M.A. Paissoni1,2,3, *, P. Waffo-Teguo2,3, W. Ma2,3,4, M. Jourdes2,3, S. Giacosa1, S. Río Segade1, L. Rolle1, P-L. Teissedre2,3
1 Dipartimento di Scienze Agrarie, Forestali e Alimentari. Università degli Studi di Torino, Grugliasco, Italy.
2 ISVV, EA 4577 Oenologie, F-33140, Université de Bordeaux, Villenave d’Ornon, France
3 INRA, ISVV, USC 1366 Oenologie, F-33140, Villenave d’Ornon, France
4 Wine School, Ningxia University, Yinchuan, Ningxia, 750021, P.R. China
* Corresponding author: mariaalessandra.paissoni@unito.it
https://doi.org/10.53144/INFOWINE.EN.2021.10.01.3
Article extracted from the presentation held during Enoforum Web Conference (23-25 February 2021)
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Introduction
In red wine, polyphenols are strictly associated with the final product quality. Among them, monomeric anthocyanins extracted from skins are responsible for young wine colour. Grape anthocyanin profile is linked to the cultivar genome, but their content is influenced, among others, by vineyard practices, climate, and soil features. During the winemaking process, they undergo several reactions, leading to monomeric anthocyanins decrease in favour of compounds combined with fermentative metabolites and other grape polyphenols. Among the latter category, monomeric, oligomeric and polymeric flavan-3-ols are the most reactive. They are extracted from skins and seeds and they are well known for their contribution to astringency and bitterness of wine. The evolution of flavan-3-ols during winemaking, including the condensation products with anthocyanins, is considered as the main responsible of wine astringency decrease found in aged wines (Ma et al., 2014; García-Estévez et al., 2017). Nevertheless, flavan-3-ol features alone are far to represent the full wine “in-mouth” complexity. Wine technological parameters, other macromolecules in wine derived from grape, yeast, and external sources as wood, can modulate the tannin interaction with salivary protein or directly elicit in-mouth sensations (Laguna et al., 2017).
Several researches were focused in understanding flavan-3-ol sensory characteristics (Ma et al., 2014; Soares et al., 2020), whereas little is known about other phenolic compounds, and, in particular, the role of anthocyanins on in-mouth characteristics is far to be fully understood. Several studies attempted to give an explanation on their involvement in taste and mouthfeel properties of wine, giving divergent results but not definitely excluding an impact. Anthocyanins have been described in sensory analysis to have a “mild taste” (Singleton & Noble, 1997) increasing astringency sub-qualities, such as “fine grain” attribute (Broussard et al., 2001; Vidal et al., 2004a; Oberholster et al., 2009; Ferrer-Gallego et al., 2015). Individual acetylated and p-coumaroylated anthocyanins are involved in both astringency and bitterness (Gonzalo-Diago et al., 2014). Chemical determination of astringency as interaction with salivary proteins has been carried out on glucoside anthocyanins, showing the ability of malvidin-3-O-glucoside to form soluble complexes with salivary proteins (Ferrer-Gallego et al. 2015) and to activate bitterness receptors (Soares et al. 2013). Anyway, Vidal et al. (2004b) found no differences neither in a model wine nor in unbuffered ethanolic solution (5%) added with glucoside or coumaroylated anthocyanins, justifying the in-mouth sensation reported previously as impurities in the isolated fractions.
The understanding of anthocyanin in-mouth properties often requires the extraction and purification of these compounds. In this study (Paissoni et al. 2018; 2020), these operations were attempted with Centrifugal Partition Chromatography (CPC) and Preparative HPLC. The advantage of using the liquid-liquid separation technique of CPC instrument is the possibility to use solvents as both stationary and mobile phases, allowing the recovery of the injected material, and to inject up to several grams of raw extract. Anthocyanins were fractionated depending on acylation in glucosides, acetylglucosides, and p-coumaroylglucosides. On the obtained fractions, chemical and sensory evaluations were carried out to highlight anthocyanins contribution to “in-mouth” properties were carried out. Moreover, the anthocyanins extract and the derived fractions were added to the tannin-based extract and the effect was evaluated following the same sensory procedure.
Materials and Methods
Grape samples and extracts
Grape samples from Vitis vinifera L. cv Nebbiolo and Barbera were collected at maturity in Piedmont region, Italy. Grape berries were peeled, and grape seeds were removed manually, then skins and seeds obtained were lyophilized and grounded. The resulting powders were used to carry out the extraction of grape polyphenols and anthocyanins.
