Antioxidant Capacity of Polar and Non-Polar Extracts of Four African Green Leafy Vegetables and Correlation with Polyphenol and Carotenoid Contents.

In sub-Saharan Africa, chronic malnutrition is often associated with intestinal inflammation and oxidative stress. African green leafy vegetables (GLVs), commonly consumed by these populations and rich in bioactive compounds, may improve the antioxidant status. The aim of this study was to measure the antioxidant capacity using complementary assays (DPPH, FRAP, ABTS, ORAC and NO scavenging) in polar and non-polar leaf extracts of four African GLVs, cassava (Manihot esculenta), roselle (Hibiscus sabdariffa), jute mallow (Corchorus olitorius), and amaranth (Amaranthus spp.), with spinach (Spinacia oleracea) chosen as a reference. Their antioxidant capacity was correlated with their total polyphenol (TPC), flavonoid (TFC), condensed tannin, lutein, and ß-carotene contents. Identification of phenolic compounds by UHPLC-DAD-MS/MS revealed the presence of three main classes of compound: flavonols, flavones, and hydroxycinnamic acids. Cassava and roselle leaves presented significantly higher TPC and TFC than amaranth, jute mallow, and spinach. They also exhibited the highest antioxidant capacity, even higher than that of spinach, which is known for its important antioxidant effect. The antioxidant capacity was 2 to 18 times higher in polar than non-polar extracts, and was more strongly correlated with TPC and TFC (R > 0.8) than with ß-carotene and lutein contents. These findings provide new data especially for cassava and roselle leaves, for which studies are scarce, suggesting an appreciable antioxidant capacity compared with other leafy vegetables.

Untargeted Metabolomics Approach Using UHPLC-HRMS to Unravel the Impact of Fermentation on Color and Phenolic Composition of Rosé Wines.

Color is a major quality trait of rosé wines due to their packaging in clear glass bottles. This color is due to the presence of phenolic pigments extracted from grapes to wines and products of reactions taking place during the wine-making process. This study focuses on changes occurring during the alcoholic fermentation of Syrah, Grenache and Cinsault musts, which were conducted at laboratory (250 mL) and pilot (100 L) scales. The color and phenolic composition of the musts and wines were analyzed using UV-visible spectrophotometry, and metabolomics fingerprints were acquired by ultra-high performance liquid chromatography-high-resolution mass spectrometry. Untargeted metabolomics data highlighted markers of fermentation stage (must or wine) and markers related to the grape variety (e.g., anthocyanins in Syrah, hydroxycinnamates and tryptophan derivatives in Grenache, norisoprenoids released during fermentation in Cinsault). Cinsault wines contained higher molecular weight compounds possibly resulting from the oxidation of phenolics, which may contribute to their high absorbance values.

Shades of Fine Dark Chocolate Colors: Polyphenol Metabolomics and Molecular Networking to Enlighten the Brown from the Black.

High-quality dark chocolates (70% cocoa content) can have shades from light to dark brown color. This work aimed at revealing compounds that discriminate black and brown chocolates. From 37 fine chocolate samples from years 2019 and 2020 provided by Valrhona,8 dark black samples and 8 light brown samples were selected. A non-targeted metabolomics study was performed based on ultra-high performance liquid chromatography-high resolution mass spectrometry/mass spectrometry experiments, univariate, multivariate, and feature-based molecular networking analyses. Twenty-seven overaccumulated discriminating compounds were found for black chocolates. Among them, glycosylated flavanols including monomers and glycosylated A-type procyanidin dimers and trimers were highly representative. Fifty overaccumulated discriminating compounds were found for brown chocolates. Most of them were B-type procyanidins (from trimers to nonamers). These phenolic compounds may be partially related to the chocolate colors as precursors of colored compounds. This study increases the knowledge on the chemical diversity of dark chocolates by providing new information about the phenolic profiles of black and brown chocolates.

Multi-method study of the impact of fermentation on the polyphenol composition and color of Grenache, Cinsault, and Syrah rosé wines.

