Nuova ricerca

Lisa SOLIERI

Professore Associato
Dipartimento di Scienze della Vita sede ex-Agraria


Home | Curriculum(pdf) | Didattica |


Pubblicazioni

2023 - Effect of Fermentation with Streptococcus thermophilus Strains on In Vitro Gastro-Intestinal Digestion of Whey Protein Concentrates [Articolo su rivista]
Helal, A.; Pierri, S.; Tagliazucchi, D.; Solieri, L.
abstract

Three Streptococcus thermophilus strains, namely RBC6, RBC20, and RBN16, were proven to release bioactive peptides during whey protein concentrate (WPC) fermentation, resulting in WPC hydrolysates with biological activities. However, these bioactive peptides can break down during gastro-intestinal digestion (GID), hindering the health-promoting effect of fermented WPC hydrolysates in vivo. In this work, the effect of simulated GID on three WPC hydrolysates fermented with S. thermophilus strains, as well as on unfermented WPC was studied in terms of protein hydrolysis, biological activities, and peptidomics profiles, respectively. In general, WPC fermentation enhanced protein hydrolysis compared to unfermented WPC. After in vitro GID, WPC fermented with S. thermophilus RBC20 showed the highest antioxidant activity, whereas WPC fermented with strain RBC06 displayed the highest angiotensin-converting enzyme (ACE)- and dipeptidyl peptidase IV (DPP-IV)-inhibitory activities. Peptidomics analysis revealed that all digested WPC samples were highly similar to each other in peptide profiles, and 85% of the 46 identified bioactive peptides were shared among fermented and unfermented samples. However, semi-quantitative analysis linked the observed differences in biological activities among the samples to differences in the amount of bioactive peptides. The anti-hypertensive peptides VPP and IPP, as well as the DPP-IV-inhibitory peptide APFPE, were quantified. In conclusion, WPC fermentation with S. thermophilus positively impacted protein hydrolysis and bioactive peptide release during GID.


2023 - Impact of Spontaneous Fermentation and Inoculum with Natural Whey Starter on Peptidomic Profile and Biological Activities of Cheese Whey: A Comparative Study [Articolo su rivista]
MOHAMED IBRAHIM HELAL, Ahmed; Nasuti, Chiara; Sola, Laura; Sassi, Giada; Tagliazucchi, Davide; Solieri, Lisa
abstract

Fermentation is a promising solution to valorize cheese whey, the main by-product of the dairy industry. In Parmigiano Reggiano cheese production, natural whey starter (NWS), an undefined community of thermophilic lactic acid bacteria, is obtained from the previous day residual whey through incubation at gradually decreasing temperature after curd cooking. The aim of this study was to investigate the effect of fermentation regime (spontaneous (S) and NWS-inoculated (I-NWS)) on biofunctionalities and release of bioactive peptides during whey fermentation. In S and I-NWS trials proteolysis reached a peak after 24 h, which corresponded to the drop out in pH and the maximum increase in lactic acid. Biological activities increased as a function of fermentation time. NWS inoculum positively affected antioxidant activity, whilst S overcame I-NWS in angiotensin-converting enzyme (ACE) and DPP-IV (dipeptidyl peptidase IV) inhibitory activities. Peptidomics revealed more than 400 peptides, mainly derived from β-casein, κ-casein, and α-lactalbumin. Among them, 49 were bioactive and 21 were ACE-inhibitors. Semi-quantitative analysis strongly correlated ACE-inhibitory activity with the sum of the peptide abundance of ACE-inhibitory peptides. In both samples, lactotripeptide isoleucine-proline-proline (IPP) was higher than valine-proline-proline (VPP), with the highest content in S after 24 h of fermentation. In conclusion, we demonstrated the ability of whey endogenous microbiota and NWS to extensively hydrolyze whey proteins, promoting the release of bioactive peptides and improving protein digestibility.


2023 - Sour Beer as Bioreservoir of Novel Craft Ale Yeast Cultures [Articolo su rivista]
Nasuti, C.; Ruffini, J.; Sola, L.; Di Bacco, M.; Raimondi, S.; Candeliere, F.; Solieri, L.
abstract

: The increasing demand for craft beer is driving the search for novel ale yeast cultures from brewing-related wild environments. The focus of bioprospecting for craft cultures is to identify feral yeasts suitable to imprint unique sensorial attributes onto the final product. Here, we integrated phylogenetic, genotypic, genetic, and metabolomic techniques to demonstrate that sour beer during aging in wooden barrels is a source of suitable craft ale yeast candidates. In contrast to the traditional lambic beer maturation phase, during the aging of sour-matured production-style beer, different biotypes of Saccharomyces cerevisiae dominated the cultivable in-house mycobiota, which were followed by Pichia membranifaciens, Brettanomyces bruxellensis, and Brettanomyces anomalus. In addition, three putative S. cerevisiae × Saccharomyces uvarum hybrids were identified. S. cerevisiae feral strains sporulated, produced viable monosporic progenies, and had the STA1 gene downstream as a full-length promoter. During hopped wort fermentation, four S. cerevisiae strains and the S. cerevisiae × S. uvarum hybrid WY213 exceeded non-Saccharomyces strains in fermentative rate and ethanol production except for P. membranifaciens WY122. This strain consumed maltose after a long lag phase, in contrast to the phenotypic profile described for the species. According to the STA1+ genotype, S. cerevisiae partially consumed dextrin. Among the volatile organic compounds (VOCs) produced by S. cerevisiae and the S. cerevisiae × S. uvarum hybrid, phenylethyl alcohol, which has a fruit-like aroma, was the most prevalent. In conclusion, the strains characterized here have relevant brewing properties and are exploitable as indigenous craft beer starters.


2022 - Characterization of Cell-Envelope Proteinases from Two Lacticaseibacillus casei Strains Isolated from Parmigiano Reggiano Cheese [Articolo su rivista]
Solieri, L; Sola, L; Vaccalluzzo, A; Randazzo, Cl; Martini, S /; Tagliazucchi, D
abstract


2022 - Fermentation of whey protein concentrate by Streptococcus thermophilus strains releases peptides with biological activities [Articolo su rivista]
Solieri, L.; Valentini, M.; Cattivelli, A.; Sola, L.; Helal, A.; Martini, S.; Tagliazucchi, D.
abstract

Cheese whey is the principal by-product of cheese making and is perceived as an environmental pollutant. Different technological procedures have been applied to convert whey into value-added products including whey protein concentrate (WPC). In the present study, 2 Lactobacillus helveticus strains and 5 Streptococcus thermophilus strains, isolated from natural whey starter used in Parmigiano Reggiano production, have been evaluated for their capacity to ferment WPC and to release bioactive peptides. WPC fermented with S. thermophilus RBC06 showed the highest antioxidant activity as well as angiotensin-converting enzyme- and dipeptidyl-peptidase-IV-inhibitory activities followed by WPC fermented by S. thermophilus RBC20 and RBN16. An untargeted peptidomics approach was applied to profile the peptides released after WPC fermentation. Several bioactive peptides, mainly angiotensin-converting enzyme inhibitors, were identified and the anti-hypertensive lactotripeptides valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) were quantified. S. thermophilus RBC06 produced the highest amount of VPP (12.80 ± 0.45 mg/L) whereas the highest quantity of IPP (0.56 ± 0.02 mg/L) was detected in WPC fermented with S. thermophilus RBN16. The present study paved the way for the further exploitation of S. thermophilus RBC06 as new bioactive-peptide producer and for the application of WPC hydrolysates as additives for designing whey-based beverages with improved healthy properties.


2022 - Insights on the bacterial composition of Parmigiano Reggiano Natural Whey Starter by a culture-dependent and 16S rRNA metabarcoding portrait [Articolo su rivista]
Sola, L.; Quadu, E.; Bortolazzo, E.; Bertoldi, L.; Randazzo, C. L.; Pizzamiglio, V.; Solieri, L.
abstract

: Natural whey starters (NWS) are undefined bacterial communities produced daily from whey of the previous cheese-making round, by application of high temperature. As a result, in any dairy plant, NWS are continuously evolving, undefined mixtures of several strains and/or species of lactic acid bacteria, whose composition and performance strongly depend on the selective pressure acting during incubation. While NWS is critical to assure consistency to cheese-making process, little is known about the composition, functional features, and plant-to-plant fluctuations. Here, we integrated 16S rRNA metabarcoding and culture-dependent methods to profile bacterial communities of 10 NWS sampled in the production area of Parmigiano Reggiano cheese. 16S rRNA metabarcoding analysis revealed two main NWS community types, namely NWS type-H and NWS type-D. Lactobacillus helveticus was more abundant in NWS type-H, whilst Lactobacillus delbrueckii/St. thermophilus in NWS type-D, respectively. Based on the prediction of metagenome functions, NWS type-H samples were enriched in functional pathways related to galactose catabolism and purine metabolism, while NWS type-D in pathways related to aromatic and branched chain amino acid biosynthesis, which are flavor compound precursors. Culture-dependent approaches revealed low cultivability of individual colonies as axenic cultures and high genetic diversity in the pool of cultivable survivors. Co-culturing experiments showed that fermentative performance decreases by reducing the bacterial complexity of inoculum, suggesting that biotic interactions and cross-feeding relationships could take place in NWS communities, assuring phenotypic robustness. Even though our data cannot directly predict these ecological interactions, this study provides the basis for experiments targeted at understanding how selective regime affects composition, bacterial interaction, and fermentative performance in NWS.


2022 - Metabolomic and Transcriptional Profiling of Oleuropein Bioconversion into Hydroxytyrosol during Table Olive Fermentation by Lactiplantibacillus plantarum [Articolo su rivista]
Vaccalluzzo, A; Solieri, L; Tagliazucchi, D; Cattivelli, A; Martini, S; Pino, A; Caggia, C; Randazzo, Cl
abstract


2022 - Microbial Consortia Involved in Traditional Sicilian Sourdough: Characterization of Lactic Acid Bacteria and Yeast Populations [Articolo su rivista]
Pino, A; Russo, N; Solieri, L; Sola, L; Caggia, C; Randazzo, Cl
abstract


2021 - An integrated peptidomics and in silico approach to identify novel anti-diabetic peptides in parmigiano-reggiano cheese [Articolo su rivista]
Martini, S.; Solieri, L.; Cattivelli, A.; Pizzamiglio, V.; Tagliazucchi, D.
abstract

Inhibition of key metabolic enzymes linked to type-2-diabetes (T2D) by food-derived compounds is a preventive emerging strategy in the management of T2D. Here, the impact of Parmigiano- Reggiano (PR) cheese peptide fractions, at four different ripening times (12, 18, 24, and 30 months), on the enzymatic activity of α-glucosidase, α-amylase, and dipeptidyl peptidase-IV (DPPIV) as well as on the formation of fluorescent advanced glycation end-products (fAGEs) was assessed. The PR peptide fractions were able to inhibit the selected enzymes and fAGEs formation. The 12-month-ripening PR sample was the most active against the three enzymes and fAGEs. Mass spectrometry analysis enabled the identification of 415 unique peptides, 54.9% of them common to the four PR samples. Forty-nine previously identified bioactive peptides were found, mostly characterized as angiotensin-converting enzyme-inhibitors. The application of an integrated approach that combined peptidomics, in silico analysis, and a structure–activity relationship led to an efficient selection of 6 peptides with potential DPP-IV and α-glucosidase inhibitory activities. Peptide APFPE was identified as a potent novel DPP-IV inhibitor (IC50 = 49.5 ± 0.5 μmol/L). In addition, the well-known anti-hypertensive tripeptide, IPP, was the only one able to inhibit the three digestive enzymes, highlighting its possible new and pivotal role in diabetes management.


2021 - Characterization of yeasts isolated from parmigiano reggiano cheese natural whey starter: From spoilage agents to potential cell factories for whey valorization [Articolo su rivista]
Martini, S.; Bonazzi, M.; Malorgio, I.; Pizzamiglio, V.; Tagliazucchi, D.; Solieri, L.
abstract

Whey is the main byproduct of the dairy industry and contains sugars (lactose) and proteins (especially serum proteins and, at lesser extent, residual caseins), which can be valorized by the fermentative action of yeasts. In the present study, we characterized the spoilage yeast population inhabiting natural whey starter (NWS), the undefined starter culture of thermophilic lactic acid bacteria used in Parmigiano Reggiano (PR) cheesemaking, and evaluated thermotolerance, mating type, and the aptitude to produce ethanol and bioactive peptides from whey lactose and proteins, respectively, in a selected pool of strains. PCR-RFLP assay of ribosomal ITS regions and phylogenetic analysis of 26S rDNA D1/D2 domains showed that PR NWS yeast population consists of the well-documented Kluyveromyces marxianus, as well as of other species (Saccharomyces cerevisiae, Wickerhamiella pararugosa, and Torulaspora delbrueckii), with multiple biotypes scored within each species as demonstrated by (GTG)5-based MSP-PCR. Haploid and diploid K. marxianus strains were identified through MAT genotyping, while thermotolerance assay allowed the selection of strains suitable to grow up to 48◦C. In whey fermentation trials, one thermotolerant strain was suitable to release ethanol with a fermentation efficiency of 86.5%, while another candidate was able to produce the highest amounts of both ethanol and bioactive peptides with potentially anti-hypertensive function. The present work demonstrated that PR NWS is a reservoir of ethanol and bioactive peptides producer yeasts, which can be exploited to valorize whey, in agreement with the principles of circularity and sustainability.


