First insights into the binding mechanism and colour effect of the interaction of grape seed 11S globulin with malvidin 3-O-glucoside by fluorescence spectroscopy, differential colorimetry and molecular modelling.

Recently, the search for alternative proteins endogenous to grapes to be used as wine colour protecting agents became an important research trend. In this study, the molecular interaction between the grape seed 11S globulin from winemaking by-product and malvidin-3-O-glucoside was investigated by fluorescence, differential colorimetry and molecular modelling. Fluorescence studies revealed the formation of grape seed protein- pigment complex whose KS was 8.5 × 104 M-1 and binding sites, n = 1.3. Malvidin-3-O-glucoside showed darker and more vivid bluish colour of in the presence of 11S globulin, suggesting the flavylium cation protection in a hydrophobic region of the protein. Docking analysis and molecular dynamics simulation indicated that malvidin-3-O-glucoside interacts mainly with the acidic subunit (40 kDa) of the 11S globulin monomer (60 kDa). An average of two hydrogen bonds and Van der Wall forces were the main interaction forces found for the protein-pigment complex, whose stability was confirmed by root-means-square deviation.

Proteomic and computational characterisation of 11S globulins from grape seed flour by-product and its interaction with malvidin 3-glucoside by molecular docking.

Grape seed flour by-product (GSBP) is an economic and renewable source of proteins, increasingly being explored due to interesting technological application such as colour protection in rich-anthocyanins beverages. Globulin-like proteins from GSBP were characterised by proteomic and computational studies. MALDI TOF/TOF analysis revealed the presence of two 11S globulins (acid and basic), whose 3D structures have been elucidated for the first time in Vitis vinifera L. grape seeds by using homology models and molecular dynamics. The secondary structure showed 11 α-helices and 25 ß-sheets for acid and 12 α-helices and 24 ß-sheets for basic 11S globulins. Molecular docking results indicate that both grape seed 11S globulins could establish different types of non-covalent interactions (π-π) with malvidin 3-O-glucoside (wine anthocyanin), which suggest a possible colour protection similar to that occurring in copigmentation phenomenon. These findings provide valuable information of globulin family proteins that could be relevant in food industry applications.

A Study of Overripe Seed Byproducts from Sun-Dried Grapes by Dispersive Raman Spectroscopy.

Overripe seeds from sun-dried grapes submitted to postharvest dehydration constitute a scarcely investigated class of vinification byproduct with limited reports on their phenolic composition and industrial applications. In this study, Raman spectroscopy was applied to characterize a selection of overripe seed byproducts from different white grapes (cv. Moscatel, cv. Pedro Ximénez and cv. Zalema) submitted to postharvest sun drying. The Raman measurements were taken using a 1064 nm excitation laser in order to mitigate the fluorescent effect and the dispersive detection scheme allowed a compactness of the optical system. Spectroscopic data were processed by a principal component analysis to reduce the dimensionality and partner recognition. The evolution of the Raman spectrum during the overripening process was compared with the phenolic composition of grape seeds, which was determined by rapid resolution liquid chromatography/mass spectrometry (RRLC/MS). A multivariate processing of the spectroscopic data allowed the classification of overripe seeds according to the grape variety and the monitoring of stages of the postharvest sun drying process.

Impact of alternative protein fining agents on the phenolic composition and color of Syrah red wines from warm climate.

Currently, the wine industry has an increasing interest in developing alternative solutions to traditional animal proteins fining agents. In this study, the impact of different protein fining agents on the turbidity, phenolic composition and color of 2-month and 12-month Syrah red wines was assessed. Wines fined with egg albumin and plant-based proteins from potato, pea, and grape seed as recent alternative, were compared to unfined control wines. Changes on turbidity, phenolic composition and color (by Differential Colorimetry) showed that animal and plant proteins differed in their clarifying efficiency and ability to interact with colorless phenolics and anthocyanins, depending on the age of wine, with important consequences on color quality and stability. Plant proteins showed lower effectiveness to reduce wine turbidity than egg albumin but modified in different way the phenolic composition, inducing lower color differences with respect to control wine and similar stability, especially potato and grape seed proteins.

Impact of a double post-fermentative maceration with ripe and overripe seeds on the phenolic composition and color stability of Syrah red wines from warm climate.

