Luciana DE VERO - personale UniMoRe

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Luciana DE VERO

Personale tecnico amministrativo
Dipartimento di Scienze della Vita sede ex-Agraria

Pubblicazioni

2023 - A Microbial Co-Culturing System for Producing Cellulose-Hyaluronic Acid Composites [Articolo su rivista]
Brugnoli, M.; Mazzini, I.; La China, S.; De Vero, L.; Gullo, M.
abstract

In this study, a co-culture system combining bacterial cellulose (BC) producers and hyaluronic acid (HA) producers was developed for four different combinations. AAB of the genus Komagataeibacter sp. and LAB of the Lactocaseibacillus genus were used to produce BC and HA, respectively. Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction were used to investigate changes in BC-HA composites chemical and morphological structure. Water absorption, uptake, and antibacterial properties were also tested. Outcomes highlighted a higher bacterial cellulose yield and the incorporation of hyaluronic acid into the composite. The presence of hyaluronic acid increased fiber dimension-nearly doubled for some combinations-which led to a decreased crystallinity of the composites. Different results were observed based on the BC producer and HA producer combination. However, water holding capacity (WHC) in all the samples improved with the presence of HA, while water uptake worsened. A thymol-enriched BC-HA composite showed high antibacterial activity against Escherichia coli DSM 30083(T) and Staphylococcus aureus DSM 20231(T). Results could contribute to opening new applications in the cosmetics or pharmaceutical fields.

2023 - Exploring the Inhibitory Activity of Selected Lactic Acid Bacteria against Bread Rope Spoilage Agents [Articolo su rivista]
Iosca, Giovanna; Ivy Irorita Fugaban, Joanna; Özmerih, Süleyman; Peter Wätjen, Anders; Sommer Kaas, Rolf; Hà, Quốc; Shetty, Radhakrishna; Pulvirenti, Andrea; DE VERO, Luciana; Heiner Bang-Berthelsen, Claus
abstract

2023 - Leuconostoc performance in soy-based fermentations – Survival, acidification, sugar metabolism, and flavor comparisons [Articolo su rivista]
Wätjen, A. P.; De Vero, L.; Carmona, E. N.; Sberveglieri, V.; Huang, W.; Turner, M. S.; Bang-Berthelsen, C. H.
abstract

Leuconostoc spp. is often regarded as the flavor producer, responsible for the production of acetoin and diacetyl in dairy cheese. In this study, we investigate seven plant-derived Leuconostoc strains, covering four species, in their potential as a lyophilized starter culture for flavor production in fermented soy-based cheese alternatives. We show that the process of lyophilization of Leuconostoc can be feasible using a soy-based lyoprotectant, with survivability up to 63% during long term storage. Furthermore, the storage in this media improves the subse-quent growth in a soy-based substrate in a strain specific manner. The utilization of individual raffinose family oligosaccharides was strain dependent, with Leuconostoc pseudomesenteroides NFICC99 being the best consumer. Furthermore, we show that all investigated strains were able to produce a range of volatile flavor compounds found in dairy cheese products, as well as remove certain dairy off-flavors from the soy-based substrate like hexanal and 2-pentylfuran. Also here, NFICC99 was strain producing most cheese-related volatile flavor com-pounds, followed by Leuconostoc mesenteroides NFICC319. These findings provide initial insights into the development of Leuconostoc as a potential starter culture for plant-based dairy alternatives, as well as a promising approach for generation of stable, lyophilized cultures.

2023 - Valorization of wheat bread waste and cheese whey through cultivation of lactic acid bacteria for bio-preservation of bakery products [Articolo su rivista]
Iosca, Giovanna; Turetta, Monica; De Vero, Luciana; Bang-Berthelsen, Claus Heiner; Gullo, Maria; Pulvirenti, Andrea
abstract

In this work, three lactic acid bacteria (LAB) strains, specifically, Lactiplantibacillus plantarum UMCC 2996, Furfurilactobacillus rossiae UMCC 3002, and Pediococcus pentosaceus UMCC 3010, were tested in new bread-whey media composed by wheat bread and sweet cheese whey, designed as an alternative to the conventional MRS medium. The medium resulting from hydrolysis with amylase and neutrase (AN) was considered the best for the growth of all the strains. This medium was particularly optimal for the strain F. rossiae UMCC 3002, which showed an increase in growth of 114% compared to that in MRS medium. Additionally, the bio-preservative ability of the selected LAB was assessed in pectin-based coated sliced bread inoculated with Aspergillus flavus ITEM 7828, Penicillium paneum ITEM 1381, and Aspergillus niger ITEM 7090. Different LAB strain behavior was observed towards the specific molds. A good bio-preservation activity was shown from F. rossiae UMCC 3002 against A. flavus ITEM 7828 with results compared to the protection by ethanol treatment. The results obtained in this study suggest a novel strategy for the cultivation of selected starters with a bio-protection activity by valorizing bread waste and cheese whey by-products, in a circular economy perspective.

2023 - Zero- and Low-Alcohol Fermented Beverages: A Perspective for Non-Conventional Healthy and Sustainable Production from Red Fruits [Articolo su rivista]
Brugnoli, Marcello; Cantadori, Elsa; Arena, Mattia Pia; DE VERO, Luciana; Colonello, Andrea; Gullo, Maria
abstract

The growing health consciousness among consumers is leading to an increased presence of functional foods and beverages on the market. Red fruits are rich in bioactive compounds such as anthocyanins with high antioxidant activity. In addition, red fruits contain sugars and are rich in phenolic compounds, vitamin C, dietary fibers, and manganese. Due to these characteristics, they are also suitable substrates for fermentation. Indeed, nowadays, microbial transformation of red fruits is based on alcoholic or lactic fermentation, producing alcoholic and non-alcoholic products, respectively. Although products fermented by acetic acid bacteria (AAB) have been thoroughly studied as a model of health benefits for human beings, little evidence is available on the acetic and gluconic fermentation of red fruits for obtaining functional products. Accordingly, this review aims to explore the potential of different red fruits, namely blackberry, raspberry, and blackcurrant, as raw materials for fermentation processes aimed at producing low- and no-alcohol beverages containing bioactive compounds and no added sugars. AAB are treated with a focus on their ability to produce acetic acid, gluconic acid, and bacterial cellulose, which are compounds of interest for developing fruit-based fermented beverages.

2022 - Anti-Spoilage Activity and Exopolysaccharides Production by Selected Lactic Acid Bacteria [Articolo su rivista]
Iosca, Giovanna; De Vero, Luciana; Di Rocco, Giulia; Perrone, Giancarlo; Gullo, Maria; Pulvirenti, Andrea
abstract

In this study, eight lactic acid bacteria (LAB) strains, previously isolated from traditional and gluten-free sourdoughs, and selected for their potential in improving the sensory and rheological quality of bakery products, were screened against some common spoilage agents. The anti-mould activity was tested using strains of the species Fusarium graminearum, Aspergillus flavus, Penicillium paneum and Aspergillus niger. Regarding the antibacterial activity, it was assessed against four strains of the species Escherichia coli, Campylobacter jejuni, Salmonella typhimurium and Listeria monocytogenes. Furthermore, LAB strains were evaluated for their ability to produce exopolysaccharides, which are gaining considerable attention for their functional properties and applicability in different food industrial applications. A strain-specific behaviour against the moulds was observed. In particular, F. graminearum ITEM 5356 was completely inhibited by all the LAB strains. Regarding the antibacterial activity, the strains Leuconostoc citreum UMCC 3011, Lactiplantibacillus plantarum UMCC 2996, and Pediococcus pentosaceus UMCC 3010 showed wide activity against the tested pathogens. Moreover, all the LAB strains were able to produce exopolysaccharides, which were preliminarily characterized. The assessed features of the LAB strains allow us to consider them as promising candidates for single or multiple starter cultures for food fermentation processes.

2022 - Batteri acetici [Capitolo/Saggio]
Gullo, Maria; De Vero, Luciana
abstract

2022 - Candidate Acetic Acid Bacteria Strains for Levan Production [Articolo su rivista]
Anguluri, Kavitha; LA CHINA, Salvatore; Brugnoli, Marcello; DE VERO, Luciana; Pulvirenti, Andrea; Cassanelli, Stefano; Gullo, Maria
abstract

In this study, twelve strains of acetic acid bacteria (AAB) belonging to five different genera were tested for their ability to produce levan, at 70 and 250 g/L of sucrose concentration, respectively. The fructan produced by the bacterial strains was characterized as levan by NMR spectroscopy. Most of the strains produced levan, highlighting intra- and inter-species variability. High yield was observed for Neoasaia chiangmaiensis NBRC 101099 T, Kozakia baliensis DSM 14400 T and Gluconobacter cerinus DSM 9533 T at 70 g/L of sucrose. A 12-fold increase was observed for N. chiangmaiensis NBRC 101099 T at 250 g/L of sucrose concentration. Levan production was found to be affected by glucose accumulation and pH reduction, especially in Ko. baliensis DSM 14400 T. All the Gluconobacter strains showed a negative correlation with the increase in sucrose concentration. Among strains of Komagataeibacter genus, no clear effect of sucrose on levan yield was found. Results obtained in this study highlighted the differences in levan yield among AAB strains and showed interdependence between culture conditions, carbon source utilization, and time of incubation. On the contrary, the levan yield was not always related to the sucrose concentration.

2022 - I batteri acetici [Capitolo/Saggio]
Gullo, Maria; De Vero, Luciana
abstract

2022 - L'aceto [Capitolo/Saggio]
Gullo, Maria; De Vero, Luciana
abstract

2021 - A Mathematical Modeling of Freezing Process in the Batch Production of Ice Cream [Articolo su rivista]
Giudici, Paolo; Baiano, Antonietta; Chiari, Paola; De Vero, Luciana; Ghanbarzadeh, Babak; Massimiliano Falcone, Pasquale
abstract

This study deals with the mathematical modeling of crystallization kinetics occurring during batch production of the ice cream. The temperature decrease was recorded in-situ through a computerized wireless system. A robust pattern-recognition algorithm of the experimental cooling curves was developed to determine the initial freezing point. The theoretical freezing point was used to calibrate the whole time-temperature profile. Finally, a modified Gompertz’s function was used to describe the main steps of crystallization kinetics. Derivative analysis of the Gompertz’s function allowed to determine the time-temperature physical markers of dynamic nucleation, ice crystal growth and air whipping. Composition and freezing properties were used as input variables in multivariate analysis to classification purposes of the ice cream mixtures as a function of their ability to produce high-quality ice cream. The numerical analysis of the whole cooling curve was used to build predictive models of the ice cream quality indices.

2021 - Design of a New Fermented Beverage from Medicinal Plantsand Organic Sugarcane Molasses via Lactic Fermentation. [Articolo su rivista]
Gadhoumi, Hamza; Gullo, Maria; DE VERO, Luciana; Martinez-Rojas, Enriqueta; Saidani Tounsi, Moufida; Akrem Hayouni, El
abstract

Functional beverages obtained using medicinal plants and fermented with lactic acidbacteria are gaining much interest from the scientific community, driven by the growing demand forfood and beverages with beneficial properties. In this work, three different batches of medicinal plantsand organic sugarcane molasses, named FB-lc, FB-sp and FB-lcsp, were prepared and fermented byusingLactobacillus acidophilusATCC 43121,Bifidobacterium breveB632 and a mix of both strains’ culture,respectively. The three fermented beverages revealed a high level of polyphenols (expressed as gallicacid equivalent), ranging from 182.50 to 315.62μg/mL. The highest content of flavonoids (152.13μgquercetin equivalent/mL) and tannins (93.602μg catechin equivalent/mL) was detected in FB-lcsptrial. The IR spectroscopy analysis showed a decrease in sugar (pyranose forms, D-glucopyranoseand rhamnosides). In addition, the aromatic compounds of the fermented beverages, detected byGC-MS headspace analysis, showed twenty-four interesting volatile compounds, which could givepositive aroma attributes to the flavor of the beverages. The highest antioxidant activity was observedin the beverage obtained by the mix culture strains. Accordingly, the production of these beveragescan be further investigated for considering their well-being effects on human health.

2021 - Functional and Healthy Features of Conventional and Non-Conventional Sourdoughs [Articolo su rivista]
De Vero, Luciana; Iosca, Giovanna; Gullo, Maria; Pulvirenti, Andrea
abstract

Sourdough is a composite ecosystem largely characterized by yeasts and lactic acid bacteria which are the main players in the fermentation process. The specific strains involved are influenced by several factors including the chemical and enzyme composition of the flour and the sourdough production technology. For many decades the scientific community has explored the microbiological, biochemical, technological and nutritional potential of sourdoughs. Traditionally, sourdoughs have been used to improve the organoleptic properties, texture, digestibility, palatability, and safety of bread and other kinds of baked products. Recently, novel sourdough-based biotechnological applications have been proposed to meet the demand of consumers for healthier and more natural food and offer new inputs for the food industry. Many researchers have focused on the beneficial effects of specific enzymatic activities or compounds, such as exopolysaccharides, with both technological and functional roles. Additionally, many studies have explored the ability of sourdough lactic acid bacteria to produce antifungal compounds for use as bio-preservatives. This review provides an overview of the fundamental features of sourdoughs and their exploitation to develop high value-added products with beneficial microorganisms and/or their metabolites, which can positively impact human health.

2021 - Kombucha Tea as a Reservoir of Cellulose Producing Bacteria: Assessing Diversity among Komagataeibacter Isolates [Articolo su rivista]
LA CHINA, Salvatore; DE VERO, Luciana; Anguluri, Kavitha; Brugnoli, Marcello; Mamlouk, Dhouha; Gullo, Maria
abstract

Bacterial cellulose (BC) is receiving a great deal of attention due to its unique properties such as high purity, water retention capacity, high mechanical strength, and biocompatibility. However, the production of BC has been limited because of the associated high costs and low productivity. In light of this, the isolation of new BC producing bacteria and the selection of highly productive strains has become a prominent issue. Kombucha tea is a fermented beverage in which the bacteria fraction of the microbial community is composed mostly of strains belonging to the genus Komagataeibacter. In this study, Kombucha tea production trials were performed starting from a previous batch, and bacterial isolation was conducted along cultivation time. From the whole microbial pool, 46 isolates were tested for their ability to produce BC. The obtained BC yield ranged from 0.59 g/L, for the isolate K2G36, to 23 g/L for K2G30—which used as the reference strain. The genetic intraspecific diversity of the 46 isolates was investigated using two repetitive-sequence-based PCR typing methods: the enterobacterial repetitive intergenic consensus (ERIC) elements and the (GTG)5 sequences, respectively. The results obtained using the two different approaches revealed the suitability of the fingerprint techniques, showing a discrimination power, calculated as the D index, of 0.94 for (GTG)5 rep-PCR and 0.95 for ERIC rep-PCR. In order to improve the sensitivity of the applied method, a combined model for the two genotyping experiments was performed, allowing for the ability to discriminate among strains.

