The aim of this investigation has been the design and validation of an oligonucleotide microarray in order to detect 17 different wine-spoilage microorganisms, i.e. 9 yeasts, 5 lactic bacteria and 3 acetic acid bacteria species. Furthermore, several strains belonging to these species has been found to produce undesirable compounds for wine consumers. Oligonucleotide probes specific for each microorganism were designed to target the intergenic spacer regions (ISR) between18S-5.8S region for yeasts and 16S-ITS1 region for bacteria. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consensus primer. Each oligonucleotide-probes exclusively recognized its target without undesired aspecific cross-hybridizations. Under our experimental condition, the microarray assay analysis was able to detect the amount of DNA equivalent to 24 (Saccharomyces cerevisiae), 160 (Lactobacillus brevis) and 124 (Gluconobacter oxydans) cells, three species chosen as experimental models for the three studied microbial classes. Moreover, a novel procedure that allowed the extraction of genomic DNA from a mixture of eukaryotic and prokaryotic cells from contaminated wine was developed. The obtained results confirm that the microarray assay is able to detect specifically different spoilage microorganisms present in mixture in contaminated wines. For the first time the microarray methodology has been applied for the simultaneous identification of different mixed population of spoilage yeast and bacteria directly isolated from wine, thus indicating the practicability of oligonucleotide microarrays as a contamination control in wine industry.

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.

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.

Numerose evidenze scientifiche incoraggiano il consumo di frutta e verdura per prevenire malattie cardiovascolari e tumori. Tuttavia il loro consumo rimane sotto il limite consigliato dall'Organizzazione Mondiale della Sanità. Per questo motivo i consumatori e le aziende alimentari sono alla ricerca di bevande salutistiche "ready-to-drink".I frutti di P. mahaleb, ricchi di composti salutistici (antocianine, flavonoli e cumarine) non sono commestibili a causa del loro sapore amaro, ma l'impiego biotecnologico della fermentazione controllata, utilizzata per migliorare le qualità organolettiche, salutistiche e di serbevolezza di frutta e verdura, potrebbe consentirne l'utilizzo come nuova fonte di probiotici e phytochemicals.

Sparkling wines contain high amount of CO2 and are the result of re-fermentation of a still wine, usually called base wine. Several ingredients, such as sucrose, selected yeasts, bentonite and some nutrients, are added to base wine in order to induce the re-fermentation. Then wines can be bottled, fermented and aged for a long period (about 9-12 months). These wines represents an important percentage of the high-quality wine market and have an important economic impact due to its high added value [1-2], justifying the increasing attention to product improvements. Modern biotechnologies can be used to improve quality of sparkling wines, and also to reduce their production time and cost. Starter cultures selected for sparkling wine production must satisfy several characteristics, in particular considering that re-fermentation is carried out in hostile environment due to several factors, such elevated ethanol content, low pH and increasing carbon dioxide pressure [3]. In addition starter cultures should have other important characteristics, such as autolysis (important for aging in close contact with lees) and flocculation (another important selection criterion that aim lees removal) [4]. Authochtonous starter cultures have an important rule on wine quality, because autochthonous yeasts i) might be well adapted to specific enviromental conditions and ii) might allow to obtain wines with typical organoleptic properties. In these context this study investigated the use of select autochthonous resources for sparkling wine production. We genetically characterized about 200 Saccharomyces cerevisiae autochthonous strains isolated from "Nero di Troia" spontaneous fermentation. After the evaluation of intraspecific diversity using interdelta analyis, we selected one represtative strain for each of the 15 genetic cluster. These strains were technological characterized at lab scale and, successively, tested in winery for both induce alcoholic fermentation in base wine and re-fermentation of sparkling wine. This research was supported by the Apulian Region Project "Innovazioni di processo e di prodotto nel comparto dei vini spumanti da vitigni autoctoni pugliesi" (IProViSP).

Traditional vinification process includes two steps performed by microorganisms, alcoholic fermentation (AF) and malolactic fermentation (MLF). The AF is carried out by yeasts, mainly by Saccharomyces cerevisiae, that however is not the only species involved in the process. In last years, in particular, there is a special interest in the study of non-Saccharomyces yeasts as they are responsible for a considerable part of the aromatic complexity of the wine. MLF is an important phase during winemaking and the co-inoculation of lactic acid bacteria (LAB) resources with yeast starter represents a promising approach to enhance the quality and safety of wine. Lactobacillus plantarum selected strains have been receiving increasing attention as starter cultures for MLF in wine. With the aim to design of mixed culture to perform AF and MLF simultaneously in a short period of time, with complex organoleptic properties, in this work we report the evaluation of the best inoculation time of L. plantarum strains in combination with autochthonous S. cerevisiae strains and non-Saccharomyces (Hanseniaspora sp.). Microvinifications were performed using must from Apulian autochthonous grape varieties. Both, yeasts S. cerevisiae and non-Saccharomyces, were co-inoculated and L. plantarum strains were co-inoculated or sequentially inoculated during AF, when ethanol content was 2%, 4%, 6%, 8%, 10% or 12% (v/v). Ethanol formation, malic acid consumption and cell viability were monitored during the vinifications. Results showed that L. plantarum cell viability and L-malic consumption after AF was strain-dependent. L. plantarum strains showed the highest populations when it was co-inoculated with S. cerevisiae and H. uvarum. Only when the L. plantarum strains were inoculated with 0% of ethanol, all L-malic acid present in grape must (3.5 g/l) was consumed. We also reported preliminary results using the same panel of L. plantarum strains in combination with other autochthonous resources. This research was supported by the Apulian Region Project "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali (NEWine)".

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

The study of interactions among different microbial populations that take place in wine microbial communities is interesting for the understanding of their impact on wines' quality. The main interactions occur among yeast Saccharomyces cerevisiae, yeast non-Saccharomyces spp. and lactic acid bacteria. In last years, there is a special interest in the study of non-Saccharomyces yeasts as they are responsible for a substantial part of the aromatic complexity. Hanseniaspora uvarum has been proved to positively modify the wine chemical composition, especially contribute to the sensory characteristics of wines. In the light of the rising request for autochthonous starters tailored for given 'terroir', the objective of this work was to select the best inoculation time of 5 autochthonous of Oenococcus oeni strains in combination with 2 S. cerevisiae and one H. uvarum strains isolated from Apulian wines. Microvinifications were performed using must from Apulian grape varieties. Both, yeasts S. cerevisiae and H. uvarum, were co-inoculated and O. oeni strains were co-inoculated or sequentially inoculated during alcoholic fermentation, when ethanol content was 2%, 4%, 6%, 8%, 10% or 12% (v/v). Ethanol formation, malic acid consumption and cell viability were monitored during the vinifications. Results showed that ethanol level at the moment of bacterial inoculation was crucial for developing malolactic fermentation. The co-inoculation with S. cerevisiae and H. uvarum was the best strategy for maintaining highest O. oeni populations and therefore for carrying out MLF in red must. Significant differences in L-malic consumption were found depending on the O. oeni strain inoculated. Among the O. oeni strains, OT4 presented the highest malolactic activity, consuming completely the malic acid in all the ethanol concentrations studied. In the case of O. oeni OT4, we studied the expression of selected genes to better understand the interaction of this strain with the yeast biotypes used in this study This research was supported by the Apulian Region Project cod. QCBRAJ6 "Biotecnologie degli alimenti per l'innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali - BIOTECA.

Ochratoxin A (OTA) is a mycotoxin with a main nephrotoxic activity contaminatingseveral foodstuffs. In the present report, five soil samples collected from OTA-contaminatedvineyards were screened to isolate microorganisms able to biodegrade OTA. When cultivated inOTA-supplemented medium, OTA was converted in OTalpha by 225 bacterial isolates. To revealclonal relationships between isolates, molecular typing by using an automated rep-PCR systemwas carried out, thus showing the presence of 27 different strains (rep-PCR profiles). The16S-rRNA gene sequence analysis of an isolate representative of each rep-PCR profiles indicatedthat they belonged to five bacterial genera, namely Pseudomonas, Leclercia, Pantoea, Enterobacter, andAcinetobacter. However, further evaluation of OTA-degrading activity by the 27 strains revealedthat only Acinetobacter calcoaceticus strain 396.1 and Acinetobacter sp. strain neg1, consistentlyconserved the above property; their further characterization showed that they were able to convert82% and 91% OTA into OTalpha in six days at 24 °C, respectively. The presence of OTalpha, asthe unique OTA-degradation product was confirmed by LC-HRMS. This is the first report onOTA biodegradation by bacterial strains isolated from agricultural soils and carried out underaerobic conditions and moderate temperatures. These microorganisms might be used to detoxifyOTA-contaminated feed and could be a new source of gene(s) for the development of a novelenzymatic detoxification system.

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.

Sparkling wines should be defined as an effervescent wine, containing a relevant concentration of CO2 (about 3-7 atmosphere of pressure in the bottle). Usually sparkling wines may be produced by two methods, traditional method (also called méthode champenoise) and charmat method. they involves, respectively, a re-fermentation in the bottle or in hermetically-sealed tanks.The traditional method involves two fermentations steps: primary fermentation and secondary fermentation. During the first step grape juice is transformed into base wine, while the second one is an in-bottle fermentation of a base wine with tirage solution (saccharose, yeasts, grape must or wine, and bentonite) that led to the sparkling wine. Then second fermentation is followed by an aging period in which the wine is in contact with dead yeast (about 9-12 months). Starter cultures selected for sparkling wine production must satisfy several characteristics, in particular considering that re-fermentation is carried out in hostile environment (elevated ethanol content, low pH, increasing CO2...). Starter cultures should have other important characteristics, such as autolysis, killer phenotype and flocculation. Autochthonous starter cultures might be well adapted to specific environmental conditions and might allow to obtain wines with typical organoleptic properties. In this context, the present study investigated the use of select autochthonous resources for sparkling wine production. We evaluated the intraspecific biodiversity of about 200 Saccharomyces cerevisiae autochthonous strains isolated from Apulian spontaneous fermentation. One representative strain from each of the 16 genetic clusters was subjected to i) polyphasic technological characterization at lab scale and, successively, ii) assessment in winery for both induce alcoholic fermentation in base wine and re-fermentation of sparkling wine. Research was supported by the Apulian Region Project "Innovazioni di processo e di prodotto nel comparto dei vini spumanti da vitigni autoctoni pugliesi" (IProViSP).

