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Raspberry ( Rubus idaeus L.) is a fruit of great interest due to its aroma, nutritional properties, and the presence of many bioactive compounds. However, differences among cultivation systems can affect its composition and, consequently, its potential bioactivity. Herein, for the first time, raspberries grown in an aeroponic system were investigated for their chemical profile and antioxidant and anti-inflammatory activity, as well as their enzyme (α-glucosidase and pancreatic lipase) inhibitory properties in comparison to wild and conventionally cultivated fruits. High-performance liquid chromatography coupled with diode array detection (HPLC-DAD) analyses revealed the presence of gallic acid, caffeic acid, chlorogenic acid, p -coumaric acid, ferulic acid, rutin, and catechin in all the samples. The extracts exhibited in vitro anti-inflammatory activity (inhibition of nitric oxide production) regardless of the cultivation method. Of particular interest is the ability of raspberries to inhibit pancreatic lipase. With the exception of the β-carotene bleaching test, the raspberries grown in conventional and aeroponic systems were more active in terms of antioxidants than wild fruits, as evidenced by the ABTS (IC 50 in the range 1.6-3.4 μg/mL), DPPH (IC 50 in the range 8.9-28.3 μg/mL), and FRAP tests (24.6-44.9 μM Fe(II)/g). The raspberries from aeroponic cultivation were generally able to exert the same bioactivity as those obtained from both conventionally cultivated and wild fruits, supporting the consideration that in the future, this technology could reshape agriculture by mitigating resource constraints, fostering sustainable practices and increasing yields., Competing Interests: Author Antonio Grisolia was employed by the company Azienda Agricola Grisolia A. Author Biagio Armentano was employed by the company Azienda Agricola Armentano F. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supercritical fluid extraction with CO 2 (SFE) is an alternative technology to conventional solvent extraction (CSE), to obtain food-grade bioactives from plants. Here, SFE and CSE extracts from carrot and pumpkin matrices, impregnated with hempseed or flaxseed oil as co-solvents, were characterized by HPLC and GC-MS, and their ability to counteract the inflammatory and oxidative phenomena underlying the onset of several pathologies was assessed in vitro . All extracts showed dose-dependent anti-inflammatory potential and demonstrated an ability to interfere with the pro-inflammatory effects of breast cancer cell-conditioned media, and to inhibit reactive oxygen species (ROS) accumulation and nitrite production (NP) in lipopolysaccharide-stimulated macrophages. Nuclear factor-erythroid-2-related factor 2 (Nrf2) is involved in these response mechanisms, as highlighted by the increased mRNA levels of its target genes revealed by quantitative real-time PCR analyses. NP and ROS concentrations negatively correlated with α-tocopherol and most carotenoids, but positively with the total tocopherol/total carotenoid ratio, suggesting an idiosyncratic effect of these bioactives on cell responses and emphasizing the need to focus on extract constituents' interactions.

Film-forming yeasts are potential sources of defects in alcoholic beverages. The aim of this study is to assess the growth capacity of Pichia and Candida film-forming yeasts in cider and wine and the effects on their chemical composition. Cider, partially and fully fermented wine were inoculated with strains of C. californica, P. fermentans, P. kluyveri, P. kudriavzevii, P. manshurica, and P. membranifaciens to simulate a post-fermentative contamination. The former three species grew only in cider. Pichia manshurica and P. kudriavzevii displayed high viability in wine up to 13.18% (v v-1) ethanol. Significant changes in odour-active molecules from different chemical groups were observed in cider and wine in the inoculated samples, compared to the non-inoculated ones. Cider is more susceptible to contamination by all of the species tested, due to its low alcohol content, while P. membranifaciens, P. manshurica, and P. kudriavzevii are additionally potential spoilage agents of wine. This study highlights the risk of cider and wine contamination by film-forming yeasts. Their impact on aroma profiles depends on their ability to grow and their metabolism. This study contributes to an understanding of the possible physiological and metabolic mechanisms responsible for film formation and chemical changes in alcoholic beverages., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Pichia and Candida species include biofilm-forming yeasts able to spoil foods and beverages. Strains belonging to 10 Pichia and Candida species isolated from apples, grape musts, and wines were analysed. They were subjected to molecular typing and characterized for their ability to grow and ferment must for cider and wine production, and for their biofilm properties. All strains grew similarly in apple and grape must. Glucose-fermenting strains displayed differentiated fermentation performances. Great variation in SO2 and ethanol sensitivity was observed among the strains. Pichia manshurica strains showed high tolerance to both molecules. Eleven and five surface-spreading biofilm (MAT) phenotypes were identified in solid and liquid media, respectively. Strains produced biofilms with variable thicknesses and widths in culture tubes. Cell adherence and aqueous-hydrocarbon biphasic hydrophobicity assays were carried out. Some Pichia manshurica and P. membranifaciens strains exhibited a high capacity to form a thick biofilm and had high cell adherence and hydrophobicity values. These strains could be more likely to colonize the internal surfaces of tanks. This study evidenced that some Pichia and Candida strains can proliferate during apple and grape must fermentation and may be detrimental the beverage quality, due to their specific biofilm properties., (© The Author(s) 2022. Published by Oxford University Press on behalf of Applied Microbiology International.)