2021 - Selection of Promising Exopolysaccharide-Producing Starter Cultures for Gluten-Free Sourdough [Abstract in Rivista]
Iosca, Giovanna; Febo, Camilla; Haghighi, Hossein; De Vero, Luciana; Gullo, Maria; Pulvirenti, Andrea
abstract

Celiac disease is an autoimmune enteropathy that affects the small intestine and is caused by the gliadin fraction of wheat gluten and other alcohol-soluble proteins (prolamines) of barley and rye in genetically predisposed subjects. Nowadays, the only recognized cure for this pathology is the use of gluten-free (GF) products. Although the market for GF products has grown in the last few years, their improvement is still a challenge for the food industry. With regard to GF bakery products, some of them can result in an unappetizing taste with a high fat content. These inconveniences can be minimized or solved by using sourdough fermentation. Sourdough is a stable culture of lactic acid bacteria (LAB) and yeast in a mixture of flour and water. In particular, GF sourdough represents a rich source of naturally occurring LAB and wild yeasts, which play important roles in food fermentation. This is mainly due to their potential in improving functional, technological, and probiotics properties, contributing to safe food with a better taste. Moreover, exopolysaccharide (EPS)-producing strains can also contribute to improving the sensory and rheological quality of the different GF products. This study aims to isolate and characterize the microbial pool from a GF sourdough made with rice flour and to evaluate the ability of selected LAB strains to produce exopolysaccharides (EPS). The yeast and LAB were isolated in selective agar media. A total of 220 isolates (110 yeasts and 110 bacteria) were obtained. They were taxonomically identified and characterized using both traditional and molecular approaches. Sequencing analysis of the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, for yeasts and bacteria, respectively, revealed the occurrence of Saccharomyces cerevisiae as predominant yeasts and Lactiplantibacillus plantarum and Pediococcus pentosaceus as dominant LAB. Regarding the EPS production, 25 strains were able to produce dextran-like EPS with a maximum yield of 2 g/L. This study allowed the isolation and selection of promising EPS-producing starter cultures for the different types of flour sourdough, in particular, GF sourdough

2021 - Yeasts and Lactic Acid Bacteria for Panettone Production: An Assessment of Candidate Strains [Articolo su rivista]
De Vero, Luciana; Iosca, Giovanna; La China, Salvatore; Licciardello, Fabio; Gullo, Maria; Pulvirenti, Andrea
abstract

The recovery of yeasts and lactic acid bacteria (LAB) involved in sourdough fermentation is the first step in the selection of starters with suitable technological aptitude and capable of producing desired aromas and/or aromatic precursors. In this work, two sourdoughs samples (MA and MB) and the derived doughs (samples A and B) were collected from a bakery during artisanal Panettone manufacture. Yeasts and bacteria were isolated at different fermentation steps on selective agar media. A total of 77 isolates were obtained and characterized. Representative strains of yeasts and LAB were identified by sequencing the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, respectively. Moreover, the volatile organic compounds (VOCs) produced in the collected samples were detected and correlated to the species found in the same samples. The results highlighted the occurrence of Kazachstania humilis in both samples A and B, while Saccharomyces cerevisiae strains were detected only in samples B. Among LAB, Fructilactobacillus sanfranciscensis was the main species detected in both sourdoughs. Furthermore, strains belonging to the species Lactiplantibacillus plantarum, Furfurilactobacillus rossiae, Lactobacillus parabuchneri, Leuconostoc citreum, and Leuconostoc mesenteroides were assessed in the dough samples.

2020 - Exploring the Microbial Community of Traditional Sourdoughs to Select Yeasts and Lactic Acid Bacteria [Articolo su rivista]
Iosca, Giovanna; Vero, Luciana De; Gullo, Maria; Licciardello, Fabio; Quartieri, Andrea; Pulvirenti, Andrea
abstract

Sourdoughs represent an awesome example of ecosystem in which yeasts and lactic acid bacteria (LAB) interact with each other, defining the characteristics of the final product in terms of composition, texture, taste and flavor. Therefore, the identification of dominant yeasts and LAB involved in the fermentation process can lead to the selection of starters with suitable fermentation aptitude and capable of producing desired aromas and/or aromatic precursors. In this work, two sourdoughs samples (A and B) for Panettone production were collected from an artisan bakery. Yeasts and bacteria were isolated at different fermentation steps on selective agar media. A total of 120 isolates were obtained and firstly characterized by conventional microbiological methods. Afterward, genomic DNA was extracted from the cultures, and (GTG)5-PCR fingerprinting analysis was carried out to reduce the redundance among the isolates. Representative yeasts and LAB strains, having a unique profile, were identified by sequencing the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, respectively. The results highlighted the occurrence of Kazachstania humilis and Fructilactobacillus sanfranciscensis in both sourdoughs. Among LAB, also some other strains belonging to Lactobacillus genus were found. Moreover, Saccharomyces cerevisiae and Staphylococcus spp. strains were detected in sample B. In this study, a pool of yeasts and LAB strains for producing starter cultures with specific technological traits for sourdoughs production was obtained.

2020 - Selection of Wine Saccharomyces cerevisiae Strains and Their Screening for the Adsorption Activity of Pigments, Phenolics and Ochratoxin A [Articolo su rivista]
Pulvirenti, Andrea; De Vero, Luciana; Blaiotta, Giuseppe; Sidari, Rossana; Iosca, Giovanna; Gullo, Maria; Caridi, Andrea
abstract

Ochratoxin A is a dangerous mycotoxin present in wines and is considered the principal safety hazard in the winemaking process. Several authors have investigated the ochratoxin A adsorption ability of Saccharomyces cerevisiae yeasts, and specifically selected strains for this desired trait. In the present work, a huge selection of wine yeasts was done starting from Portuguese, Spanish and Italian fermenting musts of dierent cultivars. Firstly, 150 isolates were collected, and 99 non-redundant S. cerevisiae strains were identified. Then, the strains were screened following a multi-step approach in order to select those having primary oenological traits, mainly (a) good fermentation performance, (b) low production of H2S and (c) low production of acetic acid. The preselected strains were further investigated for their adsorption activity of pigments, phenolic compounds and ochratoxin A. Finally, 10 strains showed the desired features. The goal of this work was to select the strains capable of absorbing ochratoxin A but not pigments and phenolic compounds in order to improve and valorise both the quality and safety of red wines. The selected strains are considered good candidates for wine starters, moreover, they can be exploited to obtain a further enhancement of the specific adsorption/non-adsorption activity by applying a yeast breeding approach.

2020 - Vinegar production from Citrus bergamia by-products and preservation of bioactive compounds [Articolo su rivista]
Di Donna, Leonardo; Bartella, Lucia; De Vero, Luciana; Gullo, Maria; Giuffrè, Angelo M.; Zappia, Clotilde; Capocasale, Marco; Poiana, Marco; D’Urso, Silvia; Caridi, Andrea
abstract

Processing citrus fruits results in significant amount of wastes; however, citrus by-products still contain high amounts of polyphenolic and other bioactive value-added compounds. In addition, bergamot (Citrus bergamia) wastes contain two compounds—brutieridin and melitidin—which exhibit statin-like properties. Recently, novel fermented products containing bioactive compounds received increasing attention because of their health-promoting functions. In this study, the bioconversion of citrus wastes in vinegars with high content in bioactive and aromatic compounds was performed, detecting a high permanence at the end of the process of the main compounds of interest, especially brutieridin and melitidin. In addition, the sensory analysis of the vinegars was performed, obtaining good performances. According to the adopted preselection procedure, 50 out of the 54 strains of acetic acid bacteria were excluded because of their low aptitude to grow in the tested conditions. The best vinegar was produced from citrus wine at pH 2.90 using the Acetobacter aceti strain DSM_3508T as microbial starter. This research has demonstrated—for the first time—the possibility to produce citrus vinegar at high content of brutieridin and melitidin and other bioactive compounds using selected microbial starters.

2019 - Preservation, Characterization and Exploitation of Microbial Biodiversity: The Perspective of the Italian Network of Culture Collections [Articolo su rivista]
De Vero, Luciana; Boniotti, Maria Beatrice; Budroni, Marilena; Buzzini, Pietro; Cassanelli, Stefano; Comunian, Roberta; Gullo, Maria; Logrieco, Antonio F.; Mannazzu, Ilaria; Musumeci, Rosario; Perugini, Iolanda; Perrone, Giancarlo; Pulvirenti, Andrea; Romano, Paolo; Turchetti, Benedetta; Varese, Giovanna Cristina
abstract

Microorganisms represent most of the biodiversity of living organisms in every ecological habitat. They have profound effects on the functioning of any ecosystem, and therefore on the health of our planet and of human beings. Moreover, microorganisms are the main protagonists in food, medical and biotech industries, and have several environmental applications. Accordingly, the characterization and preservation of microbial biodiversity are essential not only for the maintenance of natural ecosystems but also for research purposes and biotechnological exploitation. In this context, culture collections (CCs) and microbial biological resource centres (mBRCs) are crucial for the safeguarding and circulation of biological resources, as well as for the progress of life sciences. This review deals with the expertise and services of CCs, in particular concerning preservation and characterization of microbial resources, by pointing to the advanced approaches applied to investigate a huge reservoir of microorganisms. Data sharing and web services as well as the tight interconnection between CCs and the biotechnological industry are highlighted. In addition, guidelines and regulations related to quality management systems (QMSs), biosafety and biosecurity issues are discussed according to the perspectives of CCs and mBRCs.

2018 - A multi-phase approach to select new wine yeast strains with enhanced fermentative fitness and glutathione production [Articolo su rivista]
Bonciani, Tommaso; De Vero, Luciana; Mezzetti, Francesco; Fay, Justin C.; Giudici, Paolo
abstract

The genetic improvement of winemaking yeasts is a virtually infinite process, as the design of new strains must always cope with varied and ever-evolving production contexts. Good wine yeasts must feature both good primary traits, which are related to the overall fermentative fitness of the strain, and secondary traits, which provide accessory features augmenting its technological value. In this context, the superiority of “blind,” genetic improvement techniques, as those based on the direct selection of the desired phenotype without prior knowledge of the genotype, was widely proven. Blind techniques such as adaptive evolution strategies were implemented for the enhancement of many traits of interest in the winemaking field. However, these strategies usually focus on single traits: this possibly leads to genetic tradeoff phenomena, where the selection of enhanced secondary traits might lead to sub-optimal primary fermentation traits. To circumvent this phenomenon, we applied a multi-step and strongly directed genetic improvement strategy aimed at combining a strong fermentative aptitude (primary trait) with an enhanced production of glutathione (secondary trait). We exploited the random genetic recombination associated to a library of 69 monosporic clones of strain UMCC 855 (Saccharomyces cerevisiae) to search for new candidates possessing both traits. This was achieved by consecutively applying three directional selective criteria: molybdate resistance (1), fermentative aptitude (2), and glutathione production (3). The strategy brought to the selection of strain 21T2-D58, which produces a high concentration of glutathione, comparable to that of other glutathione high-producers, still with a much greater fermentative aptitude.

2018 - Lieviti enologici alto-produttori di glutatione ottenuti mediante strategia basata sull'evoluzione adattativa [Abstract in Atti di Convegno]
DE VERO, Luciana; Bonciani, Tommaso; Verspohl, Alexandra; Giudici, Paolo
abstract

Le strategie di evoluzione adattativa per il miglioramento genetico dei lieviti, sono ampiamente utilizzate in campo enologico. I vantaggi di queste strategie sono quelli di non introdurre geni ricombinanti estranei nel genoma dei microrganismi e di non richiedere una pregressa conoscenza dei geni coinvolti nell’espressione dei fenotipi desiderati. Infatti, sottoponendo i microrganismi a colture seriali o continue per diverse generazioni, in presenza di una specifica pressione selettiva, si selezionano i ceppi evoluti esprimenti il carattere d’interesse. Tuttavia, poiché lo screening dei microrganismi evoluti richiede necessariamente l’espressione di fenotipi selezionabili, ossia facilmente riconoscibili, tali strategie non sono applicabili per l’ottenimento di caratteristiche enologiche legate a variazioni e ricombinazioni genetiche non direttamente selezionabili, come nel caso della elevata produzione di glutatione (GSH), o di composti sensorialmente attivi. Ciò nonostante, questo limite è superabile individuando, nella pathway biosintetica del metabolita di interesse, uno o più step in cui applicare una pressione selettiva in modo da selezionare i ceppi evoluti potenzialmente in grado di esprimere il fenotipo desiderato. Questa strategia “evolution-based” è stata recentemente messa a punto e applicata per la selezione di ceppi di lievito alto produttori di GSH (De Vero et al., 2017). La strategia descritta si basa sulla formazione di ricombinanti attraverso la riproduzione sessuale e sulla successiva individuazione dei ricombinanti di interesse attraverso uno screening rapido e altamente selettivo basato sulla resistenza ad elevate concentrazioni di molibdato, tossico per le cellule nella forma ionica esavalente. Infatti, il molibdato applicato come pressione selettiva, consente di selezionare i ceppi evoluti con un metabolismo alterato relativamente alla pathway di assimilazione dei solfati e della biosintesi del GSH. Tale strategia ha permesso di individuare, tra i ceppi evoluti, resistenti a elevate concentrazioni di molibdato, quelli migliorati per la formazione di GSH (Mezzetti et al., 2014, 2017; Bonciani et al., 2018). Reference Bonciani, T., De Vero, L., Mezzetti, F., Fay, J.C. and Giudici, P. (2018) A multi-phase approach to select new wine yeast strains with enhanced fermentative fitness and glutathione production. Appl Microbiol Biotechnol 102, 2269–2278. De Vero, L., Bonciani, T., Verspohl, A., Mezzetti, F. and Giudici, P. (2017) High-glutathione producing yeasts obtained by genetic improvement strategies: a focus on adaptive evolution approaches for novel wine strains. AIMS Microbiol 3, 155–170. Mezzetti, F., De Vero, L. and Giudici, P. (2014) Evolved Saccharomyces cerevisiae wine strains with enhanced glutathione production obtained by an evolution-based strategy. FEMS Yeast Res 14, 977–87. Mezzetti, F., Fay, J.C., Giudici, P. and De Vero, L. (2017) Genetic variation and expression changes associated with molybdate resistance from a glutathione producing wine strain of Saccharomyces cerevisiae. PLoS One 12, e0180814.

2018 - Oxidative fermentations and exopolysaccharides production by acetic acid bacteria: a mini review [Articolo su rivista]
LA CHINA, Salvatore; Zanichelli, Gabriele; DE VERO, Luciana; Gullo, Maria
abstract

Acetic acid bacteria are versatile organisms converting a number of carbon sources into biomolecules of industrial interest. Such properties, together with the need to limit chemical syntheses in favor of more sustainable biological processes, make acetic acid bacteria appropriate organisms for food, chemical, medical, pharmaceutical and engineering applications. At current, well-established bioprocesses by acetic acid bacteria are those derived from the oxidative pathways that lead to organic acids, ketones and sugar derivates. Whereas emerging applications include biopolymers, such as bacterial cellulose and fructans, which are getting an increasing interest for the biotechnological industry. However, considering the industrial demand of high performing bioprocesses, the production yield of metabolites obtained by acetic acid bacteria, is still not satisfying. In this paper we review the major acetic acid bacteria industrial applications, considering the current status of bioprocesses. We will also describe new biotechnological advances in order to optimize the industrial production, offering also an overview on future directions.

