Publications by authors named "Gianluca Bleve"

26 Publications

  • Page 1 of 1

Susceptibility to and functional xylem anatomy in : revisiting a tale of plant-pathogen interaction.

AoB Plants 2021 Aug 21;13(4):plab027. Epub 2021 May 21.

Department of Agronomy, Food, Natural resources, Animals and Environment (DAFNAE), University of Padua, Viale dell'Università 16, 35020 Legnaro (PD), Italy.

is a xylem-limited bacterium causing the Olive Quick Decline Syndrome, which is currently devastating the agricultural landscape of Southern Italy. The bacterium is injected into the xylem vessels of leaf petioles after the penetration of the insect vector's stylet. From here, it is supposed to colonize the xylem vasculature moving against water flow inside conductive vessels. Widespread vessel clogging following the bacterial infection and causing the failure of water transport seemed not to fully supported by the recent empirical xylem anatomical observations in infected olive trees. We tested the hypothesis that the higher susceptibility to the 's infection in Cellina di Nardò compared with Leccino is associated to the higher vulnerability to air embolism of its larger vessels. Such hypothesis is motivated by the recognized ability of in degrading pit membranes and also because air embolism would possibly provide microenvironmental conditions more favourable to its more efficient aerobic metabolism. We revised the relevant literature on bacterium growth and xylem physiology, and carried out empirical field, mid-summer measurements of xylem anatomy and native embolism in olive cultivars with high (Cellina di Nardò) and low susceptibility (Leccino) to the infection by . Both cultivars had similar shoot mass traits and vessel length (~80 cm), but the highly susceptible one had larger vessels and a lower number of vessels supplying a given leaf mass. Native air embolism reduced mean xylem hydraulic conductance by ~58 % (Cellina di Nardò) and ~38 % (Leccino). The higher air-embolism vulnerability of the larger vessels in Cellina di Nardò possibly facilitates the 's infection compared to Leccino. Some important characteristics of the vector-pathogen-plant interactions still require deep investigations acknowledging both the pathogen metabolic pathways and the biophysical principles of xylem hydraulics.
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http://dx.doi.org/10.1093/aobpla/plab027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8300559PMC
August 2021

Effect of different inoculation strategies of selected yeast and LAB cultures on Conservolea and Kalamàta table olives considering phenol content, texture, and sensory attributes.

J Sci Food Agric 2020 Feb 10;100(3):926-935. Epub 2019 Dec 10.

Department of Chemistry, Section of Industrial and Food Chemistry, University of Ioannina, Ioannina, Greece.

Background: The effects were studied of different inoculation strategies for selected starters -yeasts and lactic acid bacteria (LAB) - used for the fermentation process of two Greek olive cultivars, Conservolea and Kalamàta. The LAB strains applied were Leuconostoc mesenteroides K T5-1 and L. plantarum A 135-5; the selected yeast strains were S. cerevisiae KI 30-16 and Debaryomyces hansenii A 15-44 for Kalamàta and Conservolea olives, respectively.

Results: Table olive fermentation processes were monitored by performing microbiological analyses, and by monitoring changes in pH, titratable acidity and salinity, sugar consumption, and the evolution of volatile compounds. Structural modifications occurring in phenolic compounds of brine were investigated during the fermentation using liquid chromatography / diode array detection / electrospray ion trap tandem mass spectrometry (LC/DAD/ESI-MS ) and quantified by high-performance liquid chromatography (HPLC) using a diode array detector. Phenolic compounds in processed Kalamàta olive brines consisted of phenolic acids, verbascoside, caffeoyl-6-secologanoside, comselogoside, and the dialdehydic form of decarboxymethylelenolic acid linked to hydroxytyrosol, whereas oleoside and oleoside 11-methyl ester were identified only in Conservolea olive brines.

Conclusion: Volatile profile and sensory evaluation revealed that the 'MIX' (co-inoculum of yeast and LAB strain) inoculation strategy led to the most aromatic and acceptable Kalamàta olives. For the Conservolea table olives, the 'YL' treatment gave the most aromatic and the overall most acceptable product. © 2019 Society of Chemical Industry.
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http://dx.doi.org/10.1002/jsfa.10019DOI Listing
February 2020

Bioactive Compounds and Stability of a Typical Italian Bakery Products "" Enriched with Fermented Olive Paste.

Molecules 2019 Sep 6;24(18). Epub 2019 Sep 6.

Institute of Sciences of Food Production-CNR, Via Monteroni, 73100 Lecce, Italy.