2021 - Hybridization of Saccharomyces cerevisiae Sourdough Strains with Cryotolerant Saccharomyces bayanus NBRC1948 as a Strategy to Increase Diversity of Strains Available for Lager Beer Fermentation [Articolo su rivista]
Catallo, Martina; Iattici, Fabrizio; Randazzo, Cinzia; Caggia, Cinzia; Krogerus, Kristoffer; Magalhaes, Frederico; Gibson, Brian; Solieri, Lisa
abstract

The search for novel brewing strains from non-brewing environments represents an emerging trend to increase genetic and phenotypic diversities in brewing yeast culture collections. Another valuable tool is hybridization, where beneficial traits of individual strains are combined in a single organism. This has been used successfully to create de novo hybrids from parental brewing strains by mimicking natural Saccharomycescerevisiae ale × Saccharomyceseubayanus lager yeast hybrids. Here, we integrated both these approaches to create synthetic hybrids for lager fermentation using parental strains from niches other than beer. Using a phenotype-centered strategy, S. cerevisiae sourdough strains and the S. eubayanus × Saccharomyces uvarum strain NBRC1948 (also referred to as Saccharomyces bayanus) were chosen for their brewing aptitudes. We demonstrated that, in contrast to S. cerevisiae × S. uvarum crosses, hybridization yield was positively affected by time of exposure to starvation, but not by staggered mating. In laboratory-scale fermentation trials at 20 °C, one triple S. cerevisiae × S. eubayanus × S. uvarum hybrid showed a heterotic phenotype compared with the parents. In 2 L wort fermentation trials at 12 °C, this hybrid inherited the ability to consume efficiently maltotriose from NBRC1948 and, like the sourdough S. cerevisiae parent, produced appreciable levels of the positive aroma compounds 3-methylbutyl acetate (banana/pear), ethyl acetate (general fruit aroma) and ethyl hexanoate (green apple, aniseed, and cherry aroma). Based on these evidences, the phenotype-centered approach appears promising for designing de novo lager beer hybrids and may help to diversify aroma profiles in lager beer.


2021 - Insights on life cycle and cell identity regulatory circuits for unlocking genetic improvement in Zygosaccharomyces and Kluyveromyces yeasts [Articolo su rivista]
Solieri, L; CassanelliS, ; Huff, F; Barroso, L; Branduardi, P; Louis, Ej; Morrissey, Jp
abstract


2021 - Peptidomics: new trends in food science [Articolo su rivista]
Martini, S.; Solieri, L.; Tagliazucchi, D.
abstract

In recent years, peptidomics is gaining ever-growing relevance in food science. The emerging field of food peptidomics is defined as the whole pool of peptides existing in food products or generated during food processing, storage or digestion. The recent advancement in high-resolution mass spectrometry techniques provides a more detailed picture, although not yet exhaustive, of the peptides present or derived from food. Food peptidomics techniques have been successfully applied in understanding food protein digestion, in the study of the microbial contribution to food protein hydrolysis, in the identification of food-derived bioactive peptides and peptide biomarkers. Ad-hoc efforts are still needed to improve the identification of short peptides and the analysis of the large data set generated.


2021 - The revenge of Zygosaccharomyces yeasts in food biotechnology and applied microbiology [Articolo su rivista]
Solieri, Lisa
abstract


2020 - Cultivable non-starter lactobacilli from ripened Parmigiano Reggiano cheeses with different salt content and their potential to release anti-hypertensive peptides [Articolo su rivista]
Tagliazucchi, Davide; Baldaccini, Andrea; Martini, Serena; Bianchi, Aldo; Pizzamiglio, Valentina; Solieri, Lisa
abstract

The impact of salt and fat intake on human health drives the consumer's attention towards dairy food with reduced salt and fat contents. How changes in salt and fat content modulate dairy LAB population and the associated proteolytic activities have been poorly studied. Here, non-starter LAB populations from 12 Parmigiano Reggiano (PR) cheeses (12-month ripened), clustered in low salt and fat content (LL-PR) and high salt and fat content (HH-PR) groups, were investigated and identified at specie-level with molecular assays. Lactobacillus rhamnosus was dominant in HH-PR samples, whereas Lactobacillus paracasei in LL-PR samples. (GTG)5 rep-PCR analysis discriminated 11 and 12 biotypes for L. rhamnosus and L. paracasei isolates, respectively. Screening for proteolytic activity identified L. rhamnosus strains more proteolytic than L. paracasei, and, within L. rhamnosus species, HH-PR strains were generally more proteolytic than LL-PR strains. Two L. rhamnosus representatives, namely strain 0503 from LL-PR and strain 2006 from HH-PR, were functionally characterized in cow milk fermentation assay. HH-PR strain 2006 overcame LL-PR strain 0503 in acidification performance, leading to a fermented milk with higher angiotensin I-converting enzyme inhibitory and antioxidant activities. L. rhamnosus 2006 was more prone to release VPP, while L. rhamnosus 0503 released higher amount of IPP. This study provides evidences that salt/fat content affects NSLAB cultivable fraction and the associated proteolytic ability resulting in a complex occurrence of bioactive peptides featuring health-promoting properties.


2020 - Designing New Yeasts for Craft Brewing: When Natural Biodiversity Meets Biotechnology [Articolo su rivista]
Iattici, F; Catallo, M; Solieri, L
abstract

Beer is a fermented beverage with a history as old as human civilization. Ales and lagers are by far the most common beers; however, diversification is becoming increasingly important in the brewing market and the brewers are continuously interested in improving and extending the range of products, especially in the craft brewery sector. Fermentation is one of the widest spaces for innovation in the brewing process. Besides Saccharomyces cerevisiae ale and Saccharomyces pastorianus lager strains conventionally used in macro-breweries, there is an increasing demand for novel yeast starter cultures tailored for producing beer styles with diversified aroma profiles. Recently, four genetic engineering-free approaches expanded the genetic background and the phenotypic biodiversity of brewing yeasts and allowed novel costumed-designed starter cultures to be developed: (1) the research for new performant S. cerevisiae yeasts from fermented foods alternative to beer; (2) the creation of synthetic hybrids between S. cerevisiae and Saccharomyces non-cerevisiae in order to mimic lager yeasts; (3) the exploitation of evolutionary engineering approaches; (4) the usage of non-Saccharomyces yeasts. Here, we summarized the pro and contra of these approaches and provided an overview on the most recent advances on how brewing yeast genome evolved and domestication took place. The resulting correlation maps between genotypes and relevant brewing phenotypes can assist and further improve the search for novel craft beer starter yeasts, enhancing the portfolio of diversified products offered to the final customer.


2020 - Peptide profiling and biological activities of 12- month ripened parmigiano reggiano cheese [Articolo su rivista]
Tagliazucchi, D.; Solieri, L.; Baldaccini, A.; Martini, S.; Bianchi, A.; Pizzamiglio, V.
abstract

Proteolysis degree, biological activities, and water-soluble peptide patterns were evaluated in 12 month-ripened Parmigiano Reggiano (PR) cheeses collected in different dairy farms and showing different salt and fat content. Samples classified in high-salt and high-fat group (HH) generally showed lower proteolysis degree than samples having low-salt and low-fat content (LL). This positive correlation between salt/fat reduction and proteolysis was also confirmed by the analysis of biological activities, as the LL group showed higher average values of angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities. UHPLC/HR-MS allowed the identification of 805 unique peptides: LL and HH groups shared 59.3% of these peptides, while 20.9% and 19.9% were LL and HH specific, respectively. Frequency analysis of peptides identified a core of 183 peptides typical of 12-month ripened PR cheeses (corresponding to the 22.7% of total peptides), but no significant differences were detected in peptide patterns between LL and HH groups. Forty bioactive peptides, including 18 ACE-inhibitors and 12 anti-microbial peptides, were identified, of which 25 firstly found in PR cheese. Globally, this work contributed to unraveling the potentially healthy benefits of peptides fraction in PR cheese and provided prior evidence that PR with reduced at/salt content showed the highest antihypertensive and antioxidant activities.


2020 - Sourdough derived strains of Saccharomyces cerevisiae and their potential for farmhouse ale brewing [Articolo su rivista]
Catallo, M; Nikulin, J; Johansson, L; Krogerus, K; Laitinen, M; Magalhães, F; Piironen, M; Mikkelson, A; Randazzo, Cl; Solieri, L; Gibson, B
abstract


2020 - Valorization of cheese whey using microbial fermentations [Articolo su rivista]
Zotta, T.; Solieri, L.; Iacumin, L.; Picozzi, C.; Gullo, M.
abstract

Cheese whey (CW), the liquid resulting from the precipitation and removal of milk casein during cheese-making, and the second cheese whey (SCW) derived from the production of cottage and ricotta cheeses are the main byproducts of dairy industry. The major constituent of CW and SCW is lactose, contributing to the high BOD and COD content. Because of this, CW and SCW are high-polluting agents and their disposal is still a problem for the dairy sector. CW and SCW, however, also consist of lipids, proteins, and minerals, making them useful for production of various compounds. In this paper, microbial processes useful to promote the bioremediation of CW and SCW are discussed, and an overview on the main whey-derived products is provided. Special focus was paid to the production of health-promoting whey drinks, vinegar, and biopolymers, which may be exploited as value-added products in different segments of food and pharmaceutical industries.


2019 - A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii [Articolo su rivista]
Bizzarri, Melissa; Cassanelli, Stefano; Dušková, Michala; Sychrová, Hana; Solieri, Lisa
abstract

The so‐called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T. By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase‐mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX–loxP module in Z. rouxii ATCC 42981 G418‐resistant mutants previously constructed by replacing the MATαP expression locus with the loxP–kanMX–loxP cassette.


2019 - Bioprospecting for Bioactive Peptide Production by Lactic Acid Bacteria Isolated from Fermented Dairy Food [Articolo su rivista]
Tagliazucchi, Davide; Martini, Serena; Solieri, Lisa
abstract

With rapidly ageing populations, the world is experiencing unsustainable healthcare from chronic diseases such as metabolic, cardiovascular, neurodegenerative, and cancer disorders. Healthy diet and lifestyle might contribute to prevent these diseases and potentially enhance health outcomes in patients during and after therapy. Fermented dairy foods (FDFs) found their origin concurrently with human civilization for increasing milk shelf-life and enhancing sensorial attributes. Although the probiotic concept has been developed more recently, FDFs, such as milks and yoghurt, have been unconsciously associated with health-promoting effects since ancient times. These health benefits rely not only on the occurrence of fermentation-associated live microbes (mainly lactic acid bacteria; LAB), but also on the pro-health molecules (PHMs) mostly derived from microbial conversion of food compounds. Therefore, there is a renaissance of interest toward traditional fermented food as a reservoir of novel microbes producing PHMs, and “hyperfoods” can be tailored to deliver these healthy molecules to humans. In FDFs, the main PHMs are bioactive peptides (BPs) released from milk proteins by microbial proteolysis. BPs display a pattern of biofunctions such as anti-hypertensive, antioxidant, immuno-modulatory, and anti-microbial activities. Here, we summarized the BPs most frequently encountered in dairy food and their biological activities; we reviewed the main studies exploring the potential of dairy microbiota to release BPs; and delineated the main effectors of the proteolytic LAB systems responsible for BPs release


2019 - Effect of Sequential Inoculum of Beta-Glucosidase Positive and Probiotic Strains on Brine Fermentation to Obtain Low Salt Sicilian Table Olives [Articolo su rivista]
Pino, Alessandra; Vaccalluzzo, Amanda; Solieri, Lisa; Romeo, Flora V.; Todaro, Aldo; Caggia, Cinzia; Noé Arroyo-López, Francisco; Bautista-Gallego, Joaquin; Randazzo, Cinzia L.
abstract

In the present study, the β-glucosidase positive strain Lactobacillus plantarum F3. 3 was used as starter during the fermentation of Sicilian table olives (Nocellara Etnea cultivar) at two different salt concentrations (5 and 8%), in order to accelerate the debittering process. The latter was monitored through the increase of hydroxytyrosol compound. In addition, the potential probiotic Lactobacillus paracasei N24 strain was added after 60 days of fermentation. Un-inoculated brine samples at 5 and 8% of salt were used as control. The fermentation was monitored till 120 days through physico-chemical and microbiological analyses. In addition, volatile organic compounds and sensorial analyses were performed during the process and at the end of the fermentation, respectively. Lactic acid bacteria and yeasts were, in depth, studied by molecular methods and the occurrence of the potential probiotic N24 strain in the final products was determined. Results highlighted that inoculated brines exhibited a higher acidification and debittering rate than control ones. In addition, inoculated brines at 5% of salt exhibited higher polyphenols (hydoxytyrosol, tyrosol, and verbascoside) content compared to samples at 8% of NaCl, suggesting a stronger oleuropeinolytic activity of the starter at low salt concentration. Lactobacilli and yeasts dominated during the fermentation process, with the highest occurrence of L. plantarum and Wickerhamomyces anomalus, respectively. Moreover, the potential probiotic L. paracasei N24 strain was able to survive in the final product. Hence, the sequential inoculum of beta-glucosidase positive and potential probiotic strains could be proposed as a suitable technology to produce low salt Sicilian table olives.