The post-fermentative double addition of Pedro Ximénez cv seeds obtained from natural matured grapes (ripe seeds, RS) and postharvest sun-dried grapes (overripe seeds, OS) were studied as sustainable enological alternatives to conventional vinification (CW) to improve the stability of Syrah wines produced in a warm climate. The phenolic composition was assessed by rapid resolution liquid chromatography, copigmentation/polymerization processes by spectrophotometry, and color quality and stability by Differential Colorimetry. OSW and RSW wines enriched their total phenolic content, being the effect more pronounced with overripe seeds (by 23% versus 10%). OSW differences were found for gallic acid, monomeric flavan-3-ols, and procyanidins compared to CW, and for (+)-catechin, procyanidin B2-3-O-gallate and the tetramer to RSW. Phenolic changes were related to higher color intensity in seed-added wines. OSW having higher percentage of polymeric pigments maintained for longer time the chromatic improvement, being visually darker and more intense than final CW and RSW.

Elucidation of the 3D structure of grape seed 7S globulin and its interaction with malvidin 3-glucoside: A molecular modeling approach.

Plant proteins are biopolymers with interesting technological applications for the food industry due to their ability to interact with phenolic compounds such as anthocyanins. The 3D structure of the 7S globulin from grape seed was elucidated for the first time using a homology model. The constructed 3D model showed that grape seed 7S globulin is rich in α-helices and ß-sheets stabilized by six disulfide bridges. The interaction with the major grape anthocyanin malvidin-3-glucoside was also assessed by Docking and Molecular Dynamic simulation. Theoretical results demonstrated that 7S globulin interacts with Mv3glc through hydrogen, alkyl and π-alkyl bonds and the flavylium cation is oriented towards a hydrophobic region of the protein, being protected from hydration. Results provide valuable insights for understanding the mechanisms involved in the molecular interaction of grape anthocyanins with grape seed proteins that could be relevant to use them as potential color protecting agents in food industry applications.

Impact of a post-fermentative maceration with overripe seeds on the color stability of red wines.

With the purpose of modulating the copigmentation equilibria of red wines, an environmentally sustainable process was performed based on post-fermentative addition of overripe seeds (OS). Simple (SW) and double (DW) addition were performed to produce different enrichment of phenolics from seeds, hence different copigmentation/polymerization ratios. The determination of the phenolic composition showed different global increases in OS-macerates wines (catechin, epicatechin, gallic acid and procyanidins B1 and B2). The double post-maceration (DW) was more effective than the simple post-maceration addition to improve the phenolic structure of wines. The application of Differential Tristimulus Colorimetry could assess the effects of this practice on the color characteristics and stability of wines. Results highlighted that both simple and double assays underwent colorimetric improvements against the control wines (CW, no seeds addition). DW led to the highest chromatic stability, showing lower lightness, higher chroma values and bluish hues than CW. This color difference was visually detectable.

Assessment of the color modulation and stability of naturally copigmented anthocyanin-grape colorants with different levels of purification.

Grape skins or their by-products from wine production are rich sources of anthocyanins and various colorless phenolics, depending on the grape variety. Phenolics have strong antioxidant and anthocyanin stabilizing properties and help to produce functional anthocyanin colorants with improved stability. This study aimed to assess differences in color expression and stability of anthocyanin colorants from red grape varieties naturally copigmented and with different levels of purity and to compare them to synthetic FD&C Red No. 3. Model juice systems were prepared at pH 3.5 with anthocyanins and phenolic copigments extracted from four Vitis vinifera grape varieties ('Tempranillo', 'Syrah', 'C. Sauvignon', and 'Graciano') both crude and purified by C18 solid phase extraction. Attention was focused on differential colorimetry and phenolic composition related to the color. Degradation kinetics of total color were also studied during storage of 17 days in darkness at 25 °C. Grape variety significantly influenced pigment yield, proportion of acylation, and proportion of copigments:pigments ratios in crude extracts; purification modulated the copigment:pigment ratios. This proportion was related to perceptible color variability among colorants and to different stabilities. With the same pigment content, grape varieties richer in skin copigments and higher copigment/pigment ratios ('Syrah' and 'Tempranillo') produced more intensely colored crude extracts whose tonalities ranged from reddish ('Graciano') to red-bluish ('Syrah'), depending on the proportion of acylation. Increasing the purity of the pigments diminished the color variability due to variety, making them less vivid and visually more similar to one another and also to the synthetic colorant. Degradation kinetic studies showed that unpurified grape colorants had higher color stability over time, with the greatest stabilizing effects achieved with varieties richer in skin flavonols ('Tempranillo' and 'Syrah').