Olive paste (OP) is a novel by-product of olive mill industry composed of water, olive pulp, and skin. Due to its richness in bioactive compounds, OP exploitation for human consumption has recently been proposed. Starter driven fermented OP is characterized by a well-balanced lipid profile, rich in mono and polyunsaturated fatty acids, and a very good oxidative stability due to the high concentration of fat-soluble antioxidants. These characteristics make OP particularly suitable as a functional ingredient for food/feed industry, as well as for the formulation of nutraceutical products. New types of were produced by adding 20% of fermented OP from black olives (cv Cellina di Nardò and Leccino) to the dough. The levels of bioactive compounds (polyphenols, triterpenic acids, tocochromanols, and carotenoids), as well as the fatty acid profile, were monitored during 180 days of storage and compared with control produced with the same flour without OP supplementation. enriched with fermented OP showed significantly higher levels of bioactive compounds than conventional ones. Furthermore, enriched maintained a low amount of saturated fatty acids and high levels of polyphenols, triterpenic acids, tocochromanols, and carotenoids, compared to the initial value, up to about 90 days in the usual conditions of retailer shelves.
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http://dx.doi.org/10.3390/molecules24183258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766877PMC
September 2019

Identification of Safety and Quality Parameters for Preparation of Jellyfish Based Novel Food Products.

Foods 2019 Jul 17;8(7). Epub 2019 Jul 17.

National Research Council, Institute of Sciences of Food Production, Lecce 73100, Italy.

Edible jellyfish are mainly consumed and marketed in Southeastern Countries, generally produced by a multi-phase drying process, using mixtures of salt and alum. Recently, jellyfish have become very attractive also for Western food markets. They are novel food in Europe and no recognized handling/processing steps have been set up yet. Moreover, no specific food safety and quality parameters are available. In this study, we identified a set of safety and quality parameters for jellyfish, based on standards and process hygiene criteria used in Europe for other products. These assays were tested on three different jellyfish preparations that can be used as raw materials for subsequent food processing. All jellyfish samples revealed the absence of pathogens ( spp. and ), and spp., even if a limited presence of was observed. No biogenic amine histamine was detected and negligible levels of total volatile basic nitrogen (TVB-N) were revealed. Total bacterium, yeast and mold counts were negligible or undetectable by conventional accredited methods, and conversely the results were higher when optimized saline conditions were used. This study, for the first time, established a set of quality and safety parameters necessary for first-operations and subsequent processing of jellyfish as novel food. Highlights: Jellyfish can represent a novel food in Europe. Identification of safety and quality parameters for jellyfish food products. Saline conditions are essential for improving safety and quality assessment of jellyfish as food.
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http://dx.doi.org/10.3390/foods8070263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678107PMC
July 2019

Patè Olive Cake: Possible Exploitation of a By-Product for Food Applications.

Front Nutr 2019 5;6. Epub 2019 Feb 5.

Consiglio Nazionale delle Ricerche-Istituto di Scienze delle Produzioni Alimentari, Lecce, Italy.

Patè Olive Cake (POC) is a new by-product derived from recently introduced new decanters in the olive oil production process. POC, is essentially composed of water, olive pulp and olive skin, and is rich in several valuable bioactive compounds. Moreover, it still contains about 8-12% residual olive oil. We characterized the main bioactive compounds in POC from black olives (cv. and ) and also verified the biotechnological aptitude of selected yeast and lactic acid bacteria from different sources, in transforming POC into a new fermented product. The strategy of sequential inoculum of and was successful in driving the fermentation process. In fermented POC total levels of phenols were slightly reduced when compared with a non-fermented sample nevertheless the content of the antioxidant hydroxytyrosol showed increased results. The total levels of triterpenic acids, carotenoids, and tocochromanols results were almost unchanged among the samples. Sensory notes were significantly improved after fermentation due to the increase of superior alcohols, esters, and acids. The results reported indicate a possible valorisation of this by-product for the preparation of food products enriched in valuable healthy compounds.
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http://dx.doi.org/10.3389/fnut.2019.00003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371699PMC
February 2019

Efficacy of yeast starters to drive and improve Picual, Manzanilla and Kalamàta table olive fermentation.

J Sci Food Agric 2019 Mar 12;99(5):2504-2512. Epub 2018 Dec 12.

Consiglio Nazionale delle Ricerche - Istituto di Scienze delle Produzioni Alimentari, Unità Operativa di Lecce, Lecce, Italy.

