High Power Ultrasound Treatments of Red Young Wines: Effect on Anthocyanins and Phenolic Stability Indices.

Polyphenols, especially anthocyanins, play an important role on red wine sensory qualities and their evolution during storage. High Power Ultrasound (HPU) has been recognized as one of the most promising technologies which can be applied in winemaking processes for several purposes, and it is recently officially approved for crushed grapes treatments. The effect of ultrasound amplitude (41 and 81%) and treatment time (1, 3, and 5 min) has been studied on anthocyanins, flavan-3-ols, tannins, polymerized pigments, HCl index, and the color intensity of two finished red young wines. Anthocyanins and phenols compounds were not degraded with an increase in amplitude and sonication time, and the chromatic properties of the selected wines were preserved. Amplitude and ultrasound time were also evaluated considering their effect on evolution of anthocyanin content and phenolic stability indices during the first thirty days of storage. The higher level of amplitude (81%) induced a higher percentage decrease in tannins, 15% and 40% after 15 and 30 days of storage, respectively, compared to untreated wine which did not show a significant change during storage. HPU shows a possible chemical effect on the evolution of some analytical indices during bottling maturation, but their effectiveness could be strictly linked with the initial phenolic profile and ratios between polyphenol classes.

A novel LC-HRMS method reveals cysteinyl and glutathionyl polysulfides in wine.

A novel ultra high pressure liquid chromatography combined with high resolution mass spectrometry (UHPLC-HRMS) method was developed to study glutathionyl and cysteinyl polysulfides in wine. Different HPLC columns were investigated in order to optimise the chromatographic resolution of the polysulfide standard mixtures synthesised in-house. The optimisation of the chromatographic conditions when trying to separate glutathionylated and cysteinylated species containing from 3 to 5 sulfur atoms proved particularly challenging, with the cationic exchange column IonPac CS12A-MS resulting to be the best column for this task.The synergistic application of the newly developed methods together with the synthesised reference standard mixtures allowed the identification and the detection of 11 different glutathionyl and cysteinyl polysulfides. Moreover, analysing 15 young white wines was possible to confirm the presence of GSSSG in wine (GS = glutathione). More importantly, this study allowed for the first identification of several symmetric and asymmetric new polysulfides, namely: GSSSSG, CSSSC (CS = cysteine), CSSSSC, CSSSG, and CSSSSG. These molecules have not previously been identified in wine, raising the question on their biogenesis and role on wine quality.

Thiol precursors in Vitis mould-tolerant hybrid varieties.

BACKGROUND: Over recent years, a new wave of disease-tolerant to mildew varieties has flooded the viticulture sector, for reasons of human safety and economic expediency. These hybrid grape cultivars are selected mainly on the basis of their intrinsic capability to counter the attack of the main fungal diseases that affect grape production, such as downy mildew and powdery mildew. However, their organoleptic and oenological characteristics have not yet been studied in depth for purposes of both juice and wine production, due to the high number of newly proposed germplasms and the lack of information about their adaptability to different environments. This work examines the thiol aroma precursors concentration in 64 red and white disease-tolerant hybrid varieties in the vine germplasm collections of Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria-Viticulture and Enology Research Center and Fondazione Edmund Mach, both from the north-east of Italy. RESULTS: All cultivars showed the presence of 3-mercaptohexan-1-ol (3MH) precursors, ranging from 4.4 to 1141 µg kg-1 for 3-S-glutathionyl hexan-1-ol (GSH-3MH), and from 0.3 to 136 µg kg-1 for 3-S-cysteinyl 3-hexan-1-ol. The concentration of GSH-3MH exceeded 600 µg kg-1 in eight varieties, with values comparable to those of the richest Vitis vinifera reported so far. On average, red grapes showed higher concentrations of 3MH precursors than white ones did. Only two hybrids had 4-mercapto-4-methylpentan-2-one (4MMP) precursors over the limit of quantification, albeit with a much higher concentration than those normally reported in Sauvignon Blanc. CONCLUSION: This is the first detailed survey of 3MH and 4MMP precursors carried out taking into account a considerable number of hybrid grape varieties. The results show that some of these varieties could be interesting for the production of tropical juices or tropical-aromatic wines and soft drinks, through the enzymatic liberation of thiol aromas, as well as for the production of aromatizing tannins to be used in the food industry. © 2020 Society of Chemical Industry.