2019 - Interplay of Chimeric Mating-Type Loci Impairs Fertility Rescue and Accounts for Intra-Strain Variability in Zygosaccharomyces rouxii Interspecies Hybrid ATCC42981 [Articolo su rivista]
Bizzarri, Melissa; Cassanelli, Stefano; Bartolini, Laura; Pryszcz, Leszek P.; Dušková, Michala; Sychrová, Hana; Solieri, Lisa
abstract

The pre-whole genome duplication (WGD) Zygosaccharomyces clade comprises several allodiploid strain/species with industrially interesting traits. The salt-tolerant yeast ATCC42981 is a sterile and allodiploid strain which contains two subgenomes, one of them resembling the haploid parental species Z. rouxii. Recently, different mating-type-like (MTL) loci repertoires were reported for ATCC42981 and the Japanese strain JCM22060, which are considered two stocks of the same strain. MTL reconstruction by direct sequencing approach is challenging due to gene redundancy, structure complexities, and allodiploid nature of ATCC42981. Here, DBG2OLC and MaSuRCA hybrid de novo assemblies of ONT and Illumina reads were combined with in vitro long PCR to definitively solve these incongruences. ATCC42981 exhibits several chimeric MTL loci resulting from reciprocal translocation between parental haplotypes and retains two MATa/MATα expression loci, in contrast to MATα in JCM22060. Consistently to these reconstructions, JCM22060, but not ATCC42981, undergoes mating and meiosis. To ascertain whether the damage of one allele at the MAT locus regains the complete sexual cycle in ATCC42981, we removed the MATα expressed locus by gene deletion. The resulting MATa/- hemizygous mutants did not show any evidence of sporulation, as well as of self- and out-crossing fertility, probably because incomplete silencing at the chimeric HMLα cassette masks the loss of heterozygosity at the MAT locus. We also found that MATα deletion switched off a2 transcription, an activator of a-specific genes in pre-WGD species. These findings suggest that regulatory scheme of cell identity needs to be further investigated in Z. rouxii protoploid yeast.


2018 - Draft genome sequences of the highly halotolerant strain Zygosaccharomyces rouxii ATCC 42981 and the novel allodiploid strain Zygosaccharomyces sapae ATB301T obtained using the MinION platform [Articolo su rivista]
Bizzarri, M; Cassanelli, S; Pryszcz, Lp; Gawor, J; Gromadka, R; Solieri, L.
abstract

Here, we report draft genome sequences of the halotolerant and allodiploid strains Zygosaccharomyces rouxii ATCC 42981 and Zygosaccharomyces sapae ABT301T. Illumina and Oxford Nanopore MinION sequencing revealed genome sizes of 20.9 and 24.7 Mb, respectively. This information will be useful for deciphering the genetics of hybrid adaptation to high salt and sugar concentrations in nonconventional yeasts.


2018 - Evaluation of fingerprinting techniques to assess genotype variation among Zygosaccharomyces strains [Articolo su rivista]
Dakal, Tc; Solieri, L; Giudici, P.
abstract

Molecular typing techniques are key tools in surveillance of food spoilage yeasts, in investigations on intra-species population diversity, and in tracing selected starters during fermentation. Unlike previous works on strain typing of Zygosaccharomyces spoilage species, here Zygosaccharomyces mellis and the Zygosaccharoymces rouxii complex yeasts, which include Z. rouxii, Zygosaccharomyces sapae, and a mosaic lineage (ML) of putatively hybrids, were evaluated by three typing methods for intra- and inter-species resolution. Overall these yeasts are relevant for food fermentation and spoilage, but are quite difficult to discriminate at strain and species level as they evolved by reticulation. A pool of 76 strains from different sources were typed by M13 and (GTG)5 MSP-PCR fingerprinting and PCR-RFLP of ribosomal intergenic spacer region (IGS). We demonstrated that M13 overcame (GTG)5 fingerprinting to group Z. sapae, Z. rouxii, Z. mellis and the ML isolates in congruent distinct clusters. Even if (GTG)5 primer yielded a number of DNA fingerprints comparable with those obtained by M13 primer, it failed to discriminate Z. sapae, Z. mellis and Z. rouxii at species level. Clustering of IGS RFLP patterns obtained with three endonucleases produced groups congruent with species assignment and highlighted intra-species diversity similar to that observed by M13 fingerprinting. However, IGS PCR amplification failed for 14 ML and 6 Z. mellis strains under the experimental conditions tested here, indicating that this marker could be less easy to use in fast typing protocol. Finally, our results posit that the genetic diversity within Z. sapae and Z. mellis could be shaped by isolation source. The information generated in this study would facilitate the monitoring of these yeasts during food processing and storage, and provides preliminary evidences about Z. sapae and Z. mellis intra-species diversity.


2018 - Mating-type switching in CBS 732T derived subcultures unveils potential genetic and phenotypic novelties in haploid Zygosaccharomyces rouxii. [Articolo su rivista]
Bizzarri, M; Cassanelli, S; Solieri, L.
abstract

In haploid Saccharomyces cerevisiae, a complex recombination system regulates mating-type switching and requires one MAT expression locus, two donor cassettes (HML and HMR) and the HO endonuclease that catalyses gene conversion. Zygosaccharomyces rouxii is the most distant species from S. cerevisiae with a functional HO, but with a poorly understood mating-type switching. Here, we described that two subcultures of the type strain CBS 732T underwent the α to a genotype switching leading to mixed MATα and MATa populations. Remarkably, during this event the donor cassette was copied into the MAT locus, except for its own 3΄ end, resulting in a new MATa2 gene copy different from the silenced HMRa2. Moreover, CBS 732T cells bypassed the cell-cycle control, which oversees HO transcription in S. cerevisiae, and expressed HO at the stationary phase. Despite HO dysregulation, mating-type switching seemed to occur rarely or belatedly during CBS 732T colony formation in most of the tested conditions. When morphology and mating behaviour were analysed, two subcultures displayed distinct outcross fertility responses. Overall, our data support that mating-type switching causes genotype instability and phenotypic novelties in CBS 732T, and open the question whether this mechanism is shared by other Z. rouxii haploid homothallic strains.


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.


2017 - Allodiploid Zygosaccharomyces genomes sequencing assists in deciphering the genetic basis of hybrid sterility [Abstract in Atti di Convegno]
Bizzarri, M; Pryszcz, L; Solieri, L; Cassanelli, S; Sychrová, H
abstract

In Saccharomyces cerevisiae diploid MATα/MATa cells, a1-α2 heterodimer encoded by mating-type (MAT) genes is the master sensor of diploidy, promoting the meiosis under starvation, and the repression of haploid-specific genes (h-sgs), such as HO gene, under standard growth conditions. However, how other biotech yeasts govern life cycle remains poorly understood. Halotolerant Zygosaccharomyces rouxii yeasts have applications in food spoilage and fermentation, and exhibit ploidy and karyotype variation, tendency to hybridization, and high diversity in osmostress response. Here, the allodiploid Z. rouxii ATCC42981 serves as a model to investigate how transcriptional network incompatibility affects hybrid sterility. To extend the Zygosaccharomyces genomic knowledge, we sequenced ATCC42981 and Z. sapae ABT301T genomes. We obtained high-quality assemblies (33-45 scaffolds) by combining heterozygosity reduction and assisting the assembling process with Z. rouxii CBS732T reference chromosomes. Both strains are hybrids derived from one lineage that is over 99% identical to CBS732T and another one not yet identified and ~14% diverging from Z. rouxii type-strain. Interestingly, ATCC42981 and ABT301T differ from one another for the second parent. ATCC42981 kept all reference chromosomes, while ABT301T probably lost one. Both genomes harbor chromosomes from the second parental species, suggesting that unexpectedly no reduction of one of the parental karyotypes occurred after hybridization to resolve genome incompatibility.In ATCC42981 genome, MATa1 and MATα2 genes are from two different parents (Z. rouxii and Z. sapae, respectively). The different evolutionary history of a1 and α2 subunits could generate negative epistasis accounting for ATCC42981 inability to repress HO gene and to regulate the main meiosis inducer IME4 gene. To prove this, Z. sapae MATα was disrupted by a loxP–kanMX–loxP cassette. Surprisingly, we found that the deletion mutants still actively transcribed MATα, expressed HO and were unable to sporulate by autodiploidization. Besides, MATα removal did not rescue the ability of ATCC42981 to make conjugated asci in mixture with Z. rouxii mating testers. Overall, this work suggests that 1) HO expression does not assure mating-type switching; 2) with 1 copy of MAT disrupted, ATCC42981 does not behave as a haploid; 3) MATα deletion induces HMLα loci de-silencing or, alternatively, reveals the incomplete silencing of donor cassettes in the wild type strain.


2017 - Exploration of genetic and phenotypic diversity within Saccharomyces uvarum for driving strain improvement in winemaking [Articolo su rivista]
Verspohl, Alexandra; Solieri, Lisa; Giudici, Paolo
abstract

The selection and genetic improvement of wine yeast is an ongoing process, since yeast strains should match new technologies in winemaking to satisfy evolving consumer preferences. A large genetic background is the necessary starting point for any genetic improvement programme. For this reason, we collected and characterized a large number of strains belonging to Saccharomyces uvarum. In particular, 70 strains were isolated from cold-stored must samples: they were identified and compared to S. uvarum strains originating from different collections, regarding fermentation profile, spore viability and stress response. The results demonstrate a large biodiversity among the new isolates, with particular emphasis to fermentation performances, genotypes and high spore viability, making the isolates suitable for further genetic improvement programmes. Furthermore, few of them are competitive with Saccharomyces cerevisiae and per se, suitable for wine fermentation, due to their resistance to stress, short lag phase and fermentation by-products.


2016 - Chimeric sex-determining chromosomal regions and dysregulation of cell-type identity in a sterile zygosaccharomyces allodiploid yeast [Articolo su rivista]
Bizzarri, Melissa; Giudici, P.; Cassanelli, S.; Solieri, L.
abstract

Allodiploidization is a fundamental yet evolutionary poorly characterized event, which impacts genome evolution and heredity, controlling organismal development and polyploid cell-types. In this study, we investigated the sex determination system in the allodiploid and sterile ATCC 42981 yeast, a member of the Zygosaccharomyces rouxii species complex, and used it to study how a chimeric mating-type gene repertoire contributes to hybrid reproductive isolation. We found that ATCC 42981 has 7 MAT-like (MTL) loci, 3 of which encode α-idiomorph and 4 encode a-idiomorph. Two phylogenetically divergent MATexpression loci were identified on different chromosomes, accounting for a hybrid a/α genotype. Furthermore, extra a-idimorph-encoding loci (termed MTLa copies 1 to 3) were recognized, which shared the same MATa1 ORFs but diverged forMATa2 genes. Each MATexpression locus was linked to a HML silent cassette, while the corresponding HMR loci were located on another chromosome. Two putative parental sex chromosome pairs contributed to this unusual genomic architecture: one came from an as-yet-undescribed taxon, which has the NCYC 3042 strain as a unique representative, while the other did not match any MAT-HML and HMR organizations previously described in Z. rouxii species. This chimeric rearrangement produces two copies of the HO gene, which encode for putatively functional endonucleases essential for mating-type switching. Although both a and a coding sequences, which are required to obtain a functional cell-type a1-α2 regulator, were present in the allodiploid ATCC 42981 genome, the transcriptional circuit, which regulates entry into meiosis in response to meiosis-inducing salt stress, appeared to be turned off. Furthermore, haploid and a-specific genes, such as MATα1 and HO, were observed to be actively transcribed and up-regulated under hypersaline stress. Overall, these evidences demonstrate that ATCC 42981 is unable to repress haploid a-specific genes and to activate meiosis in response to stress. We argue that sequence divergence within the chimeric a1-α2 heterodimer could be involved in the generation of negative epistasis, contributing to the allodiploid sterility and the dysregulation of cell identity.


2016 - Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex [Articolo su rivista]
Chand Dakal, T; Giudici, Paolo; Solieri, Lisa
abstract

Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5' end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding.


2016 - Differential hypersaline stress response in Zygosaccharomyces rouxii complex yeasts: a physiological and transcriptional study. [Articolo su rivista]
Solieri, Lisa; Vezzani, Veronica; Cassanelli, Stefano; Dakal, Tikam Chand; Pazzini, Jacopo; Giudici, Paolo
abstract

The Zygosaccharomyces rouxii complex comprises three distinct lineages of halotolerant yeasts relevant in food processing and spoilage, such as Z. sapae, Z. rouxii and a mosaic group of allodiploid strains. They manifest plastic genome architecture (variation in karyotype, ploidy level and Na+/H+ antiporter-encoding gene copy number), and exhibit diverse tolerances to salt concentrations. Here, we investigated accumulation of compatible osmolytes and transcriptional regulation of Na+/H+ antiporter-encoding ZrSOD genes during salt exposure in strains representative for the lineages, namely Z. sapae ABT301T (low salt tolerant), Z. rouxii CBS 732T (middle salt tolerant) and allodiploid strain ATCC 42981 (high salt tolerant). Growth curve modelling in 2 M NaCl-containing media supplemented with or without yeast extract as nitrogen source indicates that moderate salt tolerance of CBS 732T mainly depends on nitrogen availability rather than intrinsic inhibitory effects of salt. All the strains produce glycerol and not mannitol under salt stress and use two different glycerol balance strategies. ATCC 42981 produces comparatively more glycerol than Z. sapae and Z. rouxii under standard growth conditions and better retains it intracellularly under salt injuries. Conversely, Z. sapae and Z. rouxii enhance glycerol production under salt stress and intracellularly retain glycerol less efficiently than ATCC 42981. Expression analysis shows that, in diploid Z. sapae and allodiploid ATCC 42981, transcription of gene variants ZrSOD2-22/ZrSOD2 and ZrSOD22 is constitutive and salt unresponsive.