Biogenic amines in wine represent a toxicological risk for the health of the consumer, with several trade implications. In this study 26 strains of Lactobacillus plantarum were analysed for their ability to degrade biogenic amines commonly found during wine fermentation. Two strains of L. plantarum were selected in reason of their ability to degrade putrescine and tyramine. The degradation was assessed in vitro, both in presence of the biogenic amines and in presence of the specific chemical precursor and of producer bacteria. The two L. plantarum biotypes were found capable to work synergically. In addition, the survival in wine-like medium and the aptitude to degrade malic acid after alcoholic fermentation of the selected L. plantarum strains was analysed. Our results suggest the potential application of wine L. plantarum strains to design malolactic starter cultures able to degrade BA in wine.

Biogenic amines (BA) are a group of organic nitrogenous compounds formed and degraded by the metabolism of living organisms (microorganisms, plants and animals). The main BA associated with wine are putrescine, histamine, tyramine and cadaverine, followed by phenylethylamine, spermidine, spermine, agmatine and tryptamine. The variability in the BA content of wine could be explained on the basis of differences in the winemaking process, time and storage conditions, raw material quality, and possible microbial contamination during winery opera- tions. BA are formed by decarboxylation of the corresponding amino acids by microorganisms through substrate-specific decarboxylase enzymes. This property is usually strain dependent. Decarboxylase enzymes are generally induced at acidic pH and therefore they have a possible role in maintaining pH homeostasis or extending the microbial growth period by detoxification of the extracellular medium. The presence of these compounds is considered by some authors a fundamental parameter for the detriment of wine.

L'attività di ricerca è focalizzata sulle filiere viti-vinicola e olivicola pugliesi. Sono attive ricerche per la caratterizzazione biotecnologica di batteri lattici e lieviti isolati dalla microflora autoctona mediante l'applicazione di metodiche molecolari innovative, analisi delle caratteristiche fenotipiche e genotipiche, studio delle proprietà fisiologiche e tecnologiche. Obiettivo principale è la selezione e la preparazione di colture da utilizzare come starter fermentativi, che permettano di esprimere al meglio il potenziale della materie prime "uva" e "olive" e legare al territorio le bevande e i prodotti derivati, assicurando il corretto svolgimento del processo fermentativo e la salute del consumatore.

Brettanomyces spp. (main B. bruxellensis) is generally recognized as one of the main oenological spoilage microbes. These yeasts negatively affect wine organoleptic properties, due to volatile phenols production. In particular, 4-ethylphenol led to undesirable flavours, described as "phenolic", "horse sweat", "stable", "leather" or "animals" [1]. Usually contamination of B. bruxellensis is low during the first steps of winemaking, due to slow growth, furthermore the metabolic activity of stronger fermenters inhibits its development and its population remains low. Nevertheless, it may proliferate during ageing [2]. Several studies focus attention on control of B. bruxellensis, developing several methods to prevent wine depreciation, using chemicals such as of SO2, organic acids, dimethyldicarbonate (DMDC) [3]. Nevertheless, some strains of B. bruxellensis can tolerate SO2, high ethanol and low sugar concentrations, making difficult its control in wine. To counteract to these tendencies, several studies investigated the efficacy of biological methods to control B. bruxellensis in wine [2-4].The current study presented the assessment of several autochthonous microbial resources in order to control B. bruxellensis growth and volatile phenols release in wine.Several yeasts, both Saccharomyces spp. and non-Saccharomyces, can produced killer toxins that should be investigated as an alternative tool for B. bruxellensis control during winemaking and wine aging. In addition, we selected a Lactobacillus plantarum strain displaying strain-specific inhibitory activities on B. bruxellensis, suggesting potential application in wineries. This work was supported by the Apulian Region i) with the Project cod. QCBRAJ6 "Biotecnologie degli alimenti per l'innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali - BIOTECA."and i) in the framework of "FutureInResearch" program (practice code 9OJ4W81).

Il vino è il prodotto derivante dall'azione della microflora presente sull'uva, la quale utilizza come substrato di crescita le sostanze nutritive presenti nel mosto, realizzando quel processo biochimico noto come fermentazione alcolica. I lieviti vinari si suddividono in apiculati detti "cattivi fermentatori" ed ellittici detti "buoni fermentatori". La prima fase della fermentazione, ad opera dei lieviti apiculati, è caratterizzata dalla crescita di lieviti appartenenti a generi quali Kloeckera, Hanseniaspora e Candida, mentre successivamente, ma comunque a concentrazione di alcol è generalmente inferiore a 3-4 gradi alcolici, si osserva lo sviluppo di specie afferenti a generi quali Metschnikowia e Pichia. Questi lieviti apiculati assieme ad una modesta quantità di etanolo, sono responsabili della produzione di numerosi altri composti associati ai processi fermentativi. Tali composti sono spesso importanti dal punto di vista enologico, in quanto possono avere un'influenza positiva sulle qualità organolettiche finali del vino. Infine, quando la concentrazione dell'alcol etilico raggiunge circa il 4%, inizia, usualmente, la fase della fermentazione dominata in modo particolare da ceppi di Saccharomyces cerevisiae, che prendono il sopravvento su tutte le altre specie presenti fino ad esaurimento degli zuccheri, producendo grandi quantità di alcol con un relativamente inferiore apporto di prodotti secondari. Scopo di questo studio è stato la caratterizzazione molecolare e tecnologica di lieviti autoctoni appartenenti alla specie Candida zemplinina, isolati da fermentazioni spontanee di uve Negroamaro e Primitivo. L'identificazione a livello intraspecie è stata eseguita utilizzando metodiche molecolari basate sull'amplificazione genica utile a caratterizzare regioni del genoma altamente significative a livello tassonomico, in modo tale da distinguere isolati strettamente correlati dal punto di vista genetico. I ceppi identificati sono stati valutati in prove di microfermentazione in mosto, valutando le proprietà enologiche e tecnologiche di ciascuno di essi. Il prodotto della fermentazione operata da ognuno dei ceppi di C. zemplinina è stato sottoposto ad analisi dei principali parametri chimici ed aromatici, mediante spettroscopia IR a trasformata di Fourier (FT-IR) e gas cromatografia a fiamma (GC-FID) o accoppiata alla spettrometria di massa (GC-MS). I risultati prodotti dalla caratterizzazione a livello genetico e fisiologico dei ceppi di C. zemplinina identificati e le loro implicazioni a livello biotecnologico vengono discussi.

Currently, there is very little information available regarding the microbiome associated with the wine production chain. Here, we used an amplicon sequencing approach based on high-throughput sequencing (HTS) to obtain a comprehensive assessment of the bacterial community associated with the production of three Apulian red wines, from grape to final product. The relationships among grape variety, the microbial community, and fermentation was investigated. Moreover, the winery microbiota was evaluated compared to the autochthonous species in vineyards that persist until the end of the winemaking process. The analysis highlighted the remarkable dynamics within the microbial communities during fermentation. A common microbial core shared among the examined wine varieties was observed, and the unique taxonomic signature of each wine appellation was revealed. New species belonging to the genus Halomonas were also reported. This study demonstrates the potential of this metagenomic approach, supported by optimized protocols, for identifying the biodiversity of the wine supply chain. The developed experimental pipeline offers new prospects for other research fields in which a comprehensive view of microbial community complexity and dynamics is desirable.

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.

Ochratoxin A (OTA) is a mycotoxin denoted by a nephrotoxic activity contaminating several foodstuffs. Nowadays, the biological systems for OTA degradation to the less toxic OT? aroused great interest by the scientific community. In the present study, bacteria able to biodegrade OTA were isolated from soil samples collected in OTA-contaminated vineyards. Soil samples were collected from five vineyards of Negroamaro and Primitivo grape cultivars in Salento (Southern Italy). They were cultured in appropriate media added with OTA, mycotoxin degradation was determined by HPLC/FLC analysis and bacterial colonies were isolated by plating. Clonal relationships between isolates was assessed by using an automated rep-PCR system and then each strain was identified by 16S rRNA gene sequencing. A total of 225 bacterial isolates were able to convert OTA in OT?. The molecular analysis of the above isolates showed the presence of 27 different strains (rep-PCR profiles). The sequence analysis of the 16S-rRNA gene indicated that they belonged to five bacterial genera: Pseudomonas, Leclercia, Pantoea, Enterobacter and Acinetobacter. Additional assessment of OTA-degrading capacity of the 27 strains indicated that only the Acinetobacter calcoaceticus strain 396.1 and the A. sp. strain neg1 conserved the above property: both strains were further studied thus showing that they were able to convert 82% and 91% OTA into OT? in 6 days at 24°C, respectively. The occurrence of OT?, as the sole OTA-degradation product was established by LC-MS/MS.This is the first description on OTA biodegradation under aerobic conditions and moderate temperature by bacterial strains from agricultural soils. These microorganisms might be used to detoxify OTA contaminated feed and could be a resource for the development of a new enzymatic detoxification system.

