Desarrollo de novedosos biopolímeros reticulados para la estabilización protéica

de los vinos R. López1; F. Trotta2; P. Cottereau3; E. Vela1; E. Gracia-Moreno1; P.

Hernández-Orte1; J. Cacho1; V. Ferreira1

¹ Laboratorio de Análisis del Aroma y Enología. Facultad de Ciencias. Instituto de Investigación en Ingeniería de Aragón, Universidad de Zaragoza, 50.009 Zaragoza

2Dipt. Chimica, Universitá degli Studi di Torino, Italia 3Institut Français de la Vigne et du Vin, Francia

BPRs: Todos los BPRs desarrollados están

basados en esqueletos de β-ciclodextrinas entrecruzadas con

diferentes monómeros

Introducción Para eliminar las proteínas inestables del vino, cada año se utilizan en Europa cerca de 10.000 toneladas de bentonita, un tipo de arcilla proveniente en su mayor parte de otros continentes. A pesar de su eficacia y bajo coste, esta operación no gusta a los productores porque la bentonita tiende a atenuar y modificar el aroma y el sabor del vino, y el tratamiento provoca también pérdidas importantes del producto. Una posible solución puede encontrarse en el uso de biopolímeros. Se trata de compuestos de origen natural, obtenidos de productos secundarios de la industria agroalimentaria y que ya se están aprovechando en los sectores farmacéutico y cosmético.

Algunos biopolímeros reticulados (BRPs) pueden adsorber selectivamente la fracción de proteínas inestables del vino y pueden, por tanto, representar una alternativa interesante al uso extensivo de bentonitas como material adsorbente. En la presente comunicación se mostrarán los resultados preliminares obtenidos con los nuevos BRPs que se están desarrollando en el marco del proyecto europeo STABIWINE (www.stabiwine.eu)

Materiales y métodos

Conclusiones

Los BPRs desarrollados en el proyecto STABIWINE han demostrado ser competitivos con la bentonita desde el punto de vista de su eficacia en la estabilización proteica y también por su escaso impacto en el aroma. Los MIP-BPR presentan resultados prometedores para la eliminación

selectiva de compuestos responsables de malos olores en el vino. A la vista de los resultados obtenidos, los BPRs aparecen como alternativas viables y respetuosas con el medioambiente.

Resultados

Figura 2. Comparación de la eficacia en la estabilidad proteica de bentonita vs BPR para dos vinos blancos (azul oscuro Sauvignon, azul claro Sylvaner) medida con test de calor

Figura 3. Comparación impacto en aroma del tratamiento con BPR vs bentonita (azul oscuro Sauvignon, azul claro Sylvaner)

Laboratorio de Análisis del Aroma y Enología

Universidad de Zaragoza www.laae.es

STABIWINE –Deliverable D 1.1 – p. 14

synthesized are listed in table 4, along with Z potential and particle size measurements. As an example, the schematic

representation of βNS-TART-CITR is drawn in figure 15.

Table 4 - Z potential and particle size of ternary formulations saccharides – tartaric acid - citric acid.

Fig. 15 Schematic representation of βNS-TART-CITR, containing citric acid (black) and tartaric acid (red) units.

As it can be observed from the data presented in table 3, the addition of tartaric acid does not change significantly either

the particle size or the Z potential values of citric acid-based polymers. Similarly to what was observed for the binary

formulations TREHA-CITR and Linecaps-CITR, the introduction of an excess of carboxyl moieties, during the synthesis

process can impede the cross-linking reaction, leading to structures (e.g βNS-TART-CITR(1:12:8)) that show a lower

content of carboxyl groups, hence a less negative Z potential. As previously discussed, Z potential is an important

parameter that must be taken into account when studying systems for the absorption of proteins, since these

phenomena are mostly driven by electrostatic interactions. However, by crossing the data presented in this report with

the results collected by the Institut Français de la Vigne et du Vin, no clear and regular correlations can be found between

the intensity of Z potentials and the protein absorption capability of polymers, meaning that besides Z potential, other

parameters can influence, in ways that are not easily predictable, the affinity of negatively charged polymers for wine

proteins.