Anthocyanins isolation and purification
Anthocyanins were extracted from skins with acidified methanol (0.1% trifluoroacetic acid) and then purified on Amberlite XAD 16 resin. The obtained extracts, evaporated and freeze dried, represent the total anthocyanins extract (TA), which was prepared separately for the two varieties. Barbera and Nebbiolo TAs were injected separately in a 200 mL CPC apparatus to optimize the separation method, taken from Renault et al., 1997. Later, a large-scale purification was carried out on 1 L CPC instrumentation, and several fractions were collected monitoring the UV-VIS spectra at 280 and 520 nm. The CPC fractions corresponding to the two varieties were mixed depending on the anthocyanidin acylation, i.e. monoglucosides (GF), acetylglucosides (AF), and coumaroylated forms (CF), the latter composed by a mix of p-coumaroylglucosides and, in minor part, of caffeoylglucoside anthocyanins. Final TA extracts and CPC fractions were analysed through HPLC-DAD (Chira, 2009) and the fractions with lower purity (AF and CF) were then cleaned throughout Preparative-HPLC (Figure 1). The final extracts purity reached was 95%, 98%, 87%, and 91% for TA, GF, AF, and CF, respectively, calculated on the area ratio at 520 nm and 280 nm by HPLC-DAD chromatograms.
Polyphenol extracts preparation
Skin and seed polyphenols extraction was performed as previously described by González-Centeno et al. (2012) with an ASE 350 Accelerated Solvent Extraction System (Dionex Corporation. Sunnyvale. CA). The extract was then cleaned from lipophilic material with chloroform three times (Lorrain et al., 2011). These aqueous extracts were concentrated and lyophilised to obtain a dry powder, the so called SkTOT and SdTOT extracts, for skin and seeds extracts, respectively. The obtained fractions were characterized by phloroglucinolysis Lorrain et al., 2011) and mean degree of polymerization (mDP) was 15.5±0.5 and 3.7±0.1 for SkTOT and SdTOT, respectively. For SdTOT the galloylation percentage (G%) was 15.2±0.3 and for SkTOT it was 2.2±0.1, in the latter the percentage of prodelphinidins (Pd%) was 34.0±0.2, whereas prodelphinidins were not detected in the seed extract. For skins extract anthocyanin content was determined by HPLC-DAD (Chira, 2009), and accounted for 130.2±2.8 mg/g of extract. All the fractions were lyophilized twice before sensory analysis to avoid the presence of solvents.
Chemical analysis of astringency of anthocyanin extract and fractions
Barbera TA and CPC fractions were tested with human saliva to understand if precipitation occurred. Saliva collection and precipitation method were taken from Schwarz & Hofmann (2008). Interaction was evaluated as difference (delta) between the area of the saliva-treated and control samples at 520 nm in HPLC-DAD system (Ma et al., 2016).
Sensory analysis of grape extracts
Sensory analyses were conducted in the tasting room of University of Bordeaux, Oenology research unit (ISVV, France). The room fulfils the ISO 8589:2007 standard for this type of equipment. A panel of 18 volunteer assessors from the Oenology department at the University of Bordeaux (ISVV, FRANCE) took part in the experiment. Sensory analysis of extracts was done in model wine solution (12% ethanol, 4 g/L tartaric acid, pH 3.5) using black ISO 3591:1977 wine glasses.
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Figure 1. Purification scheme of grape anthocyanin extracts.
(a) Barbera TA chromatogram at 520 nm; (b) Barbera TA CPC chromatogram at 280 nm, and (c) chromatograms of glucoside fraction (GF), (d) acetylated fraction (AF), and p-coumaroylated fraction (CF) at 520 nm obtained by a mix of the fractions obtained from Nebbiolo (not shown) and Barbera extracts. Legend: 1=delphinidin-3-O-glucoside; 2=cyanidin-3-o-glucoside, 3=petunidin-3-O-glucoside, 4=peonidin-3-O-glucoside; 5=malvidin-3-O-glucoside; 6= delphinidin-3-O-acetylglucoside; 7= cyanidin-3-O-acetylglucoside; 8= petunidin-3-O-acetylglucoside; 9= peonidin-3-O-acetylglucoside; 10=malvidin-O-acetylglucoside; 11= delphinidin-3-O-coumaroylglucoside; 12= malvidin-3-O-caffeoylglucoside; 13= cyanidin-3-O-coumaroylglucoside; 14= petunidin-3-O-coumaroylglucoside; 15=peonidin-3-O-coumaroylglucoside; 16=malvidin-3-O-coumaroylglucoside
Detection thresholds of anthocyanins extract and fractions
Total anthocyanin (TA) extracts from Barbera and Nebbiolo winegrapes were tasted individually in a preliminary evaluation (sensory-triangular test), and since no differences were found on the extracts, subsequent evaluations were performed only on Barbera TA. The fractions differentiated by acylation were composed of a mix of the two different varieties. Barbera TA extract and CPC fractions GF, AF, and CF were used to determine detection thresholds using the three-alternative forced choice method (3AFC - ISO 13301:2002). “Best Estimated Threshold” (BET) was calculated as described by Meilgaard et al., 1999.