2018 - Peptidomic study of casein proteolysis in bovine milk by Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331 [Articolo su rivista]
Solieri, Lisa; DE VERO, Luciana; Tagliazucchi, Davide
abstract

Lactobacilli contain different cell envelope proteinases (CEPs) responsible for the hydrolysis of caseins and the release of various bioactive peptides. In this work, we explored the CEP activity of Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331 whole cells towards β-, αS1-, κ- and αS2-caseins in bovine milk. Mass spectrometry analysis of fermented milk hydrolysates identified a total of 331 peptides, which were mainly derived from β-caseins (59.0 and 60.1% for PRA205 and PRA331, respectively). Analysis of αS1-casein (f1–23) cleavage site specificity congruently supports that Lb. casei PRA205 and Lb. rhamnosus PRA331 exhibited a mixed-type CEPI/III activity. PRA205 and PRA331 CEPs also showed cleavage site specificity toward β-casein. These CEPs cleaved the peptide bond preferentially when hydrophobic or negatively charged amino acids were present. 13.5% and 13.7% of peptides released by Lb. casei PRA205 and Lb. rhamnosus PRA331 CEPs were found to have 100% homology with previously identified bioactive peptides.

2018 - Qualitative and quantitative screening of the β-glucosidase activity in Saccharomyces cerevisiae and Saccharomyces uvarum strains isolated from refrigerated must [Articolo su rivista]
Bonciani, T.; De Vero, L.; Giannuzzi, E.; Verspohl, A.; Giudici, P.
abstract

The aim of the present work was to screen a pool of 75 yeasts belonging to the species Saccharomyces cerevisiae and Saccharomyces uvarum in order to select the strains endowed with β-glucosidase activity. The first screening was a qualitative assay based on chromogenic substrates (arbutin and esculin). The second screening was the quantitative evaluation of the β-glucosidase activity via a p-nitrophenyl-β-d-glucopyranoside assay. The measurement was performed on three different cell preparations, including the extracellular compartment, the cell lysates and the whole cells. This study pointed out the high frequency of β-glucosidase activity in S. uvarum strains. In particular, we retrieved three promising S. uvarum strains, CRY14, VA42 and GRAS14, featuring a high enzymatic activity, exploitable for winemaking. Significance and Impact of the Study: In yeasts, β-glucosidase activity has been extensively described, especially in non-Saccharomyces species, while there is only little evidence of this activity in strains belonging to the Saccharomyces species. In winemaking, β-glucosidase plays essential roles in the hydrolysis of glyco-conjugated precursors and the release of active aromatic compounds. This study provides new insights into the β-glucosidase activity in strains belonging to Saccharomyces cerevisiae and Saccharomyces uvarum species, which are the most important strains in wine industry. Our results point out a marked enzymatic activity for the tested S. uvarum strains. These strains could be exploited for their potential ability to enhance the aroma profiles of wine. In addition, they could be potential sources for the commercial production of enzymes to be applied in winemaking.

2018 - Screening dell’attività β-glucosidasica in ceppi di Saccharomyces spp. isolati da mosto [Abstract in Atti di Convegno]
DE VERO, Luciana; Bonciani, Tommaso; Verspohl, Alexandra; Giudici, Paolo
abstract

Nei lieviti, l'attività della β-glucosidasi è stata ampiamente descritta, specialmente nelle specie non-Saccharomyces, mentre questa attività risulta essere poco evidente nei ceppi appartenente al genere Saccharomyces. In enologia l'attività β-glucosidasica è un carattere di interesse, in quanto correlata al miglioramento dell'espressione aromatica dei vini. Infatti, il suddetto enzima va ad agire sulla classe dei composti aromatici terpenici. Tali composti si trovano sotto forma di coniugati β-glucosidici nelle varietà d'uva aromatiche come Moscato e Gewürztraminer. La forma coniugata è tuttavia non volatile e, conseguentemente, non aromatica. La presente ricerca fornisce nuove conoscenze sull'attività della β-glucosidasi in ceppi di interesse enologico appartenenti alle specie Saccharomyces cerevisiae e Saccharomyces uvarum. In particolare, 75 ceppi, isolati da mosto d’uva refrigerato e appartenenti alle due specie suddette, sono stati valutati relativamente all’attività β-glucosidasica, sia dal punto di vista qualitativo che quantitativo. Lo screening qualitativo in piastra, mediante l’uso di substrati cromogenici, quali esculina e arbutina, ha messo in evidenza un maggior grado di positività dei test provati per i ceppi della specie S. uvarum rispetto a quelli della specie S. cerevisiae. Successivamente, sui ceppi positivi ad entrambi i due precedenti test, è stato condotto un test quantitativo dell’attività β-glucosidasica, mediante il metodo spettrofotomentrico basato sull’idrolisi del p‐nitrophenyl‐β‐D‐glucopyranoside (p‐NPG). La valutazione è stata effettuata su tre diverse frazioni dei campioni analizzati: il surnatante, il lisato cellulare e le cellule intere. I risultati ottenuti hanno messo in evidenza che l'attività enzimatica è principalmente associata alle cellule intere. Inoltre, tre ceppi di S. uvarum, CRY14, VA42 and GRAS14, hanno mostrato una pronunciata attività β-glucosidasica (Bonciani et al., 2018). Questi ceppi potrebbero essere sfruttati per la loro potenziale capacità di migliorare i profili aromatici del vino. Più in generale, lo studio apre un nuovo e promettente campo della ricerca dell'attività β-glucosidasica in ceppi appartenenti alla specie S. uvarum, fin'ora poco esplorata. Reference Bonciani T., De Vero L., Giannuzzi E., Verspohl A. and Giudici P. (2018). Qualitative and quantitative screening of the β‐glucosidase activity in Saccharomyces cerevisiae and Saccharomyces uvarum strains isolated from refrigerated must. Lett. Appl. Microbiol. Accepted Manuscript. doi:10.1111/lam.12891

2017 - Genetic variation and expression changes associated with molybdate resistance from a glutathione producing wine strain of Saccharomyces cerevisiae [Articolo su rivista]
Mezzetti, Francesco; Fay, Justin C; Giudici, Paolo; DE VERO, Luciana
abstract

Glutathione (GSH) production during wine fermentation is a desirable trait as it can limit must and wine oxidation and protect various aromatic compounds. UMCC 2581 is a Saccharomyces cerevisiae wine strain with enhanced GSH content at the end of wine fermentation. This strain was previously derived by selection for molybdate resistance following a sexual cycle of UMCC 855 using an evolution-based strategy. In this study, we examined genetic and gene expression changes associated with the derivation of UMCC 2581. For genetic analysis we sporulated the diploid UMCC 855 parental strain and found four phenotype classes of segregants related to molybdate resistance, demonstrating the presence of segregating variation from the parental strain. Using bulk segregant analysis we mapped molybdate traits to two loci. By sequencing both the parental and evolved strain genomes we identified candidate mutations within the two regions as well as an extra copy of chromosome 1 in UMCC 2581. Combining the mapped loci with gene expression profiles of the evolved and parental strains we identified a number of candidate genes with genetic and/or gene expression changes that could underlie molybdate resistance and increased GSH levels. Our results provide insight into the genetic basis of GSH production relevant to winemaking and highlight the value of enhancing wine strains using existing variation present in wine strains.

2017 - High-glutathione producing yeasts obtained by genetic improvement strategies: a focus on adaptive evolution approaches for novel wine strains [Articolo su rivista]
DE VERO, Luciana; Bonciani, Tommaso; Verspohl, Alexandra; Mezzetti, Francesco; Giudici, Paolo
abstract

Glutathione (GSH) is the most abundant non-protein thiol in living organisms. Due to its important antioxidant role, it is widely used in medicine, as a food additive, and in the cosmetic industry. Recently, GSH has received growing attention in winemaking because of its ability to control oxidative spoilage damage and to protect various aromatic compounds. Indeed, GSH concentration in wine is highly variable and several factors are involved in its regulation, ranging from grape must to yeast fermentation activity. This short review aims at highlighting the common genetic strategies, useful for obtaining wine yeasts with enhanced GSH production, paying particular attention to the adaptive evolution approaches. Moreover, other strategies, such as random mutagenesis, metabolic engineering and hybridization have been briefly reviewed with a stress on both their strengths and weaknesses in terms of actual feasibility and acceptance by wine consumers.

2017 - Preservation of Acetic Acid Bacteria [Capitolo/Saggio]
DE VERO, Luciana; Gullo, Maria; Giudici, Paolo
abstract

Increased knowledge on physiological, biochemical and genetic properties of acetic acid bacteria (AAB) promotes advancements in biotechnology and innovative fermentation processes. Accordingly, the microbial collections of well-characterized AAB strains are fundamental sources for selecting cultures with desired properties for biotechnological applications. Commonly, culture collections have the fundamental role of safeguarding long-term accessibility of relevant strains by ensuring the maintenance of their ‘authenticity’. Nevertheless, they provide more than just strains, but also essential data, expertise and useful services, which are fundamental for supporting industrial researches.

2017 - Role of microbial collections in the selection of starter cultures [Articolo su rivista]
De Vero, L.; Gazzotti, E.; Giudici, P.
abstract

The aim of this study was to characterize, through a polyphasic approach, a pool of 27 strains of lactic acid bacteria (genus Lactobacillus), isolated from salami and deposited in the Unimore (University of Modena and Reggio Emilia) Microbial Culture Collection (UMCC). Moreover, 15 selected strains have been used for a preliminary technological screening in order to implement starter cultures suitable for the production of sausages. The genetic improvement programmes and the selection of starter cultures suitable for specific fermentation processes are some of the main activities of UMCC. As well as the preservation of characterized microbial cultures, UMCC manages the cataloging and sharing of the information on the strains collected by transferring and updating the biological data in an online public database (http://biolomics.umcc.unimore.it).

2017 - Vinegars [Capitolo/Saggio]
Giudici, Paolo; DE VERO, Luciana; Gullo, Maria
abstract

The history of fermentation is strictly linked to vinegar production. Among fermented foods, however, vinegar is recognized as a poor product; in many cases, the raw materials have higher nutritional values than the nal product. Vinegar is generally used as a food avoring, a preservative and a beverage with healthy properties. Acetic acid has several uses in the pharmaceutical, cosmetic, chemical and textile industries, where it is produced via chemical synthesis. Vinegar is the product of double scalar fermentation, which is performed by yeasts and acetic acid bacteria (AAB) from sources of fermentable carbohydrates, such as apples, pears, grape, honey, sap of plants, cereals and hydrolyzed starch. In addition, distilled ethanol from fermented raw materials can be used to produce vinegar. In the latter case, vinegar is referred to as ‘spirit vinegar’ or ‘white vinegar’; its use is widespread in northern Europe and the Americas. In some countries, vinegar is also produced from acetic acid via wood pyrolysis; however, this practice is less common, as the fermentation of agricultural raw material is a priority for international organizations, such as the Food and Agriculture Organization (FAO), the World Health Organization (WHO), the European Union (EU) and countries that engage in sustainable production.

2016 - Fermentation strategy to produce high gluconate vinegar [Articolo su rivista]
Giudici, Paolo; DE VERO, Luciana; Gullo, Maria; Solieri, Lisa; Lemmetti, Federico
abstract

Gluconic acid is a non-volatile acid that has many applications in food, pharmaceutical and cleaning fields. Gluconic acid has been detected as main oxidation product of Acetobacter and Gluconobacter strains growing on grape must, and it plays an important role in Traditional Balsamic Vinegar. Commonly, high gluconate vinegars have a greater physical stability and a greater preference by consumers because are perceived less pungent. In fact, gluconic acid reduces the pH and increases fixed acidity of the vinegar without increasing the sensation of pungency typical of acetic acid. Its taste is acid but mild sweet and, therefore, gluconic acid has influence on the sensory complexity of the vinegar. The aim of this work is to set up a fermentation procedure that improves the quality of balsamic vinegar by using selected yeasts and acetic acid bacteria strains able to oxidize glucose in grape must-based media having a different sugars concentration. In particular, Saccharomycodes ludwigii UMCC 297 and Acetobacter pasteurianus UMCC 1754 strains were chosen as selected starter cultures for small-scale fermentation of cooked grape must, to evaluate the physical-chemical parameters affecting gluconic acid production in the obtained vinegar. The strains used and the control of all production process have been fundamental for obtaining the vinegar with the desired characteristics.

2016 - Improved wine yeasts by direct mating and selection under stressful fermentative conditions [Articolo su rivista]
Bonciani, Tommaso; Solieri, Lisa; DE VERO, Luciana; Giudici, Paolo
abstract

Hybridization of yeasts allows whole-genome modifications that can be exploited to obtain global improvements in industrial traits, such as those involved in the winemaking industry. In our work we applied direct mating to achieve the construction of hybrids and we subsequently applied these hybrids in fermentation trials under stressful conditions, in order to select hybrid strains with improved technological traits. Five hybrids, obtained from six parental strains by direct spore conjugation, were validated through PCR amplification of highly variable minisatellite-containing genes; the validation phase also revealed three meiotic derivative strains, characterized by contracted number of repeats. Analysis of the mating-type locus in the homozygous spore progeny of parental strains provided useful insights into the understanding of hybridization yields and unveiled some irregularities in yeast autodiploidization mechanism. The fermentative trials were followed by chemical analysis; afterwards principal component analysis allowed the metabolic footprinting of wine yeasts and the selection of the two best industrial candidates, which display superior phenotypes in fermentative fitness and secondary metabolite production, respectively.

2015 - Acetic Acid Bacteria of Unimore Microbial Culture Collection: from strains preservation to selected starter cultures distribution [Abstract in Atti di Convegno]
DE VERO, Luciana; Gullo, Maria; Giudici, Paolo
abstract

Increased knowledge on physiological, biochemical and genetic properties of acetic acid bacteria (AAB) is driving advancements in biotechnology and innovative fermentation processes. AAB scalable fermentations require robust strains able to maintain their functional traits over processes and to dominate unsterile environments. Accordingly, microbial collections of well-characterized AAB strains are fundamental sources to select cultures with desired properties for biotechnological applications. The University of Modena and Reggio Emilia Microbial Culture Collection (UMCC), specialised in the selection of functional microorganisms for both academic and industrial purposes, holds several AAB strains collected from must, wine, vinegars and kombucha tea. Selective strain isolation, molecular typing, polyphasic identification, and technological selection are the core of UMCC activity finalized to build up appropriate AAB starters for different industrial needs. Shift from preservation status to active AAB cultures is optimised to maintain high cell viability and to reduce genetic drift or strain instability. Technological screening of strains, design and development of selected starters at pilot scale, in static and submerged conditions, are performed to provide industrial cultures for vinegar production. Moreover, consumer demand for high added-value products, including fermented and low sour beverages indicates potential applications for novel and functional starter cultures. UMCC strain information is managed by the BioloMICS NET software (BioAware/ http://biolomics.umcc.unimore.it), which acts as a comprehensive platform able to combine phenotypic and molecular traits with industrial strain performance.