The goal of this study has been the design and the validation of an oligonucleotide microarray in order to detect 17 different wine-spoilage microorganisms, i.e. 9 yeast, 5 lactic bacteria and 3 acetic acid bacteria species. Oligonucleotide probes specific to each microorganism were designed to target the intergenic spacer regions (ISR) between18S-5.8S region for yeasts and 16S-ITS1 region for bacteria. Before hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consensus primer. Each oligonucleotide-probe solely recognized its target without undesired aspecific cross-hybridizations. Under our experimental condition, the microarray assay analysis was able to detect the amount of DNA equivalent to 24 (Saccharomyces cerevisiae), 160 (Lactobacillus brevis) and 124 (Gluconobacter oxydans) cells, three microbes chosen as experimental models. Furthermore, a new procedure that allowed the extraction of genomic DNA from a mixture of eukaryotic and prokaryotic cells from contaminated wine was developed. The microarray methodology has been applied for the first time to simultaneously identify different mixed population of spoilage yeast and bacteria directly from wine. The significance and the impact of the above findings will be discussed. This research was partially supported by the Apulian Region Projects "Biotecnologie degli alimenti per l'innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali - BIOTECA -QCBRAJ6." and "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali (NEWine)".

A yeast strain was isolated during a study on vineyard-associated yeast strains from Apulia in Southern Italy. ITS and LSU D1/D2 rDNA sequences showed this strain not to belong to any known species and was described as the type strain of Ogataea uvarum sp.nov, a close relative of O. philodendri. Several secondary peaks appeared in the sequences, suggesting internal heterogeneity among the copies of the rDNA. This hypothesis was tested by sequencing single clones of the marker region. The analyses showed different levels of variability throughout the operon with differences between the rRNA encoding genes and the internally transcribed regions. O. uvarum and O. philodendri share high frequency variants, i.e. variants frequently found in many clones, whereas there is a large variability of the low frequency polymorphisms, suggesting that the mechanism of homogenization is more active with the former than with the latter type of variation. These findings indicate that low frequency variants are detected in Sanger sequencing as secondary peaks whereas in Next Generation Sequencing (NGS) of metagenomics DNA would lead to an overestimate of the alpha diversity. For the first time in our knowledge, this investigation shed light on the variation of the copy number of the rDNA cistron during the yeast speciation process. These polymorphisms can be used to investigate on the processes occurring in these taxonomic markers during the separation of fungal species, it being a genetic process highly frequent in the complex microbial ecosystem existing in grape, must and wine.

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.

Lactobacillus brevis IOEB 9809 is able to produce both tyramine and putrescine via tyrosine decarboxylase and agmatine deiminase enzymes, respectively, when cultured on synthetic media. The aims of this study were to assess the expression of L. brevis IOEB 9809 tdc and aguA1 genes, during wine fermentation and to evaluate the effect of substrate availability and pH on tdc and aguA1 expression, as well as on biogenic amine production and L. brevis viability.Methods and Results: The relative expression of L. brevis IOEB 9809 tdc and aguA1 genes was analysed in wine by quantitative real-time RT-PCR (qRTPCR) during a period of incubation of 30 days. Cell viability, pH values, putrescine and tyramine concentration were monitored throughout the experiments. Conclusions: The wine trials indicated that L. brevis IOEB 9809 is able to produce both tyramine and putrescine during wine fermentation. Increased cell viability was also observed in wine supplemented with tyrosine or agmatine. qRT-PCR analysis suggests a strong influence of substrate availability on the expression of genes coding for tyrosine decarboxylase and agmatine deiminase in L. brevis IOEB 9809. Less evident is the relationship between putrescine and tyramine production and tolerance to wine pH.Significance and Impact of Study: To our knowledge, this study represents the first assessment of relative expression of L. brevis IOEB 9809 genes involved in biogenic amine production in wine. Furthermore, an effect of biogenic amine production on viability of L. brevis during wine fermentation was established.

The aim of this work was to study the biodiversity of yeasts isolated from the autochthonous grape variety called "Uva di Troia", monitoring the natural diversity from the grape berries to wine during a vintage. Grapes were collected in vineyards from two different geographical areas and spontaneous alcoholic fermentations were performed. Different restriction profiles of ITS-5.8S rDNA region, corresponding to Saccharomyces cerevisiae, Issatchenkia orientalis, Metschnikowia pulcherrima, Hanseniaspora uvarum, Candida zemplinina, Issatchenkia terricola, Kluyveromyces thermotolerans, Torulaspora delbrueckii, Metschnikowia chrysoperlae, Pichia fermentans, Hanseniaspora opuntiae and Hanseniaspora guilliermondii, were observed. The yeast occurrences varied significantly from both grape berries and grape juices, depending on the sampling location. Furthermore, samples collected at the end of alcoholic fermentation (AF) revealed the great predominance of Saccharomyces cerevisiae, with a high intraspecific biodiversity. This is the first report on the population dynamics of 'cultivable' microbiota diversity of "Uva di Troia" cultivar from the grape to the corresponding wine ("Nero di Troia"), and more general for Southern Italian oenological productions, allowing us to provide the basis for an improved management of wine yeasts (with both non-Saccharomyces and Saccharomyces) for the production of typical wines with desired unique traits. A certain geographical-dependent variability has been reported, suggesting the need of local based formulation for autochthonous starter cultures, especially in the proportion of the different species/strains in the design of mixed microbial preparations

Grape berries polyphenols are mainly synthesized in the skin tissues and seeds and they are extracted during the winemaking process. These substances have a potentially positive effect, on human health, thus giving to grape and red wine "functional properties" that can contribute to prevent a number of human illness. Nevertheless, the research community is showing that the real effect is a result of a combination of different factors notably daily intake, bioavailability, or in vivo antioxidant activity that are yet to be resolved. Viticulture and winemaking practices, determine the concentration of polyphenols in grape and wine. To date, reduced knowledge is existing on the effects of different yeast strains on the final concentration of polyphenols in red wine. We summarize the recent findings concerning the effects of polyphenols on human chronic disease and the future directions for research to increase the amount of these compounds in wine.

Spontaneous grapes must fermentation, induced by the indigenous micro flora, is believed to be associated with a specific vineyard and to give a distinctive style and quality to that wine. The alcohol-tolerant Saccharomyces cerevisiae strains invariably dominate the latter stage of natural wine fermentation. The S. cerevisiae population and other specific yeasts present in the vineyard niche habitats are considered autochthonous and their involvement in natural fermentation allows the production of wines with particular features in each microclimatic area. The present study was aimed to the individuation of autochthonous yeast strains useful in the improvement of oenological production of Salento, which is a very important wine-producing area of Southern Italy. Grapes were sampled from the most representative areas of Salento region (Ugento e Guagnano for "Negroamaro" and Gioia del Colle for "Primitivo") and separately subjected to natural fermentation in an experimental scale. The identification of micro biota present during the last step of wine fermentation (>1 °Bé) of grapes, was carried out to select autochthonous yeast strains for industrial wine production. Aliquots of must samples of several dilutions were isolated after spreading on YPD agar medium supplemented. As preliminary screening, averages of 1500 colonies for each of the three fermentations were assayed for H2S production on BIGGY agar. The colonies appearing white or light brown (about the 10% of initial 1500 individual clones) were selected and recognized as S. cerevisiae by a microbiological screening based on the implementation of the taxonomical keys for identification of yeasts belonging to Saccharomyces genus. Identification at strain level of S. cerevisiae isolates is a fundamental step to investigate the biodiversity of this yeast and to examine population dynamics during the fermentative process. The criterion for strain differentiation was provided by the amplification of genomic sequence blocks flanked by delta elements of retrotrasposon origin. Interdelta (ID) typing has proved to be a very convenient method that can advantageously replace the other methods for molecular characterization of S. cerevisiae strains. For the first time, a recently developed method has been applied for the ID by the use of fluorescent primers and automatic sequencers. When compared with gel-based analytical methods, the use of capillarity system and automated analysis increase data throughput, scoring and reliability, decreasing the overall experimental error. The results of the genetic characterization of S. cerevisiae strains associated to natural fermentations of must from "Primitivo" and "Negroamaro" grapes and their implications for the selection of autochthonous industrial starters will be discussed.

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.

Mycotoxins have become one of the most recognised feed chain contaminants, with hundreds of mycotoxins identified to date. Management of mycotoxins includes prevention, regulation, monitoring, decontamination, and animal treatments. Even with good management, unavoidably low levels of several mycotoxins can cause loss of feedstuffs, increased animal disease, reduced animal performance, and food residues. A promising approach to protect animals against the harmful effects of contaminated feed is based on the use of feed additives. These additives are defined as substances that, when included into contaminated feed, can adsorb or denature mycotoxins in the digestive tract of animals. Since 2009, they are officially allowed in the UE as technological feed additives. Mycotoxin adsorbents are the most studied additives and a variety of products are on the market claiming multi-toxin adsorption capacity. The efficacy of adsorbents in sequestering different mycotoxins has been poorly addressed. The aim of this study was the screening of commercial products for preparing a nutritional composition intended to reduce bioavailability of a large range of mycotoxins. 52 commercial products from 26 industrial partners, including minerals, yeast-based products and blend of components, were tested. Preliminary adsorption tests allowed the selection of 4 commercial products as effective in sequestering simultaneously aflatoxin B1, zearalenone, ochratoxin A and fumonisin B1. All products failed in adsorbing deoxynivalenol, but activated carbon. Adsorption experiments were performed with selected binders, at physiologically relevant pH values commonly found in the stomach and intestine, to determine adsorption parameters (capacity, affinity, chemisorption index). Mineralogical analysis (XRD) and ash content showed that 3 out of the 4 commercial products selected as best multi-toxin adsorbents (designated by the supplying companies as minerals) were organoclays. Organoclays are not suitable for feed ingredients due to toxicity of the interlayer quaternary alkylammonium ions. Two organoclays and one yeast cell wall product, out of 52 commercial products, were found toxic in 2 bioassays. In conclusion, multi-toxin adsorbents covering major mycotoxins are not commercially available. Most of them lack effectiveness towards trichotechenes. The identity/composition of commercial products could be counterfeit and misleading. Some commercial products can be even highly toxic in toxicity bioassays.

