Diversity of dung beetle-associated yeasts from pristine environments of Botswana.

Yeast-insect interactions are increasingly becoming an attractive source of discovery for previously unknown, unique, diverse, and industrially relevant yeast species. Despite a wealth of studies that have recently focused on yeasts in symbiotic association with Hymenopteran insects, yeasts associated with Coleopteran insects, such as lignocellulosic-rich dung-dependent beetles, remain poorly studied. Trends in yeast discovery suggest that species richness and diversity can be attributed to the ecological niche of the insect. Here, we considered the potential of dung beetles inhabiting the extreme environments of Botswana, characterized by desert-like conditions (semi-arid to arid and hot) as well as protected pristine environments, as possible attribute niches that can shape the extremophilic and diverse life history strategies of yeasts. We obtained a total of 97 phylogenetically diverse yeast isolates from six species of dung beetles from Botswana's unexplored environments, representing 19 species belonging to 11 genera. The findings suggest that the guts of dung beetles are a rich niche for non-Saccharomyces yeast species. Meyerozyma and Pichia were the most dominant genera associated with dung beetles, representing 55% (53 out of 97) of the yeast isolates in our study. Trichosporon and Cutaneotrichosporon genera represented 32% (31 out of 97) of the isolates. The remaining isolates belonged to Apiotrichum, Candida, Diutina, Naganishia, Rhodotorula, and Wickerhamiella genera (12 out of 97). We found out that about 62% (60 out of 97) of the isolates were potentially new species because of their low internal transcribed spacer (ITS) sequence similarity when compared to the most recent optimal species delineation threshold. A single isolate was unidentifiable using the ITS sequences. Using an in silico polymerase chain reaction-restriction fragment length polymorphism approach, we revealed that there was genetic diversity within isolates of the same species. Our results contribute to the knowledge and understanding of the diversity of dung beetle-associated yeasts.

Semi-Targeted Profiling of the Lipidome Changes Induced by Erysiphe Necator in Disease-Resistant and Vitis vinifera L. Varieties.

The ascomycete Erysiphe necator is a serious pathogen in viticulture. Despite the fact that some grapevine genotypes exhibit mono-locus or pyramided resistance to this fungus, the lipidomics basis of these genotypes' defense mechanisms remains unknown. Lipid molecules have critical functions in plant defenses, acting as structural barriers in the cell wall that limit pathogen access or as signaling molecules after stress responses that may regulate innate plant immunity. To unravel and better understand their involvement in plant defense, we used a novel approach of ultra-high performance liquid chromatography (UHPLC)-MS/MS to study how E. necator infection changes the lipid profile of genotypes with different sources of resistance, including BC4 (Run1), "Kishmish vatkhana" (Ren1), F26P92 (Ren3; Ren9), and "Teroldego" (a susceptible genotype), at 0, 24, and 48 hpi. The lipidome alterations were most visible at 24 hpi for BC4 and F26P92, and at 48 hpi for "Kishmish vatkhana". Among the most abundant lipids in grapevine leaves were the extra-plastidial lipids: glycerophosphocholine (PCs), glycerophosphoethanolamine (PEs) and the signaling lipids: glycerophosphates (Pas) and glycerophosphoinositols (PIs), followed by the plastid lipids: glycerophosphoglycerols (PGs), monogalactosyldiacylglycerols (MGDGs), and digalactosyldiacylglycerols (DGDGs) and, in lower amounts lyso-glycerophosphocholines (LPCs), lyso-glycerophosphoglycerols (LPGs), lyso-glycerophosphoinositols (LPIs), and lyso-glycerophosphoethanolamine (LPEs). Furthermore, the three resistant genotypes had the most prevalent down-accumulated lipid classes, while the susceptible genotype had the most prevalent up-accumulated lipid classes.

Metabolomic Workflow for the Accurate and High-Throughput Exploration of the Pathways of Tryptophan, Tyrosine, Phenylalanine, and Branched-Chain Amino Acids in Human Biofluids.