Measurement of ripening of raspberries (Rubus idaeus L) by near infrared and colorimetric imaging techniques.

This work includes the evaluation of 168 samples of raspberries 'Glen Lyon', representing whole maturation period, by colorimetric and near infrared imaging techniques, as well as the quantification of total phenols, total anthocyanins and antioxidant activity by chemical methods. Samples showed significant differences depending on the maturation stage using CIELAB colour parameters and total anthocyanins content. The application of partial least squares regression allowed predicting the chemical features from image analysis data, with coefficients of determination (R2) up to 0.75. The best prediction for total anthocyanins including colorimetric data was observed. The proposed methodology can be used as a reference method for assessing important quality attributes of raspberries. Moreover, it is useful, rapid and accurate automatic inspection method.

Optimisation of an oak chips-grape mix maceration process. Influence of chip dose and maceration time.

Oak chips-related phenolics are able to modify the composition of red wine and modulate the colour stability. In this study, the effect of two maceration techniques, traditional and oak chips-grape mix process, on the phenolic composition and colour of Syrah red wines from warm climate was studied. Two doses of oak chips (3 and 6g/L) at two maceration times (5 and 10days) during fermentation was considered. Changes on phenolic composition (HPLC-DAD-MS), copigmentation/polymerisation (spectrophotometry), and colour (Tristimulus and Differential Colorimetry) were assessed by multivariate statistical techniques. The addition of oak chips at shorter maceration times enhanced phenolic extraction, colour and its stabilisation in comparison to the traditional maceration. On contrast, increasing chip dose in extended maceration time resulted in wines with lighter and less stable colour. Results open the possibility of optimise alternative technological applications to traditional grape maceration for avoiding the common loss of colour of wines from warm climate.

Comparative Study of Phenolic Profile, Antioxidant Capacity, and Color-composition Relation of Roselle Cultivars with Contrasting Pigmentation.

Roselle is a plant that accumulates anthocyanins significantly, hence its importance as food coloring and as a source of antioxidant compounds for human health. This study was aimed to determine phenolic composition and antioxidant capacity of methanolic extracts, and beverages obtained from native roselle cultivars in Mexico (Negra, Sudan, Rosa and Blanca) with different degrees of pigmentation, and to establish the color-composition relationship. Chromatographic methods were used to determine phenolic compounds: flavanols, flavonols, benzoic, hibiscus and phenolic acids as well as two main anthocyanins (cyanidin 3-sambubioside and delphinidin 3-sambubioside). The antioxidant capacity was evaluated by ABTS and FRAP assays. Tristimulus colorimetry showed to be a useful technique to determine the color-composition relationship, leading to equations that allowed to predict anthocyanin content of roselle (R > 0.84). Also, a stepwise linear discriminant analysis (SLDA) was developed in order to classify roselle cultivars. The obtained mathematical model could be an important tool to be used in colorimetric characterization of functional compounds used in food processing.

Application of Differential Colorimetry To Evaluate Anthocyanin-Flavonol-Flavanol Ternary Copigmentation Interactions in Model Solutions.

The combined effect of anthocyanin-flavanol-flavonol ternary interactions on the colorimetric and chemical stability of malvidin-3-glucoside has been studied. Model solutions with fixed malvidin-3-glucoside/(+)-catechin ratio (MC) and variable quercetin-3-ß-d-glucoside concentration (MC+Q) and solutions with fixed malvidin-3-glucoside/quercetin-3-ß-d-glucoside ratio (MQ) and variable (+)-catechin concentration (MQ+C) were tested at levels closer to those existing in wines. Color variations during storage were evaluated by differential colorimetry. Changes in the anthocyanin concentration were monitored by HPLC-DAD. CIELAB color-difference formulas were demonstrated to be of practical interest to assess the stronger and more stable interaction of quercetin-3-ß-d-glucoside with MC binary mixture than (+)-catechin with MQ mixture. The results imply that MC+Q ternary solutions kept their intensity and bluish tonalities for a longer time in comparison to MQ+C solutions. The stability of malvidin-3-glucoside improves when the concentration of quercetin-3-ß-d-glucoside increases in MC+Q mixtures, whereas the addition of (+)-catechin in MQ+C mixtures resulted in an opposite effect.