Evidence of the Possible Interaction between Ultrasound and Thiol Precursors.

The effect of ultrasound (20 kHz, 153 µm) on the prefermentation extraction mechanisms in Sauvignon Blanc grapes was studied, focusing on 3-mercaptohexan-1-ol (3MH) and 4-mercapto-4-methyl-pentan-2-one (4MMP) precursors linked to glutathione (GSH) and cysteine (Cys). The treatment determined a positive extraction trend between the duration (untreated, 3 and 5 min) and the conductivity or the concentration of catechins and total phenols, significantly differentiated after 5 min. Nevertheless, the concentration of the thiol precursors in grape juice not only remained undifferentiated, but that of 3-S-glutathionyl mercaptohexan-1-ol showed a negative trend with the treatment time applied (168 ± 43, 156 ± 36, and 149 ± 32 µg/L, respectively, for control, 3 and 5 min). The divergence on the effect between families of compounds suggests an interaction between the sonication treatment and thiol precursor molecules. In order to evaluate the possible degradation properly, ultrasound was applied in a model solution spiked with 3MH and 4MMP precursors, reproducing the conditions of grapes. Except for Cys-3MH, the mean concentration (n = 5) for the rest of the precursors was significantly lower in treated samples, predominantly in those linked to glutathione (~-22% and ~18% for GSH-3MH and GSH-4MMP) rather than to cysteine (~-6%~-8% for Cys-3MH and Cys-4MMP). The degradation of precursors was associated with a significant increase of 3MH and 4MMP. The formation of volatile thiols following sonication is interesting from a technological point of view, as they are key aroma compounds of wine and potentially exploitable in the wine industry through specific vinification protocols.

First evidence of ethyl-hydrogen phosphonate (fosetyl) formation in winemaking.

The formation of ethylphosphonate (fosetyl) was investigated in wine matrices. In experimental conditions, alcoholic fermentation of grape juices spiked with phosphonic acid resulted in wines with detectable amounts of fosetyl. The ageing period adopted for these wines further increased the fosetyl content in the absence of yeast lees, thus indicating a chemical origin. A model wine assay was performed in order to study the main thermodynamic variables affecting the Fisher reaction. The results obtained allowed us to report evidence of ethylphosphonate formation in winemaking conditions, for the first time to the best of our knowledge.

The effects of Cissampelos pareira extract on envenomation induced by Bothropsdiporus snake venom.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophidian accidents are a serious public health problem in Argentina; the Bothrops species is responsible for 97% of these accidents, and in particular, B. diporus is responsible for 80% of them. In the northeast of the country (Corrientes Provinces), Cissampelos pareira L. (Menispermaceae) is commonly used against the venom of B. diporus; its use is described in almost all ethnobotanical literature from countries where the plant grows. AIM OF THE STUDY: In this study, the in vitro and in vivo antivenom activities of C. pareira extracts were evaluated against B. diporus venom, with a particular focus on the local effects associated with envenoming. The seasonal influence on the chemical composition of the active extracts was also studied, in order determine the associated range of variability and its influence on the antivenom activity. MATERIALS AND METHODS: This research was conducted using aerial parts (leaves, flowers, tender stems) and roots of Cissampelos pareira collected from two different phytogeographic regions of Corrientes (Argentina); Paso de la Patria and Lomas de Vallejos. In addition, to perform a seasonal analysis and to evaluate the metabolic stability, material was collected at three different growth stages. In vivo and in vitro anti-snake venom activities were tested, and a bio-guided chromatographic separation was performed in order to determine the active chemicals involved. The fractions obtained were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the chemical profile of the most active constituent was analyzed by ultra high-performance liquid chromatography coupled to quadrupole/high-resolution mass spectrometry (Q-Orbitrap). (UHPLC-MS). RESULTS: The alcoholic extract was found to be the most active The bio-guided fractionation allowed selection one fraction to be analyzed by UHPLC-MS in order to identify the components responsible for the activities found; this identified five possible flavonoids. CONCLUSIONS: Our studies of the activity of C. pareira against the venom of B. diporus have confirmed that this species possesses inhibitory effects in both in vitro and in vivo models. Moreover, the present data demonstrate that certain flavonoids may mitigate some of the venom-induced local tissue damage.