Background: Table olive fermentation is an unpredictable process 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 and fermentation driven by commercial S. cerevisiae baker's yeast strain were carried out for each of Manzanilla, Picual and Kalamàta table olive cultivars.

Results: Time of fermentation was significantly shortened to 40 days to complete the transformation process for all 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 amine reduction as well as absence of undesired microorganisms at the end of the process.

Conclusions: Autochthonous, but also non-autochthonous, yeasts can be used to start and control table olive fermentations and can significantly improve quality and safety aspects of table olives produced by many smallholder farmers. © 2018 Society of Chemical Industry.
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http://dx.doi.org/10.1002/jsfa.9460DOI Listing
March 2019

In vitro activity of antimicrobial compounds against Xylella fastidiosa, the causal agent of the olive quick decline syndrome in Apulia (Italy).

FEMS Microbiol Lett 2018 03;365(5)

Consiglio Nazionale delle Ricerche, Istituto di Scienze delle Produzioni Alimentari, 73100 Lecce, Italy.

Olive quick decline syndrome (OQDS) causes 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 an OQDS affected plant and classified as ST53. A bioassay based on agar disk diffusion method revealed that 17 out of the 32 tested antibiotics did not affect bacterial growth at a dose of 5 μg disk-1. When we assayed micro-, ultra- and nano-filtered fractions of olive mill wastewaters, we found that the micro-filtered fraction resulted to be 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.
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http://dx.doi.org/10.1093/femsle/fnx281DOI Listing
March 2018

Fermented Apulian table olives: Effect of selected microbial starters on polyphenols composition, antioxidant activities and bioaccessibility.

Food Chem 2018 May 11;248:137-145. Epub 2017 Dec 11.

Institute of Sciences of Food Production (ISPA), National Council of Research (CNR), Via G. Amendola, 122/O, 70126 Bari, Italy. Electronic address:

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 μmol hydroxytyrosol eq/100 g 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.
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http://dx.doi.org/10.1016/j.foodchem.2017.12.032DOI Listing
May 2018

Isolation, Characterization, and Selection of Molds Associated to Fermented Black Table Olives.

Front Microbiol 2017 18;8:1356. Epub 2017 Jul 18.

Consiglio Nazionale delle Ricerche-Istituto di Scienze delle Produzioni AlimentariLecce, Italy.

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 (21), (29), (1), (6), (2), (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.
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http://dx.doi.org/10.3389/fmicb.2017.01356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513898PMC
July 2017

Evaluation of bioactive compounds in black table olives fermented with selected microbial starters.

J Sci Food Agric 2018 Jan 5;98(1):96-103. Epub 2017 Jul 5.

Istituto di Scienze delle Produzioni Alimentari (ISPA)-CNR, Lecce, Italy.

Background: Table olives have been a component of the Mediterranean diet for centuries, with the trend for their consumption currently increasing worldwide. They are rich in bioactive molecules with nutritional, antioxidant, anti-inflammatory or hormone-like properties. In the present study, the concentrations of phenolics, triterpenic acids, carotenoids and vitamins, as well as fatty acid profiles and antioxidant activity, were analyzed in the edible portion of black table olives (Olea europea L.) from Italian (Cellina di Nardò and Leccino) and Greek (Kalamàta and Conservolea) cultivars fermented with selected autochthonous starters 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. No significant differences were found with respect to the levels of total triterpenic (maslinic and oleanolic) acids and vitamin E among cultivars. All table olives were characterized by high amounts of oleic, linoleic and palmitic acids. Oils were richer in lipophilic antioxidants (carotenoids and tocochromanols) than table olives, which, instead, showed a higher content of polyphenols and triterpenic acids than oils.

Conclusion: The present study demonstrates that fermented table olives are an excellent natural source of unsaturated fatty acids, as well as being nutritionally important health-promoting bioactive compounds. © 2017 Society of Chemical Industry.
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http://dx.doi.org/10.1002/jsfa.8443DOI Listing
January 2018

Simultaneous Alcoholic and Malolactic Fermentations by Saccharomyces cerevisiae and Oenococcus oeni Cells Co-immobilized in Alginate Beads.

Front Microbiol 2016 14;7:943. Epub 2016 Jun 14.

Unità Operativa di Lecce, Consiglio Nazionale delle Ricerche - Istituto di Scienze delle Produzioni Alimentari Lecce, Italy.

Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF). However, 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 similar 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) re-use of the biocatalyst.
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http://dx.doi.org/10.3389/fmicb.2016.00943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906054PMC
July 2016

New process for production of fermented black table olives using selected autochthonous microbial resources.