The modulation of host and dietary metabolites by gut microbiota (GM) is important for maintaining correct host physiology and in the onset of various pathologies. An ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for the targeted quantitation in human plasma, serum, and urine of 89 metabolites resulting from human-GM cometabolism of dietary essential amino acids tryptophan, tyrosine, and phenylalanine as well as branched-chain amino acids. Ninety-six-well plate hybrid-SPE enables fast clean-up of plasma and serum. Urine was diluted and filtered. A 15 min cycle enabled the acquisition of 96 samples per day, with most of the metabolites stable in aqueous solution for up to 72 h. Calibration curves were specifically optimized to cover expected concentrations in biological fluids, and limits of detection were at the order of ppb. Matrix effects were in acceptable ranges, and analytical recoveries were in general greater than 80%. Inter and intraday precision and accuracy were satisfactory. We demonstrated its application in plasma and urine samples obtained from the same individual in the frame of an interventional study, allowing the quantitation of 51 metabolites. The method could be considered the reference for deciphering changes in human-gut microbial cometabolism in health and disease. Data are available via Metabolights with the identifier MTBLS4399.

Application of a Target-Guided Data Processing Approach in Saturated Peak Correction of GC×GC Analysis.

Detector and column saturations are problematic in comprehensive two-dimensional gas chromatography (GC×GC) data analysis. This limits the application of GC×GC in metabolomics research. To address the problems caused by detector and column saturations, we propose a two-stage data processing strategy that will incorporate a targeted data processing and cleaning approach upstream of the "standard" untargeted analysis. By using the retention time and mass spectrometry (MS) data stored in a library, the annotation and quantification of the targeted saturated peaks have been significantly improved. After subtracting the nonperfected signals caused by saturation, peaks of coelutes can be annotated more accurately. Our research shows that the target-guided method has broad application prospects in the data analysis of GC×GC chromatograms of complex samples.

Enhanced flavour profiles through radicicol induced genomic variation in the lager yeasts, Saccharomyces pastorianus.

The yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and have acquired traits from the combined parental genomes such as ability to ferment a range of sugars at low temperatures and to produce aromatic flavour compounds, allowing for the production of lager beers with crisp, clean flavours. The polyploid strains are sterile and have reached an evolutionary bottleneck for genetic variation. Here we describe an accelerated evolution approach to obtain lager yeasts with enhanced flavour profiles. As the relative expression of orthologous alleles is a significant contributor to the transcriptome during fermentation, we aimed to induce genetic variation by altering the S. cerevisiae to S. eubayanus chromosome ratio. Aneuploidy was induced through the temporary inhibition of the cell's stress response and strains with increased production of aromatic amino acids via the Shikimate pathway were selected by resistance to amino acid analogues. Genomic changes such as gross chromosomal rearrangements, chromosome loss and chromosome gain were detected in the characterised mutants, as were single-nucleotide polymorphisms in ARO4, encoding for DAHP synthase, the catalytic enzyme in the first step of the Shikimate pathway. Transcriptome analysis confirmed the upregulation of genes encoding enzymes in the Ehrlich pathway and the concomitant increase in the production of higher alcohols and esters such as 2-phenylethanol, 2-phenylethyl acetate, tryptophol, and tyrosol. We propose that the polyploid nature of S. pastorianus genomes is an advantageous trait supporting opportunities for genetic alteration in otherwise sterile strains.

Volatile Aroma Compound Production Is Affected by Growth Rate in S. cerevisiae.

The initial growth rate of a yeast strain is a key parameter in the production of fermented beverages. Fast growth is linked with higher fermentative capacity and results in less slow and stuck fermentations unable to reach the expected final gravity. As concentrations of metabolites are in a constant state of flux, quantitative data on how growth rate affects the production of aromatic compounds becomes an important factor for brewers. Chemostats allow to set and keep a specific dilution rate throughout the fermentation and are ideal system to study the effect of growth on aroma production. In this study, we ran chemostats alongside batch and fed-batch cultures, compared volatile profiles detected at different growth rates, and identified those affected by the different feeding profiles. Specifically, we quantified six abundant aroma compounds produced in anaerobic glucose-limited continuous cultivations of S. cerevisiae at different dilution rates. We found that volatile production was affected by the growth rate in four out of six compounds assayed, with higher alcohols and esters following opposite trends. Batch and fed-batch fermentations were devised to study the extent by which the final concentration of volatile compounds is influenced by glucose availability. Compared with the batch system, fed-batch fermentations, where the yeast growth was artificially limited by a slow constant release of nutrients in the media, resulted in a significant increase in concentration of higher alcohols, mirroring the results obtained in continuous fermentations. This study paves the way to further process development optimization for the production of fermented beverages. IMPORTANCE The production of fermentation beverages will need to quickly adapt to changes in both the climate and customer demands, requiring the development of new strains and processes. Breakthroughs in the field are hindered by the limited knowledge on the interplay between physiology and aroma compound production in yeast. No quantitative data on how growth rate affects aroma profile is available in the literature to guide optimization of the complex flavors in fermented beverages. In this study, we exploited the chemostat system, alongside with batch and fed-batch cultures, to compare volatile profiles at different growth rates. We identified the aromatic compounds affected by the different feeding profiles and nutrient limitations. Moreover, we uncovered the correlation between yeast growth, esters, and higher alcohols production. This study showcases the potential of the application of feeding profiles for the manipulation of aroma in the craft beverage industry.