In conventional winemaking, grape must fermentations are carried out by a succession of different yeast species. The Saccharomyces cerevisiae completes the process, whereas its initial stage is dominated by non-Saccharomyces strains, whose by-products contribute to the composition of the wine bouquet. In this study, we evaluated the performance of two selected strains of Hanseniaspora uvarum and S. cerevisiae as multistarters for inoculation of Negroamaro must vinification.

Saccharomyces cerevisiae is the yeast species predominating the alcoholic fermentation of grape must. The aim of this research was to evaluate the impact of indigenous S.cerevisiae strains biodiversity on the aroma of wines from Negroamaro grapes. Grapes collected in two different Negroamaro producing micro districts in Salento (Southern Italy), were subjected to natural fermentation and two indigenous S.cerevisiae populations were isolated. Fifteen strains for each of the two populations were selected and tested by micro fermentation assay in order to evaluate their specific contribute to the volatiles composition and sensory impact of the produced wines. The aromatic profile of wines obtained by each selected strain was characterized by different contents of acetates, ethyl esters of fatty acids, higher alcohols, thus showing to be related to the strains geographical origin. The sensorial analysis of wines produced by the six best performing strains confirmed that they are good candidates as industrial starter cultures, This study indicates that the use of a "microarea-specific" starter culture is a powerful tool to enhance the peculiarity of wines deriving from specific areas. © 2014 Elsevier Ltd.

Phenolic coumpounds present in grape berries are extracted from the skin, seeds and flesh during the winemaking process. These substances have a potentially positive effect, on human health, thus giving to red wine "bioactive properties" that an contribute to decrease the incidence of atherosclerosis, cancer, neurodegenerative diseases. Grape variety, quality, climate, geographical origin and phythopatologies can affect the quality and quantity of phenolic compounds that accumulate in the cells. Viticulture and vinification practice, which varies in different countries, determine the concentration of phenolic compounds in wine. During the vinemaking process numerous parameters, such as temperature, presence of seeds and berry skins and addition of enzymes, have been reported to affect the extraction of phenolic compounds during grape must fermentation. Conversely, poor knowledge is available on the effects of different yeast strains on the final concentration of polyphenols in red wine. Indeed, yeasts enzymatic activity are likely to influence the extraction of phenolic substances from grape tissues. With the aim to evaluate the existence of a potential correlation between polyphenols content, antioxidant capacity of wine and yeast starter cultures, three selected autochthonous strains of Saccharomyces cerevisiae, were used to inoculate large scale must fermentation. At the end of the process, the experimental wines obtained were evaluated for the content of different class of polyphenols and antioxidant capacity. The statistical analysis of obtained results revealed a significant influence of the autochthonous strain on the concentration of several class of polyphenols and total antioxidant capacity in the produced wines. The different strain influence on the analysed parameters assumes a technological significance and it strongly contribute in determining the final quality of the wine by influencing "functional parameters" important for the preservation of human health.

The aim of this work is to study the influence of 30 different autochthonous Saccharomyces cerevisiae strains, isolated from Negroamaro grapes, sampled in the northern (NN strain) and in the southern (NS strain) of Salento (south Apulia, ITALY), on the volatile fraction of the corresponding Negroamaro wines made using the different isolated yeast strains by controlled microvinification processes. A further aim is to compare the volatile profiles of the as-obtained 30 different Negroamaro wines with the volatile profile of a Negroamaro wine produced by using a commercial yeast strain selected among the most employed by local producers. Modern wine makers prefer to employ commercial yeast strains due to their known specific yield and efficiency characteristics so as to ensure a reproducible product, reduce the risk of wine spoilage and allow a more predictable control of fermentation and quality. However, it's important to know the potential of using the autochthonous S. cerevisiae yeast strains with the best performance, in order to exploit them to improve wine quality and commercial standards. In the present work we have used two different analytical methods in order to analyze different compounds present in wine volatile fraction in a wide range of concentrations. Highly compounds concentrations (major volatiles) were directly determined on wine by GC-FID, whereas represented minor molecules were extracted by SPE method and then analysed by GC/MS. The complete volatile profile of all the wine samples was obtained together with a quantitative analysis by internal standardization method. Principal Component Analysis, applied to compound concentrations data showed that the wines, respectively produced with NN strains and strains NS, differentiated among them according to their aroma composition. The differences in the composition appear to be quantitative rather than qualitative. In fact, the formation of secondary products depends both on the yeast's enzymes and on the nitrogen nutrients and cofactors present in must so that the same yeast can produce different volatile patterns, depending on must composition.The obtained results suggest that the concentration of most of the volatile compounds was significantly influenced by the particular inoculated S. cerevisiae yeast strain. Autochthonous yeast strains have been shown to be able to produce wines with different volatile profiles. In particular most of NN strain have produced wines rich in alcohols and esters, most of NS strain have produced have produce wines rich in acids, esters and acetaldehyde.

A yeast strain was isolated during a large-scale survey yeast strains from vineyard-associated in Apulia (Southern Italy). The sequences of the ITS and LSU D1/D2 domain of the ribosomal RNA (rDNA), revealed that this strain did not fit in to any known species and was consequently described as the type strain of Ogataea uvarum sp.nov. The novel species resulted to be a close relative of O. philodendri. The sequences of the two rDNA domains showed several secondary peaks, indicating internal heterogeneity among the copies of the rDNA operon. This supposition was verified by cloning the region across the ITS1 to LSU D1/D2 and sequencing both strands of each clones. The analyses on the obtained data indicated different levels of variability throughout the operon with differences between the 5.8S and D1/D2 domain of the 26S and the internally transcribed regions (ITS). O. uvarum and O. philodendri share high frequency variants, i.e. variants frequently found in many clones, whereas great variability of the low frequency polymorphisms was found, thus suggesting that the homogenization mechanism is more active with the former than with the latter type of variation. These results indicate that low frequency variants are present in Sanger sequencing as secondary peaks and represent a sort of noise, whereas Next Generation Sequencing (NGS) approach will trace also the secondary variants, with the risk of overestimating the alpha diversity. The significance and the impact of the above finding on the use of these taxonomic markers to investigate the separation of fungal species will be discussed.This research was partially supported by the Apulian Region Projects "Biotecnologie degli alimenti per l'innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali - BIOTECA -QCBRAJ6.

The yeast Brettanomyces bruxellensis, generally considered the main oenological spoilage microbe, is able to survive during the winemaking process and it confers off-odors to wine, in reason of its ability to produce considerable amounts of volatile phenols. Forty-eight isolates of B. bruxellensis, obtained from several wines collected in Apulia (Southern Italy), were genetically characterized using an integrated approach, including a strain biodiversity analysis by Sau-PCR. Furthermore, the production of volatile phenols was assessed in wine and in synthetic medium, confirming the oenological spoilage potential of the analysed strains. Our findings indicate a remarkable genetic variability of the B. bruxellensis identified strains and corroborate the evidence of a high level of genotypic and phenotypic polymorphism within B. bruxellensis species. Moreover, the observation reported suggest that strains from wines produced in the same geographical areas often clustered differently, indicating a complex intraspecific biodiversity in the regional wine environments. Diversity in volatile phenol production reflects intraspecific biodiversity highlighted by Sau-PCR. Strains diversity linked to differences in 'spoilage potential' increase the industrial relevance of this study, allowing the design of new strategies for B. bruxellensis control in wines.

The winemaking process includes two traditional steps performed by microorganisms, alcoholic fermentation (AF) and malolactic fermentation (MLF). Even though the AF is principally carried out by the yeast Saccharomyces cerevisiae, it is not the only yeast involved in this process. Several non-Saccharomyces species, such as Hanseniaspora uvarum, have been proved to positively modify the wine chemical composition, especially contributing to the sensory characteristics of wines. The study of the Interactions between the different microbial populations present in grape must is thus important since their impact on the final quality of the wine. The main interactions occur among Saccharomyces spp. yeasts, non-Saccharomyces yeasts, and lactic acid bacteria (LAB). In the light of the rising request for autochthonous starters tailored for specific 'terroir', the objective of this work was to select the best inoculation time of autochthonous of Oenococcus oeni strains in combination with two S. cerevisiae strains and one H. uvarum strain isolated from Apulian wines. Microvinifications were performed using must derived from Apulian autochthonous grape varieties. Both, yeasts S. cerevisiae and H. uvarum, were co-inoculated and O. oeni strains were co-inoculated or sequentially inoculated during AF, when ethanol content was 2%, 4%, 6%, 8%, 10% or 12% (v/v). Ethanol formation, malic acid consumption and cell viability were monitored during the fermentative process. The obtained results indicated that the ethanol concentration at the moment of bacterial inoculation was crucial for the occurrence of a complete MLF. The co-inoculation with S. cerevisiae and H. uvarum demonstrated to be the best strategy for maintaining highest O. oeni populations and therefore for carrying out MLF in red must. This research was supported by the Apulian Region Project cod. QCBRAJ6 "Biotecnologie degli alimenti per l'innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali - BIOTECA.

