource whose use would be economically profitable and environmentally sustainable, make jellyfish a good candidate for future exploitation. However, in the attempt to introduce jellyfish as a new food product in Western market, several issues should be considered. Within the EU Project "GoJelly", we are continuing studies on "western style" jellyfish food. Protocols and treatments currently used for fish and sea-foods preparation are under consideration as a starting point to develop ad hoc new handling and storage procedures from fishing to on-boat pre-treatment and new processing methods will be developed to optimize taste, texture, flavor and shelf-life of final products acceptable for European consumers. Moreover, these new practices will be addressed also to maintain or enhance nutritional traits of formulated jellyfish products. In addition, study on the identification of strategies to reduce spoilage and to individuate possible food-borne microbial pathogens have been started up. The setting up of diagnostic and analytical assays to define consumers risk assessment and to monitor safety threat during jellyfish processing steps for preparation of food for human consumption is also in progress.

La fermentazione malolattica (FML) è la conversione dell'acido L-malico in acido L-lattico e CO2 attuata da parte dei batteri malolattici (BML) a seguito della loro crescita nel vino. Questo processo fermentativo provoca la disacidificazione del vino, in quanto un acido di-carbossilico, l'acido malico, viene trasformato in un acido mono-carbossilico quale l'acido lattico. Associate a questo processo avvengono altre trasformazioni importanti dal punto di vista organolettico quali modificazioni del colore, rivelazione di aromi e modificazioni dell'impatto gustativo. Essa può avvenire spontaneamente ad opera della flora indigena o mediante l'utilizzo di colture starter selezionate, solitamente appartenenti alla specie Oenococcus oeni. I vantaggi dell'induzione della FML con l'inoculo di BML selezionati sono un maggiore controllo sull'avvio e sul completamento della degradazione dell'acido malico ed un effetto positivo sull'aroma e sul gusto del vino. Il momento dell'inoculo batterico gioca un ruolo importante nella definizione del profilo sensoriale del vino . Generalmente si raccomanda l'inoculo dei batteri nel vino dopo la fermentazione alcolica (FA), quando la concentrazione di zuccheri è bassa. Infatti, una possibile conseguenza indesiderata del metabolismo eterofermentativo dei BML è lo spunto lattico ovvero la degradazione degli zuccheri con conseguente produzione di acido acetico e di acido D-lattico. In vini che al termine delle FA presentano particolari condizioni chimico-fisiche come elevato livello di etanolo, carenze nutrizionali, pH basso, elevati tenori di SO2 molecolare, rendono difficile lo sviluppo dei BML si consiglia il coinoculo (inoculo simultaneo di lieviti e batteri). Una conseguenza importante della tecnica del coinoculo è la notevole riduzioni dei tempi di fermentazione, che risulta vantaggiosa sia dal punto di vista economico sia dal punto di vista tecnico-pratico di gestione della cantina e riduce inoltre il rischio di alterazioni microbiologiche dei vini prodotti .Va comunque detto che la pratica della coinoculazione è un'operazione molto delicata, da gestire con cura ed estrema attenzione [6] e sulla quale poche informazioni sono disponibili in letteratura.Scopo di questo lavoro sperimentale è stato quello di studiare gli effetti a livello fisiologico e chimico dell'inoculo simultaneo in mosto d'uva del lievito Saccharomyces cerevisiae e del batterio O. oeni, per lo svolgimento in contemporanea delle fermentazioni alcolica e malolattica per una sua applicazione in condizioni difficili di vinificazione, ovvero, ovvero alto tenore alcolico e elevata acidità, tipiche dell'enologia Meridionale.

