1. Introduction
Natural polyphenolic compounds have been reported to have multiple biological activities, including cardio-protective, anti-inflammatory, anti-carcinogenic, antigenotoxic, pro-apoptotic, antiviral, and antibacterial properties attributed mainly to their antioxidant and antiradical activity [
1,
2,
3,
4,
5,
6]. Moreover, polyphenols can limit insulin resistance and type 2 diabetes (T2D) risk [
7], and act as protective agents against atherosclerosis and brain dysfunction [
8]. Natural phenols have also been reported to have excellent properties as food preservatives, due to their antimicrobial and antioxidant activities [
9], as nutraceuticals [
10], and for many other industrial applications (natural colorants for foods, production of cosmetics, etc.).
Grapes are one of the richest sources of natural polyphenols, among which flavonoids are the most abundant and important for wine quality. Flavonoids are characterized by a 15-carbon structural backbone C
6-C
3-C
6 (aryl-propyl-aryl). They are typically produced in plants as color pigments, and as a defense mechanism in response to environmental changes (exposure to ultra-violet (UV) radiations, pathogenic invasion) [
11]. Grapes flavonoids include anthocyanins, flavonols, flavanones, flavones, isoflavones, and flavan-3-ols [
12]. The flavan-3-ols exist as monomers ((+)-catechin, (+)-gallocatechin, (−)-epicatechin, (−)-epigallocatechin and (−)-epicatechin-3-
O-gallate) and also as oligomers and polymers, called flavans, condensed tannins, or proanthocyanidins, the most abundant class of soluble polyphenols in grape berries. Grape proanthocyanidins are composed by four different flavan-3-ols monomer subunits linked via 4–6 and 4–8 interflavan bonds: (+)-catechin, (−)-epicatechin, (−)-epicatechin-3-
O-gallate and (−)-epigallocatechin, the latter only in the skins. Proanthocyanidins structures vary in monomeric composition, linkage position and size (degree of polymerization), ranging from dimers to polymers with more than 40 units [
13,
14,
15]: based on the number of flavanic units, procyanidins are divided in oligomeric (from 3 to 10 units) and condensed procyanidins (more than 10 units) that have a molecular weight higher than 3000 [
12]. The condensed tannins are present in all solid parts of grape clusters (skins, seeds, stalks), with the percentage monomer composition varying with the cultivar and other geographic, agronomic and climatic factors [
15,
16,
17].
Agro-industry byproducts can still contain a large number of phenolic compounds, and one of the richest sources are grape skins and seeds as grape pomace after winemaking, both fermented (red winemaking) and unfermented (white winemaking) [
18]. Generally, grape pomace accounts for 20–30% of the initial weight of the grape [
19], and its residual content of polyphenolic compounds depends on various factors such as grape variety, vintage and winemaking technique [
20,
21,
22].
The polyphenolic varietal characterization of grapes has been and still is the subject of numerous works [
21,
23,
24], aimed mainly to oenological purposes. The winemaking process, in particular the maceration operations, can be planned in order to define “tailor-made” winemaking techniques (varietal enology): the agronomic and enological practices may be more effectively directed toward the expression of some polyphenolic classes rather than others, depending on the expected results [
25,
26]. Similarly, the knowledge of the polyphenolic profile of the grape pomace is at the basis of its industrial exploitation (pharmaceutical, biomedical, nutraceutical applications). In both cases, the chemical characterization of the polyphenolic fraction is preceded by its extraction from the solid parts of the berry. To date no standard extraction method has been defined, and different approaches were chosen by different authors. Part of the existing literature regarding the sample treatment is reviewed in
Table 1.
Solvent extraction is one of the most frequently used techniques for the isolation of phenolic compounds [
18,
42,
43]. Solvents such as methanol, ethanol, acetone, diethyl ether, ethyl acetate, and their combinations have been widely used for the extraction of phenolics, often as aqueous mixtures with different proportions of water [
28,
44,
45]. Generally, ethanol (a dietary alcohol) is preferable than methanol in view of a food application of the extracts [
38].
Despite the high number of recent publications on the various properties of the winemaking byproducts, the studies on the differences in polyphenolic composition between the fresh grape and the corresponding pomace due to the fermentative maceration are still scarce. Some interesting works [
21,
22] concerned the polyphenolic characterization of some red grape cultivars mainly cultivated in France, and of their respective pomaces remaining after vinification. In the course of the present work four red grape varieties cultivated in northern Italy have been studied: Albarossa, Barbera, Nebbiolo, and Uvalino. Barbera and Nebbiolo are the two most widely cultivated varieties in Piedmont; Uvalino is a minor variety from Piedmont that has been particularly studied for its high content in polyphenolic compounds [
46,
47]; Albarossa, obtained from the crossbreed between Barbera and Chatus (or Nebbiolo di Dronero) [
48,
49] is appreciated for its richness in anthocyanins and the intense color of wines, and its use has been spreading in recent years mainly due to its attitude to late harvests.