The winemaking process includes two key steps performed carried out by microorganisms, i.e. alcoholic fermentation (AF) and malolactic fermentation (MLF). The AF primarily produce the is essential; conversion of grape must sugars from grape must are converted into ethanol. This fermentation is carried out by yeast, mainly by Saccharomyces cerevisiae strains: However, that, however,, this is not the only yeast involved in the process. In fact,A few specificseveral non-Saccharomyces species, such as Hanseniaspora uvarum, have been proved to positively modify the wine chemical composition, contributing to the sensory characteristics of wines. On the other side, MLF is carried out by lactic bacteria (LAB), mainlyprincipally by Oenococcus oeni and Lactobacillus plantarum strains. While Although the possible interactions of enological significance between S. cerevisiae and O. oeni strains have been extensively studied, little is known about possible interactions between non-Saccharomyces and O. oeni strains. Objective: In the light of the rising request for autochthonous starters tailored for given 'terroir', the objective of this work was to select identify the best inoculation time of autochthonous of Oenococcus oeni or Lactobacillus plantarum strains in combination with two autochthonous S. cerevisiae strains and one H. uvarum strain, all isolated from Apulian wines.Methods: Both, yeasts S. cerevisiae and non-Saccharomyces, were co-inoculated in red must, and L. plantarum or O. oeni strains were co-inoculated or sequentially inoculated during AF, when ethanol content was 2%, 4%, 6%, 8%, 10% or 12% (v/v). Ethanol formation, L-malic acid consumption and bacterial cell viability were monitored during the vinifications.Conclusions: The co-inoculation of H. uvarum with S. cerevisiae did not affect the AF. In all cases AF was finished completed in about 4 days. Ethanol concentration at the moment of bacterial inoculation was a crucial factor for the developing success of MLF. The co-inoculation with S. cerevisiae and H. uvarum was the best strategy for maintaining the highest LAB populations concentration, in order to successfully and therefore for carryicarryng out the MLF in red must. Bacterial cell viability and L-malic consumption after AF were strain-dependent. This research was supported by the Apulian Region Project cod. QCBRAJ6 "Biotecnologie degli alimenti per l'innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali - BIOTECA", and by the Apulian Region Project "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali (NEWine)". Vittorio Capozzi was supported by 'Fondo di Sviluppo e Coesione 2007-2013--APQ Ricerca Regione Puglia "Programma regionale a sostegno della specializzazione intelligente e della sostenibilità sociale ed ambientale--FutureInResearch".

Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PC using gene-specific primers. Furthermore, the strain ability to produce tyramine was assayed in a syntetich media was and the biogenic amine produced was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluted in microvinifications assays using two different musts with different ethanol concentrations (10% and 12% (v/v)) and the tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine either in vitro or in vivo and that they are able to survive in wine after an acidic adaptation step.

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.

Brecve lezione all'utilizzo dei lieviti come sistema eterologo di espressione genica per la produzione di proteine ricombinanti.

Saccharomyces and non-Saccharomyces represents a heterogeneous class in the grape/must/wine environments including several yeast genera (e.g. Saccharomyces, Hanseniaspora, Pichia, Candida, Metschnikowia, Kluyveromyces, Zygosaccharomyces, Torulaspora, Dekkera and Schizosaccharomyces) and species. Since each species may differently contribute to the improvement/depreciation of wine qualities, it appears clear the reason why species belong to non-Saccharomyces are also considered a biotechnological resource in wine fermentation. Here, we briefly review the oenological significance of this specific part of microbiota associated with grapes/musts/wine. Moreover, the diversity of cultivable non-Saccharomyces genera and their contribute to typical wines fermentations will be discussed.

Microbial starter cultures represent a fundamental level of innovation in the wine sector. Selected yeast strains are routinely used to achieve the needed biomass preparation to accelerate and steering alcoholic fermentation in grape must. The use of starter cultures to induce malolactic fermentation in wine relies on the characterization and propagation of suitable strains of lactic acid bacteria. Furthermore, the selection of new strains, the renewal of management of microbial resources and new technologies allow continuous improvements in oenology, which may increase the benefit of wine. In this review, with the aim to stimulate microbial-driven, consumer-oriented advances in the oenological sector, we propose an overview of the recent trends in this field, that are reported by following the classical separation into 'product innovation' and 'process innovation'. Hence, we shall highlight i) the possible positive innovative impacts of microbial resources on the safety and on the sensorial and functional properties of wine (product innovation) and ii) the potential microbial-based improvements allowing the reduction of time/costs and the environmental impacts associated with wine-making (process innovation).

La diversità microbica associata alle fermentazioni alimentari rappresenta da millenni una preziosa risorsa per la salute, la qualità della vita e la nutrizione umana. Nell'ultimo secolo, inoltre, la biodiversità dei lieviti, dei batteri e dei funghi protecnologici associata alla produzione di alimenti fermentati è stata oggetto di una consistente valorizzazione economica nel quadro dell'industria delle colture starter. Esiste un'ampia letteratura scientifica riguardo l'isolamento e la caratterizzazione di microorganismi autoctoni/indigeni associati con fermentazioni spontanee, spesso relativamente a produzioni ad indicazione geografica, tipiche e/o artigianali. Recenti evidenze scientifiche, tendenze economiche ed istanze sociali hanno portato ad un rinnovato e crescente interesse per la gestione delle fermentazioni spontanee e per il possibile impiego di risorse microbiche autoctone/indigene selezionate per specifiche produzioni ad indicazione geografica, tipiche e/o artigianali. Proponiamo una rassegna di evidenze scientifiche, tendenze economiche ed istanze sociali in grado di fornire un'idea sui principali trend nel settore. Inoltre, utilizzando il vino come matrice 'modello', offriamo una disamina di problematiche ed opportunità associate a tali trend (es. fermentazioni spontanee in enologia, innesto di lieviti e batteri autoctoni, valorizzazione di diverse specie di lieviti e batteri enologici), con particolare attenzione ad aspetti inerenti alla qualità sensoriale ed igienico-sanitaria dei prodotti finiti. Sono discusse, infine, possibili soluzioni di management delle risorse microbiche coerenti con il quadro di riferimento oggetto di analisi ed in grado di raggiungere gli standard qualitativi assicurati dalla tecnologia delle colture starter nella produzione di alimenti fermentati.

The development of fast, reliable and culture-independent molecular tools to detect bacteria producingbiogenic amines deserves the attention of research and ultimately of the food industry in order to protectconsumers' health. Here we present the application of a simple, low-cost, fast and sensitive method toperform microdroplet-based multiplex PCR, directly on a food matrix, for the simultaneous detection ofbacterial genes involved in biogenic amine biosynthesis. After inoculating wine with Lactobacillus brevisIOEB 9809, cell lysis and DNA amplification are performed in one single step, without preliminary nucleicacid extraction or purification treatments. The assay is performed in about 30 min, requiring 150 nL ofstarting sample and it enables the detection of down to 15 bacterial cells. With respect to traditionalculture techniques, the speed, the simplicity and the cheapness of this procedure allow an effectivemonitoring of microbial cells during food-making and processing.

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.

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.

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.

The main goal of the present investigation was to assess the microbiological safety of two typical meat-derived products, i.e. raw pork sausages and entrails lamb rolls, produced in Salento (Apulia, Southern Italy). Analyses were carried out for 7 years (from 2008 to 2014) and a total number of 6720 samples was collected by specialized personnel. The presence of Listeria monocytogenes and Salmonella spp. was detected by a PCR-based assay, combined with culturing in enrichment broth. The prevalence of L. monocytogenes was assessed in 2.4% entrails lamb rolls and in 4.2% raw pork sausages samples, whereas the occurrence of Salmonella spp. was revealed in 2.7% lamb rolls and in 3.5% pork sausages. A statistically significant seasonal variation was found in the occurrence of L. monocytogenes; in fact a higher number of samples contaminated by this pathogen was recorded in spring and autumn. On the contrary, no significant seasonal changes occurred in the prevalence of Salmonella spp. The data reported indicate that, due to the presence of these pathogens, the Italian food processors need to improve the microbiological monitoring of the processing chains, in order to guarantee health safety.

Indigenous yeasts are present on the surfaces of grapes and their success in surviving and driving fermentation depends on the sum of various physical, chemical and biotic factors. The analysis of berry-resident strain diversity and the relationship between genotype and phenotype can be used in the development and identification of specific non-Saccharomyces strains provided with interesting technological properties. In this contribution, during a large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy), we isolated a new yeast species from "Negroamaro" grape berries.

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.

For three consecutive years, an Italian winery in Apulia has dealt with sudden alcoholic stuck fermentation in the early stages of vinification process, i.e., typical defects addressable tobacterial spoilage. After a prescreening trial, we assessed, for the first time, the influence of the commercial fungicide preparation Ridomil Gold® (Combi Pepite), containing Metalaxyl-M (4.85%)and Folpet (40%) as active principles, on the growth of several yeasts (Saccharomyces cerevisiae and non-Saccharomyces spp.) and lactic acid bacteria of oenological interest. We also tested, separatelyand in combination, the effects of Metalaxyl-M and Folpet molecules on microbial growth both in culture media and in grape must. We recalled the attention on Folpet negative effect on yeasts,extending its inhibitory spectrum on non-Saccharomyces (e.g., Candida spp.). Moreover, we highlighted a synergic effect of Metalaxyl-M and Folpet used together and a possible inhibitoryrole of the fungicide excipients. Interestingly, we identified the autochthonous S. cerevisiae strain E4 as moderately resistant to the Folpet toxicity. Our findings clearly indicate the urgent need forintegrating the screening procedures for admission of pesticides for use on wine grape with trials testing their effects on the physiology of protechnological microbes.