Laccases are biotechnologically interesting enzymes belonging to the polyphenol oxidases family. They are widely distributed throughout the phylogenetic scale from bacteria to mammals. In fungi the analysis of three-dimensional crystal structures of laccases indicate that ascomycete laccases are processed at their C-termini, at a conserved cleavage site, resulting in the proteolytic removal of C-terminal residues. We have isolated and cloned in expression vectors the cDNAs encoding two laccase isoforms (Ery3 and Ery4) from the basidiomycete Pleurotus eryngii. The Ery3 enzyme expressed in Saccharomyces cerevisiae, is functional, whereas the recombinant Ery4 protein does not show enzymaticactivity. In order to explain this evidence, we investigated the relationship between the structure of the C-terminal extension and laccase enzymatic activity. The tasks of the present study were to determine the biological role of laccase C-terminal, and to validate a "molecular engineering" approach for the production of recombinant laccases with novel biochemical properties. Genetically engineered mutant genes were produced from Ery4 by: i) progressive 3'-terminal deletions, ii) point mutations, iii) Ery3/Ery4 chimeras. The mutant genes were expressed in S. cerevisiae and active recombinant laccase isoforms were produced, exhibiting each a different biological behaviour. The correlations between the structural information deriving from both biochemical and bioinformatic analyses shed light on the role of Cterminal region in determining laccase functions. The obtained data also indicated that our approach could represent an efficient method for laccase genetic engineering. To our knowledge, this study has produced the first evidences obtained by biotechnological approach of the involvement of the C-terminal tail in the inactivation/activation process of a basidiomycete laccase.

The characterization of autochthonous Saccharomyces cerevisiae strains is an important step towards the conservation and employment of microbial biodiversity.The utilization of selected autochthonous yeast strains would be a powerful tool to enhance the organoleptic and sensory properties of typical regional wines. In fact, indigenous yeasts are better tailored to a particular must and because of this they are able to praise the peculiarities of the derived wine. The present study described the biodiversity of indigenous S. cerevisiae strains isolated from natural must fermentations of an ancient and recently rediscovered Apulian grape cultivar, denoted as "Susumaniello." The yeast strains denoted by the best oenological and technological features were identified and their fermentative performances were tested by either laboratory assay. Five yeast strains showed that they could be excellent candidates for the production of industrial starter cultures, since they dominated the fermentation process and produced wines characterized by peculiar oenological and organoleptic features.

DNA-based approaches were used to characterize a strain (Salento-1) of Xylella fastidiosa obtained from an olive plant suffering from the syndrome of quick decline in Apulia (South Italy). Salento-1 was indistinguishable from strain CoDiRO previously isolated from olive in Apulia and assigned to X. fastidiosa subsp. pauca. Based on our results and comparative analysis with reported data, the subspecies pauca, multiplex, and fastidiosa may invade olive throughout the world (California, Italy, Argentina and Brazil). The strain Salento-1 has been deposited in the National Collection of Plant Pathogenic Bacteria (NCPPB), England, and in the Belgian Coordinated Collections of Microorganisms (BCCM), Belgium.

Table olives are one of the most important traditional fermented vegetables in Europe and their world consumption is increasing. In the Greek system, table olives are produced by natural fermentation process, that is not predictable and strongly influenced by the physical-chemical conditions and by the presence of microorganisms contaminating the olives, In this study , we have developed and validated a novel procedure for table olive production based on the use of a mixed yeast/bacteria starter.

Table olives represent one important fermented product in Europe and, in the world, their demand is constantly increasing. At the present time, no systems are available to control black table olives spontaneous fermentation by the Greek method. During this study, a new protocol for the production of black table olives belonging to two Italian (Cellina di Nardò and Leccino) and two Greek (Kalamàta and Conservolea) cultivars has been developed: for each table olive cultivar, starter-driven fermentations were performed inoculating, firstly, one selected autochthonous yeast starter and, subsequently, one selected autochthonous LAB starter. All starters formulation were able to dominate fermentation process. The olive fermentation was monitored using specific chemical descriptors able to identify a first stage (30 days) mainly characterized by aldehydes; a second period (60 days) mainly characterized by higher alcohols, styrene and terpenes; a third fermentation stage represented by acetate esters, esters and acids. A significant decrease of fermentation time (from 8 to 12 months to a maximum of 3 months) and an significant improvement in organoleptic characteristics of the final product were obtained. This study, for the first time, describes the employment of selected autochthonous microbial resources optimized to mimic the microbial evolution already recorded during spontaneous fermentations.