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 - Homoplasmy restoration of mitochondrial DNA: a non-stochastic event in heteroplasmic Saccharomyces hybrid zygotes [Relazione in Atti di Convegno]
Verspohl, Alexandra; Solieri, Lisa; Giudici, Paolo
abstract

Homoplasmy restoration of mitochondrial DNA: a non-stochastic event in heteroplasmic Saccharomyces hybrid zygotes


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.


2016 - Recent advances in understanding yeast genetics of sex determination. [Articolo su rivista]
Cassanelli, Stefano; Bizzarri, Melissa; Solieri, Lisa
abstract

Sex determination is among the most fascinating areas of study in modern genetics and encompasses many topics, such as developmental mechanisms, behaviour, sex chromosome biology, population evolution and diversity. Yeasts from the subphylum Saccharomycotina are of great importance to humans, not only as pathogens but for numerous essential ecosystem services and serve as model to study evolution in action. The recent advent of inexpensive sequence information and other new tools has led to notable advances in our understanding of reproductive mechanisms over a broad range of species, revealing that genetic sex determination occurs in different ways with a myriad of outcomes in yeast.


2016 - Release of the Antihypertensive Tripeptides Valine-Proline-Proline and Isoleucine-Proline-Proline from Bovine Milk Caseins during in Vitro Gastrointestinal Digestion [Articolo su rivista]
Rutella, GIUSEPPINA SEFORA; Solieri, Lisa; Martini, Serena; Tagliazucchi, Davide
abstract

The aim of this study was to identify and quantify the release of antihypertensive tripeptides valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) during in vitro oro-gastro-intestinal (OGI) digestion of bovine skimmed milk. The experimental approach combined the recently developed harmonized static in vitro digestion (IVD) model and targeted mass spectrometry to monitor peptide generation. We first demonstrated that VPP and IPP are released from bovine milk proteins during in vitro OGI digestion at final concentrations of 354.3 ± 29.8 and 973.8 ± 155.7 μg/L, respectively. In silico analysis of cleavage sites and mass spectrometry revealed that tetrapeptides VPPF, IPPL, and IPPK are precursors of VPP and IPP. The release of other ACE-inhibitory peptides, such as FVAP, VAP, AW, and VY, was demonstrated, and their fate and the time course were investigated. This research underlines the suitability of an IVD system to study the release of short bioactive peptides during OGI transit.


2016 - Survival and bioactivities of selected probiotic lactobacilli in yogurt fermentation and cold storage: New insights for developing a bi-functional dairy food [Articolo su rivista]
Rutella, GIUSEPPINA SEFORA; Tagliazucchi, Davide; Solieri, Lisa
abstract

In previous work , we demon strated that two probiotic strains, name ly Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331, produce fermented milks with potent angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities. Here, we tested these strains for the survivability and the release of antihypertensive and antioxidant peptides in yogurt fermentation and cold storage. For these purposes three yogurt batches were compared: one prepared using yogurt starters alone (Lactobacillus delbrueckii subspecies bulgaricus 1932 and Streptococcus thermophilus 99), and the remaining two containing either PRA205 or PRA331 in addition to yogurt starters. Despite the lower viable counts at the fermentation end compared to PRA331, PRA205 overcame PRA331 in survivability during refrigerated storage for 28 days, leading to viable counts (>108 CFU/g) higher than the minimum therapeutic threshold (106 CFU/g). Analyses of in vitro ACE-inhibitory and antioxidant activities of peptide fractions revealed that yogurt supplemented with PRA205 displays higher amounts of antihypertensive and antioxidant peptides than that produced with PRA331 at the end of fermentation and over storage. Two ACE-inhibitory peptides, Valine-Proline-Proline (VPP) and Isoleucine-Proline-Proline (IPP), were identified and quantified. This study demonstrated that L. casei PRA205 could be used as adjunct culture for producing bi-functional yogurt enriched in bioactive peptides and in viable cells, which bring health benefits to the host as probiotics.


2015 - Ectopic recombination in sex-determination system as a source of genetic variation in the diploid yeast Zygosaccharomyces sapae. [Abstract in Atti di Convegno]
Bizzarri, Melissa; Verspohl, Alexandra; Giudici, Paolo; Cassanelli, Stefano; Solieri, Lisa
abstract

Sexual reproduction increases genetic variation, which is strongly advantageous under harsh environmental conditions, as it allows natural selection to proceed more effectively. The yeasts of the Zygosaccharomyces rouxii complex are relevant in food elaboration and spoilage due to their ability to cope with low water activity environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy (Solieri et al. 2013). The mating-type locus (MAT) is a hotspot for chromosome rearrangement in yeasts. Here, we investigated the genetic architecture of sex determinants, including MAT loci and HO endonuclease in Zygosaccharomyces sapae diploid strain ABT301T, belonging to the Z. rouxii complex. We cloned these genes through a DNA walking strategy and a characterization of the flanking regions, while the chromosome assignment was performed combining Southern blotting and PFGE-karyotyping. We identified three divergent mating type-like (MTL) α-idiomorph sequences, designated as ZsMTLα copies 1, 2, and 3, which encoded homologues of Z. rouxii CBS 732T MATα2 (aa sequence identity from 67.0 to 99.5%) and MATα1 (identity 81.5-99.5%). Cloning of MATa-idiomorph yielded one ZsMTLa locus encoding two Z. rouxii-like proteins, MATa1 and MATa2. ABT301T possesses two divergent HO genes encoding distinct endonucleases. Based on the cloned ZsMTLα and ZsMTLa idiomorphs flanking regions we discovered that Z. sapae ABT301T displays an aααα genotype lacking the HMR silent cassette. Additionally, four putative HML cassettes were identified, two harbouring the ZsMTLα copy 1 and the remaining containing ZsMTLα copies 2 and 3. In conclusion, our results show that the mating-type switching is responsible for hyper-mutation in Z. rouxii complex. The ectopic recombination underlying this process is an error-prone mechanism, which represents a possible source of genetic variation providing yeast progeny with phenotypic variability and adaptation to hostile environments.


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 - Fast method for identifying inter- and intra-species Saccharomyces hybrids in extensive genetic improvement programs based on yeast breeding [Articolo su rivista]
Solieri, Lisa; Verspohl, Alexandra; Bonciani, Tommaso; Caggia, Cinzia; Giudici, Paolo
abstract

Aims: The present work proposes a two-step molecular strategy to select inter- and intra-species Saccharomyces hybrids obtained by spore-to-spore mating, one of the most used methods for generating improved hybrids from homothallic wine yeasts. Methods and Results: As low spore viability and haplo-selfing are the main causes of failed mating, at first, we used colony screening PCR (csPCR) of discriminative gene markers to select hybrids directly on dissection plate and discard homozygous diploid colonies arisen from one auto-diploidized progenitor. Then, pre-selected candidates were submitted to recursive streaking and conventional PCR in order to discriminate between the hybrids with stable genomic background and the false-positive admixtures of progenitor cells both undergone haplo-selfing. csPCRs of internal transcribed spacer (ITS) 1 or 2, and the subsequent digestion with diagnostic endonucleases HaeIII and RsaI, respectively, were efficient to select six new Saccharomyces cerevisiae × Saccharomyces uvarum hybrids from 64 crosses. Intragenic minisatellite regions in PIR3, HSP150, and DAN4 genes showed high inter-strain size variation detectable by cost-effective agarose gel electrophoresis and were successful to validate six new intra-species S. cerevisiae hybrids from 34 crosses. Conclusions: Both protocols reduce significantly the number of massive DNA extractions, prevent misinterpretations caused by one or both progenitors undergone haplo-selfing, and can be easily implemented in yeast labs without any specific instrumentation. Significance and Impact of the Study: The study provides a method for the marker-assisted selection of several inter- and intra-species yeast hybrids in a cost-effective, rapid and reproducible manner.


2015 - Impact of non-starter lactobacilli on release of peptides with angiotensin-converting enzyme inhibitory and antioxidant activities during bovine milk fermentation [Articolo su rivista]
Solieri, Lisa; Rutella, Giuseppina Sefora; Tagliazucchi, Davide
abstract

This study aimed at evaluating non-starter lactobacilli (NSLAB) isolated from cheeses for their proteolytic activity and capability to produce fermented milk enriched in angiotensin-converting enzyme (ACE)-inhibitory and antioxidant peptides. Preliminarily, 34 NSLAB from Parmigiano Reggiano (PR) and 5 from Pecorino Siciliano cheeses were screened based on their capacity to hydrolyze milk proteins. Two NSLAB strains from PR, Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331, showed the most proteolytic phenotype and were positively selected to inoculate sterile cow milk. The fermentation process was monitored by measuring viable cell population, kinetic of acidification, consumption of lactose, and synthesis of lactic acid. Milk fermented with Lb. casei PRA205 exhibited higher radical scavenging (1184.83 ± 40.28 umol/L trolox equivalents) and stronger ACE-inhibitory (IC50 = 54.57 ug/mL) activities than milk fermented with Lb. rhamnosus PRA331 (939.22 ± 82.68 umol/L trolox equivalents; IC50 = 212.38 ug/mL). Similarly, Lb. casei PRA205 showed the highest production of ACE-inhibitory peptides Val-Pro-Pro and Ile-Pro-Pro, which reached concentrations of 32.88 and 7.52 mg/L after 87 and 96 h of milk fermentation, respectively. This evidence supports Lb. casei PRA205, previously demonstrated to possess characteristics compatible with probiotic properties, as a promising functional culture able to promote health benefits in dairy foods


2015 - Molecular two-step strategy to select inter-species Saccharomyces hybrids [Abstract in Atti di Convegno]
Verspohl, Alexandra; Solieri, Lisa; Bizzarri, Melissa; Giudici, Paolo
abstract

Hybridization is a common tool to improve yeast for wine industry. Using non-GE hybridization techniques a lot of attempts lead to failed mating which is mainly caused by low spore viability and haplo-selfing. This work proposed a two-step molecular strategy to validate inter-species Saccharomyces hybrids rapidly.


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 - The sex determination system and the role of chimeric a1/alpha2 heterodimer in the sterility of an allodiploid Zygosaccharomyces yeast [Abstract in Rivista]
Solieri, Lisa; Bizzarri, Melissa; Giudici, Paolo; Cassanelli, Stefano
abstract

In Saccharomyces cerevisiae diploids, the a1/alpha2 protein heterodimer acts as repressor of haploid-specific genes, such as MATalpha1 and HO genes, and allows a/alpha diploid cells to undergo sporulation. The regulatory routes governing the cell type and fate have been not yet studied in the yeasts belonging to the Zygosaccharomyces rouxii complex. These yeasts are relevant in foodstuff elaboration and spoilage due to a wide repertoire of tolerances to osmotic stress. Their genetic variability favors phenotypic diversity and adaptation to hostile environments by ectopic recombination of mating-type (MTL) loci through an error-prone switching mechanism. Among them, the allodiploid strain ATCC42981 exhibits hybrid vigor and multi-stress tolerance, but it is unable to undergo sexual reproduction. Aim of this work was to characterize allodiploid strain ATCC42981 for the genetic organization and the transcriptional expression of mating type-like (MTL) loci and HO genes. Genetic dissection of ATCC42981 sex determinants revealed a MATa/MATalpha genotype with a redundant number of partial divergent HMR cassettes. MATa expression locus contained Z. rouxii-related MATa1 and MATa2 genes, whereas MATalpha expression locus contained Z. sapae-related MATalpha1 and MATalpha2 genes (termed MATalpha1 and MATalpha2 copy 2, respectively). Both MAT expression loci are linked to phylogenetically congruent HML silent cassettes on non-homologous chromosomes. Other than a “hybrid” and redundant three-cassette system, ATCC42981 possesses two divergent HO genes. Consistently with this gene organization, ATCC42981 expressed MATa1 and MATalpha2 copy 2 genes under standard conditions, whereas aand α-idiomorph genes from HMR and HML cassettes were silent. Differently from S. cerevisiae diploids, ATCC42981 did not repress either MATalpha1 or HO gene transcription. Under hypersaline stress (which should be induce meiosis in Zygosaccharomyces cells by turning on the haploid gene-specific program), ATCC42981 was not able to undergo meiosis and over-expressed HO copy 2, MATa1 and MATalpha1 copy 2. We hypothesized that the partial incompatibility between ATCC42981 Z. rouxii-like a1 and Z. sapae-like alpha2 subunits in MATa1/alpha2 heterodimer causes a defective silencing of haploid-specific genes, including meiosis inhibiting factors, leading to clonality as the only possible reproduction strategy for this allodiploid yeast


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 - Adaptive response and tolerance to sugar and salt stress in the food yeast Zygosaccharomyces rouxii [Articolo su rivista]
Dakal, TIKAM CHAND; Solieri, Lisa; Giudici, Paolo
abstract

The osmotolerant and halotolerant food yeast Zygosaccharomyces rouxii is known for its ability to grow and survive in the face of stress caused by high concentrations of non-ionic (sugars and polyols) and ionic (mainly Na(+) cations) solutes. This ability determines the success of fermentation on high osmolarity food matrices and leads to spoilage of high sugar and high salt foods. The knowledge about the genes, the metabolic pathways, and the regulatory circuits shaping the Z. rouxii sugar and salt-tolerance, is a prerequisite to develop effective strategies for fermentation control, optimization of food starter culture, and prevention of food spoilage. This review summarizes recent insights on the mechanisms used by Z. rouxii and other osmo and halotolerant food yeasts to endure salts and sugars stresses. Using the information gathered from S. cerevisiae as guide, we highlight how these non-conventional yeasts integrate general and osmoticum-specific adaptive responses under sugar and salts stresses, including regulation of Na(+) and K(+)-fluxes across the plasma membrane, modulation of cell wall properties, compatible osmolyte production and accumulation, and stress signalling pathways. We suggest how an integrated and system-based knowledge on these mechanisms may impact food and biotechnological industries, by improving the yeast spoilage control in food, enhancing the yeast-based bioprocess yields, and engineering the osmotolerance in other organisms.