Assessment of the differences in the phenolic composition and color characteristics of new strawberry (Fragaria x ananassa Duch.) cultivars by HPLC-MS and Imaging Tristimulus Colorimetry.

The phenolic composition (by HPLC-DAD-MS) and color characteristics (by Imaging Tristimulus Colorimetry) of four strawberry cultivars that have shown good climate adaptation to subtropical area (Nikte, Zamorana, Jacona and Pakal) have been assessed. 24 monomeric phenolics were identified, including 15 anthocyanins, 5 phenolic acids, 1 flavanol and 4 flavonols. Nikte and Zamorana showed the highest phenolic potential mainly due to their higher content of anthocyanins, while Pakal was richer in phenolic acids. Regarding color, Nikte and Zamorana were the more similar cultivars having the lowest values of lightness and hue. On the contrary, the color of Pakal was quite different from all the rest, due to the specific distribution between pelargonidin and cyanidin. The inclusion of both phenolic and colorimetric information in the Linear Discriminant Analysis allowed reaching very good discriminations among cultivars.

Effect of salt stress in the regulation of anthocyanins and color of hibiscus flowers by digital image analysis.

The effect of salt stress (200 mM NaCl for 28 days) on physiological characteristics of Hibiscus rosa-sinensis, such as abscisic acid (ABA) content, electrolyte leakage, and photochemical efficiency in leaves, and its influence on biomass production, anthocyanin composition, and color expression of flowers were evaluated. Salinity significantly increased electrolyte leakage and ABA content in leaves and reduced the flower fresh weight. Chlorophyll fluorescence parameters were lower in salt stress condition, compared to control. Moreover, salt stress negatively affected the content of anthocyanins (mainly cyanidin-3-sophoroside), which resulted in a visually perceptible loss of color. The detailed anthocyanin composition monitored by HPLC-DAD-MS and the color variations by digital image analysis due to salt stress showed that the effect was more noticeable at the basal portion of petals. A forward stepwise multiple regression was performed for predicting the content of anthocyanins from appearance characteristics obtained by image analysis, reaching R-square values up to 0.90.

Comparative study of the enological potential of different winemaking byproducts: implications in the antioxidant activity and color expression of red wine anthocyanins in a model solution.

Different white winemaking byproducts (pomace, skins, seeds, and stems) were compared as natural sources of phenolic compounds having biological and sensory properties of enological interest. Antioxidant and copigmentation effects of these byproducts were studied in a wine-like model solution. RRLC-DAD was used to establish differences in the phenolic composition, and the ABTS method was used to compare the antioxidant activities. Spectrophotometric and colorimetric analyses were performed to assess the magnitude of copigmentation and the changes induced in the color expression of red wine anthocyanins. Antioxidant and copigmentation properties significantly varied depending on the type of byproduct, which was related to their qualitative and quantitative phenolic composition. Seeds and pomace showed the highest antioxidant potential, whereas skins and pomace led to the strongest and visually perceptible color effects on red wine anthocyanins by multiple copigmentation (darker, more saturated, and vivid bluish colors). Results open the possibility of technological applications for the wine industry based on reusing winemaking byproducts to improve the biological value and color characteristics of red wines.

A novel method for evaluating flavanols in grape seeds by near infrared hyperspectral imaging.

Chemical composition of seeds changes during grape ripening and this affects the sensory properties of wine. In order to control the features of wines, the condition of seeds is becoming an important factor for deciding the moment of harvesting by winemakers. Sensory analysis is not easy to carry out and chemical analysis needs lengthy procedures, reagents, and it is destructive and time-consuming. In the present work, near infrared hyperspectral imaging has been used to determine flavanols in seeds of red (cv. Tempranillo) and white (cv. Zalema) grapes (Vitis vinifera L.). As reference measurements, the flavanol content was estimated using the p-dimethylaminocinnamaldehyde (DMACA) method. Not only total flavanol content was evaluated but also the quantity of flavanols that would be extracted into the wine during winemaking. A like-wine model solution was used for this purpose. Calibrations were performed by partial least squares regression and they provide coefficients of determination R(2)=0.73 for total flavanol content and R(2)=0.85 for predicting flavanols extracted with model solution. Values up to R(2)=0.88 were reached when cultivars were considered individually.

Impact of adding white pomace to red grapes on the phenolic composition and color stability of Syrah wines from a warm climate.