Two approaches were developed in order to select microorganisms suitable to be used in olive millwastewaters bioremediation. By the first approach, three hundred yeasts were isolated from fiveindustrial mills and identified by molecular analysis. The different strains were selected accordingto their capacity to grow in OMW (olive mill wastewaters) as the sole carbon source and to reducephenolics, chemical oxygen demand (COD) and antimicrobial compounds. One Geotrichum candidumisolate was used to set up a whole-cell immobilization system in calcium alginate gel andthe 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, anew protocol was developed to isolate and select aerobic microorganisms from different industrialsamples and environmental niches (soils, OMWs) and able to detoxify olive mill wastewaters.

Egypt is the most important producer and consumer Country of table olives in the world. Within MARSADEV Project, a survey on table olives commercial products collected in the Northwestern region of Matrouh Governorate in Egypt was performed. Low safety conditions were observed in these products. Some of them are affected by the occurrence of high counts of Pseudomonas, Staphylococci and Enterobacteriaceae and some of them by the presence of biogenic amines. All tested samples resulted very poor in compounds related to microbial activities, such as organic acids and olives-derived phenols. Also, from the organoleptic point of view, odour profiles of tested olives are very modest. Scarce hygienic conditions were registered within domestic or small industrial plants along the whole production chain: from the harvest, to the processing and to the table olives packaging and storage. Several operations, such as the use of microbial starters, the monitoring of the fermentation process, together with some operational precautions, i.e. the heat treatment of water before olive soaking and the use of gloves during all operations, have been proposed to improve the process safety. The use of selected yeasts as starter for table olives production and the control of physical-chemical parameters during fermentation allowed to control the process, to sensitively improve organoleptic and safety traits of table olives, to reduce time required to end the transformation process from 8 to 2 months.Guidelines suggesting good practices and describing the most important operational steps to be followed for table olives processing have been produced for Bedouin rural communities living in this area, in particular for women within the concerned communities.

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.

High-level expression of the GUP1 gene in Saccharomyces cerevisiae resulted in the formation of proliferatedstructures, which hosted endoplasmic reticulum (ER), Golgi and itinerant proteins. The GUP1 over-expressionenhanced ER biogenesis, as shown by the coordinated increased transcription rate of genes involved in both ERand Golgi metabolism and in phospholipids biosynthesis. The formation of Gup1-induced proliferation revealedthat it depended on an intact unfolded protein response, because their assembly was reported to be lethal toyeast strains unable to initiate the UPR (Unfolded Protein Response) pathway. GUP1 over-expression affectedglobal ER and Golgi structure and resulted in the biogenesis of novel membrane arrays with Golgi and ER hybridcomposition. In fact, a number of ER and Golgi resident proteins together with itinerant proteins that normallycycle between ER and Golgi, were localized in the proliferated stacked membranes. The described assembling ofnovel membrane structures was affected by the functionality of the Gup1 O-acyltransferase domain, whichregulates the Gup1 protein role as remodelase in the glycosylphosphatidylinositol (GPI) anchored proteinsbiosynthesis. To our knowledge, we presented the first evidence of sub cellular modifications in response overexpressionof a GPI-anchor remodelase in S. cerevisiae.

Table olives are one of the most important traditional fermented vegetables in Europe and their world consumption is constantly increasing. In the Greek style, table olives are obtained by spontaneous fermentations, without any chemical debittering treatment. 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 technological characterization of LAB and yeast strains as possible candidate autochthonous starters for table olive fermentation from Cellina di Nardò and Leccino cultivars. The study of the main physical, chemical and aromatic parameters during fermentation helped to determine chemical descriptors that may be suitable for monitoring olive fermentation. In both the analyzed table olive cultivars, aldehydes proved to be closely related to the first stage of fermentation (30 days), while higher alcohols (2-methyl-1-propanol; 3-methyl-1-butanol), styrene, and o-cymene were associated with the middle stage of fermentation (90 days) and acetate esters and acetic acid with the final step of olive fermentation (180 days).

Table olives are one of the most important traditional fermented vegetables in Europe and their worldconsumption is constantly increasing. Conservolea and Kalam
ata are the most important table olivesGreek varieties. In the Greek system, the final product is obtained by spontaneous fermentations,without any chemical debittering treatment. This natural fermentation process is not predictable andstrongly influenced by the physical-chemical conditions and by the presence of microorganismscontaminating the olives. Natural fermentations of Conservolea and Kalam
ata cultivars black olives were studied in order to determine microbiological, biochemical and chemical evolution during the process. Following the process conditions generally used by producers, in both cultivars, yeasts were detected throughout the fermentation, whereas lactic acid bacteria (LAB) appeared in the last staged of the process. A new optimized specific protocol was developed to select autochthonous yeast and LAB isolates that can be good candidates as starters. These microorganisms were pre-selected for their ability to adapt to model brines, to have beta-glucosidase activity, not to produce biogenic amines. Chemical compounds deriving by microbiological activities and associated to the three different phases (30, 90 and 180 days) of the fermentation process were identified and were proposed as chemical descriptors to follow the fermentation progress.

Affiliazione autori: CNR-ISPA, UNISALENTOAbstract: It has been demonstrated that Agaricus bisporus tyrosinase is able to oxidize various phenolic compounds, thus being an enzyme of great importance for a number of biotechnological applications. The tyrosinase-coding PPO2 gene was isolated by reverse-transcription polymerase chain reaction (RT-PCR) using total RNA extracted from the mushroom fruit bodies as template. The gene was sequenced and cloned into pYES2 plasmid, and the resulting pY-PPO2 recombinant vector was then used to transform Saccharomyces cerevisiae cells. Native polyacrylamide gel electrophoresis followed by enzymatic activity staining with L-3,4-dihydroxyphenylalanine (L-DOPA) indicated that the recombinant tyrosinase is biologically active. The recombinant enzyme was overexpressed and biochemically characterized, showing that the catalytic constants of the recombinant tyrosinase were higher than those obtained when a commercial tyrosinase was used, for all the tested substrates. The present study describes the recombinantproduction of A. bisporus tyrosinase in active form. The produced enzyme has similar properties to the one produced in the native A. bisporus host, and its expression in S. cerevisiae provides good potential for protein engineering and functional studies of this important enzyme

Ochratoxin A (OTA) undergoes to enzymatic biodegradation by proteolytic enzymes able to hydrolyze its amide bond with consequent formation of ochratoxin ? (OT?) and L-?-phenylalanine. This mechanism can be regarded as a detoxification method since OT? and L-?-phenylalanine are considered as less and non-toxic, respectively. Different microorganisms belonging to bacterial, yeast and fungal species have been reported to degrade OTA. Several enzymes may be involved in microbiological degradation of OTA, such as carboxypeptidase A, lipase, and acid proteases. Also Aspergillus carbonarius, one of the most important fungal producer of OTA and the major responsible of OTA contamination of grapes, wine and by-products, turned out to be able to degrade OTA. In the attempt to identify the enzyme able to degrade OTA in this microorganism, a protease encoding gene, located in the genomic region recognized as OTA cluster, has driven our attention. In particular, this gene, namely Acap1 of A. carbonarius strain ITEM 5010, encodes for an aspartic protease and is located downstream of the core genes involved in OTA biosynthesis. Acap1gene was isolated and cloned for its characterization. The gene is 1367 bp long and the in silico analyses of the deduced protein sequence of 421 aa revealed that the AcAP1 protein shows the functional typical structure of aspartic protease enzymes. Aspartyl proteases are a highly specific family of proteases that tend to cleave dipeptide bond and they are optimally active at acidic pH. Heterologous recombinant production of the AcAP1 protein has been carried out in order to verify the involvement of AcAP1 in the ability of A. carbonarius in OTA degradation and to analyze its structural and functional properties for a potential biotechnological use of the enzyme. Acap1 gene was cloned in two expression vectors (p426 and pYES), carrying a constitutive and an inducible promoter, respectively, in fusion with a sequence encoding for a His-tag at the 3'-terminus. Three different strains of Saccharomyces cerevisiae, carrying diverse genotypes, have been transformed. Data concerning the protein expression by yeast, evaluation of the protease activity, and purification of the recombinant protein will be produced.