The characterization of Oenococcus oeni strains isolated from Nero di Troia wine (Apulia, Italy) sampled in two distinct production areas was carried out. The two indigenous populations, consisting of 95 and 97 isolates, displayed high genetic diversity when analyzed by amplified fragments length polymorphisms (AFLP). Based on the UPGMA dendrogram obtained by AFLP analysis, the two populations displayed similar genotypes that grouped in the same clusters with a high level of similarity (>95%). One genotype was found in only one of the two areas. Representative strains of each cluster were analyzed for their enzymatic activities (esterase, β-glucosidase, and protease), assayed in whole cells, and tested for their metabolic properties (consumption of L-malic acid, citric acid, acetaldehyde, and arginine) and growth parameters. Significant differences among strains, including the reference strain ATCC BAA-1163, were observed for all of these properties. Principal component analysis evidenced phenotypic differences among strains, and well separated some of them belonging to different genotypes. Strains exhibiting the best performances in most of these traits could be further investigated in order to select possible candidates as malolactic starters for Nero di Troia wine. This study provided insights on the population structure of O. oeni of a local winemaking area useful to the understanding of the regional diversity of this bacterium, an issue not yet completely resolved

This work aimed to investigate and compare the in vitro antioxidant and anti-inflammatory effects of Salvia officinalis L. (sage) from Italy, with the aim of raising its current knowledge in this field. Leaves and flowers (S1-S8), harvested in two areas of Southern Italy, were extracted with methanol as a solvent by maceration or ultrasound-assisted extraction. Sage extracts, analysed by high pressure liquid chromatography-diode-array detection-electrospray ionization-quadrupole-mass spectroscopy (HPLC-DAD-ESI-Q-MS), exerted a promising antioxidant activity investigated using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and β-carotene bleaching tests, and elicited a significant decrease in reactive oxygen species (ROS) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. The anti-inflammatory activity was analysed in the same in vitro model. All the extracts did not affect cell viability although they showed anti-inflammatory activity, as they induced a decrease in nitrite levels that was greater than 50%, when employed at 50 µg/mL. Furthermore, they elicited a decrease in nitrite levels, as well as a decline in pro-inflammatory cytokine expression. The NF-κB transcription factor proved to be involved in the mechanisms that underlie such effects. Interestingly, sage extracts were able to interfere with the inflammatory activity induced by breast cancer cell-conditioned media (nitrite levels were significantly decreased, p < 0.05; p < 0.01), highlighting for the first time the important role of S. officinalis in controlling inflammation processes related to neoplastic progression.