Check all that apply (CATA) for descriptors selection
Check-all-that-apply (CATA) analysis was chosen to identify most frequently reported descriptors for the investigated fractions (Varela & Ares, 2012). For the sensory evaluation, four grape extracts representative of the classes under evaluation were identified as follows: skin extract (SkTOT, 1000 mg/L), seed extract (SdTOT, 1000 mg/L); total anthocyanin extract from Barbera (TA, 400 mg/L), and glucoside fraction of anthocyanin (GF, 400 mg/L). The extracts were tasted in model wine.
Attributes were selected based on previous results obtained from tasting, and by bibliographic references on grape/wine extracts and fractions, in particular anthocyanins (Oberholster et. al., 2009; Gawel et al., 2000; Vidal et al., 2004a, 2004b, 2004c; Saenz-Navajas et al., 2017). The final descriptors (23 in total) belonged to taste category (sweet, bitter, salty, acid) and sub-qualities were selected on the basis of the mouth-feel wheel terminology proposed by Gawel et al. (2000). In detail, they were: weight (watery, viscous, dense), texture (oily), heat (hot), irritation (prickle, tingle), drying (dry), and related to astringency, such as surface smoothness (emery, silky) and particulates (dusty, grainy, chalky), complex terms (soft, round, mouthcoating, aggressive), a dynamic group attribute (adhesive), and the term “rich”.
Descriptive Analysis (DA)
From CATA analysis, selected descriptors were overall astringency, bitterness, and sub-qualities of astringency “particulate” and “surface smoothness”. Assessor training on astringency and bitterness scales was slightly modified from the one proposed by Chira et al. (2012). For overall astringency and bitterness, aluminium sulphate and quinine sulphate were used as standard references, respectively. Concerning astringency sub-qualities, the definition of “particulate” (in-mouth sensation) and “surface smoothness” (after expectoration) were discussed among assessors and the use of tactile standards was introduced. Fabric samples were selected to anchor the “surface smoothness”, and powders with different granulometry (from talc powder to withe sugar) were used for “particulate” attribute. Standard references and scales were defined by slightly modifying the scales proposed by Pickering & Demiglio (2008) for evaluation of oral sensations elicited by white wine. The rating of selected attributes was done on an 8-point scale (0 = “absence”, 1 = “very low” and 7 = “very high”), where “very low” and “very high” rates were anchored with the use of standard references for each attribute.The 0 point was included as representative of the model wine solution.
Formal DA of grape extracts was conducted for a total of 5 sessions with 4 extracts tasted per session, and one sample was repeated to evaluate assessors performance. Samples were served at room temperature and were evaluated in individual booths. All the extracts were evaluated in model wine using final concentrations of 1000 mg/L for both SdTOT and SkTOT, 400 mg/L for TA and GF, and 100 mg/L for CF and AF.
The total extract (TA) and the individual fractions of anthocyanins GF, and CF were also added to the 1000 mg/L of SkTOT and SdTOT solutions, at the same concentration they were tasted individually (400 mg/L for TA and GF, and 100 mg/L for CF).
Results and discussion
Chemical investigation of anthocyanin fractions astringency
Analyses on the CPC fractions showed anthocyanins concentration reduction in all saliva-treated samples, with -8.53% and -9.48% (p <0.05) for GF and AF, respectively, and -12.82% (p<0.001) for CF (Figure 2). In our study, the reactivity of CF fraction was the most evident. Anthocyanins precipitation is anyway limited with respect to flavan-3-ols (Ma et al., 2016), in accordance with the poor ability of small phenolic compounds to form insoluble complexes with proteins (de Freitas & Mateus, 2001). Nevertheless, interaction among proteins and anthocyanins can lead to soluble compounds in wine like solution, which may be linked to astringency onset (Ferrer-Gallego et al., 2015).
