**3. Results and discussion**

#### **3.1 Phenolic content of wine lees, grape pomace and grape stems**

In the present work, the determination of the phenolic composition and the antioxidant capacity of wine lees, grape stems, and grape pomace extracts of two grape (*Vitis vinvifera* L.) varieties (Sousão and Tinta Barroca) were performed. The phenolic content of these samples collected from Douro region (Northern Portugal) was presented in **Table 1**. As it can be observed, in general, grape stems were the WPBs with the highest content of total phenols (168.75 mg GA g−1 DW, on average), *ortho*-diphenols (166.39 mg GA g−1 DW, on average), and flavonoids (152.31 mg CAT g−1 DW, on average), followed by grape pomace and wine lees. Concerning this last winery by-product, it can be stated that the samples from Sousão variety showed the lowest content of these three studied parameters, being significantly different from the other variety and WBPs (p < 0.05). In fact, wine lees and grape stems presented the highest values of phenolic content in Tinta Barroca samples which has not been observed for the grape pomace extracts, which can be explained by the different phenolic compounds present in these WBPs.

Romero et al. obtained similar values in wine lees of total phenols (38–254 mg CAT g−1) and flavonoids (16–146 mg CAT g−1) content from the Tempranillo variety, with these ranges caused by the extraction solvent employed by these authors [16]. However, Pérez-Serradilha et al. [17] obtained higher values in this WBP of total phenols content (364 mg g−1) than those obtained in this work after a microwaveassisted extraction optimized. Our research group have analyzed the phenolic content of grape stem extracts prepared with conventional extraction methods (hydro-methanolic solvents) [3, 4, 11, 18].

The values ranged between 32 and 123 mg GA g−1 DW for total phenols, between 35 and 116 mg GA g−1 DW for *ortho*-diphenols, and from 34 to 106 mg CAT g−1 DW for flavonoids, depending on cultivar, geographical localization, crop season, among other factors [4, 18, 19]. The values of the present work were slightly higher than the values obtained in those studies, maybe due to the new efficient extraction method performed in this work (UAE). Grape pomace has been also analyzed by other authors concerning its phenolic content, obtaining values around 40 mg GA g−1 DW for total phenols and around 14 mg CAT g−1 DW for flavonoids which are significantly lower than those obtained in this work.



**9**

*Winery By-Products as Source of Bioactive Compounds for Pharmaceutical and Cosmetic…*

solvent, with particular advantageous for natural sources [8, 20].

**3.2 Antioxidant capacity of wine lees, grape pomace and grape stems**

In fact, these contents are particularly dependent of several factors, such as the agronomic conditions [3], cultivar [4] and extraction methods employed [6]. In this sense, in order to reduce production costs and optimize processes, new technologies—such as ultrasound-assisted extraction (UAE) or microwave-assisted extraction (MAE)—have been employed to decrease energy consumption and increase the product or process safety/control and quality. These techniques, already used at large scale, emerged as efficient, energy/time-saving and clean extraction methodologies, providing higher recoveries of bioactive compounds using low amounts of

The antioxidant capacity of the WBPs investigated in this work was performed by three methods (ABTS, DPPH, and FRAP), being the results presented in **Table 1**. As expected, due to the correlation of most phenolic compounds with antioxidant capacity, Tinta Barroca samples presented the highest values for all the methods concerning wine lees and grape pomace samples. In contrast, grape pomace samples from Sousão variety showed the highest antioxidant capacity for ABTS (5.54 ± 0.09 mmol Trolox g−1 DW), DPPH (1.64 ± 0.03 mmol Trolox g−1 DW), and FRAP (1.75 ± 0.01 mmol Trolox g−1 DW) methodologies, being significantly different from the Tinta Barroca