In this study, we evaluated and compared the chemical composition, the antioxidant, anti-inflammatory, and anti-proliferative effects of four methanol extracts (R1-R4), of Salvia rosmarinus Spenn. in two different sites of Southern Italy obtained by maceration or ultrasound-assisted extraction. Extracts of S. rosmarinus collected on the Ionian coast are indicated with the abbreviations R1 (maceration) and R2 (ultrasound-assisted extraction). Extracts of S. rosmarinus collected on the Tyrrhenian coast are indicated with the abbreviations R3 (maceration) and R4 (ultrasound-assisted extraction). The chemical composition was analyzed using High Pressure liquid chromatography-Diod-Array detection-Electrospray ionization-Quadrupole-Mass Spectroscopy (HPLC-DAD-ESI-Q-MS). The antioxidant activity was analyzed by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) 2,2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene bleaching, and Ferric Reducing Antioxidant Power (FRAP) assays. Antioxidant features were also assessed in lipopolysaccharide (LPS)-stimulated RAW-264.7 murine macrophages, evaluating Reactive Oxygen Species (ROS) production; in the same experimental model, the anti-inflammatory activity of the extracts was investigated. Interestingly, all extracts displayed antioxidant and anti-inflammatory properties. They exhibited significative nitrite production inhibitory activity, whith IC 50 values ranging from 3.46 to 5.53 µg/mL, without impairing cell viability. The anti-inflammatory activity was also investigated by Western Blotting and immunofluorescence assay, highlighting the R3 and R4 extracts ability to reduce NF-κB translocation, as well as to disrupt the MAPKs signaling pathway. Extracts exhibited both potential anti-proliferative activity on breast cancer cells, inducing apoptosis, without affecting non-tumorigenic cells, and the ability to inhibit MDA-MB-231 cells' motility. Finally, the rosemary extracts treatment significantly reduced the power of conditioned media, from MCF-7 or MDA-MB-231 cells to induce nitrite production on RAW 264.7 cells, confirming their promising anti-inflammatory activity., Competing Interests: The authors declare that there is no conflict of interest.

Aims: The bioactivities of Santolina corsica Jord. & Fourr. n-hexane (EHS) and methanol (EMS) extracts were evaluated in relation to their chemical profile., Main Methods: EHS and EMS were analysed by gas chromatography-mass spectrometry () and high performance liquid chromatography-diode array detection (HPLC-DAD), respectively. Antioxidant activity was determined by β-carotene bleaching, Ferric Reducing Activity Power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) tests. Nitric oxide (NO) production was assessed in LPS-stimulated RAW 264.7 cells. Anti-proliferative activity was evaluated by MTT assay on A549, HeLa, PC3, MCF-7, MDA-MB-231 cancer cells, and non-tumorigenic MCF10 A cells. Cell motility, migration and invasion were assessed by wound-healing scratch, migration and invasion assays, respectively. DNA fragmentation was tested by TUNEL assay. Cells morphology was studied by phase-contrast microscopy. Procaspase-8, -9, poly (ADP-ribose) polymerase and COX-2 expression levels were evaluated by immunoblotting analysis., Key Findings: Kaempferol-3-O-glucoside (5878.67 mg/100 g of extract), chlorogenic acid (746.11 mg/100 g), and rosmarinic acid (550.16 mg/100 g) were the dominant EMS constituents. EHS showed myrcene (18.86%) as the main compound, followed by palmitic acid methyl and ethyl esters (9.35 and 9.16%, respectively), β-phellandrene (8.48%), and ar-curcumene (5.63%). Both extracts showed promising anti-proliferative activity on all tested cancer cells, without inducing cytotoxicity in non-tumorigenic cells MCF-10 A. Moreover, extracts inhibited motility, migration, and invasion of MDA-MB-231 cells, inducing apoptosis. EHS decreased NO production, showing anti-inflammatory activity., Significance: S. corsica extracts might be potentially useful in cancer treatment, since reduce invasive and migratory potential of MDA-MB-231 cells triggering apoptosis., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Glycyrrhiza glabra cultivation and harvesting produces substantial quantities of aerial parts as waste. With the aim to prospect an innovative valorization of these byproducts, the aerial parts were harvested in May and October and analyzed for their chemical profile, antioxidant properties, and effects on viability of five cancer cell lines. Pinocembrin was the main constituent. A significant protection of lipid peroxidation was observed with the May total extract (IC 50 of 4.2 ± 0.4 μg/mL at 30 min of incubation). The effects on viability of HeLa, MCF-7, MDA-MB-231, Caco-2, and PC3 human cancer cells were investigated. All samples shown a remarkable activity with IC 50 values below 25 μg/mL. Samples from plants harvested in May exhibited greater activity than those harvested in October. MCF-7 and HeLa were the most sensitive cells with IC 50 in the range 2.73-3.01 and 3.28-5.53 μg/mL, respectively. G. glabra aerial parts represent a good source of valuable products.