Phenolic Compound Profile by UPLC-MS/MS and Encapsulation with Chitosan of Spondias mombin L. Fruit Peel Extract from Cerrado Hotspot-Brazil.

Taperebá (Spondias mombin L.) is a native species of the Brazilian Cerrado that has shown important characteristics such as a significant phenolic compound content and biological activities. The present study aimed to characterize the phenolic compound profile and antioxidant activity in taperebá peel extract, as well as microencapsulating the extract with chitosan and evaluating the stability of the microparticles. The evaluation of the profile of phenolic compounds was carried out by UPLC-MS/MS. The in vitro antioxidant activity was evaluated by DPPH and ABTS methods. The microparticles were obtained by spray drying and were submitted to a stability study under different temperatures. In general, the results showed a significant content of polyphenols and antioxidant activity. The results of UPLC-MS/MS demonstrated a significant content of polyphenols in taperebá peel, highlighting the high content of ellagic acid and quercetin compounds. There was significant retention of phenolic compounds when microencapsulated, demonstrating high retention at all evaluated temperatures. This study is the first to microencapsulate the extract of taperebá peel, in addition to identifying and quantifying some compounds in this fruit.

Effect of green-Mediterranean diet on intrahepatic fat: the DIRECT PLUS randomised controlled trial.

OBJECTIVE: To examine the effectiveness of green-Mediterranean (MED) diet, further restricted in red/processed meat, and enriched with green plants and polyphenols on non-alcoholic fatty liver disease (NAFLD), reflected by intrahepatic fat (IHF) loss. DESIGN: For the DIRECT-PLUS 18-month randomized clinical trial, we assigned 294 participants with abdominal obesity/dyslipidaemia into healthy dietary guidelines (HDG), MED and green-MED weight-loss diet groups, all accompanied by physical activity. Both isocaloric MED groups consumed 28 g/day walnuts (+440 mg/day polyphenols provided). The green-MED group further consumed green tea (3-4 cups/day) and Mankai (a Wolffia globosa aquatic plant strain; 100 g/day frozen cubes) green shake (+1240 mg/day total polyphenols provided). IHF% 18-month changes were quantified continuously by proton magnetic resonance spectroscopy (MRS). RESULTS: Participants (age=51 years; 88% men; body mass index=31.3 kg/m2; median IHF%=6.6%; mean=10.2%; 62% with NAFLD) had 89.8% 18-month retention-rate, and 78% had eligible follow-up MRS. Overall, NAFLD prevalence declined to: 54.8% (HDG), 47.9% (MED) and 31.5% (green-MED), p=0.012 between groups. Despite similar moderate weight-loss in both MED groups, green-MED group achieved almost double IHF% loss (-38.9% proportionally), as compared with MED (-19.6% proportionally; p=0.035 weight loss adjusted) and HDG (-12.2% proportionally; p<0.001). After 18 months, both MED groups had significantly higher total plasma polyphenol levels versus HDG, with higher detection of Naringenin and 2-5-dihydroxybenzoic-acid in green-MED. Greater IHF% loss was independently associated with increased Mankai and walnuts intake, decreased red/processed meat consumption, improved serum folate and adipokines/lipids biomarkers, changes in microbiome composition (beta-diversity) and specific bacteria (p<0.05 for all). CONCLUSION: The new suggested strategy of green-Mediterranean diet, amplified with green plant-based proteins/polyphenols as Mankai, green tea, and walnuts, and restricted in red/processed meat can double IHF loss than other healthy nutritional strategies and reduce NAFLD in half. TRIAL REGISTRATION NUMBER: NCT03020186.