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

La fermentazione spontanea delle uve è promosse dai lieviti indigeni, che sono in grado di conferire stile e qualità distintivi al vino prodotto. Tale processo è dapprima dominato da lieviti non Saccharomyces e, in una fase finale, il processo di fermentazione alcolica è completato dall'azione di ceppi di S. cerevisiae. I ceppi autoctoni di lievito sono associati a uno specifico habitat di nicchia del vigneto e il loro ruolo nella fermentazione naturale consente la produzione di vini con caratteristiche particolari in ogni area microclimatica. Tuttavia, al fine di evitare l'imprevedibilità della fermentazione spontanea del mosto in termini di qualità e sicurezza del vino finito, i produttori impiegano colture di lievito secco commerciale per produzioni vinicole industriali. È sempre più evidente un crescente interesse in Puglia per l'utilizzo di lieviti autoctoni per la gestione della fermentazione, in quanto colture starter formulate a partire da ceppi autoctoni possono contribuire alla conservazione e alla valorizzazione delle caratteristiche enologiche e sensoriali tipiche e rappresentative di una regione enologica, garantendo, al contempo, standardizzazione e salubrità delle produzioni. L'impiego di ceppi autoctoni di S. cerevisiae come starter, inoltre, sembra preferibile in quanto diversamente adattati ad una specifica area di produzione del vino e quindi potenzialmente in grado di dominare in modo più efficiente la microflora indigena durante il processo di fermentazione. Nel presente studio, abbiamo applicato un protocollo di selezione di starter fermentativi da una popolazione di lieviti isolata da uve Primitivo nell'area di produzione della DOC "Gioia del Colle", una delle zone di produzione più rappresentative della Puglia. La popolazione di S. cerevisiae è stata isolata dalle fermentazioni naturali dei mosti di uva e caratterizzata geneticamente tramite saggi molecolari (analisi interdelta e sequenziamento del DNA ribosomale). I ceppi individuati sono stati caratterizzati, mediante prove di micro-fermentazione in mosto, valutandone le proprietà enologiche e tecnologiche. Il prodotto della fermentazione operata da ognuno dei ceppi è stato sottoposto ad analisi dei principali parametri chimici ed aromatici, tramite Spettroscopia ad Infrarossi in Trasformata di Fourier (FT-IR) e Gas Cromatografia-Spettrometria di Massa (GC-MS). Al termine della procedura di selezione, le attitudini fermentative di tre ceppi candidati starter (ITEM14093, ITEM 14088, ITEM14090), caratterizzati da interessanti proprietà tecnologiche ed enologiche, sono state valutate mediante prove su scala pilota. Durante la successive vendemmia, il ceppo ITEM14093, risultato come quello dotato delle migliori caratteristiche enologiche, è stato utilizzato come starter per la produzione su scala industriale di vino Primitivo della DOC "Gioia del Colle". La qualità dei vini prodotti e le risultanze inerenti l'impiego di colture starter autoctone per la produzione industria

El proceso de vinificación incluye dos etapas llevadas a cabo por microorganismos, la fermentación alcohólica y la fermentación maloláctica. Esta última es llevada a cabo por bacterias lácticas, y en ella se transforma el ácido L-málico en L-láctico liberándose CO2. Lactobacillus plantarum es capaz de llevar a cabo esta fermentación, especialmente en condiciones de alto pH y cuando se co-inocula con las levaduras. Por otro lado, esta especie es capaz de acidificar el mosto antes de que se realice la FA. Estas características hacen que se considere una especie muy interesante para ser utilizada como cultivo iniciador en el proceso de vinificación. En este estudio se llevó a cabo una caracterización tecnológica de cepas de L. plantarum aisladas de vinos de Apulia con el fin de combinar las 2 propiedades (realización de la FML y acidificación del mosto). Además, los resultados mostraron que la co-inoculación de cepas seleccionadas de L. plantarum con S. cerevisiae promovían la adaptación de la bacteria a las condiciones del vino y se reducía el tiempo total de vinificación.

PRUNUS MAHALEB, L. FERMENTED FRUITS: A POTENTIAL SOURCE OF ANTIOXIDANT ACTIVITY AND PROBIOTIC MICRORGANISMSGerardi Carmela1, Tristezza Mariana1, Giordano Luca1, Maiorano Gabriele1, Blando Federica1, de Candia Silvia2, Grieco Francesco11 Institute of Sciences of Food Production, CNR, Territorial Unit of Lecce, Italy2 Institute of Sciences of Food Production, CNR, Territorial Unit of Bari, ItalyFermentation is a process traditionally adopted to preserve food which results in novel fermented products, provided with specific organoleptic properties. Prunus mahaleb L. is an autochthonous tree of the Mediterranean area. Its highly pigmented small-stone fruits are characterized for astringent and sour taste and therefore not used for fresh consumption. Recently, Prunus mahaleb L. fruits were found endowed with high bioactive compound content, especially anthocyanins, and strong antioxidant capacity [1]. With the aim to reduce their sourness, P. mahaleb fruits were dipped in water and fermented for 20 days at 25°C with four starter cultures of Lactobacillus plantarum strains alone or in mixture with a single strain of Saccharomyces cerevisiae. During this period, viable cell count of L. plantarum strains was never found lower than 107CFU/ml that is the lower concentration request to probiotic microorganisms to promote beneficial effects on human health. S. cerevisiae in all trials reached a maximum population three days after inoculation and then, cells counts slightly decreased in a range from 107 to 103 CFU/mL depending on the co-inoculated bacteria strain. Among L. plantarum strains, only the FG68 survived after an in vitro digestion protocol specifically set up for the selection of potentially probioitic lactic acid bacteria [2]. As concerns the antioxidant activity, the fermentation medium always showed higher values when L. plantarum strains were co-coltured with S. cerevisiae. Conversely, no differences were found in fermented fruits even though their antioxidant capacity values were higher than the large part of fruits included in the human diet.References1.Gerardi C, Tommasi N, Albano C, Blando F, Rescio L, Pinthus E, Mita G (2015) Prunus mahaleb L., fruit extracts: a novel source from natural food pigments. European Food Research & Technology 241: 683-695.2.Baruzzi F, Poltronieri P, Quero GM, Morea M. (2011) An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks. Applied Microbiology and Biotechnology 90: 331-342.

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.

In winemaking, the utilization of mixed starter cultures of Saccharomyces and non-Saccharomyces yeasts is a methodology that allows the enhancement of sensory quality and complexity of the final product, without compromising the general quality and safety of the oenological products. The aim of this study was to assess the oenological potential of Candida zemplinina in mixed cultures (co-inoculation) and sequential inoculation with S. cerevisiae for the industrial wine production. Three previously characterized S. cerevisiae strains [1] were separately used, as multistarter, together with an autochthonous C. zemplinina strain [2] in lab-scale microvinification trials of Negroamaro must. The fermentation course and the quality of final product indicated that C. zemplinina strain 35NC1 positively contributed to increasing the wine organoleptic quality. At the best of our knowledge, the present report is the first study regarding the utilization of a selected C. zemplinina strain in multistarter inoculation with S. cerevisiae for the production of Negroamaro wine. The results of this investigation corroborate the concept that non-Saccharomyces yeasts play a essential role in wine-bouquet formation and their action could reduce the risks for wine quality and safety often associated with uncontrolled vinification.This research was partially supported by the Apulian Region Projects "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali (NEWine)".References1.Chiriatti M., et al. (2012). Identification, characterization and application of autochthonous fermentative starter cultures for the industrial production of Negroamaro and Primitivo wine Proceedings of the III SIMTREA Congress, June 26-28, Bari, Italy, pp. 93-94.2.Tristezza M., et al. (2016). Caratterizzazione molecolare e tecnologica di lieviti vinari autoctoni appartenenti alla specie Candida zemplinina. Proceedings of the "11° Convegno Nazionale sulla Biodiversità", 9-10 giugno, Matera, Italy, p. 211.

The aim of the present study was to isolate and characterize yeast strains as good candidates for driving the industrial fermentation process, from natural must fermentations of "Primitivo" grape cultivar, grown in the PDO/DOC "Gioia del Colle" (Apulia, Southern Italy),. The selection protocol was based on parameters such as low production of acetic acid and hydrogen sulphide, complete sugar consumption during fermentation, significant production of some classes of volatile molecules responsible for wine aroma. Three Saccharomyces cerevisiae strains, named ITEM14088, ITEM14090 and ITEM14093, successfully dominated the fermentation process and contributed to increase organoleptic quality of the produced wines. The best performing strain, namely ITEM14093, was used as fermentation starter for three different industrial vinifications. The wines obtained were characterized by high levels of esters, associated to fruity nuances, as well as of alcohols responsible for vinous, sweet and floral notes. Furthermore, from a sensory point of view, all wines were positively judged, being characterized by frankness, gustatory persistence and intensity, good balance and body wine.

We report the first polyphasic characterization of native Saccharomyces cerevisiae in order to selectcandidate strains for the design of starter cultures tailored for Apulian sparkling wines obtainedfrom local grape variety. In addition, it is the first survey in our region that propose the selection ofautochthonous starter cultures for sparkling wine i) including a preliminary tailored genotypic andtechnological screening, and ii) monitoring analytical contribution during secondary fermentation interms of volatile compounds (VOCs). Furthermore, we exploit the potential contribute ofautochthonous cultures throughout the productive chain, including the possible improvement ofbase wine. One representative strain from each cluster was characterized i) for tolerance to abioticand biotic stressors peculiar of sparkling wine fermentation, ii) for the performances in base wineproduction, and iii) for the aptitudes to promote in-bottle secondary fermentation in white and rosésparkling wines, both obtained from Apulian grape varieties. Genetic characterization led to group164 S. cerevisiae in 16 genetic clusters based on interdelta profiles. Stress tolerance assays shown acertain correlation with fermentative attitude. Our evidences demonstrated a different fermentativebehavior and release of VOCs of the different strains in association with primary and secondaryfermentations and as function of wine and rosé sparkling wine. Furthermore, performances inwhite/rosé sparkling wines have been found to be strain-dependent characters. Overall, we proposedifferent strains as biotechnological resources suitable to improve the quality of regional sparklingwines and to provide a driver of innovation/segmentation in the market.

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.