Targeted and untargeted high resolution mass approach for a putative profiling of glycosylated simple phenols in hybrid grapes.

Vitis vinifera is one of the most widespread grapevines around the world representing the raw material for high quality wine production. The availability of more resistant interspecific hybrid vine varieties, developed from crosses between Vitis vinifera and other Vitis species, has generated much interest, also due to the low environmental effect of production. However, hybrid grape wine composition and varietal differences between interspecific hybrids have not been well defined, particularly for the simple phenols profile. The dynamic of these phenols in wines, where the glycosylated forms can be transformed into the free ones during winemaking, also raises an increasing health interest by their role as antoxidants in wine consumers. In this work an on-line SPE clean-up device, to reduce matrix interference, was combined with ultra-high liquid chromatography-high resolution mass spectrometry in order to increase understanding of the phenolic composition of hybrid grape varieties. Specifically, the phenolic composition of 4 hybrid grape varieties (red, Cabernet Cantor and Prior; white, Muscaris and Solaris) and 2 European grape varieties (red, Merlot; white, Chardonnay) was investigated, focusing on free and glycosidically bound simple phenols and considering compound distribution in pulp, skin, seeds and wine. Using a targeted approach 53 free simple phenols and 7 glycosidic precursors were quantified with quantification limits ranging from 0.001 to 2mgKg-1 and calibration R2 of 0.99 for over 86% of compounds. The untargeted approach made it possible to tentatively identify 79 glycosylated precursors of selected free simple phenols in the form of -hexoside (N=30), -pentoside (21), -hexoside-hexoside (17), -hexoside-pentoside (4), -pentoside-hexoside (5) and -pentoside-pentoside (2) derivatives on the basis of accurate mass, isotopic pattern and MS/MS fragmentation.

δ(15) N from soil to wine in bulk samples and proline.

The feasibility of using δ(15) N as an additional isotopic marker able to link wine to its area of origin was investigated. The whole production chain (soil-leaves-grape-wine) was considered. Moreover, the research included evaluation of the effect of the fermentation process, the use of different types of yeast and white and red vinification, the addition of nitrogen adjuvants and ultrasound lysis simulating wine ageing. The δ(15) N of grapes and wine was measured in bulk samples and compounds, specifically in proline, for the first time. Despite isotopic fractionation from soil to wine, the δ(15) N values of leaves, grapes, wine and particularly must and wine proline conserved the variability of δ(15) N in the growing soil. Fermentation and ultrasound treatment did not affect the δ(15) N values of grape must, which was therefore conserved in wine. The addition of inorganic or organic adjuvants was able to influence the δ(15) N of bulk wine, depending on the amount and the difference between the δ(15) N of must and that of the adjuvant. The δ(15) N of wine proline was not influenced by adjuvant addition and is therefore the best marker for tracing the geographical origin of wine. Copyright © 2016 John Wiley & Sons, Ltd.

Verifying the botanical authenticity of commercial tannins through sugars and simple phenols profiles.

Commercial tannins from several botanical sources and with different chemical and technological characteristics are used in the food and winemaking industries. Different ways to check their botanical authenticity have been studied in the last few years, through investigation of different analytical parameters. This work proposes a new, effective approach based on the quantification of 6 carbohydrates, 7 polyalcohols, and 55 phenols. 87 tannins from 12 different botanical sources were analysed following a very simple sample preparation procedure. Using Forward Stepwise Discriminant Analysis, 3 statistical models were created based on sugars content, phenols concentration and combination of the two classes of compounds for the 8 most abundant categories (i.e. oak, grape seed, grape skin, gall, chestnut, quebracho, tea and acacia). The last approach provided good results in attributing tannins to the correct botanical origin. Validation, repeated 3 times on subsets of 10% of samples, confirmed the reliability of this model.