Romero et al. [16] also determined the antioxidant capacity of wine lees extracts from Tempranillo variety, obtaining values ranged between 0.46 and 2.197 mmol Trolox g−1. The values obtained in the present work are in the range of those presented by these authors, concerning FRAP method. In literature, grape stem extracts have been also analyzed concerning this biological property, which present antioxidant capacities of 0.35–0.84 mmol Trolox g−1 DW, 0.15–0.76 mmol Trolox g−1 DW, and 0.33–1.03 mmol Trolox g−1 DW for ABTS, DPPH, and FRAP methodologies [4, 18, 19], which were lower than those presented in the present study essentially due to several factors, such as different extraction methods, extraction

The phenolic profile of wine lees, grape pomace and grape stems was performed by RP-HPLC-DAD, being the results presented in **Table 2**. Fifteen compounds were identified, being grape pomace samples the ones with more phenolic compounds identified, including phenolic acids, flavanols, and anthocyanins. In this study, it was possible to observe the same behavior referred above, namely the significant highest content of the phenolic compounds identified in grape pomace extracts from Sousão variety (p < 0.05). Similar compounds were identified in wine lees, namely gallic acid, catechin, epicathechin, and malvidin-3-*O*-gluside, beside others which were found only in this by-product, namely protocatechuic acid (0.337 mg g−1 DW, on average), delphinidin-3-*O*-glucoside (0.190 mg g−1 DW, on average), and petunidin-3-*O*-glucoside (0.268 mg g−1 DW, on average). All these compounds were also determined in higher concentrations in Tinta Barroca samples which is in agreement with the previous results reported above. Grape stem extracts presented three phenolic compounds which were not identified in wine lees and grape pomace, namely isorhammetin-3-*O*-(6-*O*-feruloyl)-glucoside, caftaric acid, and ԑ-viniferin, which were also present in higher concentration in Tinta Barroca

*DOI: http://dx.doi.org/10.5772/intechopen.97881*

samples (p < 0.05).

solvents or protocols, varieties, among others.

**3.3 Phenolic profile of wine lees, grape pomace and grape stems**

samples, being significantly different from Sousão samples (p < 0.05).

Several compounds identified in this work have been also identified by other authors in these WBPs, namely gallic acid, catechin, delphinidin-3-*O*-glucoside,

*Total phenols (mg GA g−1 DW),* ortho*-diphenols (mg GA g−1 DW), and flavonoids (mg CAT g−1 DW) content and antioxidant capacity (mmol Trolox g−1 DW) of WBPs from Sousão and Tinta Barroca varieties.* *Winery By-Products as Source of Bioactive Compounds for Pharmaceutical and Cosmetic… DOI: http://dx.doi.org/10.5772/intechopen.97881*

In fact, these contents are particularly dependent of several factors, such as the agronomic conditions [3], cultivar [4] and extraction methods employed [6]. In this sense, in order to reduce production costs and optimize processes, new technologies—such as ultrasound-assisted extraction (UAE) or microwave-assisted extraction (MAE)—have been employed to decrease energy consumption and increase the product or process safety/control and quality. These techniques, already used at large scale, emerged as efficient, energy/time-saving and clean extraction methodologies, providing higher recoveries of bioactive compounds using low amounts of solvent, with particular advantageous for natural sources [8, 20].

#### **3.2 Antioxidant capacity of wine lees, grape pomace and grape stems**

The antioxidant capacity of the WBPs investigated in this work was performed by three methods (ABTS, DPPH, and FRAP), being the results presented in **Table 1**. As expected, due to the correlation of most phenolic compounds with antioxidant capacity, Tinta Barroca samples presented the highest values for all the methods concerning wine lees and grape pomace samples. In contrast, grape pomace samples from Sousão variety showed the highest antioxidant capacity for ABTS (5.54 ± 0.09 mmol Trolox g−1 DW), DPPH (1.64 ± 0.03 mmol Trolox g−1 DW), and FRAP (1.75 ± 0.01 mmol Trolox g−1 DW) methodologies, being significantly different from the Tinta Barroca samples (p < 0.05).