Rosé wines show large color diversity, due to different phenolic pigment compositions. However, the mechanisms responsible for such diversity are poorly understood. The present work aimed at investigating the impact of fermentation on the color and composition of rosé wines made from Grenache, Cinsault, and Syrah grapes. Targeted MS analysis showed large varietal differences in must and wine compositions, with higher concentrations of anthocyanins and flavanols in Syrah. UV-visible spectrophotometry and size exclusion chromatography data indicated that Grenache and Cinsault musts contained oligomeric pigments derived from hydroxycinnamic acids and flavanols which were mostly lost during fermentation due to adsorption on lees. Syrah must color was mainly due to anthocyanins which were partly converted to derived pigments through reactions with yeast metabolites with limited color drop during fermentation. This work highlighted the impact of must composition, reflecting varietal characteristics, on changes occurring during fermentation and consequently wine color.

Focus on the relationships between the cell wall composition in the extraction of anthocyanins and tannins from grape berries.

Concentrations of anthocyanins and tannins after extraction from berries in wines and from skin macerations in model solutions have been studied for two grape varieties, two maturation levels and two vintages berries. Characterization of the cell wall polysaccharides has also been performed, the classical method based on the analysis of the neutral sugars after depolymerization being completed by a comprehensive microarray polymer profiling (CoMPP). Extraction was lower in model solutions than in wines, with the same ranking: non acylated anthocyanins> tannins > p-coumaroylated anthocyanins. The polysaccharidic composition suggested a role of homogalacturonans, rhamnogalacturonans and extensins in the extraction process. A global explanation of the interactions between anthocyanins, tannins and polysaccharides is proposed.

Improved Analysis of Isomeric Polyphenol Dimers Using the 4th Dimension of Trapped Ion Mobility Spectrometry-Mass Spectrometry.

Dehydrodicatechins resulting from (epi)catechin oxidation have been investigated in different foods and natural products, but they still offer some analytical challenges. The purpose of this research is to develop a method using ultra-high performance liquid chromatography coupled with trapped ion mobility spectrometry and tandem mass spectrometry (UHPLC-ESI-TIMS-QTOF-MS/MS) to improve the characterization of dehydrodicatechins from model solutions (oxidation dimers of (+)-catechin and/or (-)-epicatechin). Approximately 30 dehydrodicatechins were detected in the model solutions, including dehydrodicatechins B with ß and ε-interflavanic configurations and dehydrodicatechins A with γ-configuration. A total of 11 dehydrodicatechins B, based on (-)-epicatechin, (+)-catechin, or both, were tentatively identified in a grape seed extract. All of them were of ß-configuration, except for one compound that was of ε-configuration. TIMS allowed the mobility separation of chromatographically coeluted isomers including dehydrodicatechins and procyanidins with similar MS/MS fragmentation patterns that would hardly be distinguished by LC-MS/MS alone, which demonstrates the superiority of TIMS added to LC-MS/MS for these kinds of compounds. To the best of our knowledge, this is the first time that ion mobility spectrometry (IMS) was applied to the analysis of dehydrodicatechins. This method can be adapted for other natural products.

A genome-wide association and prediction study in grapevine deciphers the genetic architecture of multiple traits and identifies genes under many new QTLs.

To cope with the challenges facing agriculture, speeding-up breeding programs is a worthy endeavor, especially for perennial species such as grapevine, but requires understanding the genetic architecture of target traits. To go beyond the mapping of quantitative trait loci in bi-parental crosses, we exploited a diversity panel of 279 Vitis vinifera L. cultivars planted in 5 blocks in the vineyard. This panel was phenotyped over several years for 127 traits including yield components, organic acids, aroma precursors, polyphenols, and a water stress indicator. The panel was genotyped for 63k single nucleotide polymorphisms by combining an 18K microarray and genotyping-by-sequencing. The experimental design allowed to reliably assess the genotypic values for most traits. Marker densification via genotyping-by-sequencing markedly increased the proportion of genetic variance explained by single nucleotide polymorphisms, and 2 multi-single nucleotide polymorphism models identified quantitative trait loci not found by a single nucleotide polymorphism-by-single nucleotide polymorphism model. Overall, 489 reliable quantitative trait loci were detected for 41% more response variables than by a single nucleotide polymorphism-by-single nucleotide polymorphism model with microarray-only single nucleotide polymorphisms, many new ones compared with the results from bi-parental crosses. A prediction accuracy higher than 0.42 was obtained for 50% of the response variables. Our overall approach as well as quantitative trait locus and prediction results provide insights into the genetic architecture of target traits. New candidate genes and the application into breeding are discussed.