A yeast strain isolated during a large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy) was subjected to sequence analysis of the large subunit (LSU) and internal transcribed spacer (ITS) domains of its DNA operon encoding for the ribosomal RNA (rDNA). The two molecular marker sequences indicated that this strain could not be attributed to any known species and it was described as the type strain of Ogatea uvarum sp.nov. Moreover, the molecular assays showed several secondary peaks in the ITS2 sequence, but not in the LSU D1/D2. In the aim to test whether these peaks were due to the internal heterogeneity of the DNA operon encoding for the rDNA, the two domains themselves and the clones from them derived after PCR amplification were sequenced. The analyses on the internal variants of ITS and LSU showed a significant variability, although within that predictable among different strains of the same yeast species. In this Ogatea uvarum Sp. Nov., ITS was more variable than LSU especially in the ITS2 region. The heterogeneity revealed by this strain was then judged in the frame of its potential consequence in NGS-based environmental metagenomic studies, in which the variability among operons can lead to biodiversity overestimation and to incorrect identification at the species level. The above findings are discussed in the light of the diverse analytical approaches for fungi identification based on sequence similarity. The results of this study show that the internal variability of the rDNA operon requires careful consideration before being used in future NGS metagenomic investigations and emphasizes the need of specific models to interpret the concept of fungal species, when the reproductive barriers represented by exclusively sexual reproduction are not present.

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.

Traditional vinification process is undertaken with the inoculation of the lactic acid bacteria (LAB) at the end of alcoholic fermentation (AF) to induce malolactic fermentation (MLF). MLF is an important phase during winemaking and the LAB co-inoculation with yeast starter represents a promising approach to enhance the quality and safety of wine. In this investigation we have studied: i) the effect of timing of LAB inoculation on the vinification dynamics and chemical features of Negroamaro wines; ii) the interactions between two commercial yeast and two commercial Oenococcus oeni strains. The fermentations dynamics were monitored by microbial counts, quantifying L-malic acid concentration and analyzing the volatile compounds contents in the obtained wines. Our results indicate that simultaneous yeasts/bacteria inoculation at the beginning of vinification reduces the processes duration and simultaneously lowers of volatile acidity. Wine obtained after co-inoculum showed a profile dominated by red and ripe fruits notes associated to esters and to buttery and creamy notes linked to diethyl succinate and ethyl lactate. Furthermore, compatibility specification between commercial yeasts and LAB strains were observed, suggesting the importance of the assessment of microbial-compatibility before their utilization in large-scale vinification.

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

Brettanomyces bruxellensis is a common and significant wine spoilage microorganism. B.bruxellensis strains generally detain the molecular basis to produce compounds that are detrimental for the organoleptic quality of the wine, including some classes of volatile phenols thatderive from the sequential bioconversion of specific hydroxycinnamic acids such as ferulate and p-coumarate. Although B.bruxellensiscan be detected at any stage of the winemaking process, it is typically isolated at the end of the alcoholic fermentation (AF), before the staring of the spontaneous malolactic fermentation (MLF) or during barrel aging. For this reason, the endemic diffusion ofB.bruxellensisleads to consistenteconomic losses in the wine industry. Considering the interest in reducing sulphur dioxide use during wine-making, in recent years, biological alternatives, such as the use of tailored selected yeast and bacterial strains inoculated to promote AF and MLF, are actively sought as biocontrol agents to avoid the 'Bretta' character in wines. Here we review the importance of dedicated characterization and selection of starter cultures for AF and MLF in wine, in order to reduce or prevent both growth of B. bruxellensis and its production of volatile phenols in the matrix.

The winemaking process includes two traditional steps performed by microorganisms, alcoholic fermentation (AF) and malolactic fermentation (MLF). The AF is essential; sugars from grape must are converted into ethanol. This fermentation is carried out by yeast, mainly by Saccharomyces cerevisiae, however is not the only yeast involved in the process. A few specific non-Saccharomyces species, as Hanseniaspora uvarum have been proved to positively modify the wine chemical composition, especially contribute to the sensory characteristics of wines [1]. Interactions between different microbial populations present in grape must are interesting from the point of view of their impact on the final quality of the wine. The main interaction occurs among Saccharomyces spp. yeasts, non-Saccharomyces yeasts, and lactic acid bacteria (LAB) [2]. In the light of the rising request for autochthonous starters tailored for given 'terroir'[3], the objective of this work was to select the best inoculation time of autochthonous of Oenococcus oeni strains in combination with two S. cerevisiae strains and one H. uvarum strain isolated from Apulian wines. Direct interactions were also investigated monitoring cell viability on specific solid culture media. Microvinifications were performed using must from Apulian autochthonous grape varieties. Both, yeasts S. cerevisiae and H. uvarum, were co-inoculated and O. oeni strains were co-inoculated or sequentially inoculated during AF, when ethanol content was 2%, 4%, 6%, 8%, 10% or 12% (v/v). Ethanol formation, malic acid consumption and cell viability were monitored during the vinifications. This research was supported by the Apulian Region Project cod. QCBRAJ6 "Biotecnologie degli alimenti per l'innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali - BIOTECA.

The interactions among different microbial populations have interested the wine microbiology community with the final aim of understanding the impact on wines' quality. The main interaction occurs between yeast Saccharomyces cerevisiae, yeast non-Saccharomyces spp. and lactic acid bacteria (LAB) [1]. In last years, there is a special interest in the study of non-Saccharomyces yeasts as they are responsible for much of the aromatic complexity and its presence improves the final organoleptic characteristics of the wine [2]. Interactions between yeasts and LAB have been studied always combining S. cerevisiae with some LAB and in almost all cases Oenococcus oeni, but poor evidences have been reported for interactions between non-Saccharomyces and LAB in the wine environment. Lactobacillus plantarum selected strains have been receiving increasing attention as starter cultures for malolactic fermentation in wine. With the aim to design of mixed culture to perform AF and MLF simultaneously in a short period of time, with complex organoleptic properties, and also to control the indigenous microbiota and to protect wine against spoilage microorganisms, in this work we report the evaluation of the best inoculation time of L. plantarum strains (both commercial and autochthonous Apulian strains) in combination with autochthonous S. cerevisiae strains and non-Saccharomyces (Hanseniaspora sp. and Candida sp.). Interactions were also investigated monitoring cell viability on specific solid culture media. Microvinifications were performed using must from Apulian autochthonous grape varieties. Both, yeasts S. cerevisiae and non-Saccharomyces, were co-inoculated and L. plantarum strains were co-inoculated or sequentially inoculated during AF, when ethanol content was 2%, 4%, 6%, 8%, 10% or 12% (v/v). Ethanol formation, malic acid consumption and cell viability were monitored during the vinifications. This research was supported by the Apulian Region Project "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali (NEWine)".

In the last years, the interactions among different microbial populations have been studied by the scientific community in the aim of understanding their impact on the quality of wines. The main interactions during the vinification process occur among the yeast Saccharomyces cerevisiae, non-Saccharomyces yeasts species and lactic acid bacteria (LAB). A special interest has been dedicated to investigate the role non-Saccharomyces yeasts as they are responsible for much of the aromatic complexity and its presence improves the final organoleptic characteristics of the wine. Concerning LABS, selected strains belonging to Lactobacillus plantarum species have been recently investigated to produce starter cultures for malolactic fermentation in wine. The aim of the present study is to design mixed starter culture to simultaneously carry out Alcoholic fermentation (AF) and malolactic fermentation (MLF), thus obtaining wine denoted by complex organoleptic properties in a shorter period of time. We report the evaluation of the best inoculation time of L. plantarum strains in combination with autochthonous S. cerevisiae strains and non-Saccharomyces (Hanseniaspora sp.). Microvinifications were performed using must derived from Apulian autochthonous grape varieties. Both, yeasts S. cerevisiae and non-Saccharomyces strains were simultaneously or sequentially co-inoculated during the AF with the L. plantarum strains. The sequential co-inoculation was carried out at different point, when the concentration of the produced ethanol was 2%, 4%, 6%, 8%, 10% or 12% (v/v). Ethanol formation, malic acid consumption and cell viability were monitored during the vinifications. The obtained results demonstrated that L. plantarum cell viability after AF was strain-dependent. L. plantarum strains showed the highest viability when they were co-inoculated with S. cerevisiae and H. uvarum strains. This research was supported by the Apulian Region Project "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali (NEWine)".

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.

Malolactic fermentation (MLF) is a secondary fermentation in wine that usually takes place during or at the end of alcoholic fermentation. Lactobacillus plantarum is able to conduct MLF (particularly under high pH conditions and in co-inoculation with yeasts) and some strains are commercially used as MLF starter cultures. Recent evidences suggest a further use of selected L. plantarum strains for the pre-alcoholic acidification of grape must. In this study, we have carried out an integrated (molecular, technological, and biotechnological) characterization of L. plantarum strains isolated from Apulian wines in order to combine the two protechnological features (MLF performances and must acidification aptitudes). Several parameters such as sugar, pH and ethanol tolerance, resistance to lyophilisation and behaviour in grape must were evaluated. Moreover, the expression of stress gene markers was investigated and was linked to the ability of L. plantarum strains to grow and perform MLF. Co-inoculation of Saccharomyces cerevisiae and Lactobacillus plantarum in grape must improve the bacterial adaptation to harsh conditions of wine and reduced total fermentation time. For the first time, we applied a polyphasic approach for the characterization of L. plantarum in reason of the MLF performances. The proposed procedure can be generalized as a standard method for the selection of bacterial resources for the design of MLF starter cultures tailored for high pH must.