Packing a punch: understanding how flavours are produced in lager fermentations.

Beer is one of the most popular beverages in the world and it has an irreplaceable place in culture. Although invented later than ale, lager beers dominate the current market. Many factors relating to the appearance (colour, clarity and foam stability) and sensory characters (flavour, taste and aroma) of beer, and other psychological determinants affect consumers' perception of the product and defines its drinkability. This review takes a wholistic approach to scrutinise flavour generation in the brewing process, focusing particularly on the contribution of the raw ingredients and the yeasts to the final flavour profiles of lager beers. In addition, we examine current developments to improve lager beer flavour profiles for the modern consumers.

Biotechnological exploitation of Saccharomyces jurei and its hybrids in craft beer fermentation uncovers new aroma combinations.

Hybridisation is an important evolutionary mechanism to bring about novel phenotypes and may produce new hybrids with advantageous combinations of traits of industrial importance. Within the Saccharomyces genus, Saccharomyces jurei is a newly discovered species and its biotechnological potential has not yet been fully explored. This yeast was found to be able to grow well in unhopped wort and at low temperatures, qualities necessary in good candidates for fermented bevarages. Here, we analysed its fermentation and aroma profile and created novel non-GMO hybrids between S. jurei and S. cerevisiae ale yeasts to develop new starter strains with interesting flavours for the craft brewing and beverage industry in general. Pilot beer fermentations with specific hybrids showed a good fermentation performance, similar to the ale parent strain, while eliminating the hyper-attenuation characteristic and a more complex flavour profile. This study exploits the genetic diversity of yeasts and shows how inter-specific hybridisation and clone selection can be effectively used in brewing to create new products and to eliminate or increase specific traits.

On sample preparation methods for fermented beverage VOCs profiling by GCxGC-TOFMS.

INTRODUCTION: Aromas and tastes have crucial influences on the quality of fermented beverages. The determination of aromatic compounds requires global non-targeted profiling of the volatile organic compounds (VOCs) in the beverages. However, experimental VOC profiling result depends on the chosen VOC collection method. OBJECTIVES: This study aims to observe the impact of using different sample preparation techniques [dynamic headspace (DHS), vortex-assisted liquid-liquid microextraction (VALLME), multiple stir bar sorptive extraction (mSBSE), solid phase extraction (SPE), and solid phase micro-extraction (SPME)] to figure out the most suitable sample preparation protocol for profiling the VOCs from fermented beverages. METHODS: Five common sample preparation methods were studied with beer, cider, red wine, and white wine samples. After the sample preparation, collected VOCs were analyzed by two-dimensional gas chromatography coupled with time of flight mass spectrometry (GCxGC-TOFMS). RESULTS: GCxGC oven parameters can be optimized with the Box-Behnken surface response model and response measure on peak dispersion. Due to the unavoidable column and detector saturation during metabolomic analysis, errors may happen during mass spectrum construction. Profiling results obtained with different sample preparation methods show considerable variance. Common findings occupy a small fraction of total annotated VOCs. For known fermentative aromas, best coverage can be reached by using SPME together with SPE for beer, and VALLME for wine and cider. CONCLUSIONS: GCxGC-TOFMS is a promising tool for non-targeted profiling on VOCs from fermented beverages. However, a proper data processing protocol is lacking for metabolomic analysis. Each sample preparation method has a specific profiling spectrum on VOC profiling. The coverage of the VOC metabolome can be improved by combining complementary methods.

Inoculation of Lupinus albus with the nodule-endophyte Paenibacillus glycanilyticus LJ121 improves grain nutritional quality.

Metabolic changes occurring in white lupine grain were investigated in response to Plant Growth Promoting Rhizobacteria (PGPR) root inoculation under field condition. We precisely targeted lipids and phenolics changes occurring in white lupine grain in response to Pseudomonas brenneri LJ215 and/or Paenibacillus glycanilyticus LJ121 inoculation. Lipids and phenolic composition were analyzed using an Ultra High-Performance Liquid Chromatography/Tandem Mass Spectrometry Methods. As compared to grain of un-inoculated control plant, Paenibacillus glycaniliticus inoculation highly increased the total lipids content (from 232.55 in seeds of un-inoculated control plant to 944.95 mg/kg) and the relative percentage of several fatty acid such as oleic acid (+20.95%) and linoleic acid (+14.28%) and decreased the relative percentage of glycerophospholipids (- 13.11%), sterol (- 0.2% and - 0.34% for stigmasterol and campesterol, respectively) and prenol (- 17.45%) class. Paenibacillus glycaniliticus inoculation did not affect total phenolic content, while it modulated content of individual phenolic compounds and induced the accumulation of "new" phenolics compounds such as kaempferol. Paenibacillus glycanilyticus LJ121 can be a useful bio-fertilizer to enhance nutritional quality of white lupine grain.