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 - 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?


2014 - Quantitative phenotypic analysis of multistress response in Zygosaccharomyces rouxii complex. [Articolo su rivista]
Solieri, Lisa; C., Dakal T; Bicciato, Silvio
abstract

Zygosaccharomyces rouxii complex comprises three yeasts clusters sourced from sugar- and salt-rich environments: haploid Zygosaccharomyces rouxii, diploid Zygosaccharomyces sapae and allodiploid/aneuploid strains of uncertain taxonomic affiliations. These yeasts have been characterized with respect to gene copy number variation, karyotype variability and change in ploidy, but functional diversity in stress responses has not been explored yet. Here, we quantitatively analysed the stress response variation in seven strains of the Z. rouxii complex by modelling growth variables via model and model-free fitting methods. Based on the spline fit as most reliable modelling method, we resolved different interstrain responses to 15 environmental perturbations. Compared with Z. rouxii CBS 732T and Z. sapae strains ABT301T and ABT601, allodiploid strain ATCC 42981 and aneuploid strains CBS 4837 and CBS 4838 displayed higher multistress resistance and better performance in glycerol respiration even in the presence of copper. μ-based logarithmic phenotypic index highlighted that ABT601 is a slow-growing strain insensitive to stress, whereas ABT301T grows fast on rich medium and is sensitive to suboptimal conditions. Overall, the differences in stress response could imply different adaptation mechanisms to sugar- and salt-rich niches. The obtained phenotypic profiling contributes to provide quantitative insights for elucidating the adaptive mechanisms to stress in halo- and osmo-tolerant Zygosaccharomyces yeasts


2014 - Sensory analysis of traditional balsamic vinegars: current state and future perspectives [Articolo su rivista]
Lemmetti, Federico; Solieri, Lisa; Bonciani, Tommaso; Zanichelli, Gabriele; Giudici, Paolo
abstract

Quality evaluation of traditional balsamic vinegar (TBV) is primarily based on sensory analysis. For every TBV batch, official sensory panels give a final score, which determines its assessment into quality and price categories. Therefore, an effective and objective sensory analysis is a core aspect in TBV production and marketing and it should fulfill at least two conditions: i) the panelists have been properly trained on the TBV features; ii) the panelists have to be free from any psychological and physical conditions which might affect human judgments. Traditionally, a panel of trained members assesses the TBV sensory attributes evaluating visual, olfactory, gustatory and trigeminal features at the same time. The result is that visual appearance significantly affects the subsequent stages of the sensory analysis, and even the olfactory and gustatory sensations will greatly affect each other. The aim of this work was to review the procedures for the sensory analysis of TBV and to define a set of TBV attributes. A new assessment questionnaire has been proposed to establish the appropriate sensory vocabulary for a complete description of TBV sensory properties.


2014 - Sex-determination system in the diploid yeast Zygosaccharomyces sapae [Articolo su rivista]
Solieri, Lisa; Dakal, TIKAM CHAND; Giudici, Paolo; Cassanelli, Stefano
abstract

Sexual reproduction and breeding systems are driving forces for genetic diversity. The mating-type (MAT) locus represents a mutation and chromosome rearrangement hotspot in yeasts. Zygosaccharomyces rouxii complex yeasts are naturally faced with hostile low water activity (aw) environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy. Here, we investigated sex-determination system in Zygosaccharomyces sapae diploid strain ABT301(T), a member of the Z. rouxii complex. We cloned three divergent mating type-like (MTL) α-idiomorph sequences and designated them as ZsMTLα copies 1, 2, and 3. They encode homologs of Z. rouxii CBS 732(T) MATα2 (amino acid sequence identities spanning from 67.0 to 99.5%) and MATα1 (identity range 81.5-99.5%). ABT301(T) possesses two divergent HO genes encoding distinct endonucleases 100% and 92.3% identical to Z. rouxii HO. Cloning of MATA: -idiomorph resulted in a single ZsMTLA: locus encoding two Z. rouxii-like proteins MATA: 1 and MATA: 2. To assign the cloned ZsMTLα and ZsMTLA: idiomorphs as MAT, HML, and HMR cassettes, we analyzed their flanking regions. Three ZsMTLα loci exhibited the DIC1-MAT-SLA2 gene order canonical for MAT expression loci. Furthermore, four putative HML cassettes were identified, two containing the ZsMTLα copy 1 and the remaining harboring ZsMTLα copies 2 and 3. Finally, the ZsMTLA: locus was 3'-flanked by SLA2, suggesting the status of MAT expression locus. In conclusion, Z. sapae ABT301(T) displays an aααα genotype missing of the HMR silent cassette. Our results demonstrated that mating-type switching is a hypermutagenic process in Z. rouxii complex that generates genetic diversity de novo. This error-prone mechanism could be suitable to generate progenies more rapidly adaptable to hostile environments.


2014 - Tailoring the probiotic potential of non-starter Lactobacillus strains from ripened Parmigiano Reggiano cheese by in vitro screening and principal component analysis [Articolo su rivista]
Solieri, Lisa; Bianchi, Aldo; Mottolese, Giovanni; Lemmetti, Federico; Giudici, Paolo
abstract

Non-starter lactic acid bacteria (NSLAB) inhabiting fermented food have been recently revised as source of probiotic strains. Here, we in vitro assessed the potential probiotic aptitude of a de-replicated set of NSLAB previously isolated from long ripened Parmigiano Reggiano cheeses (22 Lactobacillus rhamnosus, 18 Lactobacillus paracasei, 3 Lactobacillus casei, 2 Lactobacillus harbinensis, and 2 Lactobacillus fermentum). Most strains showed moderate to good resistance to biological barriers, including bile salts, lysozyme, and simulated gastric and pancreatic juices. Antimicrobial susceptibility tests against seven antimicrobials belonging to different categories showed that most strains are susceptible towards all the antibiotics, with the exception of vancomycin and streptomycin. The strains lost the streptomycin resistance when assayed on agar medium containing 0.5% bile salts, suggesting that detergent-like properties of cholic acids increase membrane permeability and mediate streptomycin susceptibility. No isolate showed bile salt hydrolase (BSH) activities, supporting that bile salts resistance and BSH activity are unpaired traits. Finally, NSLAB strains had moderate to high auto-aggregative and hydrophobic phenotypes, whereas two subsets of 22 and 8 strains co-aggregated with Escherichia coli and Salmonella enterica s. typhimurium, respectively. A multivariate analysis was effective to segregate one L. casei and two L. rhamnosus strains showing physiological characteristics compatible with probiotic properties.


2013 - Getting insights from genomic complexities in Zygosaccharomyces rouxii complex [Abstract in Rivista]
Dakal, TIKAM CHAND; Solieri, Lisa; Giudici, Paolo
abstract

Genus Zygosaccharomyces includes osmo/halotolerant yeasts that diverged from the S. cerevisiae lineage prior to its genome duplication (WGD). Recently, we delineated a novel species, Zygosaccharomyces sapae. Z. sapae has phylogenetic importance as its conspecific strain NCYC3042 has been implicated in allodiploid nature of hybrid ATCC42981. We carry out an extensive genetic survey on some Zygosaccharomyces strains (called Z. rouxii complex) to unravel intra and inter-strain DNA sequences heterogeneity; and diversity in chromosome patterns. Intragenomic copies of rDNA genes are not homogenized by concerted evolution in them, with indels and substitutions occurring mainly in internal transcribed spacer regions. This suggests a birth and death process of evolution. Variability in protein-coding sequences and ploidy level showed a marked intergenomic diversity in diploid and aneuploid cells. Functional analysis revealed a broad range tolerance to alkali metal cations, congruent with clusters obtained through genetic survey. Life cycle and breeding system are suggested to shape these diversities in this complex, making it an interesting pre-WGD model for studying evolutionary forces driving genome plasticity in low-aw environments.


2013 - Integrated approach based on 16S rDNA PCR-DGGE and hsp60 qPCR assay for monitoring dynamics of a L. rhamnosus probiotic strain in table olive fermentation. [Abstract in Atti di Convegno]
Randazzo, CINZIA LUCIA; Solieri, Lisa; Pitino, Iole; Caggia, Cinzia
abstract

We developed an integrated approach based on 16S rDNA PCR-DGGE profiling and Lactobacillus rhamnosus-specific qPCR assay to detect the dominant species in Sicilian table olives and to monitor the implantation of the L. rhamnosus probiotic strain H25 in table olives.


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 - Next-generation sequencing and its potential impact on food microbial genomics [Articolo su rivista]
Solieri, L; Dakal, Tc; Giudici, P
abstract

Recent efforts of researchers to elucidate the molecular mechanisms of biological systems have been revolutionized greatly with the use of high throughput and cost-effective techniques such as next generation sequencing (NGS). Application of NGS to microbial genomics is not just limited to predict the prevalence of microorganisms in food samples but also to elucidate the molecular basis of how microorganisms respond to different food-associated conditions, which in turn offers tremendous opportunities to predict and control the growth and survival of desirable or undesirable microorganisms in food. Concurrently, NGS has facilitated the development of new genome-assisted approaches for correlating genotype and phenotype. The aim of this review is to provide a snapshot of the various possibilities that these new technologies are opening up in area of food microbiology, focusing the discussion mainly on lactic acid bacteria and yeasts associated with fermented food. The contribution of NGS to a system level understanding of food microorganisms is also discussed.


2013 - Unravelling genomic diversity of Zygosaccharomyces rouxii complex with a link to its life cycle [Articolo su rivista]
Solieri, Lisa; T., Chand Dakal; Croce, Maria Antonietta; Giudici, Paolo
abstract

Zygosaccharomyces rouxii and the related species Zygosaccharomyces sapae (hereafter referred to as Z. rouxii complex) are protoploid hemiascomycete yeasts relevant in the elaboration and spoilage of foodstuff. Divergence of Z. rouxii complex before whole genome duplication, leading to the genus Saccharomyces, makes these yeasts very attractive for genome evolution study. Relatively little is known, however, about the diversity in this branch at the genetic and physiological levels. In this work, we investigated Z. rouxii complex, encompassing strains that in other works have been studied separately and comparing them in a comprehensive way. We showed that the majority of strains are unusually heterogeneous in their ribosomal DNA, a signal of relaxation of concerted evolution. Further analysis showed that they have hypervariable karyotypes, different levels of ploidy, and that housekeeping markers vary both in copy number and sequence. Overall, the results provide compelling evidence that the strains considered in this study are a complex of haploid, aneuploid and diploid mosaic lineages. The reproductive mode and life cycle of Zygosaccharomyces could lead to this unsuspected diversity


2013 - Zygosaccharomyces sapae sp. nov., a novel yeast species isolated from Italian traditional balsamic vinegar. [Articolo su rivista]
Solieri, Lisa; T., Chand Dakal; Giudici, Paolo
abstract

Fourteen yeast isolates were recovered from two traditional balsamic vinegar (TBV) samples collected in the provinces of Modena and Reggio Emilia, Italy. Microsatellite-primed-PCR (MSP-PCR) was used to de-replicate the isolate collection into two representative strains, ABT301T and ABT601. Phylogenetic analysis based on the D1/D2 domains of the 26S rRNA gene indicated that these strains represented a distinct species of the Zygosaccharomyces genus, closely related to Zygosaccharomyces rouxii and Zygosaccharomyces mellis. Physiological and morphological tests supported the recognition of a novel taxon of halotolerant, osmotolerant, non-psychrotrophic, and maltose-fermenting negative yeasts showing a chain or star-shaped pattern of budding cells, which remain attached to each other. Morphological observation offered evidence of ascospore formation. In this work, we propose a novel species, Zygosaccharomyces sapae sp. nov., to accommodate these strains, with strain ABT301T (= CBS 12607T = MUCL 54092T) designated as the type strain. The MycoBank number of the new species is MB 564957. Based on D1/D2 domain phylogenetic analysis, the novel strains shared the highest sequence similarity (100%) with Zygosaccharomyces sp. strain NCYC 3042, previously isolated from sugar [James et al. (2005) FEMS Yeast Res 5, 747-755]. However, based on phylogenetic (internal transcribed spacers, ITS), PCR fingerprinting and physiological analyses, marked differences were observed between the the novel species and strain NCYC 3042, and these results are discussed in more detail.