2015 - Acetic acid bacteria from Unimore microbial culture collection: biolomics platform as a tool for their industrial exploitation [Poster]
Gullo, Maria; DE VERO, Luciana; Zanichelli, Gabriele; Giudici, Paolo
abstract

Unimore Microbial Culture Collection includes a pool of acetic acid bacteria (AAB) strains collected since 2000. Main isolation sources of the UMCC AAB strains are wine vinegars, balsamic vinegars, cereal vinegars, fermented juices and kombucha tea. During the years, methods to recover representative isolates and preserve phenotypic stability were improved within UMCC, in order to investigate the key traits useful for their industrial exploitation. Acetic acid bacteria have attractive features useful in performing conventional and innovative processes/products. They incompletely oxidize sugars, alcohols and polyols to the corresponding oxidation products and they are exploited by pharmaceutical, medical, chemical and food-grade industries for the production of acetic acid, dihydroxyacetone, gluconic acid, 2-keto-L-gulonic acid and cellulose. Phenotypic changes, mainly regarding ethanol oxidation, acetic acid resistance and cellulose production, have been detected, consequently, suitable preservation methods and check procedures are performed in UMCC. The AAB strains are identified and characterized by a polyphasic approach and molecular typing are performed by fingerprinting techniques (ERIC/PCR and GTG5). Target strain performance and fermentation assays are developed at pilot scale to provide industrial strains. The BioloMICS (BioAware) platform of UMCC allows a comprehensive data management that combines phenotypic and molecular traits with industrial strain performance.

2015 - Exploitation of an evolution strategy to select yeast strains improved in glutathione production. [Abstract in Rivista]
Solieri, Lisa; DE VERO, Luciana; Mezzetti, Francesco; Bizzarri, Melissa; Giudici, Paolo
abstract

Yeasts have been largely explored as cell factories to produce substances for food and industrial biotechnological applications. Among these chemicals, glutathione (GSH) is an important antioxidant molecule involved in several processes, including the control of redox potential, protection against oxidative stress, detoxification and transport of organic sulfur. Due to its functional roles, GSH is widely used in the pharmaceutical, food and cosmetic industries. Recently, GSH has received growing attention also in the winemaking field, to control oxidative spoilage damage; to limit the amount of browning pigments; to avoid the formation atypical aging characters; and to exert a protective effect on various aromatic compounds. At present GSH is successfully produced on an industrial scale through fermentation by high GSH-producing Saccharomyces cerevisiae strains, and several methodological tools have been reported for increasing efficiency and yield of the bioprocess. In this study, we have applied an evolution-based strategy that combines the sexual recombination of spores with the application of molybdate Mo(VI), a sulfate analogue toxic for the cells at high concentration, as specific selective pressure, to generate evolved S. cerevisiae strains with enhanced GSH production. To achieve this aim we used the 21T2 wine strain from the Unimore Microbial Culture Collection (UMCC) and we exploited its resistance to Mo(VI) as a rapid and high-throughput screening method for the selection of the evolved strains improved in GSH production. By this strategy, we obtained two evolved strains, Mo21T2-5 and Mo21T2-12, both able to enhance GSH content in wine with an increase of 100% and 36%, respectively, compared with the parental strain 21T2, and 120% and 50% compared with initial GSH content in the must. Our strategy, unlike the standard evolutionary approaches, has the advantage of not requiring multiple rounds of screening and extensive cultivation periods because the evolved strains are recognized through a selectable phenotype. The Mo(VI) resistance has proved to be effective for the selection of the desired evolved strains, probably by activating the yeast common metal response that involves sulfur assimilation and GSH biosynthesis.

2015 - I balsamici: fermentazione acetica, viscosità e parametri sensoriali [Monografia/Trattato scientifico]
De Vero, Luciana; Giudici, Paolo; Gullo, Maria; Lemmetti, Federico; Mazza, Stefano
abstract

La produzione di aceto di vino è un processo che utilizza materie prime dal valore relativamente basso per ottenere un prodotto che raggiunge prezzi relativamente alti. La categoria merceologica che maggiormente beneficia dell’uso di questi prodotti “poveri” è quella relativa ai condimenti alimentari agrodolci a base di mosto concentrato e/o cotto e aceto di vino, cioè tutti i prodotti che hanno un esplicito riferimento al termine “balsamico”. Indubbiamente la diffusione di prodotti balsamici sul mercato è in forte crescita, anche grazie allo sviluppo di nuove forme di presentazione e di utilizzo del prodotto base (come glasse, creme, spume, spray) e una continua ricerca dell’aromatizzazione distintiva e particolare (attraverso l’utilizzo di fragranze o seguendo processi di produzione peculiari). Nelle province di Modena e Reggio Emilia è concentrata l’intera produzione di aceti “balsamici” a denominazione protetta italiani: due DOP per l’aceto balsamico tradizionale di Modena (ABTM) e quello di Reggio Emilia (ABTRE) e una IGP per l’aceto balsamico di Modena (ABM). L’ABT non è aceto di vino bensì di mosto cotto, tuttavia nell’ABM l’acidità deriva dal comune aceto di vino ottenuto per fermentazione in sommerso. La linea di prodotti sviluppati coniuga gli aspetti migliori dal punto di vista qualitativo ed economico delle due tipologie, introducendo numerose innovazioni: i) le fermentazioni dirette e in condizioni statiche consentono un risparmio energetico notevole e favoriscono la formazione di composti secondari sensorialmente positivi, anche grazie all’impiego di lieviti e batteri acetici specifici e selezionati appositamente per le loro caratteristiche fermentative; ii) il processo di concentrazione del mosto tramite cottura è assente o fortemente limitato mentre l’invecchiamento è accelerato tramite l’utilizzo di tecniche di condizionamento ambientale ecosostenibile; iii) non sono presenti additivi e conservanti di nessun genere in quanto vengono impiegate tecniche di conservazione basate sull’impiego oculato degli stessi microrganismi usati nelle fermentazioni; iv) le condizioni di cottura applicate limitano al minimo la formazioni di composti tossici.

2015 - New Saccharomyces cerevisiae F1 hybrids for winemaking under unbalanced nutritional conditions [Abstract in Atti di Convegno]
Bonciani, Tommaso; Solieri, Lisa; Bizzarri, Melissa; DE VERO, Luciana; Giudici, Paolo
abstract

Hybridization to improve wine yeasts

2015 - Selection of Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP)–producer Lactobacillus strains for developing functional yogurt enriched of bioactive peptides [Abstract in Atti di Convegno]
Solieri, Lisa; Rutella, Sefora; Tagliazucchi, Davide; DE VERO, Luciana; Giudici, Paolo
abstract

Accepted poster for SESSION II: The complexity of food ecosystems: physiology of single strains in pure culture vs. complex consortia

2015 - Unimore Microbial Culture Collection: a source for providing novel yeasts for winemaking. [Abstract in Atti di Convegno]
DE VERO, Luciana; Bonciani, Tommaso; Verspohl, Alexandra; Mezzetti, Francesco; Bizzarri, Melissa; Solieri, Lisa; Giudici, Paolo
abstract

The University of Modena and Reggio Emilia (UNIMORE) Microbial Culture Collection (UMCC) supplies authenticated strains and fundamental biological data for research and biotechnological application (www.umcc.unimore.it). More than 1600 yeast strains, isolated from must, wine, beer vinegar, sourdough and other fermented products are included in UMCC and are maintained by techniques that ensure preservation and long term genetic stability.

2014 - Evolved Saccharomyces cerevisiae wine strains with enhanced glutathione production obtained by an evolution-based strategy [Articolo su rivista]
Mezzetti, Francesco; DE VERO, Luciana; Giudici, Paolo
abstract

In winemaking, the application of glutathione (GSH) has been the subject of ever-growing interest because of its important role in limiting must and wine oxidation and in protecting various aromatic compounds. Glutathione concen- tration in wine is highly variable, involving as it does several factors from must, through alcoholic fermentation, to yeast strain activity. Consequently, the development of new wine yeast strains able to improve flavor stability is in great demand. To generate evolved Saccharomyces cerevisiae strains with enhanced GSH production, we have applied an evolution-based strategy that combines the sexual recombination of spores with the application of molyb- date, which is toxic for the cells at high concentration, as specific selective pres- sure. Eight molybdate-resistant strains were selected and further screened for GSH production in synthetic grape must and in microvinification assay. By this nongenetically modified strategy, we obtained two evolved strains, Mo21T2-5 and Mo21T2-12, both able to enhance GSH content in wine with an increase of 100% and 36%, respectively, compared with the parental strain 21T2, and 120% and 50% compared with initial GSH content in the must. Introduction

2014 - Fermentative aptitude of non-Saccharomyces wine yeast for reduction in the ethanol content in wine [Articolo su rivista]
Gobbi, M.; DE VERO, Luciana; Solieri, Lisa; Comitini, F.; Oro, L.; Giudici, Paolo; Ciani, M.
abstract

Over the last few decades, there has been a progressive increase in the ethanol content in wines due to global climate change and to the new wine styles that are associated with increased grape maturity. This increased ethanol content can have negative consequences on the sen- sory properties of the wines, human health, and economic aspects. Several microbiological approaches for decreasing the ethanol content have been suggested, such as strategies based on genetically modified yeasts, the adaptive evolution of yeasts, and the use of non-Saccharomyces yeast. In the present study, we investigated the interspecies and intraspe- cies variability of some non-Saccharomyces wine yeast species under anaerobic fermentation conditions. Across different grape juices and fermentation trials, Hansenias- pora uvarum, Zygosaccharomyces sapae, Zygosaccharo- myces bailii, and Zygosaccharomyces bisporus promoted significant reductions in ethanol yield and fermentation efficiency in comparison with Saccharomyces cerevisiae. The diversion of alcoholic fermentation and the abundant formation of secondary compounds might explain the marked reduction in ethanol yield, as determined through the segregation of the majority of the strains according to their species attributes observed using principal component analysis. These data suggest that careful evaluation of inter- species and intraspecies biodiversity can be carried out to select yeast that produces low-ethanol yields.

2014 - La fermentazione del Lambrusco: modello di evoluzione biotecnologica [Articolo su rivista]
Giudici, Paolo; DE VERO, Luciana; Solieri, Lisa; Catena, M.
abstract

Il rinnovato successo del Lambrusco è legato anche all’apporto delle tecnologie e delle innovazioni enologiche. Nel caso specifico della fermentazione da mosti desolforati la selezione di lieviti ad hoc è una esigenza stringente per l’ottenimento di prodotti di qualità sensoriale elevata e completa assenza di sensazioni olfattive sgradevoli

2014 - La selezione di lieviti migliorati per la produzione di glutatione [Articolo su rivista]
Mezzetti, F.; DE VERO, Luciana
abstract

Negli ultimi anni le tendenze del mercato enologico sono state fortemente influenzate dalle esigenze dei consumatori sempre più attenti alla qualità dei vini e, sulla scia di queste tendenze, oltre che delle innovazioni tecnologiche di produzione, sono stati improntati i nuovi criteri di selezione dei lieviti enologici da utilizzare come coltura starter per i processi fermentativi.

2014 - Lieviti progettati per ottenere vini diversi [Articolo su rivista]
Giudici, Paolo; DE VERO, Luciana; Solieri, Lisa; Catena, M.
abstract

La disponibilità di strumenti tecnologici e biotecnologici a supporto della selezione di nuovi starter enologici consente di conferire complessità e specificità che sono la base per lo sviluppo di vini inediti. Un’evoluzione premiata dai trend di mercato che porta ad ottenere tipologie ben distanti da quelle tradizionali. Lasciando aperta una domanda: ha ancora senso l’equazione tra qualità e tipicità?

2013 - La mimica della selezione naturale nel miglioramento genetico dei lieviti [Articolo su rivista]
DE VERO, Luciana; Mezzetti, F.; Solieri, Lisa; Giudici, Paolo
abstract

Per le sue caratteristiche di alcool-tolleranza e per la generale purezza fermentativa, la specie d’elezione per la conduzione della fermentazione alcolica è S. cerevisiae, tuttavia, in passato sono state proposte altre specie di lievito, recentemente riconsiderate per alcuni caratteri tecnologici e sensoriali.

2013 - Significance and management of acetic acid bacteria culture collections [Articolo su rivista]
DE VERO, Luciana; Giudici, Paolo
abstract

Acetic acid bacteria (AAB) are obligate aerobic microorganisms which have large significance in human life. Traditionally, AAB species have been used to produce fermented food and beverages thanks to their ability to oxidize ethanol to acetic acid. Moreover, in the last decades, they have been extensively investigated for other industrial biotechnology applications as the development of processes for highvalue products or biosensors. The potential exploitation of AAB diversity requires the existence of microbial culture collections, which are able to supply not only strains but essential data for fundamental microbial research. Therefore, microbial collections can be helpful to provide critical insights into AAB physiology and metabolism as well as integrate sequence data with transcriptional and functional studies to better define complex traits and develop new potential microbial processes. This article reviews the significance of microbial collections, with an overview of the well-known European Biological Resources Centers (BRCs) collecting AAB, and provides an insight into their cultivability and metabolic activity. It also discusses appropriate techniques in preserving authentic strains, quality control implications, databases and BRC networking as well as connections among collections and stakeholders.

2012 - Acetic acid bacteria culture collection: significance and management [Abstract in Rivista]
DE VERO, Luciana
abstract

Acetic acid bacteria (AAB) occur in very specialized habitats such as plants, fruits and flowers, as well as several kinds of sugary or alcoholic substrates. In the last decades, some AAB species have been extensively investigated not only for foods and beverages but also others industrial biotechnology applications as the development of processes for high-value products or biosensors. The potential exploitation of AAB strain diversity requires the existence of culture collections which represent important biological resources for selection and genetic improvement of microbial cultures useful for biotechnological applications. The greatest hurdles to acetic acid bacteria study are related to their difficult isolation, thus several media and incubation conditions are necessary for successful cultivation and preservation of pure cultures. Furthermore culture collections have to evaluate microbial changes over time as well as guarantee appropriate tools in preserving ‘‘authentic’’ strains.These are also the tasks of the Food and Industrial Microbial Collection (FIMC) of the Department of Agricultural and Food Sciences (DipSAA) that, currently, holds about 300 AAB strains, above yeast and lactic acid bacteria strains, isolated from several matrices. Specifically, the AAB strains maintained in the collection have been isolated from musts, wines, various vinegars, solid-state fermentation matrix and kombucha tea. Different preservation techniques are used, ranging from continuous growth methods, for short-term maintenance, to cryopreservation methods at -80°C, for long-term maintenance and for avoiding genetic drift or instability. Full understanding of metabolic potential of the AAB strains collected is achieved by the integration of sequence data with transcriptional and functional studies. A “Strain Database” (www.renewx.org) and a Bioinformatics Software (GelCompare II - Applied Math) support all the information related to the strains starting from isolating source until the full characterization, as well as the collection management.