Two apples a day modulate human:microbiome co-metabolic processing of polyphenols, tyrosine and tryptophan.

PURPOSE: Validated biomarkers of food intake (BFIs) have recently been suggested as a useful tool to assess adherence to dietary guidelines or compliance in human dietary interventions. Although many new candidate biomarkers have emerged in the last decades for different foods from metabolic profiling studies, the number of comprehensively validated biomarkers of food intake is limited. Apples are among the most frequently consumed fruits and a rich source of polyphenols and fibers, an important mediator for their health-protective properties. METHODS: Using an untargeted metabolomics approach, we aimed to identify biomarkers of long-term apple intake and explore how apples impact on the human plasma and urine metabolite profiles. Forty mildly hypercholesterolemic volunteers consumed two whole apples or a sugar and energy-matched control beverage, daily for 8 weeks in a randomized, controlled, crossover intervention study. The metabolome in plasma and urine samples was analyzed via untargeted metabolomics. RESULTS: We found 61 urine and 9 plasma metabolites being statistically significant after the whole apple intake compared to the control beverage, including several polyphenol metabolites that could be used as BFIs. Furthermore, we identified several endogenous indole and phenylacetyl-glutamine microbial metabolites significantly increasing in urine after apple consumption. The multiomic dataset allowed exploration of the correlations between metabolites modulated significantly by the dietary intervention and fecal microbiota species at genus level, showing interesting interactions between Granulicatella genus and phenyl-acetic acid metabolites. Phloretin glucuronide and phloretin glucuronide sulfate appeared promising biomarkers of apple intake; however, robustness, reliability and stability data are needed for full BFI validation. CONCLUSION: The identified apple BFIs can be used in future studies to assess compliance and to explore their health effects after apple intake. Moreover, the identification of polyphenol microbial metabolites suggests that apple consumption mediates significant gut microbial metabolic activity which should be further explored.

Apple fruit superficial scald resistance mediated by ethylene inhibition is associated with diverse metabolic processes.

Fruits stored at low temperature can exhibit different types of chilling injury. In apple, one of the most serious physiological disorders is superficial scald, which is characterized by discoloration and brown necrotic patches on the fruit exocarp. Although this phenomenon is widely ascribed to the oxidation of α-farnesene, its physiology is not yet fully understood. To elucidate the mechanism of superficial scald development and possible means of prevention, we performed an integrated metabolite screen, including an analysis of volatiles, phenols and lipids, together with a large-scale transcriptome study. We also determined that prevention of superficial scald, through the use of an ethylene action inhibitor, is associated with the triggering of cold acclimation-related processes. Specifically, the inhibition of ethylene perception stimulated the production of antioxidant compounds to scavenge reactive oxygen species, the synthesis of fatty acids to stabilize plastid and vacuole membranes against cold temperature, and the accumulation of the sorbitol, which can act as a cryoprotectant. The pattern of sorbitol accumulation was consistent with the expression profile of a sorbitol 6-phosphate dehydrogenase, MdS6PDH, the overexpression of which in transgenic Arabidopsis thaliana plants confirmed its involvement in the cold acclimation and freezing tolerance.

Measuring the impact of olive pomace enriched biscuits on the gut microbiota and its metabolic activity in mildly hypercholesterolaemic subjects.