2012 - DIVERSITY AND DYNAMICS OF CULTIVABLEPOPULATION OF ACETIC ACID BACTERIA ANDYEASTS IN KOMBUCHA [Abstract in Rivista]
Solieri, Lisa; D., Mamlouk; L., Kallel; Gullo, Maria
abstract

In recent years functional foods promoted with healthclaims have attracted increasing attention on the market.Among them kombucha is a fermented beverage widelyconsumed in Eastern Asian countries, but little is knownabout its constituent microbial communities. In this studytwo 12 days benchmark kombucha fermentations fromgreen and black tea were carried out. A culture-dependentapproach was applied both on exopolysaccharidic andliquid phases to monitor dynamics and diversity of aceticacid bacteria (AAB) and yeasts community. Among AABone main profile was observed (86% of strains); remainingstrains were grouped in 4 profiles by 16S/RFLPbasedanalysis. Whereas by (GTG)5/PCR typing sixteenclusters were obtained. 16S rRNA gene sequencing confirmedthe occurrence of Gluconacetobacter xylinus as predominantboth in green and black samples at 0, 6 and 12days of fermentation. Mainly on ACB medium minor bacterialgroups often colonizating tea leaves (Paenibacillusspp.), plants (Plantibacter spp.) and moisturing environments(Williamsia spp.), were detected starting from 6thfermentation day. Yeast population consisted of a restrictednumber of dominant species: Dekkera sp., Schizo -saccharomyces sp., Zygosaccharomyces sp., Dekkera sp.and Pichia sp. D. anomala was prevailing in both phasesthough all black and green kombucha fermentation times.Sc. pombe was detected only within 6 days of both greenand black tea and it was not isolated after 9 days, when the high ethanol-producing species D. bruxellensis was detected.Z. bailii was isolated from exopolysaccharidic ofblack tea from 6 to 12 days. Finally P. membranifacienswas detected occasionally at the end of the fermentativeprocess. For each species the degree of diversity wasdetermined by combined M13 and OPA20-based RAPDmethod. Cluster analysis showed that one to two prevailingbiotypes occurred through all the process.


2012 - Inventory of non starter lactic acid bacteria from ripened Parmigiano Reggiano cheese as assessed by a culture dependent multiphasic approach [Articolo su rivista]
Solieri, Lisa; Bianchi, A; Giudici, Paolo
abstract

The objective of this study was to investigate microbial species diversity and strain complexity of the cultivable non starter lactic acid bacteria (NSLAB) occurring in 31 ripened Parmigiano Reggiano (PR) cheeses. Dereplication of 127 lactobacilli isolates by (GTG)(5)-PCR fingerprinting yielded a total of 51 genotypes. Phylogenetic relatedness of all the genotypes with known Lactobacillus species was determined by a novel combined amplified 16S rDNA restriction analysis (16S-ARDRA), species-specific PCR assays and 16S rRNA gene sequencing. The species Lactobacillus rhamnosus and Lactobacillus paracasei comprise the largest portions of the cultivable NSLAB community in PR cheese, with an inter-individual diversity ranging from one to four dominant genotypes per sample. Lactobacillus casei, Lactobacillus harbinensis and Lactobacillus fermentum species were also detected at low frequency. The data showed differences in cultivable NSLAB population, with an overall decrease in diversity and complexity from early to advanced stages of ripening. Finally the de-replicated collection of genotypes resulting from this work is the bases for further functional screening.


2012 - No-culture strategy for tracking AAB and yeasts in low-acidic and high polyphenolic niches [Abstract in Rivista]
Mamlouk, Dhouha; Solieri, Lisa; Gullo, Maria
abstract

Community of acetic acid bacteria (AAB) and yeasts inhabitslow acidic and high polyphenolic niches such as fermentedbeverages. In this work, we selected kombucha asmodel to optimize a no-culture strategy for tracking thedynamics of AAB and yeasts. The strategy consisted of: i)Genomic DNA extraction optimization, ii) PCR/DGGEsetup and iii) Validation of the assay. For gDNA extractionoptimization, we evaluated the suitability of the followinggDNA preparations for a standard PCR-DGGE assay: 1)CTAB/AAB; 2) CTAB/vinegar; 3) Freeze/bead/CTAB1; 4)Freeze/bead/CTAB2 methods. Different extraction methodsaffected not only the quantity and purity of DNA but alsothe complexity of PCR/DGGE profiling. Method 4 allowedus to obtain suitable DNA for PCR assays, as well as thehighest level of complexity in PCR/DGGE band patternsboth for AAB and yeasts. Moreover changes from exopolysaccharidicto liquid phases did not affect the suitability ofthis method. PCR/DGGE setup was performed consideringthe following variables: DNA template concentration, PCRconditions and specificity, effect of GC-clamp and nestedPCR. For AAB, primers pair WBAC1/WBAC2 enabled tostudy AAB dynamics as well other bacterial groups, whereas341f/518r permitted the detection of members of theGluconacetobacter genus. For yeasts, primers pair U1/U2was more efficient then NL1/LS2 in community profiling ofthe majority of kombucha samples. GC-clamp added toforward primers negatively affected gDNA suitability and anested re-PCR for yeasts and AAB was required. Finally,validation of the assay was carried out during kombuchafermentation following AAB and yeasts dynamics from theinoculum to 12 days of incubation. Within AAB species,Gluconacetobacter xylinus responsible for the exopolysaccharidicnetwork matrix, was mainly detected, whereasZygosaccharomyces spp, Dekkera spp, Pichia spp andSchizosaccharomyces pombe in the case of yeasts.


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


2012 - Traditional balsamic vinegar: a microbiological overview [Capitolo/Saggio]
Solieri, Lisa; Gullo, Maria; Giudici, Paolo
abstract

Traditional balsamic vinegar: a microbiological overview


2012 - Vinegars: history and cultures in Europe [Capitolo/Saggio]
Giudici, Paolo; Solieri, Lisa; Mazza, Stefano; Theeragool, G.
abstract

History and culture of European vinegars


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 - Traditional Balsamic Vinegar: a microbiological overviw [Capitolo/Saggio]
Solieri, Lisa; Gullo, Maria; Giudici, Paolo
abstract

Over time, the name “balsamic vinegar” has acquired an important trade value and is now being usedto generally designate sweet and sour condiments with high viscosity and dark/brown color. Balsamiccondiments could be grouped in four different categories: generic balsamic vinegars, balsamic vinegarwith appellation of origin (PDO and Protected Geographical Indication or PGI), balsamic sauces, anddressing (Giudici et al. 2009). In particular “Balsamic Vinegar of Modena” (BVM) is an industrial classof special vinegar legally recognized since 1965 and recently granted European PGI status (CommissionRegulation EC No 583/2009). Despite their similar appellation, TBV and BVM are very distinct productsdue to different manufacturing technologies and starting materials. BVM is produced by aging a blendof cooked must and wine vinegar with addition of caramel, for a short time (minimum 2 months). Due tothis different technology, BVM cannot duplicate the complexity flavor and the higher viscosity of TBVand it meets consumer’s expectations to buy cheaper vinegar compared to TBV (Giudici et al. 2009a).This chapter aims to describe the main technological and microbiological steps of TBV processing,underlying the role of microbial transformations in determining its quality.


2010 - Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations: a framework for selection of new starter cultures [Articolo su rivista]
Solieri, Lisa; M., De Paola; F., Genova; Giudici, Paolo
abstract

Aims: To characterize the genetic and phenotypic diversity of 135 lactic acidbacteria (LAB) strains isolated from Italian wines that undergone spontaneousmalolactic fermentation (MLF) and propose a multiphasic selection of newOenococcus oeni malolactic starters.Methods and Results: One hundred and thirty-five LAB strains were isolatedfrom 12 different wines. On the basis of 16S amplified ribosomal DNArestriction analysis (ARDRA) with three restriction enzymes and 16S rRNAgene sequencing, 120 O. oeni strains were identified. M13-based RAPD analysiswas employed to investigate the molecular diversity of O. oeni population.Technological properties of different O. oeni genotypes were evaluated insynthetic medium at increasing selective pressure, such as low pH (3Æ5, 3Æ2and 3Æ0) and high ethanol values (10, 11 and 13% v ⁄ v). Finally, themalolactic activity of one selected strain was assessed in wine by malolactictrial in winery.Conclusions: The research explores the genomic diversity of wine bacteria inItalian wines and characterizes their malolactic metabolism, providing an efficientstrategy to select O. oeni strains with desirable malolactic performancesand able to survive in conditions simulating the harsh wine environment.Significance and Impact of the Study: This article contributes to a betterunderstanding of microbial diversity of O. oeni population in Italian wines andreports a framework to select new potentially O. oeni starters from Italianwines during MLF.


2010 - Development of SCAR marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation. [Articolo su rivista]
Solieri, Lisa; Giudici, Paolo
abstract

Control over malolactic fermentation (MLF) is a hard goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starter (MLS) in a stressful environment such as wine. In this study we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based random amplified polymorphic DNA (RAPD) band. The 5' and 3' flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled the strain-specific detection without cross amplification of other O. oeni strains and wine species of lactic acid bacteria (LAB), acetic acid bacteria and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detects as low as 2.2 x 10(2) CFU per ml of red wine, with good quantification effectiveness, as shown by correlation of QPCR and plate counting. Therefore the cultivation-independent monitoring of a single O. oeni strain in wine based on SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF


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 - Mitochondrial inheritance in budding yeasts: towards an integrated understanding. [Articolo su rivista]
Solieri, Lisa
abstract

Recent advances in yeast mitogenomics have significantly contributed to our understanding of the diversity of organization, structure and topology in the mitochondrial genome of budding yeasts. In parallel, new insights on mitochondrial DNA (mtDNA) inheritance in the model organism Saccharomyces cerevisiae highlighted an integrated scenario where recombination, replication and segregation of mtDNA are intricately linked to mitochondrial nucleoid (mt-nucleoid) structure and organelle sorting. In addition to this, recent discoveries of bifunctional roles of some mitochondrial proteins have interesting implications on mito-nuclear genome interactions and the relationship between mtDNA inheritance, yeast fitness and speciation. This review summarizes the current knowledge on yeast mitogenomics, mtDNA inheritance with regard to mt-nucleoid structure and organelle dynamics, and mito-nuclear genome interactions.


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 - 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 - Technological and Microbiological Aspects of Traditional Balsamic Vinegar and Their Influence on Quality and Sensorial Properties [Capitolo/Saggio]
Giudici, Paolo; Gullo, Maria; Solieri, Lisa; Falcone, Pasquale Massimiliano
abstract

The term “balsamic” is widespread and popular all over the world of vinegar and fancy foods; it is used generally to refer to vinegars and sauces with a sweet and sour taste. However, the original is the European Protected Denomination, registered as “Aceto Balsamico Tradizionale of Modena, or of Reggio Emilia” that should not be confused with the “Aceto Balsamico di Modena” very similar in the name, but completely different for technology, raw material, quality, and sensorial properties. Traditional balsamic vinegar is made by a peculiar procedure, that starts with a thermal concentration of freshly squeezed grape juice, followed by alcoholic and acetic fermentations and, finally, long aging in a wooden barrel set, by a procedure which requires a partial transfer of vinegar from cask to cask with the consequential blending of vinegars of different ages. In addition, water transfer occurs across the wood of the barrels, the result being an increase of solute concentration of the vinegar. The chemical and physical transformations of the vinegar are mainly directed by the low water activity of the vinegar. High-molecular polymeric compounds are the main and characteristic constituents of original and old traditional balsamic vinegar, and the major cause of its rheological and sensorial properties.


2009 - Traditional balsamic vinegar [Capitolo/Saggio]
Giudici, Paolo; Gullo, Maria; Solieri, Lisa
abstract

The term ‘balsamic vinegar’ is frequently applied to describe sauces, condiments and dressings with particular sweet taste. In Italy there are two types of balsamic vinegar: ‘balsamic vinegar of Modena’ and ‘traditional balsamic vinegar’. The first is a flavoured wine vinegar obtained by blending cooked must and wine vinegar and, in some cases, by adding a small amount of caramel. Traditional balsamic vinegar (TBV) is made in Modena and Reggio Emilia with cooked grape must, through a three-step process: conversion of sugars to ethanol by yeasts; oxidation of ethanol to acetic acid by acetic acid bacteria (AAB); and, finally, at least 12 years of ageing. The final product is a highly dense, dark-brown aged vinegar, having a sweet and sour taste, fruity and complex in flavour.