Front Microbiol 2015 24;6:1007. Epub 2015 Sep 24.

Consiglio Nazionale delle Ricerche-Istituto di Scienze delle Produzioni Alimentari, Unità Operativa di Lecce Lecce, Italy.

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.
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http://dx.doi.org/10.3389/fmicb.2015.01007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585182PMC
October 2015

Physico-chemical characterization of natural fermentation process of Conservolea and Kalamàta table olives and developement of a protocol for the pre-selection of fermentation starters.

Food Microbiol 2015 Apr 6;46:368-382. Epub 2014 Sep 6.

Consiglio Nazionale delle Ricerche, Istituto di Scienze delle Produzioni Alimentari, Bari, Italy.

Table olives are one of the most important traditional fermented vegetables in Europe and their world consumption is constantly increasing. Conservolea and Kalamàta are the most important table olives Greek 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 and strongly influenced by the physical-chemical conditions and by the presence of microorganisms contaminating the olives. Natural fermentations of Conservolea and Kalamàta 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.
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http://dx.doi.org/10.1016/j.fm.2014.08.021DOI Listing
April 2015

Physico-chemical and microbiological characterization of spontaneous fermentation of Cellina di Nardò and Leccino table olives.

Front Microbiol 2014 28;5:570. Epub 2014 Oct 28.

Consiglio Nazionale delle Ricerche- Istituto di Scienze delle Produzioni Alimentari Bari, Italy.

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 protocol was developed and applied aimed at the technological characterization of lactic acid bacteria (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 physic-chemical parameters and volatile compounds 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 with the final step of olive fermentation (180 days).
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http://dx.doi.org/10.3389/fmicb.2014.00570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4211558PMC
November 2014

Molecular and Technological Characterization of Saccharomyces cerevisiae Strains Isolated from Natural Fermentation of Susumaniello Grape Must in Apulia, Southern Italy.

Int J Microbiol 2014 9;2014:897428. Epub 2014 Jan 9.

C.N.R.-Istituto di Scienze delle Produzioni Alimentari, Unità Operativa di Lecce, Via Provinciale Lecce-Monteroni, 73100 Lecce, Italy.

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.
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http://dx.doi.org/10.1155/2014/897428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942102PMC
March 2014

Biodiversity and safety aspects of yeast strains characterized from vineyards and spontaneous fermentations in the Apulia Region, Italy.

Food Microbiol 2013 Dec 16;36(2):335-42. Epub 2013 Jul 16.

CNR-Institute of Sciences of Food Production (ISPA), Operative Unit of Lecce, Lecce, Italy.

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 microbiological control during wine-making to reduce the potential health risk for consumer represented by these spoilage yeasts.
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http://dx.doi.org/10.1016/j.fm.2013.07.001DOI Listing
December 2013

AtSYP51/52 functions diverge in the post-Golgi traffic and differently affect vacuolar sorting.

Mol Plant 2013 May 19;6(3):916-30. Epub 2012 Oct 19.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, University of Salento, Campus Ecotekne, 73100 Lecce, Italy.

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.
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http://dx.doi.org/10.1093/mp/sss117DOI Listing
May 2013

Role of the C-terminus of Pleurotus eryngii Ery4 laccase in determining enzyme structure, catalytic properties and stability.

Protein Eng Des Sel 2013 Jan 20;26(1):1-13. Epub 2012 Sep 20.

CNR-Istituto di Scienze delle Produzioni Alimentari (ISPA), Lecce, Italy.

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.
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http://dx.doi.org/10.1093/protein/gzs056DOI Listing
January 2013

Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells.

J Ind Microbiol Biotechnol 2012 Dec 21;39(12):1875-80. Epub 2012 Sep 21.

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Prov. Lecce-Monteroni, 73100 Lecce, Italy.

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 recombinant production 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.
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http://dx.doi.org/10.1007/s10295-012-1192-zDOI Listing
December 2012

Autochthonous fermentation starters for the industrial production of Negroamaro wines.

J Ind Microbiol Biotechnol 2012 Jan 21;39(1):81-92. Epub 2011 Jun 21.

C.N.R. Institute of Sciences of Food Production (ISPA), Operative Unit of Lecce, via Provinciale Lecce-Monteroni, 73100, Lecce, Italy.