PURPOSE: Olive pomace is a major waste product of olive oil production but remains rich in polyphenols and fibres. We measured the potential of an olive pomace-enriched biscuit formulation delivering 17.1 ± 4.01 mg/100 g of hydroxytyrosol and its derivatives, to modulate the composition and metabolic activity of the human gut microbiota. METHODS: In a double-blind, controlled parallel dietary intervention 62 otherwise healthy hypercholesterolemic (total plasma cholesterol 180-240 mg/dl) subjects were randomly assigned to eat 90 g of olive pomace-enriched biscuit (olive-enriched product, OEP) or an isoenergetic control (CTRL) for 8 weeks. Fasted blood samples, 24-h urine and faecal samples were collected before and after dietary intervention for measurement of microbiota, metabolites and clinical parameters. RESULTS: Consumption of OEP biscuits did not impact on the diversity of the faecal microbiota and there was no statistically significant effect on CVD markers. A trend towards reduced oxidized LDL cholesterol following OEP ingestion was observed. At the genus level lactobacilli and Ruminococcus were reduced in OEP compared to CTRL biscuits. A trend towards increased bifidobacteria abundance was observed after OEP ingestion in 16S rRNA profiles, by fluorescent in situ hybridization and by qPCR. Targeted LC-MS revealed significant increases phenolic acid concentrations in 24-h urine following OEP ingestion and 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid, derivatives of hydroxytyrosol, were elevated in blood. A sex effect was apparent in urine small phenolic acid concentrations, and this sex effect was mirrored by statistically significant differences in relative abundances of faecal bacteria between men and women. CONCLUSION: Ingestion of OEP biscuits led to a significant increase in the metabolic output of the gut microbiota with an apparent sex effect possibly linked to differences in microbiota makeup. Increased levels of homovanillic acid and DOPAC, thought to be involved in reducing oxidative LDL cholesterol, were observed upon OEP ingestion. However, OEP did not induce statistically significant changes in either ox-LDL or urinary isoprostane in this study.

Bioactive Polyphenols Modulate Enzymes Involved in Grapevine Pathogenesis and Chitinase Activity at Increasing Complexity Levels.

The reduction of synthetic chemistry use in modern viticulture relies on either the biological control of microorganisms or the induction of pathogenesis-related proteins. In the present study, the effects of hydro-alcoholic plant extracts (PEs) (i.e., by-products of Vitis vinifera L., leaves of Olea europaea L. and Ailanthus altissima (Mill.) Swingle) were tested on purified enzymes activity involved in plant-pathogen interactions. The polyphenolic composition was assayed and analyzed to characterize the extract profiles. In addition, suspension cell cultures of grapevine were treated with PEs to study their modulation of chitinase activity. Application of grape marc's PE enhanced chitinase activity at 4 g L-1. Additionally, foliar treatment of grape marc's PE at two doses (4 g L-1 and 800 g L-1) on grapevine cuttings induced a concentration-dependent stimulation of chitinase activity. The obtained results showed that the application of bioactive compounds based on PEs, rich in phenolic compounds, was effective both at in vitro and ex/in vivo level. The overall effects of PEs on plant-pathogen interaction were further discussed by applying a multi-criteria decision analysis, showing that grape marc was the most effective extract.

Methyl Salicylate Glycosides in Some Italian Varietal Wines.

Glycosides are ubiquitous plant secondary metabolites consisting of a non-sugar component called an aglycone, attached to one or more sugars. One of the most interesting aglycones in grapes and wine is methyl salicylate (MeSA), an organic ester naturally produced by many plants, particularly wintergreens. To date, nine different MeSA glycosides from plants have been reported, mainly spread over the genera Gaultheria, Camellia, Polygala, Filipendula, and Passiflora. From a sensorial point of view, MeSA has a balsamic-sweet odor, known as Wintergreen. MeSA was found in Vitis riparia grapes, in Vitis vinifera sp. and in the Frontenac interspecific hybrid. We found that the MeSA glycosides content in Verdicchio wines and in some genetically related varieties (Trebbiano di Soave and Trebbiano di Lugana) was very high. In order to understand which glycosides were present in wine, the methanolic extract of Verdicchio wine was injected into a UPLC-Q-TOF-HDMS and compared to the extracts of different plants rich in such glycosides. Using pure standards, we confirmed the existence of two glycosides in wine: MeSA 2-O--d-glucoside and MeSA 2-O--d-xylopyranosyl (1-6) -d-glucopyranoside (gaultherin). For the first time, we also tentatively identified other diglycosides in wine: MeSA 2-O--l-arabinopyranosyl (1-6)--d-glucopyranoside (violutoside) and MeSA 2-O--d-apiofuranosyl (1-6)--d-glucopyranoside (canthoside A), MeSA 2-O--d-glucopyranosyl (1-6)-O--d-glucopyranoside (gentiobioside) and MeSA 2-O--l-rhamnopyranosyl (1-6)--d-glucopyranoside (rutinoside). Some of these glycosides have been isolated from Gaultheria procumbens leaves by preparative liquid chromatography and structurally annotated by 1H- and 13C-NMR analysis. Two of the peaks isolated from Gaultheria procumbens leaves, namely MeSA sambubioside and MeSA sophoroside, were herein observed for the first time. Six MeSA glycosides were quantified in 64 Italian white wines, highlighting the peculiar content and pattern in Verdicchio wines and related cultivars. The total concentration in bound and free MeSA in Verdicchio wines varied in the range of 456-9796 g/L and 5.5-143 g/L, respectively, while in the other wines the bound and free MeSA was below 363 g/L and 12 g/L, respectively. As this compound's olfactory threshold is between 50 and 100 g/L, our data support the hypothesis that methyl salicylate can contribute to the balsamic scent, especially in old Verdicchio wines.