2009 - Vinegars of the World [Curatela]
Solieri, L; Giudici, P
abstract

The importance of vinegars goes far beyond their merely economic aspect: they are in fact the result of environmental resources and culture, of tradition and science. The origin of vinegar is lost in the dawn of human history, together with the beginning of agriculture and the discovery of alcoholic fermentation of fruits, cereals and vegetables. This book, written by experts and scientists working in the field and enriched by several images and tables, clearly describes some of the main types of vinegar produced in the world in their peculiar aspects. In particular, vinegar technology and microbiology are dealt with extensively. The nomenclature of the microorganisms involved has been updated according to the current taxonomy.


2009 - Vinegars of the World [Capitolo/Saggio]
Solieri, Lisa; Giudici, Paolo
abstract

The story of microbial biotransformation is closely associated with vinegar production, which dates back to around 2000 years BC. However, among fermented foods, vinegar has been always considered a “poor” component: it is not a “food”, it doesn’t have high nutritional values, and it is produced by transformation of other more nutritive rich fermented foods. Vinegar is a condiment, but also a preservative and, in some countries, a healthy drink. It can be made by a two step fermentation of yeasts followed by acetic acid bacteria from almost any fermentable carbohydrate source: apples, pears, grapes, honey, syrups, cereals, hydrolysed starches, beer, and wine are all ideal substrates for the production of vinegar. Since vinegars is, with few exceptions, an inexpensive commodity, its production requires a low-cost raw material, such as sub-standard fruits, seasonal agricultural surpluses, various food processing by-products and fruit waste. However there are vinegars, so called spirit vinegars, obtained directly by acetic oxidation of alcohol obtained from distillation of fermented mashes or, in some countries, petrolchemical ethanol. Some western by-products of wood carbonization obtained by distillation of pyroligneous liquor collected during wood pyrolisis, are called “vinegar” and they are used as agricultural material, animal health product, cosmetic ingredients and folk drug in Japan and Est-Asia (Mu et al., 2003; Mu et al., 2006). These distilled solutions have not be considered as vinegar because no fermentative process occurs in their production. Other specialty vinegars are grouped as herbal or fruit vinegars. In this cases the name “X vinegar” doesn’t indicate the raw materials used in vinegar fermentation but the flavours added to obtain specific taste and characteristic sensorial features. Herbal vinegars consist of wine vinegars or white distilled vinegars, which may be seasoned with garlic, basil, tarragon, cinnamon, clove, or nutmeg. Fruit-flavoured vinegars are wine and white vinegars sweetened with fruit or fruit juice to produce a characteristic sweet-sour taste. Traditional vinegars are very expensive vinegar produced from regional foods according to well-established customs: balsamic vinegar of Modena from Italy; Sherry vinegar from Spain; Champagne vinegar from France etc. According to international definition of vinegar, in this book we considered only vinegar derived from a two stage fermentation process. However with many of the vinegars in Africa, Asia and Latin America, knowledge of the processes involved is poor and a comprehensive view of all different kinds of vinegars is impossible. Therefore it is likely that the worldspread vinegars give a very greater picture. Economy, technology and law about vinegar were described in this chapter.


2008 - Applicazione della tecnica rDNA PCR-DGGE nello studio della popolazione lattica dominante in parmigiano reggiano [Articolo su rivista]
Solieri, Lisa; E., Gala; Gullo, Maria; Giudici, Paolo
abstract

Applicazione della tecnica rDNA PCR-DGGE nello studio della popolazione lattica in parmigiano reggiano.


2008 - Chromosomal polymorphism and ploidy divergence in Zygosacharomyces rouxii strains isolated from Traditional Balsamic Vinegars [Abstract in Atti di Convegno]
Solieri, Lisa; Cassanelli, Stefano; Giudici, Paolo
abstract

The genus Zygosaccharomyces traditionally describes osmophilic yeasts that differ from the those of the genus Saccharomyces for their vegetative growth, in which sexual conjugation between cells or a cell and its bud, precedes the sporulation1. Diploidization through zygote formation means that vegetative life cycle is mainly haploid. In the last past decade phenotypical and molecular approaches based on cloning procedure, single-gene PCR amplification and karyotyping highlighted a high variability within Zygosaccharomyces rouxii species, mainly between type strain CBS 732 and some wild strains isolated from miso and traditional balsamic vinegar (ATCC 42981, ABT 301, and ABT 601, respectively)2,3,4. Recently random fragments sequencing from strain ATCC 42981 and allele-specific PCR reaction from strain ABT301 and ABT 601 have revealed that all these wild strains have two copies of several genes, including ITS-5.8S rDNA, 26S D1/D2 regions, ZSOD and HIS34,5. Genome size is one of the most fundamental genetic properties of living organisms. However no study has been attempted to evaluate genome size and ploidy for Z. rouxii strains. In this work genome size and ploidy level estimation, as well as ZSOD gene chromosome mapping were used to elucidate the taxonomic relationships among these wild strains compared to Z. rouxii CBS 732T.In particular, we applied flow cytometry to determine DNA content of cells in G0/G1 phase. The genome size was estimated 11.89±0.21 Mb (12.1±0.22 fg) for strain CBS 732T, 21.08±0.37 Mb (21.56±0.37 fg) for ATCC 42981, 25.68±1.11 Mb (26.27±1.14) for ABT 301, and 36.00±0.37 Mb (36.80±0.38 fg) for ABT 601. Moreover pulsed field gel electrophoresis (PFGE) confirmed the genome size divergence with polymorphic PFGE patterns consisting in different number of chromosomal bands: 6, 7, 10, and 11 for CBS 732T, ATCC 42981, ABT 301, and ABT 601, respectively. By densitometric analysis of PFGE profile, the haploid genome size has been obtained as sum of chromosomal bands size. These data have been combined to DNA content from flow cytometric analysis in order to calculate the ploidy level for each strain. The results showed CBS 732T has a haploid DNA content, whereas wild strains ATCC 42981, ABT 301, and ABT 601 a diploid DNA content. Finally the change in ploidy has been also confirmed by doubling of genes involved in osmotic adaptation (ZSOD, HOG, GPD, and GCY1), as well as by polymorphic rDNA genes occurring in ATCC 429815,6 and in ABT 301 and ABT 6014, but not in CBS 732T 2. For this purpose chromosome mapping of ZSOD genes (encoding Na+/H+-antiporters involved in halotolerance) showed that, differently from haploid CBS 732T, diploid strains ATCC 42981, ABT 301 and ABT 601 have two partially divergent ZSOD copies located on different chromosomes. In conclusion genome size and ploidy are two fundamental and informative traits mainly for yeasts with complex diploid/haploid shift in their life cycle. Our complementary molecular strategies highlighted a naturally occurring variation in genome size and ploidy level within Z. rouxii, suggesting that there is a complex picture in genome organization and the strains now recognized as Z. rouxii could belong to more than one species. References1. Barker, B.T.P., Trans. R. Soc. Lond. B., 194, 467–485 (1901).2. Kinclova, O., et al.,. J. Biotechnol., 88, 151–158. (2001)3. Pribylova, L., et al., Yeast, 24, 171-180 (2007)4. Solieri, L., et al., Yeast, 24, 403–417 (2007)5. Gordon, J., Wolfe, K.H. Yeast, 25, 449-456 (2008) 6. Iwaki T., et al., Microbiol. 145, 241-248 (1999).


2008 - Diversity of lactic acid bacteria population in ripened Parmigiano Reggiano cheese [Articolo su rivista]
Gala, Elisabetta; Landi, Sara; Solieri, Lisa; Nocetti, M; Pulvirenti, Andrea; Giudici, Paolo
abstract

The diversity of dominant lactic acid bacteria population in 12 months ripened Parmigiano Reggiano cheeses was investigated by a polyphasic approach including culture-dependent and independent methods. Traditional plating, isolation of LAB and identification by 16S rDNA analysis showed that strains belonging to Lactobacillus casei group were the most frequently isolated. Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus parabuchneri, and Lactobacillus buchneri species were detected with lower frequency. PCR-denaturing gradient gel electrophoresis (DGGE) applied to DNA extracted directly from cheese samples and sequencing of rDNA amplicons confirmed the complex microbiological pattern of LAB in ripened Parmigiano Reggiano cheeses, with the significant exception of the Lactobacillus fermentum species, which dominated in several samples, but was not detected by cultivation. The present combination of different approaches can effectively describe the lactic acid bacteria population of Parmigiano Reggiano cheese in advanced stages of ripening, giving useful information for elucidating the role of LAB in determining the final cheese quality.


2008 - Genome size and ploidy level: New insights for elucidating relationships in Zygosaccharomyces species [Articolo su rivista]
Solieri, Lisa; Cassanelli, Stefano; Croce, Ma; Giudici, Paolo
abstract

Ploidy is a fundamental genetic trait with important physiological and genomic implications. We applied complementary molecular tools to highlight differences in genome size and ploidy between Zygosaccharomyces rouxii strain CBS 732T and other related wild strains (ATCC 42981, ABT 301, and ABT 601). The cell cycle analysis by flow cytometry revealed a genome size of 12.7 ± 0.2 Mb for strain CBS 732T, 21.9 ± 0.2 Mb for ATCC 42981, 28.1 ± 1.3 Mb for ABT 301, and 39.00 ± 0.3 Mb for ABT 601. Moreover, karyotyping analysis showed a high variability, with wild strains having a higher number of chromosomal bands than CBS 732T. The ploidy level was assessed comparing genome size from flow cytometry with the average haploid size from electrophoretic karyotyping. Strain CBS 732T showed an haploid DNA content, whereas the wild strains a diploid DNA content. In addition gene probe-chromosome hybridization targeted to ZSOD genes showed that wild strains with a diploid DNA content have two ZSOD copies located on different chromosomes.


2008 - I lieviti ibridi nell'era dell'ingegneria genetica [Articolo su rivista]
Giudici, Paolo; Solieri, Lisa
abstract

Breeding fra specie o fra ceppi diversi di Saccharomycescerevisiae rappresenta una delle prime strategie di miglioramento genico applicate ai lieviti.La combinazione di tecniche classiche e di tecnologie whole genome, come i DNA microarray, possonoessere integrate con successo per contribuire all’analisidelle basi genetiche e funzionali dei caratteri fenotipici complessi e per incrementare il controllo neiprocessi di costituzione di lieviti di interesse enologico


2008 - Mitochondrial inheritance and fermentative: Oxidative balance in hybrids between Saccharomyces cerevisiae and Saccharomyces uvarum [Articolo su rivista]
Solieri, Lisa; Antúnez, O; PÉREZ ORTÍN, Je; Barrio, E; Giudici, Paolo
abstract

Breeding between Saccharomyces species is a useful tool for obtaining improved wine yeast strains, combining fermentative features of parental species. In this work, 25 artificial Saccharomyces cerevisiae × Saccharomyces uvarum hybrids were constructed by spore conjugation. A multi-locus PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, targeting six nuclear gene markers and the ribosomal region including the 5.8S rRNA gene and the two internal transcribed spacers, showed that the hybrid genome is the result of two chromosome sets, one coming from S. cerevisiae and the other from S. uvarum. Mitochondrial DNA (mtDNA) typing showed uniparental inheritance in all hybrids. Furthermore, sibling hybrids, obtained by repeated crosses between the same parental strains, showed the same mtDNA, suggesting that the mitochondrial transmission is not stochastic or species-specific, but dependent on the parental strains. Finally four hybrids, two of which with S. cerevisiae mtDNA and two with S. uvarum mtDNA, were subjected to transcriptome analysis. Our results showed that the hybrids bearing S. cerevisiae mtDNA exhibited less expression of genes involved in glycolysis/fermentation pathways and in hexose transport compared to hybrids with S. uvarum mtDNA. Respiration assay confirmed the increased respiratory activity of hybrids with the S. cerevisiae mtDNA genome. These findings suggest that mtDNA type and fermentative : respiratory performances are correlated in S. cerevisiae × S. uvarum hybrids and the mtDNA type is an important trait for constructing new improved hybrids for winemaking.


2008 - Strategy for yeast improvement [Capitolo/Saggio]
Giudici, Paolo; Solieri, Lisa
abstract

Selection and genetic improvement of yeast starter cultures are based on the ability to achieve a specific task or to perform a precise function. The process can be summarized into few steps: i) the knowledge of yeast microbiota in a specific ecological niche to yield species suitable to grow on specific substrates; ii) clonal selection for obtaining a wide genetic background; iii) definition of desired traits for yeast selection. However the selection of wild strains having complete combination of all traits is not so easy to obtain. There are many strategies for improving yeasts and increasing the recombination of all desired traits within an unique genome. The choice of most suitable strategy is mainly based on three factors: the genetic nature of traits (monogenic or polygenic), the knowledge of the genes involved, the aim of the genetic manipulation.Recent developments in genetic dissection of yeast quantitative traits highlighted how the several interesting traits can be complex and influenced by several genes. In a context of low phenotype-genotype correlation, both the rational approach involving DNA recombinant technologies and the traditional random techniques based on mutagenesis meet serious obstacles. Otherwise, different blind strategies, involving the cell entire genome, can be applied in order to obtain quickly strains with interesting recombinant traits. Laboratory sexual hybridization and genome shuffling are accelerated evolutionary approaches that allow to obtain desired complex phenotypes more rapidly than any normal rational methods.