The polyphenolic extracts from skins and seeds of the four red grape varieties sampled from different commercial wineries were prepared starting either from the fresh grapes and from the corresponding fermented grape pomace. The work was also aimed at studying the variations in the polyphenolic composition of the grapes caused by the fermentative maceration, in order to assess whether the varietal differences between cultivars observed at harvest remained after the fermentative maceration or, rather, disappeared, thus losing any importance when processing the winemaking byproducts for the extraction of polyphenolic compounds.
4. Discussion
The present work concerned the study of the polyphenolic composition of skins and seeds of 4 Italian red grape cultivars (Albarossa, Barbera, Nebbiolo, and Uvalino), derived from both the fresh grapes and the corresponding pomace at the end of the fermentative maceration.
The comparison between the polyphenolic composition of skins and seeds from fresh grapes allowed the evaluation of their oenological potential. The data used are those referring to the weight of the grapes (
Supplementary Table S1), which take into account the size of the grapes and the quantity of must in which the polyphenolic fraction is extracted.
As regards the grape skins, the total anthocyanins content for Barbera was about 2.5 times higher than for Nebbiolo, according to what reported by [
56] (ratios between 1.5 and 2.6), and it ranged between the values reported by [
56] and [
57] and those reported by [
58]. Regarding Nebbiolo, the total anthocyanins content was similar to the values reported by [
56] (480–550 mg/kg), and in our previous work [
59] (476–658 mg/kg). Albarossa skins had the highest total anthocyanin content among all cultivars, ranging between the values reported by [
48] (1978 mg/kg) and those reported by [
49] (2660 mg/kg). For Uvalino, [
46] found an average anthocyanins content of 900 mg/kg, lower than what observed during the present work, but, according to our results, higher than for Nebbiolo grapes.
As regards the polyphenolic profile of the grape skins, Albarossa was the richest in total flavonoids and total polyphenols, with concentrations similar to those reported by [
48]. Nebbiolo and Uvalino, on the other hand, were the richest in flavanic compounds (condensed tannins determined with phloroglucinolysis, flavans reactive with vanillin and proanthocyanidins measured by spectrophotometry). Regarding the spectrophotometric data, for Nebbiolo the values were similar to those reported by [
60], while for Uvalino the concentrations of proanthocyanidins and flavans reactive with vanillin were higher than those reported by [
47]. Finally, Barbera skins were the poorest in polyphenolic compounds, and the total flavonoids content was in accordance with [
56].
As regards the grape seeds, Nebbiolo and Uvalino were the richest in polyphenolic compounds, followed by Albarossa and Barbera, in accordance with the data reported in the bibliography, but in our case the measured concentrations of polyphenolic compounds were averagely higher than those reported in the bibliography. In the present work, the use of a 50% hydroalcoholic solution resulted in an increase in the extraction yields of the polyphenolic fraction from the seeds, compared to a 12% ethanol solution acidified at pH 3.4 and treated with 2 g/L of potassium metabisulphite, used by the authors who studied the same cultivars [
47,
48,
49,
56,
57,
58,
59]. During a previous work [
45] we observed that the extraction yields of the polyphenolic fraction from grape seed flour doubled when the ethanol concentration in the extracting solution increased from 25% to 50%. The same trend was observed by [
28] for grape skin extracts. The fact that only for the skins our results agreed with the cited works that used the SO
2 added 12% hydroalcoholic solution probably depends on the synergistic effect of sulfur dioxide. In fact, it is known that SO
2 has an extracting effect of polyphenols from the skin cells [
12]. On the contrary, SO
2 has no extracting effect on grape seeds.
Finally, as regards the composition in condensed tannins of skins and seeds, determined with the phloroglucinolysis method, there are no reference data in the literature about the fresh grapes of the four studied cultivars. As already described in detail in the results, the cultivar effect is evident also on the concentration and composition of condensed tannins. The cultivar effect on the variability of polyphenolic composition concerns also other plant species, such as olive leaves [
61]. To compare the polyphenolic composition of fresh grape skins and seeds with that of the corresponding fermented pomace (sampled after the end of fermentative maceration), the concentrations of the different classes of compounds were referred to the DW of flour. This method of presenting the results is currently used in the studies of chemical characterization of the oenological byproducts.
Considering the values reported by [
21], the total polyphenol content (GAE) of fresh grape skins, referred to the dry weight of flour, was averagely higher in the four studied cultivars than in Grenache, Carignan, and Mourvedre, but similar to Syrah. Regarding fresh grape seeds, the GAE values were averagely 2–3 times higher; Nebbiolo and Uvalino seeds were the richest in polyphenolic compounds.