Elucidating the Color of Rosé Wines Using Polyphenol-Targeted Metabolomics.

The color of rosé wines is extremely diverse and a key element in their marketing. It is due to the presence of anthocyanins and of additional pigments derived from them and from other wine constituents. To explore the pigment composition and determine its links with color, 268 commercial rosé wines were analysed. The concentration of 125 polyphenolic compounds was determined by a targeted metabolomics approach using ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS) analysis in the Multiple Reaction Monitoring (MRM) mode and the color characterised by spectrophotometry and CieLab parameters. Chemometrics analysis of the composition and color data showed that although color intensity is primarily determined by polyphenol extraction (especially anthocyanins and flavanols) from the grapes, different color styles correspond to different pigment compositions. The salmon shade of light rosé wines is mostly due to pyranoanthocyanin pigments, resulting from reactions of anthocyanins with phenolic acids and pyruvic acid, a yeast metabolite. Redness of intermediate color wines is related to anthocyanins and carboxypoyranoanthocyanins and that of dark rosé wines to products of anthocyanin reactions with flavanols while yellowness of these wines is associated to oxidation.

Study of the relationship between red wine colloidal fraction and astringency by asymmetrical flow field-flow fractionation coupled with multi-detection.

Macromolecules including condensed tannins and polysaccharides impact wine taste and especially astringency. Asymmetrical Flow-Field-Flow-Fractionation (AF4) coupled to UV detection (UV), multi-angle light scattering (MALS) and refractive index detection (dRI) has been proposed to separate red wine colloids. The present work aimed at relating AF4-mutidetection profiles with red wine astringency. Fifty commercial red wines characterized by a trained sensory panel were analysed by AF4-UV-MALS-dRI and UV-visible spectroscopy. The analytical data set was built by selecting the three variables most predictive of the astringency score from each table (UV, dRI, MALS, Mw distribution, and UV-visible spectra of whole wine, permeate and retentate A4F fractions) and analysed by principal component analysis. Red wine astringency was more related to variables extracted from the AF4 data than to UV- absorbance of the wine or permeate, confirming the relevance of AF4-multidetection for analysis of the colloidal fraction involved in this perception.

Multi-block classification of chocolate and cocoa samples into sensory poles.

The present study aims at developing an analytical methodology which allows correlating sensory poles of chocolate to their chemical characteristics and, eventually, to those of the cocoa beans used for its preparation. Trained panelists investigated several samples of chocolate, and they divided them into four sensorial poles (characterized by 36 different descriptors) attributable to chocolate flavor. The same samples were analyzed by six different techniques: Proton Transfer Reaction-Time of Flight-Mass Spectrometry (PTR-ToF-MS), Solid Phase Micro Extraction-Gas Chromatography-Mass Spectroscopy (SPME-GC-MS), High-Performance Liquid Chromatography (HPLC) (for the quantification of eight organic acids), Ultra High Performance Liquid Chromatography coupled to triple-quadrupole Mass Spectrometry (UHPLC-QqQ-MS) for polyphenol quantification, 3D front face fluorescence Spectroscopy and Near Infrared Spectroscopy (NIRS). A multi-block classification approach (Sequential and Orthogonalized-Partial Least Squares - SO-PLS) has been used, in order to exploit the chemical information to predict the sensorial poles of samples. Among thirty-one test samples, only two were misclassified.

Fractionation and characterization of polyphenolic compounds and macromolecules in red wine by asymmetrical flow field-flow fractionation.