2012 - Acetobacter pasteurianus strain AB0220: cultivability andphenotypic stability over 9 years of preservation [Articolo su rivista]
Gullo, Maria; Mamlouk, Dhouha; De Vero, Luciana; Giudici, Paolo
abstract

Acetobacter species are members of the a-subclassof Proteobacteria, which harbors a large number of bacteriarecalcitrant to cultivation. Strain AB0220 was isolatedfrom a superficial acetification system and preserved for9 years by short and long time methods. Under short timepreservation it was estimated that 540.54 number of generationsoccurred, whereas in long time preservation conditionsthe number of generations was 17.40. Ethanoloxidation to acetic acid was stable and confirmed, as wellas acetate assimilation during long time preservation.Cultivability checks showed persistence of phenotypictraits (growth on ethanol and methanol, growth on differentcarbon sources and cellulose production) over the extendedpreservation time. 16S rRNA gene sequences analysisshowed 100 % of similarity with A. pasteurianus (Accessionnumber GQ240636). Stability of subcultures related tothe culture age and subcultures frequency, tested by ERIC/PCR, confirmed the suitability of long term preservationat least over a period of 9 years.

2012 - Oxidation of sugars and polyalcohols by acetic acid bacteria during surface culture fermentation [Abstract in Rivista]
Giudici, Paolo; Gullo, Maria; DE VERO, Luciana
abstract

Acetic Acid Bacteria (AAB) are well known for their ability to oxidize alcohols, aldehydes, sugars, polyalcohols and others molecules with ketonic or aldehydic functional groups. Species of Acetobacter and Gluconobacter lack a functional Embden-Meyerhof-Parnas pathway and are unable to metabolize hexose sugars by this route. Hexose and pentose sugars are oxidatively metabolized by the hexose monophosphate pathway to acetic and lactic acids. In some cases, hexose sugars may be directly oxidized to gluconate and ketogluconates without further catabolism, leading to an accumulation of these end products in the culture medium. Furthermore, Gluconobacter oxydans has been used to oxidize various sugars and sugar alcohols to substances of industrial significance, such as sorbose, gluconic acid and ketogluconic acids. Dihydroxyacetone, 2,3-butanediol, and acetoin are also significant products of carbohydrate metabolism evolved by AAB Our trials of surface static fermentation was carried out on complex media such as base wine (BW) for Traditional Balsamic Vinegar, where sugars and alcohols are present in different amount. In this conditions AAB sequentially oxidized the several carbon sources, first ethanol, then glucose and glycerol. In particular the oxidation of glucose to gluconate occurred when the ethanol was exhausted, then high amount of gluconate was accumulated in the medium. BW had an initial composition of around 7% (v/v) of ethanol and 25% of sugars with glucose and fructose in the ratio 1/1. At the end of the fermentation the glucose/fructose ratio was in favour of fructose, titratable acidity was very high due to the gluconate formed, while volatile acidity decreased. In summary, when surface fermentation is extended afterward the ethanol run out, other substrates are oxidized and give origin to a vinegar with a completely different composition. The extension of the oxidative step is a tool for increasing vinegar’s sweetness and acidity without the acetic acid sensorial pungency. (empty line)Keywords: gluconic acid, sugar oxidation, surface static acetification.

2012 - Starter enologici ottimizzati: dalle collezioni microbiche alla system biology [Articolo su rivista]
Giudici, Paolo; DE VERO, Luciana; Solieri, Lisa
abstract

I progressi nel campo della biologia molecolare e dell’ingegneria fermentativa offrono sempre maggiori opportunità d’innovazione. L’applicazione di modelli matematici capaci di descrivere e predire il metabolismo di lieviti nel mosto consentiranno di valorizzare le collezioni microbiche e di disegnare in breve tempo nuovi ceppi con migliorati fenotipi enologici

2011 - Complex nature of Zygosaccharomyces wild strains: a genotypic and functional characterization [Relazione in Atti di Convegno]
Solieri, Lisa; Dakal, TIKAM CHAND; DE VERO, Luciana
abstract

Genotypic and functional characterization of Zygosaccharomyces wild strains

2011 - Evolution-based strategy to generate non-genetically modified organisms Saccharomyces cerevisiae strains impaired in sulfate assimilation pathway [Articolo su rivista]
DE VERO, Luciana; Solieri, Lisa; Giudici, Paolo
abstract

Aims: An evolution-based strategy was designed to screen novel yeast strains impaired in sulfate assimilation. Specifically molybdate and chromate resistance was used as selectable phenotype to select sulfate permease deficient variants unable to produce sulfites and hydrogen sulfide (H2S).Methods and Results: Four Saccharomyces cerevisiae parent strains were induced to sporulate. After tetrad digestion, spore suspensions were observed under the microscope to monitor the gametes conjugation. Then the cell suspension was inoculated in tubes containing YPD medium supplemented with ammonium molybdate or potassium chromate. Forty-four resistant strains were obtained and then tested in microvinifications. Three strains with a low sulfites production (SO2 <10 mg l-1) and unable to produce H2S in grape must without added sulfites, were selected.Conclusions: Our strategy enabled the selection of improved yeasts with desired oenological characteristics. Particularly resistance to toxic analogues of sulfate allowed to detect strains unable to assimilate sulfates.Significance and Impact of the Study: This strategy, that combines spores sexual recombination and application of a specific selective pressure, provides a rapid screening method to generate genetic variants and select improved wine yeast strains with an impaired metabolism regarding the production of sulfites and H2S.

2011 - Mancata presa di spuma del lambrusco, il ruolo della fermentazione lattica nel vino base [Articolo su rivista]
Giudici, Paolo; DE VERO, Luciana
abstract

Indagini sperimentali mostrano come il difetto della mancata rifermentazione del lambrusco, frequente nel prodotto gestito direttamente dal consumatore, sia legato a ritardi nell’imbottigliamento e allo sviluppo di batteri lattici

2011 - Selezione di lieviti con alterata riduzione dei solfati e solfiti per la produzione di vini senza SO2 [Articolo su rivista]
DE VERO, Luciana; Giudici, Paolo
abstract

Nella presente ricerca sono stati selezionati ceppi di S. cerevisiae dotati di ottima attitudine fermentativa ed enologica e con alterata riduzione dei solfati e solfiti per la produzione di vini senza SO2. I ceppi selezionati sono stati ottenuti da parentali di S. cerevisiae mediante un approccio nonogm basato sulla coniugazione di spore e successiva esposizione a specifiche pressioni selettive. I ceppi sono stati testati su mosto d’uva ed in simil-mosto a composizione definita, in assenza o presenza di SO2. I risultati hanno chiaramente messo in evidenza che esistono delle differenze significative tra i ceppi e che la scelta del ceppo idoneo alla realizzazione degli obiettivi stabiliti è, in parte, dipendente dalle caratteristiche del mosto. In particolare, il ceppo Mo163-9 ha ottime attitudini tecnologiche, non riduce i solfati, mentre riduce sensibilmente la quantità dei solfiti aggiunti, portandoli a valori inferiori a 10 mg/L. Un comportamento analogo è dato dal ceppo Mo163-1. Il ceppo 21-T2 ha ottima attitudine tecnologica e una ridotta attività su solfati e solfiti riducendo sempre il contenuto di SO2 iniziale ma con produzione di solfuri contenuta.

2010 - Acetic Acid Bacteria, Biotechnological Applications [Voce in Dizionario o Enciclopedia]
DE VERO, Luciana; Gullo, Maria; Giudici, Paolo
abstract

2010 - Collezione di microrganismi e loro impiego per la selezione delle colture starter [Articolo su rivista]
DE VERO, Luciana; Giudici, Paolo
abstract

Il ruolo delle collezioni di microrganismi è quello di raccogliere non soltanto dei ceppi ma informazioni approfondite sulle loro caratteristiche fisiologiche, biochimiche e molecolari al fine di implementarne le potenziali applicazioni biotecnologiche nei più svariati settori. In particolare, nel settore alimentare è semprepiù importante controllare e standardizzare i processi fermentativi, ricorrendo a colture starter di specifici microrganismi in grado di garantire sicurezza e qualità dei prodotti finiti. Dal 1999, il laboratorio di Microbiologia delDipartimento di Scienze Agrarie e degli Alimenti (DIPSAA)dell’Università di Modena e Reggio Emilia dispone di una collezionedi microrganismi costituita da diversi ceppi di lieviti, batteri latticie batteri acetici isolati da diverse matrici alimentari, oggetto di studi approfonditi al fine di individuare quelli più idonei alla preparazione di colture starter.

2010 - I microrganismi del balsamico ed aspetti della qualità [Esposizione]
Giudici, Paolo; Gullo, Maria; DE VERO, Luciana; F., Lemmetti; U., Rangone
abstract

Il convegno tratta gli aspetti della scalarità del proceso fermentativo: fermentazione alcolica ed aciteca, con particolare attenzione alla resa del processo ed agli aspetti qualitativi.

2010 - LA SELEZIONE E IL MITO DEI LIEVITI AUTOCTONI [Articolo su rivista]
Giudici, Paolo; Solieri, Lisa; DE VERO, Luciana
abstract

La scelta del ceppo di lievito influenza il successo della fermentazionealcolica dei vini e modula profondamente la qualità delprodotto finale. Analisi molecolari hanno portato ad una profondarevisione della tassonomia dei lieviti ed hanno facilitato gli studiecologici delle fermentazioni spontanee dei mosti, evidenziandol’esistenza della specie Saccharomyces cerevisiae in habitat naturalinon antropici. Le specie riscontrate sulle uve sono in numeroelevato e maggiore che non nei mosti e nei vini, dove esiste unaforte pressione selettiva del mezzo e delle condizioni tecnologicheimpiegate. In generale, più stringenti sono le condizioni di crescita,minore è il numero di specie in grado sviluppare. S. cerevisiae èla specie più alcool tollerante ed è per questa ragione che a finefermentazione è sempre la più rappresentata, nonostante sia pocofrequente sulle uve. Queste osservazioni hanno suggerito la tesidella domesticazione della specie S. cerevisiae e quella dei lievitiautoctoni, ma le evidenze a supporto non possono essere consideratesufficienti. In particolare, la tesi dei lieviti autoctoni è debole,specie nel passaggio che attribuisce ai lieviti autoctoni un contributosignificativo nella qualità dei vini corrispondenti. I buoniceppi di lievito sono il risultato di un programma di selezione emiglioramento genetico e, solo raramente, l’oggetto di un isolamentocasuale. In questo articolo discutiamo il significato deitermini “domesticazione” ed “autoctono” in biologia, suggerendoche il termine “autoctono” marca troppo il rapporto tra luogo,tempo e ceppo, lasciando intendere una dipendenza non dimostrata.Il termine “indigeni” è molto più appropriato per definire i lievitipresenti in un mosto, in una cantina o in un vigneto, e con esso siintendono i lieviti presenti in un determinato momento, senzapretese di continuità temporale.

2010 - Rivisitare una tecnica antica [Articolo su rivista]
DE VERO, Luciana; Solieri, Lisa; Giudici, Paolo
abstract

L’uso delle colture starter nella produzione di alimenti e bevande fermentate è consolidato e non è possibile ipotizzare la produzione di alimenti fermentati di qualità senza il loro uso. In campo enologico, i primi esempi empirici di fermentazione guidata ante litteram si possono ricondurre alla pratica di inoculare il mosto con una piccola quantità di mosto preparato in anticipo e già in fermentazione, dove la pressione selettiva del mezzo ha favorito la crescita dei lieviti indigeni più idonei per le condizioni in essere. I lieviti indigeni sono spesso ed erroneamente definiti autoctoni, lasciando intendere che vi siano lieviti specifici nei diversi territori e nelle singole cantine, ma ciò non risponde al vero. I lieviti non conoscono la geografi a! Essi rispondono solo ai parametri chimico/fisici del mosto e del vino, fra i quali i più importanti sono: temperatura, pH, concentrazione zuccherina, grado alcolico, potenziale redox e anidride solforosa. L’effetto combinato di queste variabili rende il mosto molto selettivo e perciò consente la crescita di alcune specie di lievito e di poche specie di batteri lattici acidofili e di batteri acetici. All’interno della specie la pressione selettiva si estende a livello di ceppo e quelli più idonei prendono il sopravvento. La massa in fermentazione così ottenuta è universalmente nota come pied de cuve ovvero una coltura starter ottenuta senza l’aggiunta volontaria di lieviti, e contenente un elevato numero di cellule di ceppi di lievito dotati di un’elevata velocità di crescita nel mezzo considerato.

2010 - Tassonomia, identificazione fenotipica e molecolare dei lieviti [Capitolo/Saggio]
Tofalo, R.; DE VERO, Luciana; Giudici, Paolo; Suzzi, G.
abstract

2010 - Techiche biotecnologiche per la produzione di vini a basso tenore alcolico [Articolo su rivista]
Giudici, Paolo; Solieri, Lisa; DE VERO, Luciana
abstract

Lieviti Gm: affidabili ma poco accettati.Ceppi nonSaccharomyces: accattivanti ma con un forte impatto sensoriale.Ceppi flor di S.cerevisiae: alternativa promettente ma ancora poco sperimentata. Dosare in modo attento microbiologia, tecniche di cantina e pratiche agronomiche rimane l’unica strada per ridurre l’alcol nei vini nel breve periodo.

2009 - Succession of Selected Strains of Acetobacter pasteurianus and Other Acetic Acid Bacteria in Traditional Balsamic Vinegar [Articolo su rivista]
Gullo, Maria; DE VERO, Luciana; Giudici, Paolo
abstract

The application of a selected Acetobacter pasteurianus strain for traditional balsamic vinegar production was assessed. Genomic DNA was extracted from biofilms after enrichment cultures on GYC medium (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) and used for PCR/denaturing gradient gel electrophoresis, 16S rRNA gene sequencing, and enterobacterial repetitive intergenic consensus/PCR sequencing. Results suggested that double-culture fermentation is suitable for traditional balsamic vinegar acetification.

2008 - Genus-specific profile of acetic acid bacteria by 16S rDNA PCR-DGGE [Articolo su rivista]
DE VERO, Luciana; Giudici, Paolo
abstract

An effective method for grouping acetic acid bacteria (AAB) genera was defined and evaluated as a tool for preliminary screening of the majorAAB species involved in vinegar production.Acetobacter, Gluconobacter, Gluconacetobacter, Asaia, Neoasaia, Saccharibacter, Frateuria and Kozakia AAB strains were screened on thebasis of the 16S rDNA sequences using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique.The DGGE profile of all the strains tested, consisted of one single band of approximately 330 bp for each strain and allowed their clustering.The results obtained clearly reflected in silico phylogenetic analysis of the AAB species used in this study, in fact, the species with a higher 16SrDNA sequence homology showed a similar electrophoretic profile. In particular almost all the species belonging to the genus Gluconacetobactershowed a DGGE pattern nearly identical and well distinct from all the other AAB genera. Furthermore by PCR-DGGE it was possible to clearlygroup the species more frequently recovered from vinegar fermentation which are mainly distributed in the genera Acetobacter, Gluconobacterand Gluconacetobacter.