The analyses of the polyphenolic profile were repeated for the fermented pomace. The fermentative maceration caused a strong reduction in the total polyphenol content (GAE) of skins and seeds. Regarding pomace skins, GAE values dropped by 80-90%. Higher GAE losses were observed for pomace seeds, in particular for Nebbiolo, Uvalino, and Albarossa. The total polyphenol contents of the seeds were much lower than those reported by [
21].
The correlations between the variables describing the polyphenols and anthocyanins content of the extracts and the ABTS index that measures the radical scavenging activity of the extracts were studied in order to verify which compositional parameter could be associated with the radical scavenging activity of the extracts. The correlation matrices (
Supplementary Tables S2 and S3) were calculated separately for skins and seeds, respectively.
The degree of multicollinearity among variables, referred to the skins, was low (
Supplementary Table S2), due to the characteristics of the cultivars considered: Albarossa and Barbera are rich in anthocyanins and poor in tannins while Nebbiolo and Uvalino are poor in anthocyanins and rich in tannins. This particular condition allowed the highlighting of differences between the variables regarding the correlations with the ABTS parameter that measures the radical scavenging capacity of the extracts. The ABTS parameter was closely related to the condensed tannins content (Pearson’s
r = 0.866), and to the content of proanthocyanidins (
r = 0.742) and vanillin-reactive flavans (
r = 0.748). On the contrary, no statistically significant correlations were observed between the ABTS parameter and the GAE index and the total flavonoid content, while the correlation coefficient between ABTS and total and monomer anthocyanins was negative. On the contrary, in the case of seeds the variables that describe the polyphenolic content were all highly correlated with each other and to the ABTS index. As already reported by other authors [
62], the absence of significant correlations between the GAE and ABTS values of the skin extracts may depend on the fact that the Folin-Ciocalteu method is not specific for the polyphenolic component and is therefore influenced by the presence of other compounds that do not necessarily have antiradical activity. The presence or absence of correlations between GAE and ABTS can therefore depend on the nature of the matrix used, differently from the analysis of condensed tannins which is specific for the flavan fraction.
The data matrix related to the polyphenolic profile of the skin extracts was subjected to the Principal Components Analysis (PCA). This technique, based on a reduced number of variables (Principal Components) calculated from the linear combination of the original variables, allows visualization of the spatial distribution of the skin samples of the 4 cultivars before and after fermentative maceration. The Principal Components are uncorrelated with each other and are numbered according to the decreasing order of the data variability they describe. In our case, the information contained in the original data matrix was described by 3 Principal Components, associated with a higher variability than the average variability of an original variable.
Figure 1 and
Figure 2 show the distribution of the skin extracts from fresh grapes and pomace in the space respectively defined by the 1st and 2nd and the 1st and 3rd Principal Components (PCA).
The 1st Principal Component, which describes the 71.06% of the total data variability, distinguishes the fresh grape skins from the pomace skins (effect of fermentative maceration on the polyphenolic composition of the skins). It is positively associated with the total polyphenol content (GAE, total flavonoids, total and monomer anthocyanins, vanillin-reactive flavans, proanthocyanidins, condensed tannins), the mean degree of polymerization (mDP) of condensed tannins, and the percentage weights of (+)-catechin, (−)-epicatechin and (−)-epicatechin-3-O-gallate present as extension units of condensed tannins. Furthermore, the 1st Component is negatively associated with the percentage weights of the monomers present as terminal units of the condensed tannins. Pomace skins are distinguished from fresh grape skins by the lower content of anthocyanins and tannins, the lower mDP, the higher percentage of monomeric units in terminal position and the higher total percentage content of (+)-catechin. Maceration caused a decrease in the anthocyanins and tannins content of the skins and, in parallel, some modifications of the condensed tannins composition (decrease of mDP and changes in the percentage weight of some monomers that make up the chains).
The 2nd and 3rd Principal Components respectively describe the 15.0% and 11.1% of the total data variability. The 2nd Component mainly distinguishes the Albarossa pomace skins from that of the other cultivars by the highest percentage concentration of (+)-catechin and (−)-epicatechin-3-O-gallate in condensed tannins. These differences could be due to different maceration conditions, in particular the duration.
The 3rd Principal Component distinguishes the different cultivars from one another. The distinction mainly concerns the fresh grape skins, while pomace skins are similar to each other. Barbera and Albarossa grapes are distinguished from Nebbiolo and Uvalino grapes, in particular for the total and monomer anthocyanins content, higher in Barbera and Albarossa, and for the content of flavans reactive with vanillin, proanthocyanidins, and condensed tannins, higher in Nebbiolo and Uvalino. The total polyphenol content (GAE), depending on both the anthocyanins and tannins contents, is similar for all cultivars. Moreover, among the extension units of condensed tannins, EGC is more abundant in Uvalino and Nebbiolo, while (+)-catechin is more abundant in Barbera and Albarossa.