The advantages offered by citric acid-based polymers can be summarized as follows:

• The syntheses are conducted in water, so no organic solvents are required.

• The catalysts used are non-toxic inorganic salts, easily removed through filtration with water.

• No further purification, via Soxhlet extraction, is necessary.

Polymer Z potential Particle size

βNS-TART-CITR(1:4:8) -14,37 ± 1,54 mV 526,1 ± 16,4

βNS-TART-CITR(1:8:8) -27,77 ± 2,97 mV 538,74 ± 35,4

βNS-TART-CITR(1:12:8) -16,66 ± 1,76 mV 527,63 ± 42,8

CNN-TART-CITR(1:4:8) -14,37 ± 1,54 mV 546,4 ± 54,7

LineCaps-TART-CITR(1:4:8) -24,38 ± 1,27 mV 538,1 ± 47,4

Figure 1. Representación esquemática de β-ciclodextrinas entrecruzadas con ácido cítrico (negro) y funcionalizadas

con ácido tartárico (rojo)

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Métodos de análisis: 69 compuestos volátiles se

analizaron mediante GC-FID (Ortega et al, 2001) y GC-MS

(Lopez et al, 2002). La estabilidad proteica se evaluó mediante tests de calor y análisis electroforéticos.

Figura 4. BPR impreso molecularmente (MIP) para retirar selectivamente 4-vinilfenol. Comparación frente a control y bentonita en vino blanco

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STABIWINE –Deliverable D 1.1 – p. 9

3. CITRIC ACID-BASED POLYMERS A second kind of hyper cross-linked polyesters, were obtained from sugar-based molecules and citric acid through polycondensation reactions. Contrary to pyromellitic NSs, citric acid-based polymers are characterized by a more compact structure and show a lower water absorption capability.

Briefly, the syntheses were conducted in water in the presence of inorganic salts, which act as catalysts. The water produced by the cross-linking process, as well as the water used as solvent, was removed by heating the solutions. After the synthesis, all the polymers were ground in a mortar and rinsed with a great amount of water through Buchner filtration. In order to optimize the synthesis process and to enhance the reaction yields, different synthesis routes were explored. Since potential industrial applications are involved, the details of the synthesis reactions are kept confidential and are not reported in the following pages.

3.1 Synthesis and characterization of citric acid-based Nanosponges Citric acid-based NSs were obtained by cross-linking β-CD and CNN with citric acid and named βNS-CITR and CNN-CITR, respectively. The reactions were carried out at different molar ratios. The synthesized NSs are listed below:

βNS-CITR(1:4), βNS-CITR(1:16), βNS-CITR(1:28)

CNN-CITR(1:6), CNN-CITR(1:12), CNN-CITR(1:16)

(the numbers in brackets represent the molar ratio β−CD, CNN : citric acid respectively)

In figure 12 the infrared spectra of citric acid-based NSs are reported. The absorption peaks placed at approximately 1715-1720 cm-1 indicate the presence of carbonyl groups (C=O stretching), introduced by citric acid. As expected, these signals are not observed in the spectra of β-CD and CNN (the weak absorption detected at ~1645 cm-1 in both β-CD and CNN spectra is due to the deformation of O-H moieties). The intensity of this peak changes according to the cross-linker concentration: the greater the amount of citric acid, the higher the absorption intensity. A further evidence of the presence of citric acid in the structure of βNS-CITR and CNN-CITR is represented by the peak at 1150-1170 cm-1, mostly caused by C-OH stretching vibrations; this peak, although already visible in the spectra of β-CD and CNN, shows an intensity that grows proportionally to the cross-linker concentration.

+

β-CD or CNN

Citric acid

Heat

Fig. 11 Synthesis of βCD-AcCITR.

Inorganic salts, water

Referencias: Lopez et al. (2002) Determination of minor and trace volatile compounds in wine by solid-phase extraction and gas chromatography with mass spectrometric detection. J Chrom A 966, 167–177. Ortega et al (2001) Fast analysis of important wine volatile compounds Development and validation of a new method based on gas chromatographic–flame ionisation detection analysis of dichloromethane microextracts. J Chrom A 923, 205–214.

The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/ 2007-2013) under the grant agreement n° 314903

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