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Figure 2. Saliva test results on glucoside, acetylated, and coumaroylated fractions.
Results are expressed as mg/L of malvidin-3-O-glucoside. All data are expressed as average value ± standards deviation (n=3). Sign: *,**,***, and ns indicate significance at p<0.05,0.01, 0.001, and not significant, respectively, for difference between each compound for control and treated sample according to ANOVA. Legend: dp= delphindin, cy=cyanidin, pt=petudinidin, pn=peonidin, mv=malvidin in a) -3-O-glucoside, in b) -3-O-acetylglucoside, and in c) -3-O-coumaroylglucoside, mv caf= malvidin-3-O-caffeoylglucoside. Adapted from Paissoni et al., 2018.
Sensory analysis: detection threshold of anthocyanins
Sensory analysis results agreed with chemical analysis since CF, which is the most reactive towards proteins, is also the fraction with lower perception threshold (BET= 58 mg/L), followed by AF (BET= 68 mg/L). Moreover, the higher perception threshold of GF alone (BET= 297 mg/L) than that of TA extract (BET= 255 mg/L) may highlight that the presence of a percentage of acetylated and cinnamoylated anthocyanins on the total extract has a higher impact on sensory properties, lowering the TA BET. Results were confirmed by triangular test and judges were also asked to express one (or more) descriptor(s) that helped them to discriminate among the samples.
Descriptors selection by CATA approach
Table 1 summarizes the frequencies of each descriptor individually, and descriptors grouped in arbitrarily chosen categories based on bibliography (Gawel et al., 2000). A combination of the higher cited attributes and their ability to discriminate among samples was taken into consideration to proceed in further sensory assessment of grape derived fractions.
Concerning frequencies, the descriptors “bitter” (13.3%, 1st), “aggressive” (7.1%, 2nd), “salty” (6.6%, 3rd ), and “hot” (6.2%, 4th) were found to be the highest cited ones. Bitterness and “hot” were reported often in extracts evaluation and did not allow differentiation of samples (p = 0.464 and p= 0.651, respectively). In contrast, “salty” and “aggressive” were relevantly used for GF and Sd-TOT samples, respectively, and allowed a higher samples separation (p = 0.045 and p = 0.071, for “salty” and “aggressive”, respectively). The first two descriptors can be related to the wine model solution composed by 12% ethanol, which is reported to elicit these sensations (Vidal et al., 2004a), and therefore “bitter” and “hot” were reported for all samples. Nevertheless, phenolic compounds are considered as major contributors of wine bitterness (Ma et al., 2014). Regarding astringency sub-qualities, the related terms accounted for the 22.1% of total citations. The “dusty” and “emery” descriptors were highly cited accounting for 5.8% (5th) and 5.3% (6th), respectively, of the total citation frequencies, followed by “chalky” and “silky” (4.4%, 8th and 4.0%, 9th, respectively).
Therefore, the highest cited term “bitter” was considered relevant to be investigated. As well, astringency sub-qualities, as “surface-smoothness” and “particulate”, were found to be both highly cited as category, and able to discriminate the samples. In detail, “particulate” was chosen as representative of “in-mouth” astringency, whereas surface smoothness as “after expectoration” perceived astringency. Moreover, “overall astringency” was chosen as descriptor to summarize both in-mouth and after expectoration astringency intensities.
Table 1. Frequencies of CATA analysis.
aSubqualities group taken from Gawel et al. (2000). bp-value according to the Cochran’s Q test for descriptors in discrimination of samples. Value in bold indicates relevant terms based on Cochran’s Q test (p < 0.1) or rank of citation (1st to 8th most cited terms). Adapted from Paissoni et al., 2020.