Novel technological strategies to enhance tropical thiol precursors in winemaking by-products.

Grape pomace is a winemaking by-product that can be used to extract oenological tannins. Recently, some grape skin tannins were shown to contain very high amounts of two polyfunctional thiol precursors (3-S-glutathionylhexan-1-ol, 3-S-cysteinylhexan-1-ol) whose free forms are responsible for appreciated tropical-like flavours. This study shows that an oxidative treatment (no SO2) of white grape pomace and the presence of grape leaves and stems can increase the content of the above mentioned precursors. Moreover, it shows significant differences between Sauvignon Blanc, Gewuerztraminer and Mueller-Thurgau grape pomace for the 3-mercaptohexan-1-ol precursors and 4-S-cysteinyl-4-methylpentan-2-one. The grape cultivar is crucial, but the technological ability of enhancing the level of the volatile thiol precursors simply by treating the grape marc in different ways is a promising and powerful tool for the production of potentially flavouring tannins intended for food and beverage industry.

Pre-fermentation addition of grape tannin increases the varietal thiols content in wine.

The recent finding that grape tannin may contain significant amount of S-glutathionylated (GSH-3MH) and S-cysteinylated (Cys-3MH) precursors of the varietal thiols 3-mercapto-1-hexanol and 3-mercaptohexyl acetate, characteristic of Sauvignon blanc wines, offers new opportunities for enhancing the tropical aroma in fermented beverages. In this study this new hypothesis was investigated: Müller Thurgau (17 samples) and Sauvignon blanc (15 samples) grapes were fermented with and without addition of a selected grape tannin. As expected, the tannin-added juices were higher in precursors, and they produced wines with increased free thiols. Preliminary informal sensory tests confirmed that in particular the Sauvignon wines produced with the tannin addition were often richer with increased "fruity/green" notes than the corresponding reference wines. This outcome confirms that grape tannin addition prior to fermentation can fortify the level of these compounds.

Silicification of wood adopted for barrel production using pure silicon alkoxides in gas phase to avoid microbial colonisation.

The paper presents a new approach, covering wood with silica-based material in order to protect it from spoilage due to microbial colonisation and avoiding the loss of the natural features of the wood. Wood specimens derived from wine barrels were treated with methyltriethoxysilane in gas phase, leading to the deposition of a silica nanofilm on the surface. (29)Si and (13)C solid state Nuclear Magnetic Resonance and Scanning Electron Microscope-Energy Dispersive X-ray analysis observations showed the formation of a silica polymeric film on the wood samples, directly bonding with the wood constituents. Inductively Coupled Plasma-Mass Spectroscopy quantification of Si showed a direct correlation between the treatment time and silica deposition on the surface of the wood. The silica-coated wood counteracted colonisation by the main wine spoilage microorganisms, without altering the migration from wood to wine of 21 simple phenols measured using a HPLC-Electrochemical Coulometric Detection.

Botanical traceability of commercial tannins using the mineral profile and stable isotopes.

Commercial tannins are natural polyphenolic compounds extracted from different plant tissues such as gall, the wood of different species and fruit. In the food industry they are mainly used as flavourings and food ingredients, whereas in winemaking they are classified as clarification agents for wine protein stabilisation, although colour stabilisation, metal removal, unpleasant thiol removal and rheological correction are also well-known and desired effects. Due to their particular technical properties and very different costs, the possibility of correct identification of the real botanical origin of tannins can be considered a primary target in oenology research and in fulfilling the technical and economic requirements of the wine industry. For some categories of tannins encouraging results have already been achieved by considering sugar or polyphenolic composition. For the first time this work verifies the possibility of determining the botanical origin of tannins on the basis of the mineral element profile and analysis of the (13) C/(12) C isotopic ratio. One hundred two commercial tannins originating from 10 different botanical sources (grapes, oak, gall, chestnut, fruit trees, quebracho, tea, acacia, officinal plants and tara) were analysed to determine 57 elements and the (13) C/(12) C isotopic ratio, using inductively coupled plasma mass spectrometry and isotope-ratio mass spectrometry, respectively. Forward stepwise discriminant analysis provided good discrimination between the 8 most abundant groups, with 100% correct re-classification. The model was then validated five times on subsets of 10% of the overall samples, randomly extracted, achieving satisfactory results. With a similar approach it was also possible to distinguish toasted and untoasted oak tannins as well as tannins from grape skin and grape seeds.

Use of Fourier transform infrared spectroscopy to create models forecasting the tartaric stability of wines.

Tartaric instability of wines still represents a serious problem in terms of the commercial value of bottled wines, particularly whites, leading consumers to be suspicious as regards the effective healthiness or wholesomeness of products. The study, carried out on 536 Italian wines, investigated the potential of using Fourier Transform Infrared Spectroscopy, distinguishing between white and red/rosé wines, to create models predicting the instability of wines, assessed in comparison to two of the most widespread methods of reference: the "mini-contact test" (10 min, 0 °C, KHT) and the "cooling test" (5 days, -4 °C). The models proposed, constructed using 80% of the samples and based on Partial Least Squares-Regression and Artificial Neural Networks, were shown to work well in terms of correct classification (from 89% to 97%) of the external validation subset (20%). As regards the more problematical question of technical management of wines before bottling, in the worst cases only 4-6% of unstable samples were erroneously classified as stable.

First evidence of the presence of S-cysteinylated and S-glutathionylated precursors in tannins.

Tannins are widely used in winemaking and food and beverage preparation for the many different contributions they can give to the overall characteristics of the product (e.g., colour stability, mouthfeel and aromatic composition). Varietal thiols and their precursors are one of the most interesting research areas in food science and a lot of effort has been put to further the current understanding on their formation and on the impact of different production strategies on their concentration in the final product. This paper reports the identification of two important thiol precursors (Cys-3MH and GSH-3MH) in commercial grape oenological tannins and, to the best of our knowledge, this information is reported here for the first time. This finding allows potential new perspectives in the winemaking and the food industry, offering the possibility of controlled additions of thiol precursors in pre-fermentative stages in order to tune the aroma profile of fermented products.

Arsenic present in the soil-vine-wine chain in vineyards situated in an old mining area in Trentino, Italy.

The present study follows arsenic (As) transfer through the chain of soil-vine-leaves-grapes-wine to assess the possible risk of arsenic intake related to consuming grapes and wines produced in 10 vineyards located in a mining area rich in this element. The results are compared with date from 18 uncontaminated areas. In the soil, the content of As extracted with acqua regia and that extracted with ammonium acetate, were analyzed. Leaves and berries were analyzed after washing with acidified aqueous solution and acid mineralization in a closed vessel, whereas wines were simply diluted before analysis. All analyses were performed using an inductively coupled plasma mass-spectrometer. The aqua regia extractable As concentration in soil ranged from 3.7 to 283 mg/kg, whereas available As varied from 18 to 639 µg/kg, and As total concentration ranged from 16.3 to 579 µg/kg dry weight in leaves and from <0.1 to 36.8 µg/kg dry weight in grapes. Arsenic levels in wines were always below 1.62 µg/L, with higher concentration in red wines than in white wines. Significant and positive correlations between the As concentrations in soils, leaves, and berries are highlighted, with the samples collected near the mining area having significantly higher values. Nevertheless, As levels in wines were always well below the limit (200 µg/L) suggested by the International Organization of Vine and Wine.

Study of Sangiovese Wines Pigment Profile by UHPLC-MS/MS.

The metabolic pigment composition of Sangiovese wines produced from grapes harvested at 20 different vineyards in Montalcino over three consecutive years (2008-2010) on a semi-industrial scale and of 55 commercial Brunello di Montalcino wines (2004-2007) was studied, using a targeted method capable of analyzing 90 pigments in an 11 min UHPLC-MS/MS chromatographic run. Interesting correlations were shown between various pigments formed during wine aging and those present in Sangiovese grapes. Vitisin B-like pigment and vitisin A-like pigment concentrations would seem to have a good correlation with ethyl-linked and direct-linked flavanol-anthocyanin concentrations, respectively. Moreover, the anthocyanic pattern recognition, genetically controlled by the plant variety, was shown to be inherited by the pigments formed during wine aging.

H, C, N and S stable isotopes and mineral profiles to objectively guarantee the authenticity of grated hard cheeses.

In compliance with the European law (EC No. 510/2006), geographical indications and designations of origin for agricultural products and foodstuffs must be protected against mislabelling. This is particularly important for PDO hard cheeses, as Parmigiano Reggiano, that can cost up to the double of the no-PDO competitors. This paper presents two statistical models, based on isotopic and elemental composition, able to trace the origin of cheese also in grated and shredded forms, for which it is not possible to check the logo fire-marked on the rind. One model is able to predict the origin of seven types of European hard cheeses (in a validation step, 236 samples out of 240 are correctly recognised) and the other specifically to discriminate the PDO Parmigiano Reggiano cheese from 9 European and 2 extra-European imitators (260 out of 264 correct classifications). Both models are based on Random Forests. The most significant variables for cheese traceability common in both models are δ(13)C, δ(2)H, δ(15)N, δ(34)S and Sr, Cu, Mo, Re, Na, U, Bi, Ni, Fe, Mn, Ga, Se, and Li. These variables are linked not only to geography, but also to cow diet and cheese making processes.

Accumulation and distribution pattern of macro- and microelements and trace elements in Vitis vinifera L. cv. Chardonnay berries.

This paper describes the accumulation pattern of 42 mineral elements in Vitis vinifera L. berries during development and ripening and their distribution in berry skin, seeds, and flesh around harvest time. Grape berries were sampled in two different vineyards with alkaline soil and analyzed using a ICP-MS. Although elemental amounts were significantly different in the grapes from the two vineyards, the accumulation pattern and percentage distribution in different parts of the berries were generally quite similar. Ba, Eu, Sr, Ca, Mg, Mn, and Zn accumulate prior to veraison. Al, Ce, Dy, Er, Ga, Gd, Ho, La, Nd, Pr, Sm, Sn, Zr, Th, Tm, U, Y, and Yb accumulate mainly prior to veraison but also during ripening. Ag, As, B, Cd, Cs, Cu, Fe, Ge, Hg, K, Li, Na, P, Rb, Sb, Se, and Tl accumulate progressively during growth and ripening. With regard to distribution, Ba, Ca, Eu, Fe, Mn, P, Sr, and Zn accumulate mainly in the seeds, Al, B, Ga, Sn, and the rare earths analyzed, except for Eu, accumulate mainly in the skin, and Ag, As, Cd, Cs, Cu, Ge, Hg, K, Li, Mg, Na, Rb, Sb, Se, Th, Tl, U, and Zr accumulate mainly in the flesh. A joint representation of the accumulation and distribution patterns for the elements in the berry is also given.

Traceability along the production chain of Italian tomato products on the basis of stable isotopes and mineral composition.

The paper shows the variability of stable isotope ratios and mineral composition in tomato and derivatives along the production chain (juice, passata and paste) in order to evaluate the possibility of tracing their geographical origin. The ratios (13)C/(12)C, (15)N/(14)N, (18)O/(16)O, D/H, (34)S/(32)S and the content of Li, Be, B, Na, Mg, Al, P, K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Y, Mo, Ag, Cd, Sn, Sb, Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Dy, Ho, Tm, Yb, Ir, Tl, Pb, U and of nitrates, chlorides, sulphates and phosphates were measured by Isotope Ratio Mass Spectrometry, Inductively Coupled Plasma Mass Spectrometry and Ion Chromatography, respectively. The tomato products were from three Italian regions - Piedmont, Emilia Romagna, and Apulia. By applying linear discriminant analysis on 17 of these parameters (Gd, La, Tl, Eu, Cs, Ni, Cr, Co, δ(34)S, δ(15)N, Cd, K, Mg, δ(13)C, Mo, Rb and U) excellent discrimination among products from the three regions was achieved. Irrespective of the processing technology, over 95% of the samples were correctly reclassified in cross-validation into the production site. The use of these parameters will allow the development of analytical control procedures that can be used to check the geographical provenance of Italian tomatoes and products derived from them.