The ERY4 laccase gene of Pleurotus eryngii is not biologically active when expressed in yeast. To explain this finding, we analysed the role of the C-terminus of Ery4 protein by producing a number of its different mutant variants. Two different categories of ERY4 mutant genes were produced and expressed in yeast: (i) mutants carrying C-terminal deletions and (ii) mutants carrying different site-specific mutations at their C-terminus. Investigation of the catalytic properties of the recombinant enzymes indicated that each novel variant acquired different affinities and catalytic activity for various substrates. Our results highlight that C-terminal processing is fundamental for Ery4 laccase enzymatic activities allowing substrate accessibility to the enzyme catalytic core. Apparently, the last 18 amino acids in the C-terminal end of the Ery4 laccase play a critical role in enzyme activity, stability and kinetic and, in particular biochemical and structural data indicate that the K532 residue is fundamental for enzyme activation. These studies shed light on the structure/function relationships of fungal laccases and will enhance the development of biotechnological strategies for the industrial exploitation of these enzymes.

The yeast population dynamics in olive wastewaters (OMW), sampled in five mills from Salento (Apulia, Southern Italy), were investigated. Three hundred yeasts were isolated in five industrial mills and identified by molecular analysis. Strains belonging to Geotrichum, Saccharomyces, Pichia, Rhodotorula and Candida were detected. Five G. candidum strains were able to grow in OMW as the sole carbon source and to reduce phenolics, Chemical Oxygen Demand (COD) and antimicrobial compounds. One G. candidum isolate was selected for whole cell immobilization in calcium alginate gel. The COD and phenolic reduction obtained with immobilized cells showed a 2.2- and 2-fold increase compared to the removal obtained with free cells, respectively. The immobilization system enhanced yeast oxidative activity by avoiding the presence of microbial protease in treated OMW. To our knowledge, this is the first report on G. candidum whole-cell immobilization for OMW bioremediation.

Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF). 27However, the inoculation of lactic acid bacteria together with yeast starter cultures is a promising system to enhance the quality and safety of wine. In recent years, the use of immobilized cell systems has been investigated, with interesting results, for the production of different fermented foods and beverages. In this study we have carried out the simultaneous immobilization of Saccharomyces cerevisiae and Oenococcus oeni in alginate beads and used them in microvinifications tests to produce Negroamaro wine. The process was monitored by chemical and sensorial analyses and dominance of starters and cell leaking from beads were also checked. Co-immobilization of S. cerevisiae and O. oeni allowed to perform an efficient fermentation process, producing low volatile acidity levels and ethanol and glycerol concentrations comparable with those obtained by cell sequential inoculum and co-inoculum of yeast and bacteria cells in free form. More importantly, co-immobilization strategy produced a significant decrease of the time requested to complete AF and MLF. The immobilized cells could be efficiently reused for the wine fermentation at least three times without any apparent loss of cell metabolic activities. This integrated biocatalytic system is able to perform simultaneously AF and MLF, producing wines improved in organoleptic traits in comparison with wines fermented following traditional sequential AF and MLF with free cell starters. The immobilized-cell system, that we here describe for the first time in our knowledge, offers many advantages over conventional free cell fermentations, including: (i) elimination of non-productive cell growth phases; (ii) feasibility of continuous processing; (iii) regeneration and re-use of the biocatalyst.

The sparkling wine market has expanded in recent years, boosted by the increasing demand of the global market. As for other fermented beverages, technological yeasts and bacteria selected to design commercial starter cultures represent key levers to maximize product quality and safety. The increasing economic interest in the sector of sparkling wine has also implied a renewed interest in microbial resource management. In this review, after a brief introduction, we report an overview of the main characterization criteria in order to select Saccharomyces cerevisiae strains suitable for use as starter cultures for the production of base wines and to drive re-fermentation of base wines to obtain sparkling wines. Particular attention has been reserved to the technological characterization aspects of re-fermenting phenotypes. We also analysed the possible uses of selected non-Saccharomyces and malolactic strains in order to differentiate specific productions. Finally, we highlighted the main safety aspects related to microbes of enological interest and underlined some microbial-based biotechnological applications helpful to pursue product and process innovations. Overall, the sparkling wine industry may find a relevant benefit from the exploitation of the wide resources associated with vineyard/wine microbial diversity

Celiac disease is an immune-mediated enteropathy precipitated by the ingestion of gluten-containing foodsin genetically predisposed children and adults. After a positive diagnosis for celiac disease, the only availabletreatment is to adopt a gluten-free diet, and evaluation of the absence of gluten in foods is crucial for the health ofceliac patients. In the present study, a recombinant glutamine-binding protein (GlnBP) from Escherichia coli showedits ability to recognize peptides deriving from digested wheat flour. GlnBP and the commercially available 4F3monoclonal antibody, raised against a region of the ?-gliadin peptide 33-mer from wheat, demonstrated the abilityto detect gliadin extracted from wheat flour. Recombinant GlnBP and 4F3 monoclonal antibody were used as newcapture agents for the development of a protein chip able to detect gluten in foods. The protein microarray systemhas proven to detect the presence of gliadin in a range of concentrations between 500 and 5 ppm.