Romero et al. [16] also determined the antioxidant capacity of wine lees extracts from Tempranillo variety, obtaining values ranged between 0.46 and 2.197 mmol Trolox g−1. The values obtained in the present work are in the range of those presented by these authors, concerning FRAP method. In literature, grape stem extracts have been also analyzed concerning this biological property, which present antioxidant capacities of 0.35–0.84 mmol Trolox g−1 DW, 0.15–0.76 mmol Trolox g−1 DW, and 0.33–1.03 mmol Trolox g−1 DW for ABTS, DPPH, and FRAP methodologies [4, 18, 19], which were lower than those presented in the present study essentially due to several factors, such as different extraction methods, extraction solvents or protocols, varieties, among others.

#### **3.3 Phenolic profile of wine lees, grape pomace and grape stems**

The phenolic profile of wine lees, grape pomace and grape stems was performed by RP-HPLC-DAD, being the results presented in **Table 2**. Fifteen compounds were identified, being grape pomace samples the ones with more phenolic compounds identified, including phenolic acids, flavanols, and anthocyanins. In this study, it was possible to observe the same behavior referred above, namely the significant highest content of the phenolic compounds identified in grape pomace extracts from Sousão variety (p < 0.05). Similar compounds were identified in wine lees, namely gallic acid, catechin, epicathechin, and malvidin-3-*O*-gluside, beside others which were found only in this by-product, namely protocatechuic acid (0.337 mg g−1 DW, on average), delphinidin-3-*O*-glucoside (0.190 mg g−1 DW, on average), and petunidin-3-*O*-glucoside (0.268 mg g−1 DW, on average). All these compounds were also determined in higher concentrations in Tinta Barroca samples which is in agreement with the previous results reported above. Grape stem extracts presented three phenolic compounds which were not identified in wine lees and grape pomace, namely isorhammetin-3-*O*-(6-*O*-feruloyl)-glucoside, caftaric acid, and ԑ-viniferin, which were also present in higher concentration in Tinta Barroca samples, being significantly different from Sousão samples (p < 0.05).

Several compounds identified in this work have been also identified by other authors in these WBPs, namely gallic acid, catechin, delphinidin-3-*O*-glucoside,

*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

**8**

**Phenolic content**

*Ortho***-diphenols**

**Flavonoids**

**ABTS**

**Total phenols**

**Sousão**

Wine

Z15.44 ± 1.25a

125.39 ± 1.12b

118.91 ± 0.95a

136.03 ± 1.10b

18.50 ± 0.89a

128.34 ± 1.07b

1.71 ± 0.01a

3.28 ± 0.03b

1.24 ± 0.03a

1.58 ± 0.03c

1.54 ± 0.01a

1.96 ± 0.03b

lees

Grape

153.70 ± 0.53d

135.32 ± 2.76c

151.78 ± 1.89c

138.70 ± 1.42b

144.81 ± 1.75c

129.93 ± 0.93b

5.54 ± 0.09d

4.01 ± 0.03c

1.64 ± 0.03d

1.59 ± 0.01c

1.75 ± 0.01d

1.61 ± 0.02b

pomace

Grape

156.81 ± 1.29e

180.68 ± 2.77f

162.53 ± 1.01d

170.24 ± 1.88e

143.90 ± 2.13c

160.71 ± 1.44d

5.62 ± 0.02d

8.02 ± 0.02e

1.49 ± 0.07e

1.85 ± 0.02c

1.69 ± 0.01c

2.02 ± 0.01e

stems

*P*-value

Y\*\*\* *YLevel of significance: N.s.: not significant ( > 0.05);*

∗*significant at < 0.05;*

∗∗*significant at < 0.01;*

∗∗∗*significant at < 0.001.*

**Table 1.**

*Barroca varieties.*

*Total phenols (mg GA g−1 DW),* ortho*-diphenols (mg GA g−1 DW), and flavonoids (mg CAT g−1 DW) content and antioxidant capacity (mmol Trolox g−1 DW) of WBPs from Sousão and Tinta* 