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DA results
The grape derived fractions tasted individually were significantly different for overall astringency, particulate, and surface smoothness, but not for bitterness (Figure 3). Concerning overall astringency, fractions were sensory evaluated different being SkTOT the highest in astringency, even if not significantly different from SdTOT and TA. As expected, anthocyanin fractions (GF, AF, and CF) were those less astringent. Astringency sub-qualities ratings agreed with the perceived overall astringency results. In fact, SkTOT was perceived as the highest in scale corresponding to the anchored references “fine emery” for surface smoothness, and close to “grainy” for particulate attribute. TA, GF, and CF fractions did not differ with respect to polyphenols extracts in “particulate” attributes, although they were lower rated. In a previous study, the addition of total anthocyanins to white juice prior to fermentation led to an increase in “fine grain” attribute of final wine (Oberholster et al., 2009). Acetylated fraction (AF) was the lower rated in “particulate”, significantly different from the others, evoking chalky sensation, which was a descriptor previously reported in literature in anthocyanins sensory analysis (Vidal et al., 2004a; 2004b). For surface smoothness, AF and CF were closer to a “silky” sensation and TA and GF corresponded to a “velvety” one, whereas the SkTOT was in correspondence with “fine emery”. Concerning glucoside anthocyanins, these results are in agreement with a previous study, which described as “velvety” a wine added of glucoside anthocyanins at the same concentration (400 mg/L, Ferrer-Gallego et al., 2015).
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Figure 3. Descriptive analysis (DA) of grape extracts.
Data are expressed as mean of assessors’ ratings and error bars are calculated as s/(n)1/2, where s is standard deviation and n the number of assessors. p-value are reported according to ANOVA, and different Latin letters indicate significant differences according to HSD Tukey test (p < 0.05).
Legend: SdTOT= seed polyphenol extract; SkTOT= skin polyphenol extract; TA = total anthocyanins extract; GF= glucoside anthocyanin fraction; AF = acetylated anthocyanin fraction; CF= p-coumaroylated anthocyanin fraction. Adapted from Paissoni et al., 2020.
Although the contribution of anthocyanin fractions alone, which owned sensibly lower rating with respect to other phenol fractions, it is interesting to analyse how the addition of total anthocyanin extract (TA) and the derived fractions GF and CF may influence other polyphenols extract sensory perceptions (Table 2). In the SkTOT sample added of GF, the surface smoothness was rated significantly lower. On the contrary, a significant increase in subqualities and overall astringency ratings was found when GF was added to SdTOT. Therefore, surface smoothness descriptor showed an inverse trend when anthocyanins were added depending on the polyphenols origin (skins vs seeds), since it decreased when glucoside anthocyanins are added to skin polyphenols and increased when added to seed polyphenols
Table 2. Descriptive Analysis (DA) of mixed extracts.
Data are expressed as means of assessors rating and error bars are calculated as s/(n)1/2, where s is standard deviation and n the number of assessors. Sign: *, **, and ns indicate significance at p < 0.05, 0.01, and not significant, respectively, for the differences among samples according to ANOVA. Different Latin letters indicate significant differences according to HSD Tukey test (p< 0.05).
Legend: SdTOT= seed polyphenol extract; SkTOT= skin polyphenol extract; TA = total anthocyanin extract; GF= glucoside anthocyanin fraction; CF= p-coumaroylated anthocyanin fraction.
Adapted from Paissoni et al., 2020.
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Conclusion
The aim of this study was to investigate the grape anthocyanins contribution on the “in-mouth” perception. The combined use of Centrifugal Partition Chromatography and preparative HPLC allowed to obtain anthocyanin extracts from grape skins in good quantity and purity. The anthocyanin fractions were collected based on anthocyanin acylation, therefore in glucoside, acetylated, and p-coumaroylated fractions. On the initial total anthocyanin extract from cultivar Barbera and on CPC fractions, an investigation on astringency was done through saliva test precipitation. The reactivity was measured as difference in saliva-treated and control sample, showing a higher precipitation of p-coumaroylated anthocyanins with respect to the other fractions.
Detection thresholds were evaluated as best estimated thresholds (BET) and the results showed the lowest BET for p-coumaroylated fractions, followed by acetylated and glucoside fraction anthocyanins. The sensory analysis of attributes with Check-all-that-apply (CATA) analysis and their intensity with descriptive analysis (DA) revealed grape anthocyanins involvement in “in-mouth” perception of model wine solutions, although their contribution is less relevant than polyphenolic classes such as condensed tannins. At wine level, glucoside anthocyanins are related to velvety sub-qualities sensations, whereas their mixture with other acylation classes showed higher overall astringency and harsher sub-qualities than glucosides alone. Interestingly, the addition of glucoside fraction to skin and seed polyphenol extracts led to decrease in the astringency for the former and an increase for the latter. This different trend suggests the possibility of further studies on different fractions interactions, with a suppression or enhancing effect on sensory attributes.
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