Alginate micro beads containing Lactobacillus kefiri (the principal bacteria present in the kefir probiotic drink) were produced by a novel technique based on dual aerosols spaying of alginate based solution and CaCl 2 as cross linking agent. Carboxymethylcellulose (CMC) has been also added to the alginate in order to change the physic-chemical properties (viscosity and permeability) of the microbeads. Calcium alginate and CMC are biopolymers that can be used for developing oral drug-delivery systems. These biopolymers have been reported to show a pH-dependent swelling behaviour. Calcium alginate and CMC have also been known to possess an excellent mucoadhesive property. The loaded microbeads have been characterized in terms of morphology, chemical composition and stability in different conditions mimicking the gastric environment. In this study, we demonstrate the feasibility of a continuous fabrication of alginate microbeads in a range of 50-70μm size, encapsulating L. kefiri as active ingredient. The technique involves the use of a double aerosols of alginate based solution and CaCl 2 as crosslinking agent. Moreover, the encapsulation process was proved to be effective and not detrimental to bacteria viability. At the same time, it was verified the protective efficacy of the microcapsules against the gastric environment using both SGF pH1.2 (fasted state) and pH2.2 (feed state)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the last two decades knowledge on lactic acid bacteria (LAB) associated with wine has increased considerably. Investigations on genetic and biochemistry of species involved in malolactic fermentation, such as Oenococcus oeni and of Lactobacillus have enabled a better understand of their role in aroma modification and microbial stability of wine. In particular, the use of molecular techniques has provided evidence on the high diversity at species and strain level, thus improving the knowledge on wine LAB taxonomy and ecology. These tools demonstrated to also be useful to detect strains with potential desirable or undesirable traits for winemaking purposes. At the same time, advances on the enzymatic properties of wine LAB responsible for the development of wine aroma molecules have been undertaken. Interestingly, it has highlighted the high intraspecific variability of enzymatic activities such as glucosidase, esterase, proteases and those related to citrate metabolism within the wine LAB species. This genetic and biochemistry diversity that characterizes wine LAB populations can generate a wide spectrum of wine sensory outcomes. This review examines some of these interesting aspects as a way to elucidate the link between LAB diversity with wine aroma and flavour. In particular, the correlation between inter- and intra-species diversity and bacterial metabolic traits that affect the organoleptic properties of wines is highlighted with emphasis on the importance of enzymatic potential of bacteria for the selection of starter cultures to control MLF and to enhance wine aroma., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Filamentous fungi are the main pathogens of withered grapes destined for passito wine production. Knowledge of which species inhabit these post-harvest fruits and their pathogenicity is essential in order to develop strategies to control infection, but is still scarce. This study investigated the predominant mycobiota of withered grapes through a cultivation-dependent approach. Strain and species heterogeneity was evidenced on examining isolates collected over three consecutive years. Colony morphology and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis revealed the occurrence of several phenotypes and haplotypes, respectively. Strains were phylogenetically analyzed based on sequence typing of different genes or regions (e.g. calmodulin, β-tubulin and internal transcribed spacer region). Beside the most common necrotrophic-saprophytic species of Penicillium, Aspergillus, Alternaria and Botrytis species responsible for fruit rot, other saprobic species were identified (e.g. Trichoderma atroviride, Sarocladium terricola, Arthrinium arundinis and Diaporthe eres) generally not associated with post-harvest fruit diseases. Species such as Penicillium ubiquetum, Cladosporium pseudocladosporioides, Lichtheimia ramosa, Sarocladium terricola, Diaporthe nobilis, Bipolaris secalis, Paraconiothyrium fuckelii and Galactomyces reessii that had never previously been isolated from grapevine or grape were also identified. Moreover, it was not possible to assign a species to some isolates, while some members of Didymosphaeriaceae and Didymellaceae remained unclassified even at genus level. This study provides insights into the diversity of the epiphytic fungi inhabiting withered grapes and evidences the importance of their identification to understand the causes of fruit diseases. Finally, phylogenetic species delimitation furnished data of interest to fungal taxonomy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aims: There is scarce information on the occurrence of several fungi that infect withered grapes to produce passito wine. Isolation and characterization of Neofusicoccum parvum strains and evaluation of their effects on withered grape and wine were carried out., Methods and Results: Nine isolates were phenotypically characterized by colony morphology and genetically discriminated by molecular methods. Two representative strains were identified as N. parvum according to the phylogenetic analysis of internal transcribed spacer (ITS), and a part of translation elongation factor 1-alfa (TEF) and β-tubulin DNA sequences. The pathogenicity of both strains on grape berries varied according to the inoculation and incubation conditions. Under withering conditions, infected berries showed browning and shrivelling and some berries showed pycnidial development on the surface. The infection affected laccase, esterase, β-glucosidase and tannase on grape juice as well as the content of several aroma molecules on resulting wines. Strain-specific effects on wine composition were also observed., Conclusions: Neofusicoccum parvum occurred in withered grapes and was able to infect grapes under withering condition changing the aroma wine., Significance and Impact of the Study: This study reports for the first time the N. parvum isolation in fruit-drying rooms and indicates its important role on postharvest grape infection., (© 2015 The Society for Applied Microbiology.)