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 H(2)S and then assayed by microfermentation 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, strains 6993 and 6920, are 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 wines' 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.
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http://dx.doi.org/10.1007/s10295-011-1002-zDOI Listing
January 2012

Over-expression of functional Saccharomyces cerevisiae GUP1, induces proliferation of intracellular membranes containing ER and Golgi resident proteins.

Biochim Biophys Acta 2011 Mar 15;1808(3):733-44. Epub 2010 Dec 15.

Istituto di Scienze Delle Produzioni Alimentari del Consiglio Nazionale Delle Ricerche (ISPA), Unità di Lecce, via Provinciale Lecce, Monteroni, 73100 Lecce, Italy.

High-level expression of the GUP1 gene in Saccharomyces cerevisiae resulted in the formation of proliferated structures, which hosted endoplasmic reticulum (ER), Golgi and itinerant proteins. The GUP1 over-expression enhanced ER biogenesis, as shown by the coordinated increased transcription rate of genes involved in both ER and Golgi metabolism and in phospholipids biosynthesis. The formation of Gup1-induced proliferation revealed that it depended on an intact unfolded protein response, because their assembly was reported to be lethal to yeast strains unable to initiate the UPR (Unfolded Protein Response) pathway. GUP1 over-expression affected global ER and Golgi structure and resulted in the biogenesis of novel membrane arrays with Golgi and ER hybrid composition. In fact, a number of ER and Golgi resident proteins together with itinerant proteins that normally cycle between ER and Golgi, were localized in the proliferated stacked membranes. The described assembling of novel membrane structures was affected by the functionality of the Gup1 O-acyltransferase domain, which regulates the Gup1 protein role as remodelase in the glycosylphosphatidylinositol (GPI) anchored proteins biosynthesis. To our knowledge, we presented the first evidence of sub cellular modifications in response over-expression of a GPI-anchor remodelase in S. cerevisiae.
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http://dx.doi.org/10.1016/j.bbamem.2010.12.005DOI Listing
March 2011

An optimized procedure for the enological selection of non-Saccharomyces starter cultures.

Antonie Van Leeuwenhoek 2011 Feb 24;99(2):189-200. Epub 2010 Jun 24.

CNR-Institute of Sciences of Food Production (ISPA), Operative Unit of Lecce, via Provinciale Lecce-Monteroni, Lecce, Italy.

The apiculate yeasts are the species predominating the first stage of grape must alcoholic fermentation and are 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 Hanseniaspora 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 possessed 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 Saccharomyces cerevisiae.
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http://dx.doi.org/10.1007/s10482-010-9475-8DOI Listing
February 2011

Molecular cloning and heterologous expression of a laccase gene from Pleurotus eryngii in free and immobilized Saccharomyces cerevisiae cells.

Appl Microbiol Biotechnol 2008 Jul 29;79(5):731-41. Epub 2008 Apr 29.

Istituto di Scienze delle Produzioni Alimentari del CNR, Unità di Lecce, via Prov le Lecce-Monteroni, Lecce, Italy.

A full length cDNA encoding an extracellular laccase was isolated by reverse transcription polymerase chain reaction from the mycelia of the mushroom Pleurotus eryngii. The isolated sequence, denoted Ery3, encodes for a mature laccase isoenzyme of 531 amino acid residues with a predicted molecular weight of 56.6 kDa. All sequence motifs, being the signature sequences used to identify the laccases, were found in the Ery3 protein sequence. The Ery3 cDNA was expressed in Saccharomyces cerevisiae and the effects of copper concentration and cultivation temperature were investigated. S. cerevisiae cells were immobilized in calcium alginate gel and the optimal immobilization parameters for the enhanced production of laccase were determined. The immobilization was most effective with 3% sodium alginate, 0.1 M calcium chloride and an initial biomass of 4.5 x 10(8) cells. The enzyme yield obtained with immobilized cells (139 mU ml(-1)) showed a 1.6-fold increase compared to the highest yield obtained with free cells. The alginate beads showed good stability and retained 84% capacity of enzyme production after seven repeated cycles of batch fermentation. The immobilization system proved to increase the proteolytic stability of the recombinant Ery3 protein. To our knowledge, this is the first report on S. cerevisiae whole-cell immobilization for recombinant laccase production.
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http://dx.doi.org/10.1007/s00253-008-1479-1DOI Listing
July 2008

Isolation of epiphytic yeasts with potential for biocontrol of Aspergillus carbonarius and A. niger on grape.

Int J Food Microbiol 2006 Apr 27;108(2):204-9. Epub 2006 Jan 27.