Lipid Profiling and Stable Isotopic Data Analysis for Differentiation of Extra Virgin Olive Oils Based on Their Origin.

To differentiate extra virgin olive oils (EVOO) according to the origin of purchase, such as monocultivar Italian EVOO with protected denomination of origin (PDO) and commercially-blended EVOO purchased in supermarkets, a number of samples was subjected to the analysis of various lipid species by liquid chromatography/mass spectrometry (LC-ESI-MS/MS, LC-ESI-IT-MS) and proton nuclear magnetic resonance analysis (1H-NMR). Many putative chemical markers were extracted as differentiators by uni- and multivariate statistical analysis. Commercially-blended EVOO contained higher concentrations of the majority of minor lipids, including free fatty acids, their alkyl (methyl and ethyl) esters, monoglycerides, and diglycerides, which may be indicative of a higher degree of triglyceride lipolysis in these than in monocultivar PDO EVOO. Triterpenoids and particular TAG species were also found in higher proportions in the samples from the commercially-blended EVOO class, suggesting a possible influence of factors such as the cultivar and geographical origin. The largest differences between the classes were determined for the concentrations of uvaol and oleanolic acid. The results of the analysis by isotopic ratio mass spectrometry (IRMS) were reasonably consistent with the information about the geographical origin declared on the labels of the investigated EVOOs, showing considerable variability, which possibly also contributed to the differences in lipid composition observed between the two investigated classes of EVOO.

Complementary Untargeted and Targeted Metabolomics for Differentiation of Extra Virgin Olive Oils of Different Origin of Purchase Based on Volatile and Phenolic Composition and Sensory Quality.

In order to differentiate the extra virgin olive oils (EVOO) of different origin of purchase, such as monovarietal Italian EVOO with protected denomination of origin (PDO) and commercial-blended EVOO purchased in supermarkets, a number of samples was subjected to the analysis of volatile aroma compounds by both targeted gas chromatography/mass spectrometry (GC-MS) and untargeted profiling by comprehensive two-dimensional gas chromatography/mass spectrometry (GC×GC-TOF-MS), analysis of phenols by targeted high-performance liquid chromatography/mass spectrometry (HPLC-DAD-ESI/MS), and quantitative descriptive sensory analysis. Monovarietal PDO EVOOs were characterized by notably higher amounts of positive LOX-derived C6 and C5 volatile compounds, which corresponded to the higher intensities of all the assessed positive fruity and green odor sensory attributes. Commercial-blended EVOOs had larger quantities of generally undesirable esters, alcohols, acids, and aldehydes, which coincided with the occurrence of sensory defects in many samples. Many minor volatile compounds that were identified by GC×GC-TOF-MS were found to differentiate each of the two investigated groups. The differences between the groups with respect to phenols and taste characteristics were evident, but less pronounced. The results that were obtained in this study have undoubtedly confirmed the existence of the large heterogeneity of oils that are sold declared as EVOO. It was shown that GC-MS, GC×GC-TOF-MS, and HPLC-DAD-ESI/MS analyses have complementary outputs, and that their use in combination has advantages in supporting the results of sensory analysis and objectively differentiating these groups of EVOO.

In Planta Recapitulation of Isoprene Synthase Evolution from Ocimene Synthases.

Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated. We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated invivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause invivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms. Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance.