1H-NMR Metabolomics as a Tool for Winemaking Monitoring

The chemical composition of wine is known to be influenced by multiple factors including some viticulture practices and winemaking processes. ¹H-NMR metabolomics has been successfully applied to the study of wine authenticity. In the present study, ¹H-NMR metabolomics in combination with multivariate analysis was applied to investigate the effects of grape maturity and enzyme and fining treatments on Cabernet Sauvignon wines. A total of forty wine metabolites were quantified. Three different stages of maturity were studied (under-maturity, maturity and over-maturity). Enzyme treatments were carried out using two pectolytic enzymes (E1 and E2). Finally, two proteinaceous fining treatments were compared (vegetable protein, fining F1; pea protein and PVPP, fining F2). The results show a clear difference between the three stages of maturity, with an impact on different classes of metabolites including amino acids, organic acids, sugars, phenolic compounds, alcohols and esters. A clear separation between enzymes E1 and E2 was observed. Both fining agents had a significant effect on metabolite concentrations. The results demonstrate that ¹H-NMR metabolomics provides a fast and robust approach to study the effect of winemaking processes on wine metabolites. These results support the interest to pursue the development of ¹H-NMR metabolomics to investigate the effects of winemaking on wine quality.     

Evaluation of the feasibility of the electronic tongue as a rapid analytical tool for wine age prediction and quantification of the organic acids and phenolic compounds. The case-study of Madeira wine

A set of fourteen Madeira wines comprising wines produced from four Vitis vinifera L. varieties (Bual, Malvasia, Verdelho and Tinta Negra Mole) that were 3, 6, 10 and 17 years old was analysed using HPLC and an electronic tongue (ET) multisensor system. Concentrations of 24 organic acids, phenolic and furanic compounds were determined by HPLC. The ET consisting of 26 potentiometric chemical sensors with plasticized PVC and chalcogenide glass membranes was used. Significance of the effects of age and variety on the ET response and wine composition with respect to the organic acids, phenolics and furanic derivatives were evaluated using ANOVA—Simultaneous Component Analysis (ASCA). Significance of the effects was estimated using a permutation test (1000 permutations). It was found that effects of age, grape variety and their interaction were significant for the HPLC data set and only the effect of age was significant for the ET data. Calibration models of the HPLC and ET data with respect to the wine age and of the ET data with respect to the concentration of the organic acids and phenolics were calculated using PLS1 regression. Models were validated using cross-validation. It was possible to predict wine age from HPLC and ET data with the accuracy in cross-validation of 2.6 and 1.8 years respectively. The ET was capable of detecting the following components (mean relative error in cross-validation is shown in the parentheses): tartaric (8%), citric (5%), formic (12%), protocatehuic (5%), vanillic (18%) and sinapic (14%) acids, catechin (6%), vanillin (12%) and trans-resveratrol (5%). The ET capability of predicting Madeira wine age with good accuracy (1.8 years) as well as quantify of some organic acids and phenolic compounds was demonstrated.     

Anthocyanin Monitoring in Four Red Grape Skin Extract Varieties Using RP‐HPLC‐ED

Abstract

Anthocyanin compounds, cyanin, peonidin, myrtillin, malvin, kuromanin, oenin, petunidin and pelargonidin, present in four varieties of red grape skin extracts were quantified by reversed‐phase high‐performance liquid chromatography with electrochemical detection (RP‐HPLC‐ED). Several extraction procedures were evaluated for anthocyanin extraction from red grape skins of varieties of the Dão wine demarcated region in Portugal. The developed method was successfully applied to the analysis of the anthocyanins in skins extracts of red grapes during the 2005 harvest campaign, and monitoring these compounds can give great insight toward choosing the best time for the grape harvest for achieving a better wine quality.     

Prediction of phenolic compounds in red wine fermentations by visible and near infrared spectroscopy

Near infrared (NIR) spectroscopy was used to simultaneously predict the concentrations of malvidin-3-glucoside (M3G), pigmented polymers (PP) and tannins (T) in red wine. A total of 495 samples from 32 commercial scale red wine fermentations over two vintages using two grape varieties (Cabernet Sauvignon and Shiraz), and also including as additional variables two types of fermenters, two different yeasts, and three fermentation temperatures were used. Samples were scanned in transmission mode (400-2500 nm) using a monochromator instrument (NIRSystems6500). Calibration equations were developed from high performance liquid chromatography (HPLC) and NIR data using partial least squares (PLS) regression with internal cross validation. Using PLS regression, very good calibration statistics (R_cal²>0.80) were obtained for the prediction of M3G, PP and T with standard deviation (S.D.)/standard error in cross validation (SECV) ratio (residual predictive deviation, RPD)) ranging from 1.8 to 5.8. It was concluded that near infrared spectroscopy could be used as rapid alternative method for the prediction of the concentration of phenolic compounds in red wine fermentations.     

Potential Associations among Bioactive Molecules,Antioxidant Activity and Resveratrol Production in Vitis vinifera Fruits of North America

Grapes (Vitis vinifera L.) are rich in bioactive molecules contributing to health benefits. Consumption of grapes is linked to reduced incidence of cardiovascular diseases. Studies on table grape cultivars are limited although much attention in research was focused on the wine industry. Bioactive effects of grapes as anti-inflammatory, anticarcinogenic, cardioprotective, vasorelaxant, phytoestrogenic and neuroprotective have also been reported. For example, resveratrol is a natural food ingredient present in grapes, with high antioxidant potential. Here we conducted an exploratory study to investigate bioactive molecules, antioxidant activity and the association between constitutive stilbene synthase (STS) gene expression and the resveratrol biosynthesis in selected table grape varieties in North America. The phenolic compounds, fatty acid composition and antioxidant activity of four grape varieties were compared. Red Globe variety was rich in unsaturated fatty acids as well as phenolic compounds such as caffeic acid, quercetin and resveratrol. Meanwhile, the constitutive expression of grape stilbene synthase gene was higher in Flame and Autumn Royal where resveratrol content of these cultivars was relatively low compared to the Red Globe variety. This study shows the potential links in grape antioxidant activity and resveratrol production, but more studies are necessary to show the association.     

Grapevine as a Rich Source of Polyphenolic Compounds

Grapes are rich in primary and secondary metabolites. Among the secondary metabolites, polyphenolic compounds are the most abundant in grape berries. Besides their important impacts on grape and wine quality, this class of compounds has beneficial effects on human health. Due to their antioxidant activity, polyphenols and phenolic acids can act as anti-inflammatory and anticancerogenic agents, and can modulate the immune system. In grape berries, polyphenols and phenolic acids can be located in the pericarp and seeds, but distribution differs considerably among these tissues. Although some classes of polyphenols and phenolic acids are under strict genetic control, the final content is highly influenced by environmental factors, such as climate, soil, vineyard, and management. This review aims to present the main classes of polyphenolic compounds and phenolic acids in different berry tissues and grape varieties and special emphasis on their beneficial effect on human health.     

Untargeted metabolomic analysis using liquid chromatography quadrupole time-of-flight mass spectrometry for non-volatile profiling of wines

The current study presents a method for comprehensive untargeted metabolomic fingerprinting of the non-volatile profile of the Graciano Vitis vinifera wine variety, using liquid chromatography/electrospray ionization time of flight mass spectrometry (LC–ESI-QTOF). Pre-treatment of samples, chromatographic columns, mobile phases, elution gradients and ionization sources, were evaluated for the extraction of the maximum number of metabolites in red wine. Putative compounds were extracted from the raw data using the extraction algorithm, molecular feature extractor (MFE). For the metabolite identification the WinMet database was designed based on electronic databases and literature research and includes only the putative metabolites reported to be present in oenological matrices. The results from WinMet were compared with those in the METLIN database to evaluate how much the databases overlap for performing identifications. The reproducibility of the analysis was assessed using manual processing following replicate injections of Vitis vinifera cv. Graciano wine spiked with external standards. In the present work, 411 different metabolites in Graciano Vitis vinifera red wine were identified, including primary wine metabolites such as sugars (4%), amino acids (23%), biogenic amines (4%), fatty acids (2%), and organic acids (32%) and secondary metabolites such as phenols (27%) and esters (8%). Significant differences between varieties Tempranillo and Graciano were related to the presence of fifteen specific compounds.     

The Impact of Grape Varieties to Wine Isotopic Characterization

This paper highlights the results of a study regarding the impact of geo-climatic conditions, the influence of grape varieties, and harvest year on the isotopic characterization of a wine from a specific vineyard area, using stable isotope ratio analysis by continuous flow mass spectrometry CF-IRMS. This analytical technique has made a significant contribution on wine characterization, by proving the botanical and geographical origin of the wine raw material.

A great variability was observed to sap ¹⁸O and ²H isotopic ratios for different types of vine (Vitis vinifera plant) from the studied area, Dealu Mare-Valea Calugareasca, reflecting the significant influence of variety on isotopic features. On the other hand, the comparison of stable isotopic fingerprints for rainfall water and sap revealed a close approximation value for ¹⁸O isotopic ratio, and an insignificant variation coefficient of ¹³C isotopic ratio in wine varieties (about 2.85%).

The results of isotopic characterization for grape vine sap and original wines for different varieties existing in Dealu Mare-Valea Calugareasca vineyard indicates the relevance of meteorological and viticulture parameters like precipitation, date of vintage, and grape cultivar for wine authentication process.     

Varietal flavour compounds of four grape varieties producing Madeira wines

Boal, Malvasia, Sercial and Verdelho are the main white grape varieties used in Madeira wine production. To estimate the free fraction of varietal aroma compounds of these varieties, 39 samples of musts were analysed to determine their content of monoterpenols and C₁₃ norisoprenoids (terpenoids), using dynamic headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The r-values for linearity studies of the analytical method used, varied between 0.977 (nerolidol) and 0.999 (linalool). The repeatability for each compound varied between 2.5% (citronellol) and 11.8% (β-ionone).The mean values from three vintages (1998, 1999 and 2000) confirmed that these musts have differentiated contents of terpenoids. In opposition to Verdelho musts, Malvasia showed the highest free terpenoids content. In order to establish relations between the compounds and the varieties under investigation, principal component analysis and linear discriminant analysis were applied to the data, revealing a good separation and classification power between the four groups as a function of varietal origin.     

Analysis of terpenes in white wines using SPE-SPME-GC/MS approach

Terpenes contribute to some white wines aroma, especially these produced from Muscat grapes and others aromatic ones of high terpene contents (Gewürtztramminer, Traminer, Huxel, Sylvaner). Terpenes are present in wine in free and bound (in a form of glycosides) forms. Analyses of bound terpenes are usually performed using solid phase extraction after hydrolysis of glycosides. A new method for determination of terpenes from wine, focused on determination of terpenes released after acidic hydrolysis, based on solid phase extraction (SPE) followed by solid phase microextraction (SPME) was developed. Non-polar (free) and polar (bound terpenes) fractions were separated on 500 mg C18 cartridges. Bound terpenes were sampled using SPME immediately after acidic hydrolysis in non-equilibrium conditions. Application of combined SPE-SPME approach allowed quantification of selected terpenes in lower concentrations than in SPE approach and added a selectivity to the method, which enabled detection of compounds non-detectable in SPE extracts. Results obtained by SPE and SPE-SPME approach were correlated for free terpenes and those released after acid hydrolysis 20 white wines obtained from different grape varieties (R² = 0.923). Although developed for wine terpenes analysis, SPE followed by SPME approach has a great potential in analysis of other bound wine flavor compounds, especially those potent odorants present in trace amounts.     

Near infrared spectroscopy as a rapid tool to measure volatile aroma compounds in Riesling wine: possibilities and limits

Volatile chemical compounds responsible for the aroma of wine are derived from a number of different biochemical and chemical pathways. These chemical compounds are formed during grape berry metabolism, crushing of the berries, fermentation processes (i.e. yeast and malolactic bacteria) and also from the ageing and storage of wine. Not surprisingly, there are a large number of chemical classes of compounds found in wine which are present at varying concentrations (ng L⁻¹ to mg L⁻¹), exhibit differing potencies, and have a broad range of volatilities and boiling points. The aim of this work was to investigate the potential use of near infrared (NIR) spectroscopy combined with chemometrics as a rapid and low-cost technique to measure volatile compounds in Riesling wines. Samples of commercial Riesling wine were analyzed using an NIR instrument and volatile compounds by gas chromatography (GC) coupled with selected ion monitoring mass spectrometry. Correlation between the NIR and GC data were developed using partial least-squares (PLS) regression with full cross validation (leave one out). Coefficients of determination in cross validation (R ²) and the standard error in cross validation (SECV) were 0.74 (SECV: 313.6 μg L⁻¹) for esters, 0.90 (SECV: 20.9 μg L⁻¹) for monoterpenes and 0.80 (SECV: 1658 μg L⁻¹) for short-chain fatty acids. This study has shown that volatile chemical compounds present in wine can be measured by NIR spectroscopy. Further development with larger data sets will be required to test the predictive ability of the NIR calibration models developed.     

Glucose and fructose levels on grape skin: interference in Lobesia botrana behaviour

The vineyard produces one of the most important crops in Portugal and is affected by various pests, such as the Lobesia botrana moth, which can be attracted chemically by compounds on the grape skin.In this study, the sugar contents (glucose and fructose) on grape skin were analysed for five Portuguese Vitis vinifera grape varieties and compared in grape juice and whole grapes. Samples were harvested in July, August and September of 2001 and 2002.The quantification was attained employing a new methodology, which comprised the preparation of a methanol solution to extract the compounds from the grape skin and further chromatographic analyses by liquid chromatography (LC) with refractive index (RI) detection. The samples designated by whole grapes were homogenised, centrifuged and filtrated, while the juice was obtained by crushing and filtration.Grape skin has shown higher levels of glucose than fructose, but both compounds increased from green berry (July) to veraison (August) and had a slight decrease from veraison to harvest (September).As expected, the glucose and fructose contents (total sugar) were higher in the whole grape than in the juice and much higher than in the grape skin itself. For Loureiro and Trajadura varieties, the levels of both sugars on grape skin were very similar. This resemblance strengthened the statement of the decisive role of this parameter in female L. botrana oviposition behaviour.     

Multivariate analysis of the polyphenol composition of Tempranillo and Graciano red wines

Vitis vinifera L. cv Graciano is often used as a blending partner of Tempranillo based wines because it is considered to contribute significantly to the quality. The aim of this study is to discriminate between Tempranillo and Graciano monovarietal wines, and those made by the incorporation of 20% of Graciano variety in two different stages (at the end of malolactic fermentation and mixing the two grape varieties in the pre-fermentative maceration stage) of the winemaking process of the Tempranillo variety. To achieve this, supervised and unsupervised pattern recognition tools were applied to the data obtained in the study of the detailed polyphenolic composition, colour and other oenological parameters (143 variables). Patterns related to stages in the winemaking and ageing process, different wines and vintages can be observed using principal component analyses. Furthermore, linear discriminant analysis has been applied in order to characterise the wine samples. From the 143 variables, flavan-3-ols have exerted a profound influence on wine differentiation.     

Optimisation of solid-phase microextraction combined with gas chromatography-mass spectrometry based methodology to establish the global volatile signature in pulp and skin of Vitis vinifera L. grape varieties

The volatiles (VOCs) and semi-volatile organic compounds (SVOCs) responsible for aroma are mainly present in skin of grape varieties. Thus, the present investigation is directed towards the optimisation of a solvent free methodology based on headspace-solid-phase microextraction (HS-SPME) combined with gas chromatography-quadrupole mass spectrometry (GC-qMS) in order to establish the global volatile composition in pulp and skin of Bual and Bastardo Vitis vinifera L. varieties. A deep study on the extraction-influencing parameters was performed, and the best results, expressed as GC peak area, number of identified compounds and reproducibility, were obtained using 4 g of sample homogenised in 5 mL of ultra-pure Milli-Q water in a 20 mL glass vial with addition of 2 g of sodium chloride (NaCl). A divinylbenzene/carboxen/polydimethylsiloxane fibre was selected for extraction at 60 °C for 45 min under continuous stirring at 800 rpm. More than 100 VOCs and SVOCs, including 27 monoterpenoids, 27 sesquiterpenoids, 21 carbonyl compounds, 17 alcohols (from which 2 aromatics), 10 C₁₃ norisoprenoids and 5 acids were identified. The results showed that, for both grape varieties, the levels and number of volatiles in skin were considerably higher than those observed in pulp. According to the data obtained by principal component analysis (PCA), the establishment of the global volatile signature of grape and the relationship between different part of grapes—pulp and skin, may be an useful tool to winemaker decision to define the vinification procedures that improves the organoleptic characteristics of the corresponding wines and consequently contributed to an economic valorization and consumer acceptance.     

Comparison of micro-scale simultaneous distillation-extraction and stir bar sorptive extraction for the determination of volatile organic constituents of grape juice

Traditional micro-scale simultaneous distillation-extraction (SDE) and stir bar sorptive extraction (SBSE) were compared for their effectiveness in the extraction of volatile organic compounds in a synthetic grape juice and a real grape juice (Huxelrebe, a variety of half Muscat ancestry) from an English vineyard. The novel immersion-mode SBSE method, using stir bars with PDMS sorbent, was optimised using the synthetic grape juice. Although mean percent relative recoveries and reproducibilities (%CV) of the SBSE method were inferior to SDE (28.4 and 8.5%, respectively, against 86.9 and 6.3%), the former method proved to be significantly more sensitive: 126 aroma compounds in Huxelrebe grape juice were identified using SBSE, against 98 using SDE. This allowed the identification of a number of volatile components that have not been reported previously in the juice or wine from the grapes of Muscat varieties.     

Chemometric Optimization of Biologically Active Compounds Extraction from Grape Marc: Composition and Antimicrobial Activity

The article focuses on the optimization of the extraction process of biologically active compounds (BAC) from grape marc—a by-product of the wine industry. The influence of temperature, specifically 30 °C, 45 °C and 65 °C, and ethanol concentration in solutions, specifically 0–96% (v/v) on the extraction yield of polyphenols, flavonoids, tannins and anthocyanins, were investigated. The composition of individual polyphenols, anthocyanins and organic acids, antioxidant activity (DPPH and ABTS) and CIELab chromatic characteristics of the grape marc extracts (GME), were characterized. The microbiostatic and microbicidal effects in direct contact of GME with pathogenic microorganisms, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, were determined in vitro. The influence of extraction parameters on the total polyphenol content (TPC), total flavonoid content (TFC), tannin content (TC), total anthocyanin content (TAC) and their interdependencies were studied using information analysis. A mathematical model was developed on cubic spline functions. The analysis of individual compounds showed the presence of a wide range of flavonoids (procyanidin B2, procyanidin B1, hyperoside and quercetin), flavones (catechin), hydroxybenzoic acid derivatives (gallic, protocatechuic, p-hydroxybenzoic acids, m-hydroxybenzoic acid, syringic acid), hydroxycinic acid derivatives and ferulic acid methyl ester. The malvidol-3-glucoside was the main anthocyanin identified in the extract. A high amount of tartaric acid was also found. GME showed significant antimicrobial activity against Gram-positive bacteria and lower activity against Gram-negative bacteria.     

Characterization of the aromatic composition of some liquid foods by nuclear magnetic resonance spectrometry and liquid chromatography with nuclear magnetic resonance and mass spectrometric detection

This paper describes the first application of NMR spectroscopy and LC-NMR/MS to the direct analysis of the aromatic composition of beer, grape juice and a wine phenolic extract. NMR spectroscopy provides non-invasive information on the overall aromatic profile and enables the identification of some compounds. However, a more comprehensive assignment is hindered by the low peak intensity and strong signal overlap in the low-field spectral region, as well as by the inherent lack of scalar coupling information for many aromatic compounds present. LC-NMR/MS can overcome these problems and is shown to aid significantly in the identification of aromatic compounds composing all samples analyzed. Some examples are the identification of several cinnamic acids (e.g. p-coumaric, trans-coutaric and trans-caftaric) in grape juice, the identification of 2-phenylethanol, tyrosol and tryptophol in beer and the detection of phenolics such as catechin, epicatechin, trans-resveratrol, tyrosol and caffeic acid in the wine extract.     

Effect of Extraction Solvent and Temperature on Polyphenol Profiles,Antioxidant and Anti-Inflammatory Effects of Red Grape Skin By-Product

A fully-detailed LC-MS qualitative profiling of red grape skin, extracted with a mixture of ethanol and water (70:30 v:v) has permitted the identification of 65 compounds which can be classified into the following chemical classes: organic and phenolic acids (14 compounds), stilbenoids (1 compound), flavanols (21 compounds), flavonols (15 compounds) and anthocyanins (14 compounds). The extraction yield obtained with water at different temperatures (100 °C, 70 °C, room temperature) was then evaluated and the overall polyphenol content indicates that EtOH:H₂O solvent is the most efficient and selective for polyphenol extraction. However, by analyzing the recovery yield of each single polyphenol, we found that water extraction under heating conditions is effective (extraction yield similar or even better in respect to the binary solvent) for some polyphenolic classes, such as hydrophilic procyanidins, phenolic acids, flavonol glucosides and stilbenoids. However, according to their lipophilic character, a poor yield was found for the most lipophilic components, such as flavonol aglycones, and in general for anthocyanins. The radical scavenging activity was in accordance with the polyphenol content, and hence, much higher for the extract obtained with the binary solvent in respect to water extraction. All the tested extracts were found to have an anti-inflammatory activity in the R3/1 cell line with NF-kb reporter challenged with 0.01 µg/mL of IL-1α, in a 1 to 250 µg/mL concentration range. An intriguing result was that the EtOH:H₂O extract was found to be superimposable with that obtained using water at 100 °C despite the lower polyphenol content. Taken together, the results show the bioactive potentialities of grape skin extracts and the possibility to exploit this rich industrial waste. Water extraction carried out by heating is an easy, low-cost and environmentally friendly extraction method for some polyphenol classes and may have great potential for extracts with anti-inflammatory activities.     

Phenolic concentrations and antioxidant properties of wines made from north american grapes grown in china

The characteristics of wine phenolics found in several North American and (for comparison) European grape cultivars grown in China were analyzed. This was done to find non-Vitis vinifera wines with prominent features in order to diversify the kinds of wines. The phenolic richness and antioxidant activity decreased in the order: red > rose > white wines. In the red wines, the American grape 'Cynthiana' had the highest total concentrations of phenols, anthocyanins, flavonols and phenolic acids, as well as antioxidant capacity, followed by the French hybrid 'Chambourcin', the lowest were detected in two European grape varieties, 'Merlot' and 'Cabernet Sauvignon', while the total flavon-3-ols levels were reversed among these red grape cultivars. The highest concentration of stilbenes out of all the wines analyzed was found in the 'Merlot' variety. There were significant differences among wine phenolic compositions between North American and European grape cultivars. The antioxidant activities were significantly related to the concentrations of total phenols (r2 = 0.996), anthocyanins (r2 = 0.984), flavonols (r2 = 0.850) and gallic acid (r2 = 0.797). The prominent features of wine aroma and nutrition could make the American grape wines attractive to consumers. It is therefore necessary to perform further research on cultural practices and wine making involving these grapes.     

Simultaneous determination of polyphenolic compounds in red and white grapes grown in Sardinia by high performance liquid chromatography–electron spray ionisation-mass spectrometry

A new method for the identification and the quantification of nonanthocyanin phenolic compounds from six Vitis Vinifera grape varieties native to Sardinia (three native: Vermentino, Malvasia and Cannonau and three non-native types: Chardonnay, Sauvignon and Cabernet Sauvignon; Argiolas vineyard) was developed. This rapid and selective method employs LC/ESI-MS in negative mode. Different solvents extraction and different sorbents for purification were compared to the direct analysis of the initial extracts without further sample preparation. A total of 54 phenolic compounds were identified either in the freeze-dried skins or seeds, including nonflavonoids (hydroxybenzoic and hydroxycinnamic acids and their derivatives, stilbenes) and flavonoids (flavanols, flavonols, dihydroxyflavonols).