Table olives are one of the most important traditional fermented vegetables in Europe and their world consumption is constantly increasing. In the Greek style, table olives are obtained by spontaneous fermentations, without any chemical debittering treatment. Evolution of sugars, organic acids, alcohols, mono, and polyphenol compounds and volatile compounds associated with the fermentative metabolism of yeasts and bacteria throughout the natural fermentation process of the two Italian olive cultivars Cellina di Nardò and Leccino were determined. A protocol was developed and applied aimed at the technological characterization of lactic acid bacteria (LAB) and yeast strains as possible candidate autochthonous starters for table olive fermentation from Cellina di Nardò and Leccino cultivars. The study of the main physic-chemical parameters and volatile compounds during fermentation helped to determine chemical descriptors that may be suitable for monitoring olive fermentation. In both the analyzed table olive cultivars, aldehydes proved to be closely related to the first stage of fermentation (30 days), while higher alcohols (2-methyl-1-propanol; 3-methyl-1-butanol), styrene, and o-cymene were associated with the middle stage of fermentation (90 days) and acetate esters with the final step of olive fermentation (180 days).

The variation in the coding capacity within Oenococcus oeni can have a significant impact on wine quality. The detection of several genes involved in important metabolic pathways (i.e. citrate, sulphur and arginine metabolisms) was performed on 10 indigenous O. oeni strains from Negroamaro wine, a red table wine (Apulia, Italy). These strains were selected from 95 isolates, collected during spontaneous malolactic fermentation, according to the results of an Amplified Fragment Length Polymorphism (AFLP) analysis. A total of 16 genes were screened, most (11) of which had never previously been assayed on O. oeni. All strains possessed 10 genes encoding enzymes such as malolactic enzyme (mleA), esterase (estA), citrate lyase (citD, citE and citF), citrate transporter (maeP), α-acetolactate decarboxylase (alsD), α- acetolactate synthase (alsS), S-adenosylmethionine synthase (metK) and cystathionine β-lyase (metC) and resulted negative in the detection of genes encoding cystathionine γ-lyase (metB), ornithine transcarbamylase (arcB) and carbamate kinase (arcC). The sequence of PCR fragments of 11 genes of a representative strain (ITEM 15929) was compared to those of three reference O. oeni strains. The indigenous strain was phylogenetically more similar to PSU-1 and ATCC BAA1163 than AWRI B429. This study describes new genetic markers useful for detecting the genetic potential of O. oeni strains to contribute to aroma production and for investigating the population structure of the species., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A simple procedure is proposed for selective protein solubilization and trypsin digestion, followed by off-line liquid chromatography-matrix assisted laser desorption ionization mass spectrometry (LC-MALDI MS) analysis of Oenococcus oeni (O. oeni) bacterium. Peptides were identified from tryptic digests using sequencing by tandem mass spectrometry and database searches. Cytoplasmic and membrane related proteins (MRP) were identified in the O. oeni bacterium. MS/MS data analysis points out 13 peptides having one point mutation from 9 proteins. The major microheterogeneity was found for Zn-dependent alcohol dehydrogenase (Zn-ADH, Q04GE6) and 60 kDa chaperonin (GroEL, Q04E64) that are involved in methionine catabolism and post-translational protein folding, respectively. MS/MS data processing also leads to the identification of 34 unique phosphorylation sites from 19 phosphoproteins.