Red wine is a complex matrix containing macromolecules such as condensed tannins and polysaccharides. Wine macromolecular components and their interactions have been reported to impact taste properties such as astringency but the colloidal systems formed in wine are not well known. A key prerequisite to characterize these systems is the ability to work under analytical conditions as close as possible to the colloid environment, preserving the sample structure and limiting the denaturation of macromolecular complexes. A method of Asymmetric Flow Field-Flow Fractionation (AF4) coupled with UV detection, multi-angle light scattering (MALS), and differential refractometer index (dRI) (AF4-UV-MALS-dRI) has been developed to analyse macromolecules, including tannins and polysaccharides, and macromolecular complexes, in red wine. This method separates objects according to their hydrodynamic radius and does not require calibration to determine molecular weight (Mw). AF4 can provide native separation of wine colloidal matter while working with simulated wine as mobile phase. The channel was equipped with a 350-µm spacer and the membrane made in regenerated cellulose had a cut-off of 5kDa. Different parameters of crossflow rate were investigated using a generic red wine to optimize separation conditions. Then, purified fractions of polysaccharides and tannins were analysed using the selected AF4 parameters. The comparison of the peaks obtained for these fractions and for the wine sample allowed us to determine the retention time associated with these macromolecules. The AF4 fractogram of wine was divided into four fractions. The first three were assigned to higher Mw tannins coeluted with lower Mw polysaccharides such as rhamnogalacturonan II (F1), to intermediate Mw polysaccharides (F2), and to higher Mw mannoproteins (F3) whereas the last fraction (F4) was not identified. Furthermore, our results have shown that AF4-UV-MALS-dRI could be an efficient technique to separate large size tannins as well as polysaccharides and macromolecular complexes.

Multiblock Analysis to Relate Polyphenol Targeted Mass Spectrometry and Sensory Properties of Chocolates and Cocoa Beans.

Chocolate quality is largely due to the presence of polyphenols and especially of flavan-3-ols and their derivatives that contribute to bitterness and astringency. The aim of the present work was to assess the potential of a quantitative polyphenol targeted metabolomics analysis based on mass spectrometry for relating cocoa bean polyphenol composition corresponding chocolate polyphenol composition and sensory properties. One-hundred cocoa bean samples were transformed to chocolates using a standard process, and the latter were attributed to four different groups by sensory analysis. Polyphenols were analyzed by an ultra-high-performance liquid chromatography (UPLC) system hyphenated to a triple quadrupole mass spectrometer. A multiblock method called a Common Component and Specific Weights Analysis (CCSWA) was used to study relationships between the three datasets, i.e., cocoa polyphenols, chocolate polyphenols and sensory profiles. The CCSWA multiblock method coupling sensory and chocolate polyphenols differentiated the four sensory poles. It showed that polyphenolic and sensory data both contained information enabling the sensory poles' separation, even if they can be also complementary. A large amount of variance in the cocoa bean and corresponding chocolate polyphenols has been linked. The cocoa bean phenolic composition turned out to be a major factor in explaining the sensory pole separation.

Effect of polyvinylpolypyrrolidone treatment on rosés wines during fermentation: Impact on color, polyphenols and thiol aromas.

Fining treatment with polyvinylpolypyrrolidone (PVPP) is often used during winemaking of rosé wines. It can modulate the intensity and hue of their pink color and prevent some organoleptic degradations. In this paper, the effect of PVPP treatments on rosé wine during fermentation was investigated by measuring color, polyphenol content and thiol aromas. As expected, colorimetry results showed a decrease in color, indicating some adsorption of anthocyanins and other pigments. This was confirmed by UPLC-ESI-MS/MS analyses. Specific adsorption of certain families of polyphenols was evidenced. Flavonols, flavanols and anthocyanins, especially coumaroylated anthocyanins were preferentially adsorbed by PVPP. The thiol content (3-sulfanylhexyl acetate (3SHA) and 3-sulfanylhexan-1-ol (3SH)) was usually higher after PVPP treatments, in a dose dependent manner. A possible explanation is that the partial adsorption of some polyphenols at an early stage of fermentation would later limit the amount of quinone compounds able to trap thiol aromas.