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.


2008 - Yeasts associated to Traditional Balsamic Vinegar: Ecological and technological features [Articolo su rivista]
Solieri, Lisa; Giudici, Paolo
abstract

Traditional Balsamic Vinegar (TBV) is an Italian homemade vinegar made with cooked grape must through a three-step process: conversion of sugars to ethanol by naturally occurring yeasts; oxidation of ethanol to acetic acid by acetic acid bacteria (AAB); and, finally, at least 12-years ageing. The cooked must is a selective and stressful medium for yeasts growth, due to its high sugar content and low pH values. Recent studies have shown that a large number of yeast species are involved in the fermentation, among them there are Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Zygosaccharomyces pseudorouxii, Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces lentus, Hanseniaspora valbyensis, Hanseniaspora osmophila, Candida lactis-condensi, Candida stellata, Saccharomycodes ludwigii and Saccharomyces cerevisiae. Nevertheless, the TBV-associated yeast population could be even more complex and many other slow-growing or poorly cultivable species might contribute to cooked must fermentation. In this review the main TBV yeast species are described, pointing out their role in TBV production and their influence on final product quality. Finally, both future developments in TBV yeast community studies (culture-independent and metagenomic techniques) and technological advances in TBV making (use of starter culture) are discussed.


2007 - A new putative Zygosaccharomyces yeast species isolated from traditional balsamic vinegar. [Articolo su rivista]
Solieri, Lisa; Cassanelli, Stefano; Giudici, Paolo
abstract

The taxonomic status and species number of the genus Zygosaccharomyces have rapidly changed in the last years. In this study, two new osmotolerant Zygosaccharomyces strains isolated from traditional balsamic vinegar, viz. ABT301 and ABT601, were investigated to elucidate their taxonomic relationships with Zygosaccharomyces rouxii species. A multi-gene sequence approach was employed, including regions of the rDNA repeat [5.8S, two internal transcribed spacers (ITS) and the 26S D1/D2 domain], COX2 mitochondrial gene and two nuclear genes (SOD2 and HIS3). Cloning and sequence analysis of 5.8S-ITS rDNA revealed that these strains bear an unusual polymorphism for this region. Three highly divergent 5.8S-ITS sequences were detected, one identical to Z. rouxii, the other two showing some relatedness to Z. mellis. Sequence and gene number polymorphism was also observed for the protein-encoding nuclear genes SOD2 and HIS3, as two copies for each gene different from those found in Z. rouxii were detected. Analysis of the D1/D2 26S domain showed that ABT301 and ABT601 have only one type of D1/D2 sequence statistically different from that of Z. rouxii. The findings obtained in this work suggest that the genomic background of strains ABT301 and ABT601 is different from the other Zygosaccharomyces species. We speculated that they could belong to a new putative species related to Z. rouxii.


2007 - Determinazione dei batteri lattici nel vino mediante tecniche coltura-indipendenti. [Articolo su rivista]
Solieri, Lisa; DE PAOLA, M.
abstract

Nel presente studio abbiamo utilizzato una tecnica coltura-indipendente, laPCR-DGGE, per il monitoraggio della popolazione microbica in campioni divino inoculati con differenti starter lattici di Oenococcus oeni. La PCR-DGGEapplicata direttamente alla matrice vino ha messo in evidenza come lo starterOenococcus oeni fosse la specie dominante in tutti i campioni esaminati.


2007 - Mitochondrial DNA transmission in Saccharomyces sensu stricto hybrids constructed by spore conjugation. [Abstract in Atti di Convegno]
Solieri, Lisa; Giudici, Paolo; Rainieri, Sandra
abstract

The Saccharomyes sensu stricto group contains the most important species for the Fermentation Industry. Interspecifichybridisation among these yeasts is a useful tool for obtaining starter cultures with improved fermentative traits. Several S.cerevisiae x S. uvarum hybrids have been constructed and successfully employed in wine fermentations. Studies demonstratedthat they inherit mtDNA from either parental species. Recently, however, the interest for using other Saccharomyces species inoenology has been growing and the prospective of constructing hybrids with other species is being explored. We investigated theinheritance of mtDNA in hybrids constructed by spore conjugation between S. cerevisiae and other Saccharomyces sensu strictospecies. We obtained three hybrids for each type of crossing: S. cerevisiae x S. cariocanus; S. cerevisiae x S. mikatae; S.cerevisiae x S. kudriavzevii; S. cerevisiae x S. paradoxus; S. cerevisiae x S. uvarum. By analysing the RFLP of the mtDNA andsequencing COXII gene we observed that: 1) in all the Saccharomyces hybrids mtDNA is inherited by only one of the parentalstrains; 2) the parental strain transmitting the mtDNA can belong to either parental species; 3) for each combination of parentalstrains the same type of mtDNA is always inherited. These findings suggest that mtDNA is not inherited accidentally and thisinformation can be exploited when constructing starter cultures by interspecific hybridisation.


2007 - Physiological and genotypic characterization of a novel Zygosaccharomyces species [Abstract in Atti di Convegno]
Solieri, Lisa; Cassanelli, Stefano; Giudici, Paolo
abstract

A pool of new Zygosaccharomyces strains was isolated in Traditional Balsamic Vinegar samples. Recently an our multi-genestudy [including genes of the rDNA repeat and two nuclear genes, SOD2 and HIS3] has highlighted that: i) rDNA units areunusually polymorphic with respect to ITS regions (with three highly divergent ITS sequences), but the D1/D2 26S rDNA regionis unique and monomorphic; ii) two different copies of nuclear genes SOD2 and HIS3 are present. In this study a physiologicaland genotypic characterization of two of these strains, namely ABT301 and ABT601, was performed. Physiological tests showedthat these strains are osmophilic and halophilic yeasts able to grow at 60% glucose and 16% NaCl and with a assimilative profilepartially similar to that of Z. rouxii species. Chromosomal DNA separation by PFEG was performed in order estimate the numberand size of their chromosomes. A SOD2-22-based Real Time PCR assay was designed to determine the ploidy of ABT301 andABT601, compared to diploid Z. rouxii CBS732 type strain. Finally three transcriptional assays were carried out: the first wasaimed to 5.8S-ITS region of 35S precursor rRNA, the others to the two different SOD2 and HIS3 copies. According to ourpreliminary results, ABT301 and ABT601 showed: i) a different electrophoretic karyotype compared to CBS732T strain; ii) adiploid status; iii) both copies of SOD2 and HIS3 genes actively transcribed, as well as the three 5.8S-ITS regions.


2007 - Wine colour adsorption phenotype: An inheritable quantitative trait loci of yeasts [Articolo su rivista]
Caridi, A; Sidari, R; Solieri, Lisa; Cufari, A; Giudici, Paolo
abstract

AbstractAims: In this work, a population of 88 descendants derived from three wine strains of Saccharomyces cerevisiae was tested for the enological trait 'wine colour adsorption' (WCA) to evaluate its inheritability.Methods and Results: The WCA phenotype was tested on plate agar medium specifically formulated for the purpose. After 10 days of anaerobic incubation at 28°C, a computer-assisted assessment of WCA aptitude of the yeasts was carried out. The biomass colour – ranging from white to dark brown – reflects the adsorption of grape pigments: white and dark brown biomass colour corresponds to low and high adsorption, respectively. In order to confirm biomass colour results, microvinification trials using red must were performed, and the obtained wines were analysed.Conclusions: The analysis of the progeny demonstrated that the enological trait WCA is inheritable and polygenic.Significance and Impact of the Study: A way to describe the polygenic effect of the WCA trait has been found, also showing that this trait is inheritable. The impact of the work revolves more around the large-scale screening method, which could then assist in breeding wine yeast, and can also be used as a scientific tool to investigate WCA trait.


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 - GLUCOSE TOLERANCE AS DISTINCTIVE TRAITS OF ACETIC ACID BACTERIA [Relazione in Atti di Convegno]
Gullo, Maria; Solieri, Lisa; Giudici, Paolo
abstract

Vinegar production as well other fermented foods and beverages is based on the use of starter cultures which permit a higher degree of control and standardisation of the final product compared to spontaneous fermentation. The first task to optimise fermented foods process is the knowledge of microorganism physiological and metabolic traits. Acetic acid bacteria species occurring in vinegar production are well known, but species monitoring and strains selection are not common procedures. Then there are not extensive studies on significant traits of strains involved in vinegar oxidation process. Concerning to traditional balsamic vinegars few researches are available about occurring species and their physiological traits. In this study strains isolated from traditional balsamic vinegar samples were investigated by enzymatic digestion of ITS1 regions using RsaI and HaeIII restriction endonucleases and by sequencing of 16S rDNA and ITS1 rDNA. Since elevated sugar concentration resistance and growing is an important trait for traditional balsamic vinegar acetic acid bacteria, the isolated strains were tested for glucose tolerance. On the basis of technological results glucose concentration is an acetic acid bacteria limiting-factor and Acetobacter malorum species is more resistant to high glucose concentration than other species occurred in balsamic vinegar.Keywords: acetic acid bacteria, glucose tolerance, strains selection


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 - 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 - 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

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 - 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.


2006 - Molecular dissection of the 5.8S-ITS region variability in Zygosaccharomyces spp. [Abstract in Atti di Convegno]
Solieri, Lisa; Cassanelli, Stefano; S., Rainieri; Giudici, Paolo
abstract

Molecular dissection of the 5.8S-ITS region variability in Zygosaccharomyces spp.


2006 - The Yeasts of sourdough [Relazione in Atti di Convegno]
Pulvirenti, Andrea; Solieri, Lisa; Sara, Landi; Giudici, Paolo
abstract

III International Symposium on Sourdough. The yeasts of Sourdough


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 - 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 - Lieviti starter per aceto balsamico tradizionale. [Articolo su rivista]
Solieri, Lisa; Giudici, Paolo
abstract

La fermentazione alcolica del mosto cottorappresenta la prima fase microbiologica nellaproduzione di ABT (Aceto Balsamico Tradizionale).La popolazione di lieviti che conducono questoprocesso fermentativo influenza direttamente eindirettamente le proprietà organolettiche delprodotto finale, nonché le sue caratteristichemacroscopiche. La fermentazione spontanea delmosto cotto è un processo incontrollato e nonprogrammabile, che spesso si interrompe senzache venga prodotta una quantità sufficiente dietanolo per consentire la successiva ossidazioneacetica. La disponibilità di colture di lievito starterper ABT risolverebbe queste problematichema la loro messa a punto è imprescindibile dalsoddisfacimento di tre presupposti: lo studioapprofondito della microflora dell’ABT; lacostituzione di un patrimonio genetico medianteselezione clonale; la definizione dei caratterispecifici che una coltura starter per ABT devepossedere. Nel presente lavoro definiamo iprincipali caratteri di fitness e di qualità che,sulla base delle conoscenze attuali, un lievitoselezionato per ABT deve possedere e descriviamoper le principali specie di lievito riscontratenell’ABT, il loro comportamento relativo ad alcunidi questi caratteri. Infine prendiamo in esame lepossibilità offerte dalle tecniche di miglioramentogenetico per la combinazione di caratteridesiderati.


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 - Molecular procedure for yeasts identification of Traditional Balsamic Vinegar [Relazione in Atti di Convegno]
Solieri, Lisa; L., Castellari; A. R., Battagliola; Pulvirenti, Andrea; S., Landi; Giudici, Paolo
abstract

Molecular procedure for yeasts identification of Traditional Balsamic Vinegar


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 - Strategies and perspectives for genetic improvement of wine yeasts [Articolo su rivista]
Giudici, Paolo; Solieri, Lisa; Pulvirenti, Andrea; Cassanelli, Stefano
abstract

Recent developments in expression profile and proteomic techniques illustrated that the main oenological traits of wine yeasts are complex and influenced by several genes, each of them identified as absolutely essential. Only for monogenic properties the genetic improvement programmes of wine yeasts can be performed by alteration of individual genes. Ideally the most productive way of improving the whole-cell biocatalysts is by evolution of the entire cell genome. In this article we briefly review the main genetic improvement techniques applied in new and optimised wine strains construction, paying particular attention to blind and whole genome strategies, such as the sexual recombination and genome shuffling.


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 - The yeasts of Traditional Balsamic Vinegar [Abstract in Atti di Convegno]
Solieri, Lisa; L., Castellari; A. R., Battagliola; Pulvirenti, Andrea; S., Landi; Giudici, Paolo
abstract

Proocedng of Congress: International Symposium on Vinegars and Acetic Acid Bacteria


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 - 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 - 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 - 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 - Metodi indiretti per lo studio dei microrganismi dell’ABT. [Relazione in Atti di Convegno]
Pulvirenti, Andrea; Solieri, Lisa; Gullo, Maria; Giudici, Paolo
abstract

metodi coltura-indipendenti per la caratterizzazione della microflora dell'ABT


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.


2003 - Dominant yeast species in traditional sourdoughs [Abstract in Atti di Convegno]
Solieri, Lisa; Pulvirenti, Andrea; Gullo, Maria; Giudici, Paolo
abstract

Dominant yeast species in traditional sourdoughs


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 - 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