CHEMICAL CHARACTERIZATION OF GRAPE STALKS FROM RED GRAPE POMACES

SÓNIA O. PROZIL§ ‡, JOANA A. MENDES§ ‡, DMITRY V. EVTUGUIN§, LUÍS T. DE LEMOS‡, LUÍSA P. LOPES‡

§CICECO and Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal ‡ CI&DETS and Department of Environment, Polytechnic Institute of Viseu, 3504-510 Viseu, Portugal

1. Introduction

The vineyard culture is spread out through all Continents, in which the European Union (EU)

represents 60% of the world production of wine. Portugal, with an annual production of about 7

million hL, is tenth in the world ranking and fifth at the European level (Figure 1) [1]. From the

winemaking process comes the wine, as well as its different by-products (grape skins, grape

stalks, grape seeds, lees and tartar). Among by-products of wine production 100 litters of wine

originates 25 to 31 kg of by-products, depending if we're talking about red or white wine making;

and 1 hL of wine generates 3 to 4 Kg of grape stalks. Regarding the average annual production of

wine, Portugal produces around 24 500 tons of grape stalks per year [2].

2. Chemical Composition

5572

5023

3638

2500

1351

1083

1052

978

761

745

603

533

435

425

400

0 1000 2000 3000 4000 5000 6000

France

Italy

Spain

USA

Argentina

China

Australia

Germany

South Africa

Portugal

Chile

Romania

Hungary

Greece

Russia

1000 Tons

Figure 1: Main world producers of wine [1].

Parameters Content (%)

Ashes 7.0

Extractives

2.3

1.0

23.7

Proteins 6.1

Tannins 15.9

Klason Lignin 17.4

Cellulose Kürscher and Höffer 30.3

Hemicelluloses 21.0

Acetone

Dichloromethane

Water

Table III: Quantitative estimation of different structural

elements in grape stalks dioxane lignin from 13C NMR

spectrum.

Aiming a better understanding of grape stalks and the evaluation of its potential as a source of

new materials, particularly for the acquiring of cellulosic pulp the general chemical composition

have been evaluated. Table I presents the grape stalk’s chemical composition and Table II the

results of sugar's analysis.

The chemical characterization showed that grape stalks are lignocellulosic materials, in which

cellulose is the major component (with around 30%), followed by insolvent hemicelluloses in

hot water (21%) and lignin (17.4%). Regarding other components, it’s important to highlight

the high presence of tannins (15.9%) and extractatives in water (23.7%). Taking into account

the monosaccharide’s analysis (Table II), it can be verified that, after cellulose, xylans are the

second major polysaccharide component present in grape stalks (with around 12%).

References [1] - Vitivinicultura - Diagnóstico Sectorial, Gabinete de planeamento e políticas, 2007, Ministério da Agricultura e do Desenvolvimento Rural e das

Pescas.

[2] - Costa, M., Aproveitamento de Subprodutos da Vinificação. O Bagaço como Matéria-Prima da Indústria de Óleos e de Fabrico de Rações para

Gado, in 1º Congresso Nacional das Industrias Agro-Alimentares, 1983, Ministério da Agricultura Comércio e Pescas.

[3] - Evtuguin, D.V., et al., Comprehensive Study on the Chemical Structure of Dioxane Lignin from Plantation Eucalyptus globulus Wood. J. Agric.

Food Chem., 2001, 49(9): pp. 4252-4261.

3. Cellulose

4. Lignin

5. Xylans

6. Conclusions

Cellulose, the most abundant polymer of grape stalks, was isolated by the Kürscher and Höffer

method and characterized by X-ray diffraction to acquire information regarding its crystalline

structure. The diffractogram (Figure 2) demonstrates that this cellulose is Cellulose I with

characteristic reflexes at ca. 2θ =16 and 22º. The degree of crystallinaty obtained was of about

75%. The dimensions of the unitary cell (a, b and c) and the γ angle are presented below.

In this work the chemical composition and the structure of main macromolecular components of

grape stalks from red grape pomaces have been evaluated aiming to provide the new

strategies for their processing to value added products.

[ -D-Xylp]-(1g4)- [ -D-Xylp]4-(1g4)- [ -D-Xylp]57 -(1g4)- [ -D-Xylp]19 -(1g4)- [ -D-Xylp]14-(1g4)- [ -D-Xylp]6-(1g

2

1 4-O-Me- -D-GlcpA

3

Ac

3

Ac

2 3 2

Ac Ac Ac

Lignin was isolated by acidolysis using a solution

of dioxane-water (9:1 v/v) under acidic conditions

[3]. The nuclear magnetic resonance spectroscopy

is a technique used for the characterization,

classification and structure determination of

lignins. Figure 3 presents the HSQC spectrum of

dioxane lignin, which shows the predominance of

β-O-4, β-5, β-β and β-1 linkages in lignin’s

structure. The 13C NMR analysis allowed the

estimation of the amounts of particular structures.

Table III presents different structural elements in

grape stalks dioxane lignin as reveaved from

13CNMR spectrum.

Monosaccharide’s %(wmon/ wt mon)

Rhamnose 1.7

Fucose <0.2

Arabinose 5.5

Xylose 20.4

Mannose 4.8

Galactose 4.9

Glucose 62.7

Structural element Number / aromatic

group C6

β-O-4 0.36

β-β + β-5 0.08

Ar-O 2.07

Ar-C 1.66

Ar-H 2.27

G:S 73:27

Unit Cell Dimensions

a – 8.0 nm

b – 1.03 nm

c – 7.9 nm

γ - 91º

Table II: Neutral sugars in grape stalks.

Table I: Chemical composition of grape stalks (% dry material).

Figure 3: Aliphatic side-chain region of HSQC spectrum

of grape stalks dioxane lignin.

Ackowledgment Sónia Prozil wish to thank to TAVFER group for supplying the grape stalks samples and for the financial support.

The xylans were extracted with DMSO from the holocelluloses obtained by delignification of grape

stalks with peracetic acid. The DMSO-extracted xylan was characterized by 1D and 2D NMR, which

allowed the proposition of an empiric structure of the grape stalks O-acetyl-(4-O-methyl- -D-

glucurono)xylan as presented below:

The β-O-4 structures are the most

predominant, representing about 36% of the

linkages, followed by the β-β e β-5 structures

with about 8%. It was not possible to quantify

the β-1structure by 13C NMR, but its presence

was confirmed by HSQC. Lignin was also

characterized by nitrobenzene oxidation, from

which the ratio between the main structural

units (H:G:S) was estimated. The found ratio

was of 7:71:26, concordant with the results

obtained by 13C NMR (Table III).

This work allowed a deeper knowledge regarding the chemical composition of grape stalks to

evaluate its potentialities towards cellulosic pulp and biocomposites production. The cellulose

content in grape stalks was relatively low though unusually high degree of its crystallinity was

detected. The heteroxylan was the second in abundance after cellulose polysaccharide in grape

stalks possessing a low degree of the ramification with uronic moieties. The grape stalks lignin is a

HGS type lignin with predominance of G units, very condensed and structurally associated to

tannins.

ppm (t2)

3.003.504.004.505.005.506.006.50

50

60

70

80

90

ppm (t1)

Cβ in β-1

Cβ in β-5

Cβ in β-β

Cγ in β-1

Cβ in β-5

Cγ in β-O-4

Sugars

Cγ in β-β

Cα in β-O-4

Cα in β-1 Cα in β-O-4

Cβ in β-5 Cγ in β-β

Ic Ic

Iam

2θ (º)

Inte

ns

ity

Figure 2: X-ray difractogram of cellullose isolated by the Kürscher and Höffer method.