Fast Discrimination of Chocolate Quality Based on Average-Mass-Spectra Fingerprints of Cocoa Polyphenols.

This work aims to sort cocoa beans according to chocolate sensory quality and phenolic composition. Prior to the study, cocoa samples were processed into chocolate in a standard manner, and then the chocolate was characterized by sensory analysis, allowing sorting of the samples into four sensory groups. Two objectives were set: first to use average mass spectra as quick cocoa-polyphenol-extract fingerprints and second to use those fingerprints and chemometrics to select the molecules that discriminate chocolate sensory groups. Sixteen cocoa polyphenol extracts were analyzed by liquid chromatography-low-resolution mass spectrometry. Averaging each mass spectrum provided polyphenolic fingerprints, which were combined into a matrix and processed with chemometrics to select the most meaningful molecules for discrimination of the chocolate sensory groups. Forty-four additional cocoa samples were used to validate the previous results. The fingerprinting method proved to be quick and efficient, and the chemometrics highlighted 29 m/ z signals of known and unknown molecules, mainly flavan-3-ols, enabling sensory-group discrimination.

New flavanol O-glycosides in grape and wine.

The presence of monomeric and dimeric flavan-3-ol monohexosides was investigated in grapes and wines. Polyphenol extracts were prepared from grape seeds and skins (Syrah, Merlot, and Cabernet-Sauvignon) sampled at three different developmental stages. Different wines (Tannat, Alicante, Syrah, Merlot, and Grenache) were also studied. The different samples obtained were analyzed by UPLC-ESI-IT-MS. Specific molecular ions corresponding to flavan-3-ol hexosides were detected by using targeted molecular ions with specific m/z values: 451 for (epi)catechin hexosides, and 739 for procyanidin dimer hexosides. 4'-O-ß-glucosyl-(+)-catechin and 7-O-ß-glucosyl-(+)-catechin were obtained by using a glucosyl transferase from apple, UGT71A15, and their structures determined by NMR. These glucosylated monomers and the dimers were identified in all analyzed grape seed and several skin extracts at the different stages and in wines made from different varieties.

Characterization of new flavan-3-ol derivatives in fermented cocoa beans.

Two series of compounds showing mass signals at m/z 605 and 893 (negative ionization mode) have been detected in fermented cocoa beans. This study objective is to identify these mass signals and characterize their structure in fermented cocoa samples. Our hypothesis is that these signals may correspond to ethyl-bridged flavan-3-ols resulting from flavan-3-ol condensation with acetaldehyde which is a microbial metabolite. Mass spectrometry was used to compare the retention times and mass fragmentation patterns between a model solution using epicatechin and procyanidin dimer B2, the major flavan-3-ols of cocoa, as precursors and extracts of fermented cocoa. Their identification was confirmed: four isomers of ethyl-linked epicatechin as well as several isomers of epicatechin-ethyl-procyanidin B2, in which B2 was mostly linked through its upper unit, were characterized in cocoa. This study demonstrates the presence of flavan-3-ol acetaldehyde condensation products in fermented cocoa beans and provides the first report of epicatechin-ethyl-procyanidin B2.

Quantification of hydroxycinnamic derivatives in wines by UHPLC-MRM-MS.