The influence of the fermentative addition of Pedro Ximenez grape pomace (PXGP, white variety) on the phenolic composition and color of Syrah red wines from a warm climate was studied. Changes on phenolic composition (HPLC), copigmentation/polymerization (spectrophotometry), and color (tristimulus colorimetry) allowed differences among the maceration treatments to be established. PXGP additions at the rates studied increased the extraction of total phenolics, phenolic acids, and monomeric flavanols. However, the effect on the anthocyanins, copigmentation, and polymerization depended on the doses applied, with important consequences on the color. PXGP addition at 10% led to wines with higher polymerization, more stable colors, and bluish hues. in contrast, perceptibly lighter and less intense wines were obtained with PXGP addition at 20%. Thus, the use of white grape byproducts as wine additives at appropriate levels (10% w/w) could improve the phenolic potential of red young wines from a warm climate, contributing to preserve their color characteristic.

Application of the differential colorimetry and polyphenolic profile to the evaluation of the chromatic quality of Tempranillo red wines elaborated in warm climate. Influence of the presence of oak wood chips during fermentation.

The effect of adding American oak wood chips during fermentation on Tempranillo red wines elaborates in a warm climate has been studied. Our attention was focused on the tristimulus colorimetry, differential colorimetry and phenolic compounds related to wine colour. This technique was applied as an oenological alternative to the conventional winemaking for avoiding the common fall of colour of red wines elaborated in warm climates. The addition of oak wood chips promoted the colour enhancement and stabilisation, producing wines with a notably darker colour and with more bluish tonality. This fact was also related to the significantly higher content of some phenolic compounds. On the basis of the results, it could be affirmed that the addition of oak wood chips during fermentation induced visually perceptible colour changes (by the analysis of ΔEab(*), %Δ(2)L, %Δ(2)C and %Δ(2)H), mainly in a quantitative way, and also a lower percentage of diminution of colour.

Comprehensive colorimetric study of anthocyanic copigmentation in model solutions. Effects of pH and molar ratio.

New colorimetric variables have been defined in the uniform CIELAB color space to assess the quantitative and qualitative color changes induced by copigmentation and their incidence on visual perception. The copigmentation process was assayed in model solutions between malvidin 3-glucoside and three phenolic compounds (catechin, epicatechin, and caffeic acid) as a function of the pH and the pigment/copigment molar ratio. Along the pH variation, the greatest magnitude of copigmentation was obtained at pH 3.0, being significantly higher with epicatechin and caffeic acid. At high acidic pH, the main contribution of copigmentation to the total color was qualitative, whereas between pH 2.0 and 4.0, the main colorimetric contribution was quantitative. The contribution of epicatechin and caffeic acid to the color changes was more marked for the quantitative characteristics. On contrast, particularly at higher pH values, the qualitative contribution was more important in catechin copigmented solutions. Increasing copigment concentration induced perceptible color changes at molar ratios higher than 1:2, consisting in a bluish and darkening effect of the anthocyanin solutions. Among the different CIELAB attributes, hue difference was the best correlated parameter with the increase of copigment concentration, proving the relevance of this physicochemical phenomenon on the qualitative changes of anthocyanin color.

Influence of prefermentative cold maceration on the color and anthocyanic copigmentation of organic Tempranillo wines elaborated in a warm climate.

The stabilization of red wine color by the copigmentation phenomenon is a crucial process that does not always proceed favorably under natural conditions during the first stages of vinification. The impact of the prefermentative cold maceration technique on the phenolic composition and magnitude of the copigmentation level of organic Tempranillo wines elaborated in a warm climate have been studied as an enological alternative to the traditional maceration for obtaining highly colored wines. Tristimulus colorimetry was applied to study the color of wines during vinification, and a high-performance liquid chromatography (HPLC) procedure was used for the analysis of phenolic compounds. Spectrophotometric and colorimetric analyses were also performed to evaluate the copigmentation level of the wines. Significant chemical and color differences were found depending on the maceration technique applied. Prefermentative cold macerated wines were richer in those compounds accounting directly for the color of red wine (anthocyanins) and those involved in anthocyanin stabilization through copigmentation reactions (phenols), which was in accordance with the higher copigmentation degree and darker, more saturated and vivid bluish colors. The evaluation of the copigmentation based on colorimetric parameters in the CIELAB color space showed that prefermentative cold maceration caused greater effectiveness of copigmentation than traditional maceration since it induces more important and hence more easily perceptible color changes.