Institute of Sciences of Food Production (ISPA), Lecce Section, CNR, via Provinciale Lecce-Monteroni, 73100 Lecce, Italy.

Antagonistic yeasts were isolated from the epiphytic flora associated with grape berries cv. Negroamaro and identified at species level using molecular methods. A total of 144 yeast isolates were tested in a preliminary screening on agar to select isolates showing a killer activity against Aspergillus carbonarius and A. niger, the main species responsible for the accumulation of ochratoxin A in grape. Twenty-eight yeast isolates were selected for their inhibitory effects on the above fungal species and assayed by an in vitro nutritional competition test for their antagonistic capacity towards three selected ochratoxigenic strains. Six yeast isolates belonging to five species, namely 2 isolates of Issatchenkia orientalis and one each of Metschnikowia pulcherrima, Kluyveromyces thermotolerans, Issatchenkia terricola and Candida incommunis, were finally selected and screened on wounded grape berries for their ability to inhibit infection by ochratoxigenic moulds. With the exception of the K. thermotolerans isolate, when inoculated at 10(9) CFU/wound, the other five challenger yeasts reduced the A. carbonarius and A. niger colonization on grape berry (P<0.05). In particular, the best antagonistic activity was shown by the two I. orientalis isolates. Results suggest that antagonist yeasts with the potential to control A. carbonarius and A. niger on grape can be found among the microflora associated with the berries.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2005.12.004DOI Listing
April 2006

Subcellular localization and functional expression of the glycerol uptake protein 1 (GUP1) of Saccharomyces cerevisiae tagged with green fluorescent protein.

Biochem J 2005 Aug;390(Pt 1):145-55

Istituto di Scienze delle Produzioni Alimentari Sezione di Lecce, CNR, 73100 Lecce, Italy.

GFP (green fluorescent protein) from Aequorea victoria was used as an in vivo reporter protein when fused to the N- and C-termini of the glycerol uptake protein 1 (Gup1p) of Saccharomyces cerevisiae. The subcellular localization and functional expression of biologically active Gup1-GFP chimaeras was monitored by confocal laser scanning and electron microscopy, thus supplying the first study of GUP1 dynamics in live yeast cells. The Gup1p tagged with GFP is a functional glycerol transporter localized at the plasma membrane and endoplasmic reticulum levels of induced cells. The factors involved in proper localization and turnover of Gup1p were revealed by expression of the Gup1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaerical protein was targeted to the plasma membrane through a Sec6-dependent process; on treatment with glucose, it was endocytosed through END3 and targeted for degradation in the vacuole. Gup1p belongs to the list of yeast proteins rapidly down-regulated by changing the carbon source in the culture medium, in agreement with the concept that post-translational modifications triggered by glucose affect proteins of peripheral functions. The immunoelectron microscopy assays of cells expressing either Gup1-GFP or GFP-Gup1 fusions suggested the Gup1p membrane topology: the N-terminus lies in the periplasmic space, whereas its C-terminal tail has an intracellular location. An extra cytosolic location of the N-terminal tail is not generally predicted or determined in yeast membrane transporters.
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http://dx.doi.org/10.1042/BJ20042045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1184570PMC
August 2005

Development of reverse transcription (RT)-PCR and real-time RT-PCR assays for rapid detection and quantification of viable yeasts and molds contaminating yogurts and pasteurized food products.

Appl Environ Microbiol 2003 Jul;69(7):4116-22

Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada le Grazie 15, 37134 Verona, Italy.

Reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays have been used to detect and quantify actin mRNA from yeasts and molds. Universal primers were designed based on the available fungal actin sequences, and by RT-PCR they amplified a specific 353-bp fragment from fungal species involved in food spoilage. From experiments on heat-treated cells, actin mRNA was a good indicator of cell viability: viable cells and cells in a nonculturable state were detected, while no signal was observed from dead cells. The optimized RT-PCR assay was able to detect 10 CFU of fungi ml(-1) in pure culture and 10(3) and 10(2) CFU ml(-1) in artificially contaminated yogurts and pasteurized fruit-derived products, respectively. Real-time RT-PCR, performed on a range of spoiled commercial food products, validated the suitability of actin mRNA detection for the quantification of naturally contaminating fungi. The specificity and sensitivity of the procedure, combined with its speed, its reliability, and the potential automation of the technique, offer several advantages to routine analysis programs that assess the presence and viability of fungi in food commodities.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165170PMC
http://dx.doi.org/10.1128/AEM.69.7.4116-4122.2003DOI Listing
July 2003
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