A UHPLC-MS/MS method was developed for the quantification of the main compounds involved in oxidation reactions occurring in white musts and wines such as hydroxycinnamic acids, their glutathione and cysteinylglycine adducts (GRP, GRP2, 5-(S-glutathionyl)-trans-caftaric acid, 2-(S-cysteinylglycyl)-trans-caftaric acid, and 2-(S-glutathionyl)-trans-caffeic acid), and reduced and oxidized glutathione (GSH, GSSG) in wine. Since oxidation is the main concern in white wine-making, directly affecting its quality, the developed method was then applied in a series of white wines made with different pre-fermentation treatments to limit oxidation at must stage. The glucose esters and/or glucosides of hydroxycinnamic acids were quantified as glucogallin equivalent. The developed method led to an overall improvement in the limits of detection (LODs) and quantification (LOQs) for all the compounds studied in comparison to other methods such as high-performance liquid chromatography with fluorescence detection (HPLC-FLD) or diode array UV detection (HPLC-DAD). LOD values ranged from 0.0002 to 0.0140 mg/L and LOQs from 0.0005 to 0.0470 mg/L. The recoveries ranged between 80 and 110% in wines, and the relative standard deviation (RSD) for precision intra- and inter-day was below 15%. The accuracy and intra- and inter-day precision met the acceptance criteria of the AOAC international norms. As far as we know, this study is the first report of quantification of GRP, 2-(S-cysteinylglycyl)-trans-caftaric acid, and 2-(S-glutathionyl)-trans-caffeic acid using these non-commercially available compounds as external standards. Those compounds represent a significant proportion of hydroxycinnamic acid derivatives in wines. The methodology described is suitable for the analysis of hydroxycinnamic derivatives in wines.

Cultivar Diversity of Grape Skin Polyphenol Composition and Changes in Response to Drought Investigated by LC-MS Based Metabolomics.

Phenolic compounds represent a large family of plant secondary metabolites, essential for the quality of grape and wine and playing a major role in plant defense against biotic and abiotic stresses. Phenolic composition is genetically driven and greatly affected by environmental factors, including water stress. A major challenge for breeding of grapevine cultivars adapted to climate change and with high potential for wine-making is to dissect the complex plant metabolic response involved in adaptation mechanisms. A targeted metabolomics approach based on ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS) analysis in the Multiple Reaction Monitoring (MRM) mode has been developed for high throughput profiling of the phenolic composition of grape skins. This method enables rapid, selective, and sensitive quantification of 96 phenolic compounds (anthocyanins, phenolic acids, stilbenoids, flavonols, dihydroflavonols, flavan-3-ol monomers, and oligomers…), and of the constitutive units of proanthocyanidins (i.e., condensed tannins), giving access to detailed polyphenol composition. It was applied on the skins of mature grape berries from a core-collection of 279 Vitis vinifera cultivars grown with or without watering to assess the genetic variation for polyphenol composition and its modulation by irrigation, in two successive vintages (2014-2015). Distribution of berry weights and δ13C values showed that non irrigated vines were subjected to a marked water stress in 2014 and to a very limited one in 2015. Metabolomics analysis of the polyphenol composition and chemometrics analysis of this data demonstrated an influence of water stress on the biosynthesis of different polyphenol classes and cultivar differences in metabolic response to water deficit. Correlation networks gave insight on the relationships between the different polyphenol metabolites and related biosynthetic pathways. They also established patterns of polyphenol response to drought, with different molecular families affected either positively or negatively in the different cultivars, with potential impact on grape and wine quality.

Rosé Wine Fining Using Polyvinylpolypyrrolidone: Colorimetry, Targeted Polyphenomics, and Molecular Dynamics Simulations.

Polyvinylpolypyrrolidone (PVPP) is a fining agent polymer used in winemaking to adjust rosé wine color and to prevent organoleptic degradations by reducing polyphenol content. The impact of this polymer on color parameters and polyphenols of rosé wines was investigated, and the binding specificity of polyphenols toward PVPP was determined. Color measured by colorimetry decreased after treatment, thus confirming the adsorption of anthocyanins and other pigments. Phenolic composition was determined before and after fining by targeted polyphenomics (Ultra Performance Liquid Chromatography (UPLC)-Electrospray Ionization(ESI)-Mass Spectrometry (MS/MS)). MS analysis showed adsorption differences among polyphenol families. Flavonols (42%) and flavanols (64%) were the most affected. Anthocyanins were not strongly adsorbed on average (12%), but a specific adsorption of coumaroylated anthocyanins was observed (37%). Intermolecular interactions were also studied using molecular dynamics simulations. Relative adsorptions of flavanols were correlated with the calculated interaction energies. The specific affinity of coumaroylated anthocyanins toward PVPP was also well explained by the molecular modeling.