The lysozyme of hen's egg white is used in winemaking to control spontaneous lactic acid bacteria (LAB). A total of eight LAB strains, isolated from grape must and wine, were used to assess the inhibitory effects of wine phenolics on lysozyme activity. The presence of phenolics, extracted from grape pomace, in growth medium reduced the mortality rate due to the lysozyme activity. This effect was especially clear in the case of strains belonging to Lactobacillus uvarum, Pediococcus parvulus and Oenococccus oeni, which are more sensitive to lysozyme than L. plantarum and L. hilgardii strains. Cell lysis assays carried out on four strains sensitive to lysozyme and Micrococcus lysodeikticus ATCC 4698, used as a reference strain, confirmed the inhibition of grape pomace phenolics on the muramidase. There was no interference from non-flavonoids, flavanols and flavonol compounds, when they were tested individually, on the lysozyme activity against the strains. Anthocyanins extracted from grape skins slightly inhibited the activity only against M. lysodeikticus. However, proanthocyanidins extracted from seed berries, strongly inhibited the lysozyme. In this extract, dimers were the predominant oligomers of flavan-3-ol. The study demonstrated that the effectiveness of lysozyme against LAB in red winemaking is related to the amount of low molecular weight proanthocyanidins that are released when the grapes are macerating., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The diversity of indigenous Oenococcus oeni strains was investigated by molecular and biochemical characterization of isolates from Malvasia Nera wine, an economically important red wine of the Salento Region (Apulia, Italy), during spontaneous malolactic fermentation (MLF). A total of 82 isolates of this species, identified by species-specific PCR and 16S rDNA sequence analysis, was molecularly characterized by the amplified fragment length polymorphism (AFLP) technique. Three main groups resulted from cluster analysis and showed intraspecific homology higher than 50%, and a total of seven subgroups, with similarity values ranged from 80% to 98%, were obtained within these groups. Enzymatic activities, such as esterase, β-glucosidase, protease, and the consumption rate of l-malic acid, citric acid, acetaldehyde and arginine were assessed in the representative strains, according to AFLP analysis. The results showed different enzymatic activities and consumption rates of the tested metabolites among the strains. No correlation between molecular and biochemical data was observed. The evidence of biochemical variability observed among Malvasia Nera strains demonstrated that the wine aroma can be modulated depending on the strains involved in MLF. Hence, the heterogeneity existing within natural O. oeni populations represents an interesting ecological source that can be useful for technological purposes., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO₂ and temperature) useful for industrial application., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The Amplified Fragment Length Polymorphism (AFLP) technique was applied for the first time to investigate the genotyping of Oenococcus oeni, the most important species involved in malolactic fermentation (MLF) in wine. A total of 87 out of 220 lactic acid bacteria, isolates from "Primitivo" wine (Apulia, Italy) undergoing MLF, identified as O. oeni by species-specific PCR and 16S rRNA sequence analysis, were studied by AFLP analysis. Four main clusters were distinguished and three of them showed intraspecific homology higher than 60%. A total of 28 strains, representative of AFLP clusters, were tested for malate metabolism in order to gain information on their malolactic performances. Significant differences were observed among strains for malic acid consumed, biomass produced and specific malic acid consumption rate. These findings indicated that AFLP technique is reliable for typing O. oeni strains and that, together with metabolism studies it may be used to individuate possible candidates as industrial malolactic starters.