Considering the seeds, the PCA identified only two Principal Components (93.42% of the total data variability);
Figure 3 shows the distribution of seed extracts in the space defined by the two Principal Components. The 1st Principal Component, which describes the 78.64% of the total data variability, distinguishes the grape seeds from the pomace seeds (effect of fermentative maceration on the polyphenolic composition of the seeds). Maceration caused a marked decrease in polyphenolic content of the seeds, and some changes in the condensed tannins composition. In fresh grape seeds the condensed tannins have a higher mean degree of polymerization (mDP) than in pomace seeds, and a consequent lower percentage of monomers in terminal position (C, EC, and ECG). After fermentative maceration, the percentage of monomers as extension units decreased, in particular EC, while the total percentage of C increased.
The second Component (14.78% of the total data variability) distinguishes Barbera and Albarossa from Nebbiolo and Uvalino, in particular for the EGC content, higher in the former than in the latter for both grape seeds and pomace seeds. On the contrary, the differences between cultivars due to the polyphenolic content of the seeds varied after fermentative maceration, and the ranking order of the cultivars for the analyzed parameters changed: Nebbiolo and Uvalino seeds, with the highest tannins content as fresh grapes, show after maceration significantly lower tannins concentrations than Barbera seeds, initially much poorer in tannins.
5. Conclusions
During the work, the characterization of the polyphenolic profile of skins and seeds of Albarossa, Barbera, Nebbiolo, and Uvalino, four cultivars extensively cultivated in northern Italy (Piedmont), was carried out. In particular, Barbera and Albarossa grapes had a similar polyphenolic composition and were significantly distinguished from Nebbiolo and Uvalino grapes for many of the analyzed parameters. In particular, regarding grape skins Barbera and Albarossa had a higher content of total and monomer anthocyanins and a lower content of flavans reactive with vanillin and condensed tannins; regarding grape seeds, Barbera and Albarossa had a lower content in flavans reactive with vanillin, proanthocyanidins, and condensed tannins than Nebbiolo and Uvalino. As regards the composition of condensed tannins (phloroglucinolysis results), a lower mDP and a lower percentage of EGC were observed in Barbera and Albarossa skins than in Nebbiolo and Uvalino skins.
The differences in polyphenolic profile observed between the fresh grapes of the different cultivars disappeared after fermentative maceration for almost all the analyzed parameters: the respective pomaces had a composition much more similar to one another. In addition to the duration of the contact between the solid parts of grapes and the fermenting must, the higher homogeneity of polyphenolic composition between the pomaces of different grape cultivars is also the consequence of the management of the fermentative maceration performed by the various wineries: for example, the modalities of the pomace mixing operations (intensity and duration), the adsorbent effect of the yeast strain, the use of maceration enzymes, the temperature, the amount of oxygen supplied, and so on. All these factors have been deeply studied with reference to the polyphenolic composition and color of the wines produced [
63], and only sporadically to the composition of exhausted pomace [
64]. Unlike the skins, the polyphenolic content of the seeds was extremely reduced and of little interest for the extraction of the polyphenolic fraction. On the contrary, the seeds separated from the grapes before fermentation or during the initial phases of the maceration process could represent an important source of polyphenolic compounds, and at this level the effect of the cultivar on the extraction yields would also be relevant.
As regards the evaluation of the polyphenolic composition of skin and seed extracts, the use of the total polyphenolic index (GAE) alone could be insufficient to discriminate different cultivars from one another: for a better knowledge of the raw material used, in view of the exploitation of byproducts for multiple purposes, it would be advisable to additionally analyze at least the total anthocyanin content by spectrophotometry and the flavanic fraction by spectrophotometry or HPLC. Furthermore, in the case of the skin extracts, the antiradical activity determined with the ABTS method resulted highly and positively correlated only with the flavans content, particularly when determined by phloroglucinolysis and not with the GAE parameter.
For red berry cultivars, the interest in the use of the winemaking byproducts mainly concerns the pomace collected at the end of fermentative maceration (with the sole exception of the pressing pomace derived from the production of white or rosé sparkling wines). Therefore, the prosecution of the work will concern the polyphenolic characterization of a high number of pomace samples of different varieties and provenience, in order to further deepen the knowledge of the potential and the compositional heterogeneity of this raw material, aimed at its industrial exploitation. Conversely, regarding the seeds, a study is in progress on the polyphenolic composition and the antioxidant properties of samples with different varietal origin, sampled during the first days of maceration (
délestage technique, [
63]), when the alcohol content is still low and the losses of polyphenolic compounds are limited.