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.

Table olives are one of the most important traditional fermented vegetables in Southern European (Italy, Greece and Spain) countries. In the Greek-style production system, the fruits are placed directly into the brine, thus allowing the natural fermentation to take place. The spontaneous fermentations, that can last 8-12 months, are driven by mixed populations of microorganisms, mainly the epiphytic microbial population of yeasts and lactic acid bacteria (LAB) (Romero et al., 2004). At present, the industrial table olive process is not predictable and depends on the empirical experience of the producers. In order to avoid the unpredictability of the olive spontaneous fermentation, to improve the productive process and to constantly produce high-quality final products, the use of strains of LAB as starter cultures for olive production has been proposed (Sabatini and Marsilioet al., 2008; Panagou et al., 2008; Blana et al., 2014). However, in the last years, the importance and the potential applications of yeasts as starters for table olive processing has been recognized (Arroyo-López et al., 2008, 2012; Bevilacqua et al., 2012). Objectives: In the present work, we have studied the main physical, chemical and aromatic parameters of natural fermentations of Cellina di Nardò, Leccino, Kalamata and Conservolea table olives in order to determine chemical descriptors correlated to microbiological activities and the dynamics of microorganisms in order to select LAB and yeast strains as candidate autochthonous starter cultures. Conclusions: The identified chemical descriptors can be suitable to follow the trend and to control the outcome of the fermentation and a new protocol aimed to the selection of LAB and yeast strains as candidates autochthonous starters has been developed and applied (Bleve et al. 2014 a, b). Selected microbial starters have been successfully used to ferment olives in pilot and industrial-scale and a new method for table olive production has been set up (Bleve et al. 2103). The use of selected autochthonous starter cultures produced fermented table olives with improved organoleptic, sensorial and nutritional characteristics.

Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF), but winemakers has shown great interest about co-inoculation of yeast and malolactic bacteria at the beginning of AF. In this study we have produced a mixed starter bu co-immobilization of Saccharomyces cerevisiae and Oenococcus oeni in alginate beads and used it in microvinifuications tests

Xylella fastidiosa is a gram-negative, xylem-limited, bacterium which is responsible, in Italy, for the Olive Quick Decline Syndrome (OQDS). The disease is caused by the subspecies pauca and emerged a few years ago in the Apulia province of Lecce, in the Salento peninsula, on Olea europaea plants. X. fastidiosa can infect different plant species and is well known in California as the causal agent of Pierce's disease on grape. Infections of susceptible hosts with X. fastidiosa are known to result in xylem vessel occlusions, water movement impairment, and accordingly to induce the typical desiccation symptoms. In the present study, we investigated xylem vessel occlusions in healthy and naturally infected O. europaea plants grown in open field by analyzing three olive cultivars widespread in the region that show different degree of susceptibility to the disease: the susceptible cultivars "Ogliarola salentina" and "Cellina di Nardò", and the tolerant cultivar "Leccino". Our results show that occlusions were caused by tyloses and gums/pectin gels, and not by bacterial cell aggregates. Our data also indicate that occlusions are not responsible for the symptomatology of the OQDS and, as observed in Leccino plants, they are not a marker of tolerance/resistance to the disease.

The invention relates to a method for table olive fermentation comprising the steps of: a. soaking the olives in brine;b. inoculating the product obtained in step a. with a yeast culture;c. incubating the product obtained in step b. in order to perform the alcoholic fermentation; d. inoculating the fermented product obtained in step c. using a Lactic Acid Bacterium (LAB) culture;e. incubating the product obtained in step d. to in order to perform the lactic fermentation.Procedures for the selection of starter cultures and their use in fermentation of two cultivars of table olives are described. Some claims are directed to specific starter cultures and the uses thereof for preparing fermented table olives.