\*\*\*

\*\*\* *ZData presented as Mean (n = 3) ± SD values for the same parameter evaluated followed by different superscript lowercase letters are significantly different at p < 0.001, according to Tukey's test.*

\*\*

\*\*

\*\*

**Tinta** 

**Sousão**

**Tinta** 

**Sousão**

**Tinta** 

**Sousão**

**Tinta** 

**Sousão**

**Tinta** 

**Sousão**

**Tinta** 

**Barroca**

**Barroca**

**Barroca**

**Barroca**

**Barroca**

**Barroca**

**Antioxidant capacity**

**DPPH**

**FRAP**


**Table 2.**

**11**

**Author details**

**Acknowledgements**

Irene Gouvinhas and Ana Barros\*

Agri-Food Production, Vila Real, Portugal

provided the original work is properly cited.

\*Address all correspondence to: abarros@utad.pt

Centre for the Research and Technology of Agro-Environmental and Biological Sciences, University of Trás-os-Montes and Alto Douro (CITAB-UTAD), Inov4Agro - Institute for Innovation, Capacity Building and Sustainability of

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Winery By-Products as Source of Bioactive Compounds for Pharmaceutical and Cosmetic…*

epicatechin, *p*-coumaric acid, petunidin-3-*O*-glucoside, malvidin-3-*O*-glucoside, among others [16, 17, 21, 22]. However, it is well known that the extraction method, the cultivars, the geographical conditions, the growing season, the plant diseases, among others, affect the chemical composition of WBPs, namely the secondary metabolites

Nowadays, it is well established in the scientific community that wine has an important role in the prevention of some cardiovascular diseases, resulting from their content in bioactive phytochemicals with antioxidant capacity. Many of these compounds are derived from the solid parts of the grape cluster (stem, pomace, trimmed vine shoots, and wine lees). However, during the winemaking process, a complete extraction of these compounds to the juice/wine does not occur, and they may remain at high concentrations in certain wastes, such as in the stems. Indeed, the wine industry involves the production of large quantities of by-products, characterized by a valuable composition in phytochemicals with putative health-promoting qualities. Additionally, in light of the biological properties revealed recently, the search for natural bioactive

compounds has paid attention on these materials as promising alternatives.

conventional extraction methods employed by the research group.

industries, giving rise to sustainable agro-industrial activities.

In this work, it was possible to observe the high content of phenolic compounds and the high antioxidant capacities demonstrated by several winery by-products, namely wine lees, grape pomace, and grape stems which were subjected to an ultrasound assisted extraction, obtaining higher values than those obtained by

In this sense, the phenolics present in winery by-products may have an addedvalue to be used as an alternative to synthetic substances employed in distinct

This work is supported by National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020 (CITAB). We also acknowledge Eng. Manuel Henrique from ROZÉS who kindly provided the samples.

*DOI: http://dx.doi.org/10.5772/intechopen.97881*

which are highly present in these matrices.

**4. Conclusions**

∗*significant at < 0.05;*

∗∗*significant at < 0.01;*

∗∗∗*significant at < 0.001. nd: not detected.*

*Content of individual phenolics (mg g−1 dw) of WBPs from Sousão and Tinta Barroca varieties. Statistical treatment notes: Data were subjected to analysis of variance (ANOVA) and multiple range test (Tukey's test) with a significance of p < 0.05.*

*Winery By-Products as Source of Bioactive Compounds for Pharmaceutical and Cosmetic… DOI: http://dx.doi.org/10.5772/intechopen.97881*

epicatechin, *p*-coumaric acid, petunidin-3-*O*-glucoside, malvidin-3-*O*-glucoside, among others [16, 17, 21, 22]. However, it is well known that the extraction method, the cultivars, the geographical conditions, the growing season, the plant diseases, among others, affect the chemical composition of WBPs, namely the secondary metabolites which are highly present in these matrices.