2008 - Improvement of traditional vinegar production by selected acetic acid bacterium strain: Acetobacter pasteurianus as acetification starter [Poster]
Gullo, Maria; DE VERO, Luciana; Giudici, Paolo
abstract

Recent microbiological studies investigated the acetic acid bacteria microflora of traditional balsamic vinegar (TBV) highlighting the occurrence of Gluconacetobacter europaeus as widespread indigenous species, followed by Acetobacter pasteurianus, Acetobacter aceti and Acetobacter malorum1,2. However no correlation between occurring species and quality of product have been examined. In this study a selected starter culture (SSC) was designed, implemented at laboratory scale and applied to vinegar factory by scale up procedure. A. pasteurianus strain AB0220 was selected as SSC on the basis of phenotypical and technological traits suitable for TBV must oxidation. The SSC was implemented through three stages: starting steps in laboratory conditions (Stage 1); scale-up performed through a tanks system (Stage 2) and a barrels system (Stage 3) in factory conditions. Main analytical parameters were monitored by pH, titrable acidity, ethanol and soluble solids trend. Moreover molecular identification based on 16S rDNA region analysis (PCR-DGGE and sequencing) and ERIC-PCR were performed respectively to assess species occurrence and evaluate strain persistence during the whole process. In particular the dominant AAB species population was estimated by PCR-DGGE analysis allowing to distinguish two different species groups along the 3 stages. 16S rDNA sequencing confirmed DGGE results, showing high percentage of sequence homology (99 and 100%) with A. pasteurianus at stage 1 and 2 and Ga. europaeus at Stage 3. Finally, ERIC-PCR fingerprinting assay performed at the end of each acetification stage showed an electrophoretic profile similar to that of AB0220 at stage 1 and 2, whereas a different pattern at stage 3. This result supports DGGE data suggesting a change in the population during the 3 acetification stages. On the basis of these evidences, our hypothesis is that the persistence of inoculated strain during stages 1 and 2 was assured by the scale-up procedure. New must was periodically added to increase the SSC volume and microbial population was not exposed to constant effect of acetic acid. Instead, the static conditions of stage 3 resulted in a constant increase of acetic acid concentration, negatively affected AB0220 growth. In this environment, indigenous Ga. europaeus cells, which is less sensitive to physiological stress caused by acetic acid3, found optimal growth conditions to become dominant. In this study for the first a SSC for TBV production was implemented and applied at factory scale. Results demonstrated that selected A. pasteurianus strains are suitable to start the acetification of TBV ensuring the acetification course in unsuitable conditions for other AAB such as Ga. europaeus. On the contrary, Ga. europaeus strains are able to oxidise cooked must in presence of constant of acetic acid concentration corresponding to the final stages of TBV oxidation. Results suggest that the introduction of SSC could be a valid innovation in TBV production, contributing to safety and quality of the product and improving the reliability and stability of technological process.

2008 - Studio dell’evoluzione della popolazione di batteri lattici durante la produzione di Parmigiano Reggiano mediante 16S rDNA PCR-DGGE [Articolo su rivista]
DE VERO, Luciana; Solieri, Lisa; Gala, Elisabetta; Pulvirenti, Andrea; Giudici, Paolo
abstract

Il formaggio Parmigiano Reggiano è stato oggetto di numerosi studi microbiologici, in particolare sulle prime fasi di produzione e sul siero innesto. Nella presente ricerca, è stata studiata l’evoluzione delle specie di batteri lattici (BL) durante tutto il processo compresa la stagionatura. A tal fine è stata utilizzata una tecnica coltura indipendente, che prevede l’estrazione del DNA batterico direttamente dalla matrice, senza pertanto l’isolamento e la coltura dei batteri. Successivamente, parte della regione 16S del DNA ribosomale (rDNA) è stata amplificata con primers specifici per alcune specie di BL e gli amplificati ottenuti sono stati separati mediante gel elettroforetico di poliacrilammide in gradiente denaturante (PCR-DGGE). In letteratura è riportato che, sia nella produzione di Parmigiano Reggiano che di Grana Padano, Lactobacillus helveticus è la specie microbica prevalente nel siero innesto, fin oltre il 70% della popolazione microbica. Al contrario, nella nostra ricerca, abbiamo osservato che Lb. helveticus è prevalente nel siero maturo ma non nel siero a fine lavorazione. In particolare, durante il processo in caldaia si ha un recupero di Lactobacillus delbrueckii che numericamente prevale sulla specie Lb. helveticus. Successivamente, il siero raccolto e conservato per la preparazione della coltura starter della lavorazione successiva, favorisce la crescita di Lb. helveticus, ripristinando la dominanza di quest’ultima specie. In generale, la tecnica della PCR-DGGE può essere ritenuta maggiormente attendibile nella descrizione delle specie maggioritarie rispetto ai metodi coltura dipendente, in quanto consente di superare i problemi relativi alla non coltivabilità delle cellule batteriche presenti nel mezzo pur essendo esse in uno stato vitale.

2006 - Application of denaturing gradient gel electrophoresis (DGGE) analysis to evaluate acetic acid bacteria in traditional balsamic vinegar [Articolo su rivista]
DE VERO, Luciana; Gala, Elisabetta; Gullo, Maria; Solieri, Lisa; Landi, Sara; Giudici, Paolo
abstract

Acetic acid bacteria (AAB) are fastidious micro-organisms to isolate and cultivate despite of the great number of growth media available. Moreover, conventional techniques used to study AAB populations are time consuming and not completely reliable. In this study, we tested the usefulness of the polymerase chain reaction-denaturing gradient gel electophoresis (PCR-DGGE) as a rapid and cost effective method for the screening of AAB in traditional balsamic vinegar (TBV). DGGE analysis was applied to 19 AAB strains isolated by agar plating from three different samples of TBV. DGGE was also used for the analysis of PCR products obtained from DNA extracted directly from the TBV samples. A tentative species identification was achieved comparing the PCR-DGGE patterns of the isolated strains and the TBV samples to those of 15 AAB reference strains. The results support that DGGE is functional to monitor vinegar´s AAB population.

2006 - Aspetti microbiologici nella gestione di una batteria di Aceto Balsamico Tradizionale [Articolo su rivista]
DE VERO, Luciana; Gullo, Maria; Gala, Elisabetta; Giudici, Paolo
abstract

2006 - Characterization of acetic acid bacteria in traditional balsamic vinegar [Articolo su rivista]
Gullo, Maria; C., Caggia; DE VERO, Luciana; Giudici, Paolo
abstract

This study evaluated the glucose tolerance of acetic acid bacteria strains isolated from Traditional Balsamic Vinegar. The results showed that the greatest hurdle to acetic acid bacteria growth is the high sugar concentration, since the majority of the isolated strains are inhibited by 25% of glucose. Sugar tolerance is an important technological trait because Traditional Balsamic Vinegar is made with concentrated cooked must. On the contrary, ethanol concentration of the cooked and fermented must is less significant for acetic acid bacteria growth. A tentative identification of the isolated strains was done by 16S-23S-5S rDNA PCR/RFLP technique and the isolated strains were clustered: 32 strains belong to Gluconacetobacter xylinus group, two strains to Acetobacter pasteurianus group and one to Acetobacter aceti.

2006 - Detection of Acetic Acid Bacteria in Traditional Balsamic Vinegar by PCR-DGGE analysis [Relazione in Atti di Convegno]
DE VERO, Luciana; Gullo, Maria; Landi, Sara; Giudici, Paolo
abstract

The denaturing gradient gel electrophoresis (DGGE) applied to PCR products of relatively conserved regions in the genome is a well documented culture-independent method to study microbial communities. In the present study we used DGGE to evaluate acetic acid bacteria population of Traditional Balsamic Vinegar which is, to our knowledge, a system not investigated with this approach previously. The results obtained suggest that PCR-DGGE is clearly a suitable tool for a rapid and cost effective screening of acetic acid bacteria in vinegar’s samples.

2006 - I Microrganismi dell'aceto balsamico. Atti del convegno: "Anche la tradizione va studiata" Risultati di un biennio di indagini per l'individuazione e il possibile impiego di starter per Aceto Balsamico Tradizionale. [Curatela]
Licciardello, Fabio; Falcone, Pasquale Massimiliano; Tagliazucchi, Davide; Giudici, Paolo; Solieri, Lisa; Gullo, Maria; DE VERO, Luciana
abstract

Il ruolo e l'importanza dei microrganismi sulla qualità dell'aceto balsamico tradizionale.

2006 - I microrganismi dell'aceto balsamico. [Esposizione]
Giudici, Paolo; Solieri, Lisa; Gullo, Maria; Pulvirenti, Andrea; DE VERO, Luciana; Falcone, Pasquale Massimiliano
abstract

Il processo di produzione dell’aceto balsamico tradizionale prevede quattro fasi distinte: cottura del mosto, fermentazione alcolica, fermentazione acetica ed invecchiamento. Ognuna di queste fasi va gestita attentamente sulla base di fattori sia di tipo chimico-fisico che microbiologico. Data la scalarità delle fasi, le anomalie presenti a monte del processo si ripercuotono sul prodotto finale. Ad esempio, le fermentazioni spontanee sono tra le principali cause di variabilità e di insuccesso ed è per tal motivo che occorre un accurato monitoraggio della batteria che possa preventivamente correggere eventuali irregolarità nel sistema. In particolare, i parametri da monitorare durante la gestione della batteria sono: l’acidità titolabile, la concentrazione alcolica, il pH, la temperatura e l’ossigeno. L’impiego di colture starter di lieviti e batteri acetici selezionate sulla base dei caratteri desiderabili può essere utile per la standardizzazione del processo e per l’ottenimento di un prodotto che presenti le proprietà chimico-organolettiche richieste. L’età dell’aceto balsamico tradizionale è difficile da definire e da calcolare per il particolare modo di produzione: aliquote di aceto sono trasferite annualmente da un barile all’altro. La conduzione delle batterie rende difficoltoso il calcolo dell’età, tuttavia in questo lavoro abbiamo sviluppato un modello algebrico per il calcolo dell’età. In particolare abbiamo definito i limiti di età teoricamente raggiungibili in ogni barile della batteria.

2006 - La gestione microbiologica delle batterie [Relazione in Atti di Convegno]
DE VERO, Luciana; Landi, Sara; Gala, Elisabetta
abstract

2006 - Le Fermentazioni dell’Aceto Balsamico Tradizionale [Capitolo/Saggio]
Giudici, Paolo; Gullo, Maria; Solieri, Lisa; DE VERO, Luciana; Sara, Landi; Pulvirenti, Andrea; Sandra, Rainieri
abstract

Scritto a più mani dall'équipe di ricerca dell'Università di Modena e Reggio Emilia che fa capo al professor Paolo Giudici, il libro presenta al pubblico degli amanti dell'aceto balsamico tradizionale i più recenti risultati delle ricerche svolte in ambito microbiologico nelle varie fasi di produzione di questo straordinario prodotto della nostra tradizione, amato e apprezzato in tutto il mondo. Il professor Giudici in questo volumetto articola con limpida chiarezza divulgativa parte dei risultati conseguiti dalla sua équipe, trasmettendo alcune nozioni necessarie per la conduzione delle fermentazioni. A ciò si aggiunga una sezione di letteratura specialistica particolarmente abbondante e curata sulla microbiologia dell'aceto balsamico.

2006 - Le fermentazioni dell’aceto balsamico tradizionale [Monografia/Trattato scientifico]
Giudici, Paolo; Gullo, Maria; Solieri, Lisa; DE VERO, Luciana; Landi, Sara; Pulvirenti, Andrea; Rainieri, Sandra
abstract

2006 - Mitochondrial DNA and Mitochondrial proteins in interspecific Saccharomyces hybrids [Relazione in Atti di Convegno]
Pulvirenti, Andrea; Solieri, Lisa; DE VERO, Luciana; Giudici, Paolo
abstract

Mitochondrial DNA and Mitochondrial proteins in interspecific Saccharomyces hybrids

2006 - Molecular assessment of indigenous yeast population from traditional balsamic vinegar [Articolo su rivista]
Solieri, Lisa; Landi, Sara; DE VERO, Luciana; Giudici, Paolo
abstract

Aims: To identify and describe the indigenous yeast population involved in traditional balsamic vinegar (TBV) fermentation. Methods and Results: Using the restriction analysis of the ribosomal region 5(.)8S (5.8S rRNA) and the internal transcribed spacers 1 and 2 (5(.)8S-ITS region) we were able to group 133 strains isolated from 17 cooked grape must samples into 10 different yeast species, included into 4 genera. Moreover, we sequenced the D1/D2 domains of the 26S rRNA and confirmed the reliability of each identification at species level. Most strains belonged to the genus Zygosaccharomyces. In particular, Zygosaccharomyces bailii was found in 41% of the samples, followed by Saccharomyces cerevisiae, Zygosaccharomyces pseudorouxii and Candida stellata. Strains belonging respectively to Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces rouxii, Hanseniaspora valbyensis, Hanseniaspora osmophila and Candida lactis-condensi species were also detected. Despite the great number of species recovered, the mtDNA restriction profiles showed low variability at strain level. Saccharomyces cerevisiae isolates with an higher degree of intraspecific variance were considered an exception. Conclusions: Many different indigenous yeast species were recovered and TBV yeasts population seems to be far more complex than what was reported in previous literature. Significance and Impact of the Study: This study has allowed us to gain a better understanding of the indigenous yeast species of TBV cooked must.

2005 - Acetic acid bacteria in Traditional Balsamic Vinegar by PCR-DGGE analysis [Relazione in Atti di Convegno]
Gala, Elisabetta; DE VERO, Luciana; Solieri, Lisa; Gullo, Maria
abstract

Aims: The greatest limits in the study and selection of acetic acid bacteria are due to the difficultyof isolating and cultivating them. A culture-independent molecular technique, PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electophoresis), was used in order to study the acetic acid bacteria of Traditional Balsamic Vinegar.Methods and results: Primers WBAC1 and WBAC2 were successfully used with DNA extractedfrom both acetic acid bacteria strains and vinegar’s samples and they gave PCR products that allowed differentiation by DGGE. The results obtained indicate that acetic acid bacteria species involved in spontaneous oxidation process can be represented by only one dominant species.Conclusion: A change in acetic acid bacteria species during fermentation process could be supposed: Ga. xylinus was the main representative species in cooked must while A. pasteurianusbecame more prevalent in vinegar’s samples.Significance and impact of the study: This study clearly indicated that PCR-DGGE is a suitable tool for monitoring TBV microbial population and specifically acetic acid bacteria which are difficult to isolate applying conventional microbiological techniques.