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

Aims: Isolation and characterization of indigenous Saccharomyces cerevisiae strains from 12 grape varieties grown in an experimental vineyard of Apulia., Methods and Results: Thirty to 40 colonies from each of the 12 fermentations were obtained at the end stage of spontaneous fermentation. By using morphological and physiological methods and by the PCR analysis of internal transcribed ITS1-5,8S-ITS2, the isolates belonging to Saccharomyces genus were identified. These isolates were further characterized by amplification with S. cerevisiae species- and delta element-specific primers, thus allowing the identification of S. cerevisiae strains selected from each of the 12 fermentations. By means of RFLP analysis of mtDNA, each S. cerevisiae population isolated from a single fermentation appeared to constitute a genetically homogenous group. The comparison of the 12 cultivar-specific mtDNA RFLP patterns, allowed classifying the 12 S. cerevisiae populations into three genetically homogenous groups. The isolated strains fermented vigorously in synthetic and grape juice medium and showed high alcohol and sulphur dioxide (SO(2)) resistance and low hydrogen sulphite (H(2)S) production., Conclusions: The molecular analysis, in conjunction with the traditional morphological and physiological methods, was useful in discriminating at strain level the indigenous population of S. cerevisiae present in a vineyard of Apulia. The dominant S. cerevisiae strains identified in the 12 fermented musts showed potentially important oenological characteristics., Significance and Impact of the Study: The characterization of natural S. cerevisiae strains from several typical Italian grapes grown in a restricted experimental vineyard is an important step towards the preservation and exploitation of yeast biodiversity of Apulia, a relevant wine-producing region. The close relationship between the S. cerevisiae strains from different grapes grown in the same vineyard indicated that the occurrence of native strains is representative of the area rather than of the variety of grapes.

Background: Almond proteins can cause severe anaphylactic reactions in susceptible individuals. The aim of this study was the identification of IgE-binding proteins in almonds and the characterisation of these proteins by N-terminal sequencing., Methods: Five sera were selected from individuals with a positive reaction to food challenge. Sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting were performed on almond seed proteins. Purified IgE-binding proteins were tested for immunoblot inhibition with sera pre-incubated with extracts of hazelnut and walnut., Results: N-terminal sequences of the 12-, 30- and 45-kD proteins were obtained. The 45- and 30-kD proteins shared the same N terminus, with 60% homology to the conglutin gamma heavy chain from lupine seed (Lupinus albus) and to basic 7S globulin from soybean (Glycine max). The sequences of the N-terminal 12-kD protein and of an internal peptide obtained by endoproteinase digestion showed good homology to 2S albumin from English walnut (Jug r 1). Immunoblot inhibition experiments were performed and IgE binding to almond 2S albumin and conglutin gamma was detected in the presence of cross-reacting walnut or hazelnut antigens., Conclusions: Two IgE-binding almond proteins were N-terminally sequenced and identified as almond 2S albumin and conglutin gamma. Localisation and conservation of IgE binding in a 6-kD peptide obtained by endoproteinase digestion of 2S albumin was shown., (Copyright 2002 S. Karger AG, Basel)

Twenty-nine strains of Lactic Acid Bacteria isolated from the typical Pecorino cheese of the Salento area of Italy, were identified and grouped according to their genetic similarity. A preliminary characterisation of the strains was conducted by means of morphological and biochemical analysis, but molecular approaches were necessary for the clear identification of the species. For the species detection, the amplification and sequencing of the 16S rDNA gene was employed In addition, restriction analysis of amplified rDNA (ARDRA) and PCR and AFLP fingerprinting enabled inter- and intra-specific variation to be estimated UPGMA cluster analysis was used to divide the strains into distinct clusters which corresponded with the species delineation obtained by molecular identification. The data obtained show that the community of lactobacilli responsible for the fermentation and aging of Pecorino cheese is composed of a limited number of species. The main identified strains were Lactobacillus brevis, L. plantarum, L. casei, L. sakei, L. pentosus, L. farciminis and Leuconostoc mesenteroides.