Electrochemical Estimation of the Polyphenol Index in Wines Using a Laccase Biosensor

Abstract

The use of a laccase biosensor, under both batch and flow injection (FI) conditions, for a rapid and reliable amperometric estimation of the total content of polyphenolic compounds in wines is reported. The enzyme was immobilized by cross-linking with glutaraldehyde onto a glassy carbon electrode. Caffeic acid and gallic acid were selected as standard compounds to carry out such estimation. Experimental variables such as the enzyme loading, the applied potential, and the pH value were optimized, and different aspects regarding the operational stability of the laccase biosensor were evaluated. Using batch amperometry at -200 mV, the detection limits obtained were 2.6 x 10(-3) and 7.2 x 10(-4) mg L(-1) gallic acid and caffeic acid, respectively, which compares advantageously with previous biosensor designs. An extremely simple sample treatment consisting only of an appropriate dilution of wine sample with the supporting electrolyte solution (0.1 mol L(-1) citrate buffer of pH 5.0) was needed for the amperometric analysis of red, rosé, and white wines. Good correlations were found when the polyphenol indices obtained with the biosensor (in both the batch and FI modes) for different wine samples were plotted versus the results achieved with the classic Folin-Ciocalteu method. Application of the calibration transfer chemometric model (multiplicative fitting) allowed that the confidence intervals (for a significance level of 0.05) for the slope and intercept values of the amperometric index versus Folin-Ciocalteu index plots (r = 0.997) included the unit and zero values, respectively. This indicates that the laccase biosensor can be successfully used for the estimation of the polyphenol index in wines when compared with the Folin-Ciocalteu reference method.

Biosensors for the Polyphenolic Content of Wine Determination

“…HRP was used as the recognition enzyme, at pH 6 -9 [12]. Tyrosinase, at pH 7.4 was used in [6] and laccase at pH 5.0 in [4]. Tyrosinase and buffer at pH 8.5 were used in [3] .…”

“…Most laboratories determine the level of total phenol in wines by the FolinCiocalteu method, which is not an absolute measurement of the amount of phenolic materials but is based on their chemical reducing capacity relative to an equivalent reducing capacity of gallic acid in most situations [2]. However, caffeic acid is one of the phenols present in a high amount in wine and has been used as reference by some authors [3,4], when measuring the total content of polyphenols in wine. Caffeic acid has been studied by several groups [3][4][5][6][7], who found that it was the preferred substrate among other polyphenols analyzed, being redox recycled between the adsorbed laccase (enzymatic oxidation reaction) and the electrode (electrochemical reduction reaction).…”

“…However, caffeic acid is one of the phenols present in a high amount in wine and has been used as reference by some authors [3,4], when measuring the total content of polyphenols in wine. Caffeic acid has been studied by several groups [3][4][5][6][7], who found that it was the preferred substrate among other polyphenols analyzed, being redox recycled between the adsorbed laccase (enzymatic oxidation reaction) and the electrode (electrochemical reduction reaction). However, apart from our group, none of these authors studied the system at the pH of importance for application in wine, which is 3.5.…”

“…However, apart from our group, none of these authors studied the system at the pH of importance for application in wine, which is 3.5. In fact, buffer at pH 8.5 was studied in [3], and the groups in [6,7] used buffers at pH 5.0, while those in [4] worked at pH 7.4. The aim of the present work was to study matrices at a pH value closer to the average pH of red wines (ca 3.5) than the previous authors did.…”

Wood production, wood technology, and biotechnological impacts

“…Enzyme immobilisation is an integral part in the production of laccase-based biosensors constructed to measure concentrations of phenolic compounds in waste waters and other samples taken from the environment, to determine the phenol contents in wines, teas, and other beverages, to define concentrations of compounds in pharmaceutical formulations and medical samples, to screen plant extracts for phenolic compounds, and to detect toxic phenolic chemicals including xenobiotic compounds in various other materials (Ghindilis et al 1992, Yaropolov et al 1994, Leite et al 2003, Gomes et al 2004, JaroszWilkolazka et al 2004, Gamella et al 2006, Vianello et al 2006; see also Chapter 12 of this book). In addition, the involvement of molecular oxygen in laccase catalysis allows to measure oxygen with such biosensors, even at rather low levels (Gardiol et al 1996).…”