2005 - Acetic acid bacteria of traditional balsamic vinegar. [Relazione in Atti di Convegno]
DE VERO, Luciana; Gullo, Maria; Landi, Sara; Solieri, Lisa
abstract

Aims: Traditional Balsamic Vinegar has been made by the ageing of cooked grape must into wooden barrels arranged in set of 5-7 casks each of varying capacity. Every year a small amount of vinegar is transferred from a bigger to a smaller barrel and new cooked must is added to the first barrel of the set. In this way the sugary concentration is increased from the biggest to the smallest barrel due to water evaporation across the wood.Methods and results: Different molecular techniques were applied to study acetic acid bacteriain Traditional Balsamic Vinegar. In particular 16S-23S-5S rDNA PCR/RFLP analysis, DGGE and sequencing techniques were performed on both acetic acid bacteria type strains and isolated strains from vinegars.Conclusion: Gluconacetobacter xylinus is the main species of Traditional Balsamic Vinegar and the greatest hurdle to the growth of acetic acid bacteria is high sugar concentration.Significance and impact of the study: The results suggest new technological approach to vinegar production.

2005 - Acido succinico quale causa della mancata rifermentazione spontanea in bottiglia del Lambrusco [Articolo su rivista]
S., Landi; Gullo, Maria; DE VERO, Luciana; Ulrici, Alessandro; R., Sidari; Giudici, Paolo
abstract

La rifermentazione in bottiglia dei Lambruschiè una pratica consolidata, che conferisce alvino caratteristiche sensoriali apprezzate daiconsumatori. Recentemente, e con sempremaggiore frequenza, è stata osservata la mancatarifermentazione del prodotto in bottiglia. Studipreliminari hanno messo in evidenza che lafrequenza della mancata rifermentazione èmaggiore nei vini imbottigliati precocementee senza aggiunta di lieviti. Inoltre, tutti icampioni esaminati che manifestano mancatarifermentazione contestualmente presentano valoridi acido succinico pari a 3 g/L. Va sempre tenutoconto che la crescita microbica è condizionatanon da un singolo fattore, ma da più fattori,perciò abbiamo allestito delle sperimentazioni chetenessero conto dei parametri più significativi,quali pH, densità di inoculo, concentrazione diacido succinico e concentrazione di etanolo. Unaprova di rifermentazione in bottiglia in condizionidi cantina ha definitivamente confermato la forteazione inibitrice dell’acido succinico sullo sviluppocellulare di Saccharomyces cerevisiae. I risultatisono coerenti con l’ipotesi che l’acido succinico,nell’ambito dei valori di pH e di concentrazionidi inoculo studiati, sia la causa della mancatarifermentazione.The spontaneous secondary alcoholicfermentation of the Lambrusco wine in bottle isa well-established practice that confer it someappreciated sensorial characteristics. Recently, andwith more frequency, it was observed the lack ofspontaneous secondary alcoholic fermentation inbottle. Preliminary studies have put in evidencethat the frequency of this problem is greater inearly bottled wines, produced without the additionof selected yeasts. Moreover, all the samples withthis problem have, at the same time, a succinicacid concentration of 3 g/L. These observationshave allowed the AA. to formulate the hypothesisthat succinic acid, in the presence of other limitingconditions, could be the cause of the lackedspontaneous secondary alcoholic. Since microbialgrowth is influenced by many factors, the AA. haveperformed several experiments considering themost significant parameters: pH, inoculum density,succinic acid content, and ethanol concentration.A secondary alcoholic fermentation test, carriedout under winery conditions, has confirmed thestrong inhibiting action of the succinic acid onSaccharomyces cerevisiae growth. The resultsare consistent with the hypothesis that, for thetested range, succinic acid is the reason of lackedspontaneous secondary alcoholic fermentation ofthe Lambrusco wine in bottle

2005 - Colture starter per la produzione di Aceto Balsamico Tradizionale: aspetti pratici ed applicativi [Articolo su rivista]
Giudici, Paolo; DE VERO, Luciana; Landi, Sara; Gullo, Maria; Solieri, Lisa
abstract

I batteri acetici sono un gruppo di microrganismia nicchia molto specializzata, difficili da coltivaree mantenere in coltura pura. I batteri acetici daimpiegare nella produzione di Aceto BalsamicoTradizionale devono possedere caratteri tecnologicidifferenti da quelli richiesti per altri tipi di aceto. Unmodo empirico ma efficace per il mantenimentodelle colture miste è quello di praticare frequentirinnovi senza attendere che i batteri aceticiesauriscano l’etanolo. Le colture, mantenute in modostatico, hanno una attività metabolica rallentata perla scarsa disponibilità di ossigeno e questo consentedi dilatare l’intervallo tra un rinnovo ed il successivo.In ogni caso, va sempre controllata la concentrazionedi etanolo che non deve scendere al di sotto del3% v/v al fine di garantire la pronta attività dellacoltura. La presenza di batteri acetici vitali ed attivinella coltura starter è condizione necessaria ma nonsufficiente a garantire il successo dell’ossidazioneacetica del mosto cotto fermentato, processoche trova nella composizione stessa del mosto ilsuo limite maggiore. Condizioni non permissivedella crescita batterica annullano completamentei benefici dell’impiego di colture starter. L’elevataconcentrazione zuccherina è una delle cause piùdiffuse dell’inibizione della crescita dei batteri acetici.

2005 - Homogeneity of interspecific hybrids between Saccharomyces cerevisiae and Saccharomyces uvarum by phenotypic and transcriptional analysis. [Articolo su rivista]
Solieri, Lisa; Gullo, Maria; DE VERO, Luciana; Antunez, O; PEREZ ORTIN J., E; Giudici, Paolo
abstract

Oenological traits, such as temperature profile and production of certain metabolites, were tested for four interspecifc hybrids obtained by “spore to spore” crossing between Saccharomyces cerevisiae and Saccharomyces uvarum strains and uniformity of their inheritance was found. PCR/RFLP analysis of ITSregions was carried out to confirm the hybrid nature of the strains. They showed an additive profile with five bands of the respective 325, 230, 170 and 125 bp.Finally gene expression study was performed by comparative DNA macroarray analysis of the hybrids and the preliminary results showed that the global geneexpression patterns of hybrids are remarkably similar to one another.In conclusion, the data obtained by two different approaches, such as metabolic and transcriptomic strategies, suggest a large degree of homogeneity amonginterspecific hybrids between S. cerevisiae and S. uvarum. Moreover, theuniformity of F1 hybrids advises that the oenological trait inheritance mechanismis highly constant and reproducible

2005 - I batteri acetici. [Capitolo/Saggio]
Giudici, Paolo; Gullo, Maria; DE VERO, Luciana
abstract

I batteri acetici. Tassonomia, Ecologia, fisiologia, esigenze nutrizionali

2005 - Limitations on the use of polymerase chain reaction - Restriction fragment length polymorphism analysis of the rDNA NTS2 region for the taxonomic classification of the species Saccharomyces cerevisiae [Articolo su rivista]
Pulvirenti, Andrea; Solieri, Lisa; DE VERO, Luciana; Giudici, Paolo
abstract

Different molecular techniques were tested to determine which was the most effective in the identification of Saccharomyces cerevisiae strains. In particular, polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions and the nontranscribed spacer 2 (NTS2) region, sequencing of the D1/D2 domain, and electrophoretic karyotyping were applied to 123 yeast strains isolated from different sourdoughs and tentatively attributed to the species S. cerevisiae. All of the strains tested showed an identical PCR-RFLP pattern for the ITS regions, an identical nucleotide sequence of the D1/D2 domain, and the typical electrophoretic karyo type of S. cerevisiae. In contrast, 14 out of the 123 strains tested showed some polymorphism with BanI restriction analysis of the NTS2 region. Our results indicate that while the sequencing of the D1/D2 domain, the PCR-RFLP analysis of the ITS regions, and the electrophoretic karyotype can be employed successfully to identify S. cerevisiae strains, PCR-RFLP analysis of the NTS2 region does not allow a consistent and accurate grouping for S. cerevisiae strains. The fact that the NTS2 region of a small number of strains (8.78% of the total strains tested) is different from that of the other S. cerevisiae strains confirms that molecular methods should always be tested on a great number of strains.

2005 - Proceedings of International Symposium on Vinegars and Acetic Acid Bacteria [Curatela]
Giudici, Paolo; Solieri, Lisa; DE VERO, Luciana
abstract

Abstracts collection of posters and lectures during International Symposium on Vinegars and Acetic Acid Bacteria, Reggio Emilia, Italy May 8-12, 2005

2005 - Sugar consumption by Yeast in cooked must for Traditional Balsamic Vinegar. [Relazione in Atti di Convegno]
S., Landi; Solieri, Lisa; DE VERO, Luciana; Giudici, Paolo
abstract

2005 - Theoretical approach to age determination of Traditional BalsamicVinegar [Abstract in Atti di Convegno]
Landi, S; Gullo, Maria; Solieri, Lisa; DE VERO, Luciana; Giudici, Paolo
abstract

Theoretical approach to determine the age of Traditional Balsamic Vinegar

2005 - vinegars and acetic acid bacteria international symposium [Esposizione]
Giudici, Paolo; Gullo, Maria; Solieri, Lisa; DE VERO, Luciana; Pulvirenti, Andrea; W., Ludwig; F., Barja; J., Bourgeois; J. M., Guillamon; A., Mas; P., Raspor
abstract

The key objective in planning the scientific programme was to ensure that the event would achieve the highest levels of scientific interest and topicality. Hence, the programme combines plenary session lectures, symposium sessions, as well as poster session.It would give a comprehensive overview of the most recent development in field of acetic acid bacteria and vinegar production and would surely stimulate fruitful discussions between attendees.One of the purposes of the meeting is also to create an international network for all the laboratories working in this field.

2004 - ANCHE LA TRADIZIONE VA STUDIATA: RISULTATI PRELIMINARI PER L’INDIVIDUAZIONE DI “STARTER” PER ACETO BALSAMICO TRADIZIONALE [Esposizione]
Giudici, Paolo; Gullo, Maria; DE VERO, Luciana; Solieri, Lisa
abstract

L’uso dei microrganismi nei processi di trasformazione degli alimenti è antecedente la loro scoperta e la comprensione del loro ruolo, che risale a soli due secoli fa. Da allora la microbiologia applicata alla trasformazione alimentare è stata oggetto di studi scientifici approfonditi che hanno consentito ricadute tecnologiche consistenti. Tuttavia, per quanto riguarda i prodotti tipici l’atteggiamento di riverenza verso ciò che si ritiene tradizione non consente un approccio razionale e, gli aspetti microbiologici sono i primi ad essere trascurati ed immolati sull’altare della tipicità. Questo tipo di atteggiamento ha fatto si che un prodotto antico come l’Aceto Balsamico Tradizionale, di fatto, non sia stato studiato approfonditamente nei suoi aspetti microbiologici ed oggi non è disponibile una sufficiente letteratura scientifica adeguata.

2004 - Batteri dell’Aceto Balsamico Tradizionale: caratteristiche fenotipiche, molecolari e tecnologiche. [Relazione in Atti di Convegno]
Gullo, M.; Solieri, Lisa; DE VERO, Luciana; Landi, S.; Giudici, Paolo
abstract

2004 - Cooked ham as a model system to predict shelf-life [Articolo su rivista]
De Vero, L.; Taccogna, M.; Solieri, L.; Puglisi, M. L.; Fava, P.; Giudici, P.; Gullo, M.
abstract

Some preservation techniques are here investigated in order to evaluate their influence on the cooked ham microflora evolution. Cooked ham has been considered as reference system in order to study the shelf-life of food products that are "not permissive" towards microorganisms growth. To predict the shelf-life of sliced cooked ham is very difficult because of both the potential casual contamination after cooking and the hygienic conditions of the slicing and packaging areas; such factors make the system extremely aleatory. In this research, ham samples have been inoculated with specific spoilage microorganisms previously isolated by a spoiled cooked ham. In a second test, nitrogen treatments have been applied with the aim of decreasing the system aleatority caused by the casual contamination.

2004 - Cristallizzazione dell'aceto balsamico tradizionale [Articolo su rivista]
Giudici, Paolo; Pulvirenti, Andrea; DE VERO, Luciana; S., Landi; Gullo, Maria
abstract

Il processo di produzione dell’Aceto BalsamicoTradizionale (ABT) è complesso e dipendente davariabili fisiche, chimiche e biologiche. Durantel’invecchiamento si assiste ad una rilevanteconcentrazione dei soluti che possono dare luogoalla formazione di cristalli, in quantità tale daoccupare l’intero volume del contenitore. Nelpresente lavoro sono stati analizzati campioni diABT prelevati da barili contenenti il prodotto piùvecchio e che presentavano precipitato solido ingrande quantità. Il precipitato è risultato esserecomposto prevalentemente da glucosio ed inmisura minore da fruttosio, con un rapportofra i due zuccheri diverso da quello osservatonelle uve. La presenza di cristalli di zuccheronell’ABT è spiegato da due cause concomitanti:l’elevata concentrazione dei soluti e il rapportoglucosio/fruttosio a favore del primo. Lamaggiore presenza percentuale di glucosio èimputabile al tipo di lieviti che hanno condotto lafermentazione.Traditional Balsamic Vinegar (ABT) production iscomplex and influenced by physical, chemical,and biological parameters. During the aging,a remarkable concentration of soluble solids isobserved. These solids can make a great amountof crystals that fills the whole volume of thebottle. In the present work, the precipitatedsoluble solids from oldest ABT samples wereanalyzed.The precipitate is mainly glucose and in smalleramount is fructose. Therefore the ratio betweenthe two sugars is different from the one in thegrapes. The sugar crystals observed in aged ABTare explainable on the basis of two associatedcauses: the elevated soluble solids concentrationand the ratio glucose/fructose pro glucose. Thegreater amount of glucose is due to the yeastspecies that ferment the cooked must.

2004 - I batteri acetici dell’Aceto Balsamico Tradizionale: caratterizzazione fenotipica e molecolare. [Relazione in Atti di Convegno]
M., Gullo; Pulvirenti, Andrea; DE VERO, Luciana; Giudici, Paolo
abstract

2004 - I lieviti dell’Aceto Balsamico Tradizionale dopo tre lustri. [Relazione in Atti di Convegno]
Solieri, Lisa; Pulvirenti, Andrea; A. R., Battagliola; M., Gullo; DE VERO, Luciana; Giudici, Paolo
abstract

2004 - I microrganismi dell'aceto balsamico. Atti convegno:"anche la tradizione va studiata" ricerche preliminari per l'individuazione di starter per l'Aceto Balsamico Tradizionale. [Curatela]
Giudici, Paolo; Gullo, Maria; Pulvirenti, Andrea; DE VERO, Luciana; Solieri, Lisa
abstract

Il ruolo e l'importanza dei microrganismi sulla qualità dell'aceto balsamico tradizionale.