To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach.Methods and Results: Oenococcus oeni strains were isolated from Nero di Troia wines undergoing spontaneous MLF. Samples were taken at the end of alcoholic fermentation and during MLF. Wine samples were diluted in a sterile physiological solution and plated on MRS and on modified FT80. Identification of O. oeni strains was performed by a polymerase chain reaction (PCR) experiment using strain-specific primers. Strains were further grouped using a multiplex RAPD-PCR analysis. Then, six strains were inoculated in two winelike media with two different ethanol concentrations (11 and 13% vol D vol) with a view to evaluate their capacity to grow and to perform MLF. In addition, a quantitative PCR (qRT-PCR) approach was adapted to monitor the physiological state of the strains selected. Conclusion: A positive correlation between the malolactic activity performance and the ability to develop and tolerate stress conditions was observed for two selected O. oeni strains.Significance and Impact of the Study: The results reported are useful for the selection of indigenous MLF starter cultures with desired oenological traits from typical regional wines. It should be the base for the improvement in organoleptic quality of typical red wine.

Il processo tradizionale di produzione dei vini rossi prevede lo svolgersi consecutivo di due processi fermentativi, ovvero la fermentazione alcolica (FA) e la fermentazione malolattica (FML). La FA viene naturalmente condotta da differenti specie di lieviti, tra le quali la principale è Saccharomyces cerevisiae. Tuttavia quest'ultima non è l'unica specie di interesse protecnologico associata al processo. Negli ultimi anni, infatti, si registra un crescente interesse del mercato per ceppi selezionati di lieviti non-Saccharomyces, in ragione del loro potenziale contributo alla qualità enologica e alla complessità aromatica del vino. La FML è promossa da batteri lattici, principalmente appartenenti alle specie Oenococcus oeni e Lactobacillus plantarum. Anche per questi ultimi il mercato dei ceppi selezionati è in continua evoluzione, sebbene la FML controllata sia nel complesso più difficile da gestire in cantina. E' quindi altamente desiderabile poter disporre di formulazioni di starter misti allo scopo di ottenere il simultaneo svolgimento della FA e FML, massimizzando la tipicità delle produzioni e diminuendo il tempo di produzione. A tale scopo, nell'abito di questa ricerca è stato effettuato uno studio della tempistica di inoculazione di ceppi autoctoni malolattici in associazione con saccaromiceti autoctoni e non-Saccharomyces. Le microvinificazioni sono state eseguite utilizzando mosto ottenuto da vitigni autoctoni pugliesi (Negroamaro). La FA è stata promossa mediante inoculazione contemporanea di lieviti S. cerevisiae e non-Saccharomyces autoctoni. Quindi, due ceppi autoctoni di O. oeni e L. plantarum sono stati inoculati sequenzialmente alla coppia di lieviti starter, in mosto in fermentazione con contenuto di etanolo pari a 2%, 4%, 6%, 8%, 10% o 12 % (v /v). Le vinificazioni sperimentali sono stati monitorati giornalmente monitorate e sono stati analizzati una serie di parametri chimici e microbiologici quali la formazione di etanolo, il consumo di acido malico e la vitalità cellulare. I risultati ottenuti e le loro implicazioni sull'utilizzo di starter microbici misti per la produzione industriale di vini tipici in Puglia saranno discussi.Questa ricerca è stata supportata dal Progetto Regione Puglia "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali" (NEWINE).

The Italian group of wine microbiology (GMV) was reconstituted in 2014 with a mission to collect all the skills concerning wine making. The partners of the group GMV are 19 universities and two research centers. The main purpose is to build a benchmark for science and wine industry, able to offer applicative solutions and disseminate the numerous activities carried out by different Italian research centers on issues of "Wine Microbiology". At present, the group is working both for research dissemination, with the publication of joint articles in the journal Frontiers in Microbiology, and for a common experiment involving 17 partners. The purpose of this experiment is to validate a synthetic medium, similar to grape must, for the characterization of wine strains belonging to Saccharomyces cerevisiae species. For this reason, a common protocol has been developed in order to assess its effectiveness to obtain reproducible and statistically valid results. Furthermore, this multicenter experiment will assess the extent of experimental differences due to the fermentation in different laboratories with the same strain. The development of a validated medium and of the confidential limits for fermentation data are expected to improve the comparison of experimental data obtained in different centers and to obtain a shared protocol for wine strainevaluation. The first preliminary results will be elucidated and discussed.

In oenology, the utilization of mixed starter cultures composed by Saccharomyces and non-Saccharomyces yeasts is an approach of growing importance for winemakers in order to enhance sensory quality and complexity of the final product without compromising the general quality and safety of the oenological products. In fact, several non-Saccharomyces yeasts are already commercialized as oenological starter cultures to be used in combination with Saccharomyces cerevisiae, while several others are the subject of various studies to evaluate their application. Our aim, in this study was to assess, for the first time, the oenological potential of H. uvarum in mixed cultures (co-inoculation) and sequential inoculation with S. cerevisiae for industrial wine production. Three previously characterized H. uvarum strains were separately used as multi-starter together with an autochthonous S. cerevisiae starter culture in lab-scale micro-vinification trials. On the basis of microbial development, fermentation kinetics and secondary compounds formation, the strain H. uvarum ITEM8795 was further selected and it was co- and sequentially inoculated, jointly with the S. cerevisiae starter, in a pilot scale wine production. The fermentation course and the quality of final product indicated that the co-inoculation was the better performing modality of inoculum. The above results were finally validated by performing an industrial scale vinification The mixed starter was able to successfully dominate the different stages of the fermentation process and the H. uvarum strain ITEM8795 contributed to increasing the wine organoleptic quality and to simultaneously reduce the volatile acidity. At the best of our knowledge, the present report is the first study regarding the utilization of a selected H. uvarum strain in multi-starter inoculation with S. cerevisiae for the industrial production of a wine. In addition, we demonstrated, at an industrial scale, the importance of non-Saccharomyces in the design of tailored starter cultures for typical wines.

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

Ochratoxin A (OTA) is a nephrotoxic and potentially carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium, contaminating grapes, wine and a variety of food products.We recently isolated from OTA contaminated soil vineyard a novel free-livingstrain of Acinetobacter sp. neg1, ITEM 17016, able to degrade OTA into the nontoxic catabolic product OTalpha (OT?). Biochemical studies suggested that the degradation reaction proceeds via peptide bond hydrolysis with phenylalanine (Phe) release. In order to identify genes responsible for OTA degradationwe performed a differential gene expression analysis of ITEM 17016 grown in the presence or absence of the toxin. Among the differentially expressed genes, 6 peptidases up-regulated at 6 hours were identified. The degrading activity of the carboxypeptidase PJ_1540 was confirmed in vitro in a heterologous system. The enrichment analysis for Gene Ontology terms confirmed that OTA degradation proceeds through peptidase activities and revealed the over-representation of pathways related to Phe catabolism. These results indicate that Phe may represent an energy source for this Acinetobacter sp. neg1strain and that OTA degrading reaction triggers the modulation of further catabolic activities.

Lactobacillus plantarum is one of the most versatile species extensively used in the food industry both as microbial starters and probiotic microorganisms. Several L. plantarum strains have been shown to produce different antimicrobial compounds such as organic acids, hydrogen peroxide, diacetyl, and also bacteriocins and antimicrobial peptides, both denoted by a variable spectrum of action. In the recent decades, the selection of microbial molecules and/or bacterial strains able to produce antagonistic molecules to be used as antimicrobials and preservatives has been attracting scientific interest, in order to eliminate or reduce chemical additives, because of the growing attention of consumers for healthy and natural food products. The aim of this work was to investigate the antimicrobial activity of several food-isolated L. plantarum strains, analyzed against the pathogenic bacteria Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7 and Staphylococcus aureus. Antagonistic activity was assayed by agar spot test and revealed that strain L. plantarum 105 had the strongest ability to contrast the growth of L. monocytogenes, while strains L. plantarum 106 and 107 were the most active microorganisms against E. coli O157:H7. The antimicrobial ability was also screened by well diffusion assay and broth micro-dilution method using cell-free supernatants (CFS) from each Lactobacillus strain. Moreover, the chemical nature of the molecules released in the CFS, and possibly underlying the antagonistic activity, was preliminary characterized by exposure to different constraints such as pH neutralization, heating, catalase and proteinase treatments. Our data suggest that the ability of L. plantarum cultures to contrast pathogens growth in vitro depends, at least in part, on a pH-lowering effect of supernatants and/or on the presence of organic acids. Cluster analysis was performed in order to group L. plantarum strains according to their antimicrobial effect. This study emphasizes the tempting use of the tested L. plantarum strains and/or their CFS as antimicrobial agents against food-borne pathogens.

The spoilage potential of Brettanomyces bruxellensis in wine is strongly connected with the aptitude ofthis yeast to enter in a Viable But Non Culturable (VBNC) state when exposed to the harsh wineconditions. In this work, we characterized the VBNC behaviour of seven strains of B. bruxellensisrepresenting a regional intraspecific biodiversity, reporting conclusive evidence for the assessment ofVBNC as a strain-dependent character. The VBNC behaviour was monitored by fluorescein diacetatestaining/flow cytometry for eleven days after addition of 0.4, 0.6, 0.8, 1 and 1.2 mg/L of molecular SO2(entrance in the VBNC state) and after SO2 removal (exit from the VBNC state). Furthermore, onerepresentative strain was selected and RNA-seq analysis performed after exposure to 1.2 mg/L SO2 andduring the recovery phase. 30 and 1634 genes were identified as differentially expressed followingVBNC entrance and 'resuscitation', respectively. The results reported strongly suggested that theentrance in the SO2-induced VBNC state in B. bruxellensis is associated with both, sulfite toxicity andoxidative stress response, confirming the crucial role of genes/proteins involved in redox cell homeostasis. Among the genes induced during recovery, the expression of genes involved in carbohydrate metabolism and encoding heat shock proteins, as well as enriched categories including amino acid transport and transporter activity was observed. The evidences of a general repression of genes involved in DNA replication suggest the occurrence of a true resuscitation of cell rather than a simple regrowth.

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.