2004 - Il prosciutto cotto quale sistema modello per lo studio della previsione della shelf-life. [Articolo su rivista]
DE VERO, Luciana; M., Taccogna; Solieri, Lisa; Gullo, Maria; Puglisi, Maria Laura; Fava, Patrizia; Giudici, Paolo
abstract

Nella presente ricerca si è inteso valutare comealcune tecniche di conservazione possanoincidere sull’evoluzione della complessiva del prosciutto cotto, scelto qualesistema modello per lo studio della inprodotti alimentari non restrittivi per la crescitadei microrganismi. Predire la shelf-life delprosciutto cotto affettato risulta molto difficilein quanto la contaminazione casuale dopo lacottura e le condizioni igieniche delle zonedi affettamento e confezionamento rendonol’intero sistema estremamente aleatorio. Nellaricerca effettuata, i campioni di prosciutto sonostati inoculati con microrganismi specifici dialterazione precedentemente isolati da prosciuttocotto alterato. In una seconda prova sono statiimpiegati trattamenti con azoto allo stesso finedi diminuire l’aleatorietà del sistema dovuta allacontaminazione casuale.

2004 - Influenza della composizione del mezzo sull’attività biologica dell’ABT. [Relazione in Atti di Convegno]
DE VERO, Luciana; Landi, Sara; Gullo, Maria; Giudici, Paolo
abstract

La composizione del mosto cotto esercita una forte pressione selettiva sui microrganismi presenti determinando la prevalenza di lieviti o batteri acetici in funzione della combinazioni di variabili quali, il pH, la concentrazione in solidi solubili, la presenza di etanolo o di acido acetico. In sei differenti composizioni del mezzo ottenuti variando la percentuale di mosto cotto e mosto fresco d’uva, sono stati osservati andamenti fermentativi significativamente diversi, ma riconducibili a due tipologie: una a prevalente attività fermentativa ad opera di lieviti fruttosofili e l’altra a prevalente attività ossidativa ad opera di batteri acetici. Ciò che sembra maggiormente determinare lo sviluppo di lieviti o di batteri è la concentrazione zuccherina iniziale. Concentrazioni del mezzo superiori ai 30 °Brix, in presenza di acido acetico (1% circa) e valori di pH bassi, imprimono una forte pressione selettiva che consente lo sviluppo di poche specie di lievito, prevalentemente fruttosofile, ma non dei batteri acetici. Al contrario concentrazioni di solidi solubili inferiori a 30 °Brix non inibiscono lo sviluppo dei batteri acetici. Inoltre, alcuni prodotti del metabolismo microbico possono essere impiegati come indicatori indiretti della prevalenza di un determinato gruppo nel processo fermentativo.

2004 - Lieviti e zuccheri nell’Aceto Balsamico Tradizionale. [Relazione in Atti di Convegno]
Landi, S.; Castellari, L.; Gullo, Maria; Solieri, Lisa; DE VERO, Luciana; Giudici, Paolo
abstract

2004 - Lieviti osmofili: differenze fenotipiche e molecolari. [Relazione in Atti di Convegno]
Pulvirenti, P.; Battagliola, A. R.; Gullo, M.; DE VERO, Luciana; Landi, S.; Giudici, Paolo
abstract

2004 - Limits of r-DNA-NTS2 regions on the taxonomy of Saccharomyces genus [Relazione in Atti di Convegno]
Pulvirenti, Andrea; Solieri, Lisa; DE VERO, Luciana; Gullo, Maria; Giudici, Paolo
abstract

The aim of this work was to test different molecular techniques for determining which was the most effective in the identification of Saccharomyces cerevisiae strains. In particular, polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions and the nontranscribed spacer 2 (NTS2) region, sequencing of the D1/D2 domain, and electrophoretic karyotyping were applied to 123 yeast strains isolated from different sourdoughs and tentatively attributed to the species S. cerevisiae. All of the strains tested showed an identical PCR-RFLP pattern for the ITS regions, an identical nucleotide sequence of the D1/D2 domain, and the typical electrophoretic karyo type of S. cerevisiae. In contrast, 14 out of the 123 strains tested showed some polymorphism with BanI restriction analysis of the NTS2 region. Our results indicate that while the sequencing of the D1/D2 domain, the PCR-RFLP analysis of the ITS regions, and the electrophoretic karyotype can be employed successfully to identify S. cerevisiae strains, PCR-RFLP analysis of the NTS2 region does not allow a consistent and accurate grouping for S. cerevisiae strains. The fact that the NTS2 region of a small number of strains (8.78% of the total strains tested) is different from that of the other S. cerevisiae strains confirms that molecular methods should always be tested on a great number of strains.

2004 - MODELLO TEORICO PER LA VALUTAZIONE DELL’ ETÀ E DELLA PRODUTTIVITÀ MASSIMA DI UNA BATTERIA DI ACETO BALSAMICO TRADIZIONALE DI REGGIO EMILIA. [Articolo su rivista]
Giudici, Paolo; Gullo, Maria; DE VERO, Luciana; G., Masini
abstract

processo di produzione dell’Aceto Balsamico Tradizionale di Reggio Emilia (ABT) richiede almeno 12 anni di invecchiamento e necessita di trasferimenti tra un barile e l’altro con conseguente miscelazione di prodotti di diversa età. Tutto ciò rende molto difficile effettuare un bilancio preciso della resa di trasformazione del mosto cotto in ABT, della stima della reale età dell’ABT nonché della massima produttività ammissibile di una batteria tale per cui l’età media dell’ABT sia sempre superiore ai 12 anni previsti dal disciplinare di produzione. Nel presente lavoro è stato sviluppato un modello teorico, che a partire dal volume della batteria e del flusso in ingresso di mosto cotto necessario ad alimentare la batteria stessa, consente di calcolare l’età dell’ABT e stimare la massima produzione ammissibile di ABT. Il modello è applicabile a batterie in equilibrio che abbiano almeno 12 anni dalla data di costituzione. L’età dell’ABT in uscita è in funzione del rapporto tra la massa di soluti nella batteria ed il flusso di soluti in ingresso.The production of Traditional Balsamic Vinegar of Reggio Emilia (ABT) requires at least 12 years of aging. The vinegar is transferred from a barrel to another resulting in a mix of products with different ages. Therefore it is very difficult to assess the age and the precise yield of ABT from cooked must. The real age of the ABT, as well as the maximum admissible productivity for barrels, are important quality parameters. The ABT good manufacturing practices manual requires at least 12 years aging. In this work the AA have developed a theoretical model which allows to calculate the age and the maximum admissible productivity of ABT for barrels. The model is based on the volume of the barrels and on the incoming flow of the cooked must needed to feed the same barrels. The model is valid for barrels at the equilibrium which are at least 12 years old. The age of the ABT in exit is a function of the mass of solutes in the barrels and the flow of solutes in entry.

2004 - Nuove frontiere nello studio dei batteri acetici: la DGGE [Relazione in Atti di Convegno]
DE VERO, Luciana; Gullo, Maria; Solieri, Lisa; Landi, Sara; Giudici, Paolo
abstract

I batteri acetici (B.A.) sono microrganismi difficili da isolare e conservare in coltura pura nonostante i moltissimi mezzi di crescita e le condizioni di incubazione impiegate. Il rischio maggiore che si incontra con le procedure convenzionali adottate nello studio di popolazioni microbiche, è quello di isolare non tanto le specie o i ceppi che hanno condotto l’ossidazione, ma quelli più facilmente isolabili anche se numericamente meno importanti. A causa della non-coltivabilità dei B.A. si è reso necessario studiare le popolazioni selvagge degli aceti con l’ausilio di tecniche che non prevedano l’isolamento di questi microrganismi. Una tecnica molecolare coltura-indipendente è risultata essere la PCR-DGGE (Polymerase Chain Reaction - Denaturing Gradient Gel Electophoresis).I risultati ottenuti sono di supporto a due considerazioni, una di interesse più generale che conferma l’utilità della DGGE nello studio delle popolazioni batteriche dell’aceto. L’altra più specifica e da confermare con studi ulteriori, è relativa alle specie di batteri acetici riscontrabili in una ossidazione spontanea; tali specie possono essere in numero molto limitato e, in alcuni casi, addirittura riconducibili ad una sola specie dominante.

2004 - Occurrence and dominance of yeast species in sourdough [Articolo su rivista]
Pulvirenti, Andrea; Solieri, Lisa; Gullo, Maria; DE VERO, Luciana; Giudici, Paolo
abstract

Aims: The aim of this work is to identify the dominant yeast species in homemade sourdoughs. Methods and Results: PCR/restriction fragment length polymorphism analysis of internal transcribed spacer regions was used for the identification of isolates and the data were confirmed with phenotypic tests. The strains belonging to Saccharomyces cerevisiae were identified to strain level by analysis of inter-delta regions. Conclusion: This work shows that the dominant species in homemade sourdoughs can differ from each other. Saccharomyces cerevisiae was found to be the dominant species, followed by the Candida milleri, C. humilis, S. exiguus and Issatchenkia orientalis. The inter-delta regions of S. cerevisiae strains showed high polymorphism. Significance and Impact of the Study: Occurrence of single, non-Saccharomyces species and S. cerevisiae polymorphism in the yeast populations of sourdough samples.

2004 - Pressione selettiva del mosto cotto sull’attività biologica dell’ABT. [Articolo su rivista]
Gullo, Maria; DE VERO, Luciana; S., Landi; Giudici, Paolo
abstract

La composizione del mosto cotto, utilizzato per la produzione di Aceto Balsamico Tradizionale (ABT), esercita una forte pressione selettiva sui microrganismi presenti. La prevalenza di lieviti o batteri acetici è in funzione delle combinazioni di variabili quali, pH, concentrazione in solidi solubili, presenza di etanolo e acido acetico. In sei differenti tesi ottenute variando la composizione del mezzo, sono stati osservati andamenti fermentativi significativamente diversi ma riconducibili a due tipologie: una a prevalente attività fermentativa ad opera di lieviti fruttosofili e l’altra a prevalente attività ossidativa ad opera di batteri acetici (B.A.). Inoltre, alcuni ceppi di B.A. isolati da mosti per la produzione di ABT sono stati saggiati per la loro capacità di crescere in presenza di concentrazioni crescenti di glucosio ed etanolo. Dai risultati si evince che l’ostacolo maggiore allo sviluppo dei B.A. è rappresentato dall’elevata concentrazione zuccherina dei mosti cotti e concentrati.The composition of the cooked must has a strong selective pressure on the microorganisms. The prevalence of yeasts or of acetic acid bacteria is determined by the combinations of some parameters such as pH, concentration in soluble solids, presence of ethanol or acetic acid. Significantly different fermentative processes have been observed in six different trials, obtained varying the composition of the medium. The processes are referable to two typologies: one with a prevailing fermentative activity of fructosophilic yeasts and the other one with a prevailing oxidative activity due to the acetic acid bacteria (B.A.). Furthermore, some B.A. strains isolated from musts used in the Traditional Balsamic Vinegar production, have been tested for their ability to grow in presence of increasing concentrations of glucose and ethanol. On the basis of the results it is evident that the greatest hurdle to the growth of B.A. is the elevated sugary concentration of the cooked and concentrated musts.

2004 - Ricerche finalizzate alla tutela e tipicità dell'Aceto Balsamico Tradizionale di Reggio Emilia [Esposizione]
Giudici, Paolo; Gullo, Maria; DE VERO, Luciana; Pulvirenti, Andrea; U., Rangone
abstract

La valorizzazione e la tutela dell’Aceto Balsamico Tradizionale, al pari di qualsiasi altro prodotto alimentare tradizionale, non può prescindere dalla conoscenza delle ragioni che lo rendono così specifico e particolare. La ricerca scientifica, oltre ad essere lo strumento di conoscenza necessario alla comprensione della complessità dell’ABT, è anche veicolo di promozione, nel rispetto della chiarezza, dell’autenticità e dell’oggettività, senza ricorrere a spiegazioni aleatorie e fantasiose, che nulla hanno a che fare con la concretezza dell’ABT, frutto dell’acquisizione empirica della conoscenza.

2003 - Microflora Y La Tecnología De Producción Del Vinagre Balsámico Tradicional. [Relazione in Atti di Convegno]
Giudici, Paolo; Gullo, Maria; Pulvirenti, Andrea; Solieri, Lisa; DE VERO, Luciana
abstract

Acetic acid bacteria of vinegar

2003 - Sorting of mitocondrial DNA and proteins in the progeny of Saccharomyces interspecific hybrids [Articolo su rivista]
De Vero, Luciana; Pulvirenti, Andrea; Gullo, Maria; Bonatti, Piera; Giudici, Paolo
abstract

The mitochondrial transmission to the progeny of interspecific hybrids Saccharomyces cerevisiae x Saccharomyces uvarum was investigated. It was observed that la progenie degli hybrids ha un solo tipo di mitocondri parentali come è stato osservato attraverso l’analisi RFLP dell’intero mtDNA. The mitochondrial proteins, invece, sono il risultato della ricombinazione del genoma dei due parentali. We suppose an important role of mitochondrial proteins in the process of mitochondrial transmission in yeast and hypothesize that inheritance of mtDNA may depend upon nuclear DNA genes. It seems to be probable that uniparental inheritance is independent on position of mitochondria in the buds of zygotes when the haploid cells fuse.

2003 - Studio molecolare e fenotipico di lieviti in paste acide tradizionali [Articolo su rivista]
Solieri, Lisa; DE VERO, Luciana; Gullo, Maria; Pulvirenti, Andrea
abstract

In questo lavoro è stato condotto uno studiodelle specie di lievito più rappresentatein impasti acidi tradizionali, preparatisenza l’aggiunta di lieviti starter. Tali speciesi contraddistinguono per importanzatecnologica in quanto numericamente prevalentie maggiormente responsabili delprocesso fermentativo. Le procedure diidentificazione adottate, basate su tecnichesia molecolari che fenotipiche, hannoconsentito di evidenziare una grande biodiversitànegli impasti acidi esaminati, ciascunodei quali rappresenta un unicuumsotto il profilo eumicetico.In this work a study on dominant yeastsspecies was carried out, using phenotypicand molecular techniques. These speciesare of technological interest since theyare the main responsible of the fermentationprocess. The obtained identificationsindicate that the examined home-madesourdoughs were characterized by a greatbiodiversity: each has got a distinguishingand unique yeast profile. The prevalenceof different yeast species in sourdoughssuggests to consider in additionto S. cerevisiae, also C. humilis, C. milleri,I. orientalis, and S. exiguus as speciessuitable for bread making.

2003 - Transmission of mt DNA and mitochondrial proteins in the progeny of Saccharomyces hybrids [Abstract in Atti di Convegno]
DE VERO, Luciana; Pulvirenti, Andrea; Gullo, Maria; Solieri, Lisa; Giudici, Paolo
abstract

Transmission of mt DNA and mitochondrial proteins in the progeny of Saccharomyces hybrids

2003 - Transmission of mtdna and mitochondrial proteins in the progeny of Saccharomyces interspecific hybrids. [Relazione in Atti di Convegno]
DE VERO, Luciana; Pulvirenti, Andrea; Gullo, Maria; Solieri, Lisa; Giudici, Paolo
abstract

2002 - Uniparental Mitochondrial Inheritance in Saccaromyces spore coniugation. [Abstract in Atti di Convegno]
Pulvirenti, Andrea; Gullo, Maria; DE VERO, Luciana; Giudici, Paolo
abstract

Uniparental Mitochondrial Inheritance in Saccaromyces spore coniugation.

Università degli studi di Modena e Reggio Emilia

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