1. ELECTROCHEMICAL BIOSENSING OF POLYPHENOLS CONTENT OF WINES
Juliusz ADAMSKI1, Jolanta KOCHANA1, Paweł NOWAK2, Andrzej PARCZEWSKI1
1 Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, Krakow, Poland
2 Institute of Catalysis and Surface Chemistry PAN, Niezapominajek 8, Krakow, Poland
INTRODUCTION
The important group of biologically active compounds of wines are phenols and polyphenols. Nutritional importance of phenolic constituents is related to their antioxidant power. They are known as anti-carcinogenic and anti-inflammatory substances, when they are regularly consumed. Therefore it is important to estimate the antioxidant capacity of wines related to phenols content.
The aim of this study was to exam the usefulness of simple electrochemical sensor in determination of phenolic compounds in wine samples. For this purpose tyrosinase and laccase, separately, were employed. These enzymes posses ability to oxidise mono- and diphenols to corresponding quinones which can be reduced at electrode surface at relatively low potentials.
The influence of sample matrix components on the sensor response was studied according to Plackett-Burman experimental design. The potential interferences: ethanol, tartaric acid, sorbate, sulfate(IV), putrescine, Fe(III) and glucose, which are usually encountered in wines, were taken into account in the examination. Because of significant matrix effect found, analyses of wine samples towards polyphenol contents were carried out using standard addition method (SAM) and expressed as gallic acid equivalent. For comparative quantification of phenolic compounds well-established Folin-Ciocalteau spectrophotometric method was employed.
Modelled structure of Streptomyces tyrosinase [1]
CONSTRUCTION OF A SENSOR
Modelled structure of Rigidoporus lignosus laccase [2]
MEASURING PROCEDURE
A small droplet of the solution of analyte and enzyme was placed on the surface of a circular glassy carbon electrode [3].
Glass tubing ended with the glass frit was positioned over the glassy carbon electrode (GCE) electrode in such a manner that the solution formed a thin layer between the surface of the GC electrode and the surface of the glass frit.
Into glass tubing with saturated KCl solution a high-area Ag/AgCl electrode was placed. GCE and Ag/AgCl reference electrode were connected through a resistor and current flowing in the circuit was measured as a potential drop on the it.
The scheme of the sensor is presented in Figure 1.
CHRONOAMPEROMETRIC MEASUREMENTS
100 μl of wine sample (without and with gallic acid) was shaken (1 min) with enzyme: 1 μl of 5mg/mL tyrosinase or 5 μl of laccase solutions;
then it was poured into crevice between GCE and reference electrode;
chronoamperometric response was recorded within 10 minutes and numerically integrated. Calibration curves were constructed as the Q = f(c) dependence, where Q [mC] was charge corrected for blank signal;
between measurements electrode surface was cleaned with double distilled water and methanol.
Analyses of wine samples were carried out using standard additions method (SAM).
Because white wines contain phenolic compounds at lower concentration narrower range of gallic acid (0-136 mg GA/L) compared to rose and red ones (0-272 mg GA/L) was used for construction of calibration curves.
SPECTROPHOTOMETRIC ANALYSIS of the total phenolic content by means of Folin-Ciocalteau method was performed according to [4].
Fig. 1 Scheme of experimental setup.
1 - WE (GC disc), 2 - poly(methyl metacrylate) block, 3 - Cu wire, 4 - Glass tubing, 5 - Ag /AgCl/sat. KCl electrode, 6 - sample
ANALYSIS OF WINE SAMPLES
Wine samples
Contents of phenols expressed as gallic acid [mg/L]
Sensor
Folin-Ciocalteau spectrophotometric method
tyrosinase
laccase
Red
Brandvlei Pinotage Cinsault
Sutter Home Zinfandel
Rose
Tribal Semi–Dry Rose
White
Solaria Zawisza
Michal Schneider Riesling
4769,8
9426,7
1863,5
1750,0
123,7
3836,6
5794,7
1138,6
1003,8
1763,6
2212,8
2322,3
473,4
239,0
218,8
STUDY OF MATRIX INFLUENCE ON SENSOR RESPONSE.
Plackett-Burman design for seven factors (concentration of interferents) and eight trials. Concentration of gallic acid: 272 mg/L; concentration of tyrosinase: 50 µg/L.
Solution
Factors (concentrations of interferents)
Mean response
[mC]
C2H5OH
[% vol.]
C4H8O6
[g/L]
SO32-
[mg/L]
Fe3+
C6H7O2-
C4H12N2
C6H12O6
TYR
LACC 1 15
7,5
280 5
500
0,5
0,2
0.055
0.065 2 25
2,5
0.073
0.088 3
12,5 30
0.063
0.068 4
0.083
0.064
0.014
0.023 6
0.027
0.040 7
0.012 8
0.035 E
-0.005
0.0195
-0.010
0.0001
-0.009
-0.006
tyrosinase
t
2.29
9.25
4.95
0.06
4.48
2.43
2.87
-0.002
0.020
-0.001
0.003
0.004
laccase
0.86
7.22
0.21
0.93
0.77
1.59
2.13
E – main effect of a factor; t – t-Student’s value for a factor; critical t-values for f = 8 degrees of freedom at significance levels  = 0.01 and =0.05: t(8;0.01) = 3.36 and t(8;0.01) = 2.31, respectively.
CONCLUSIONS
INFLUENCE OF MATRIX COMPOSITION
For both sensors, regardless of enzyme used, the most significant influence (signal increase, bj > 0) was observed for tartaric acid (C4H8O6).
For tyrosinase sensor, additional interferences (signal decrease, bj < 0) were observed in the presence of sulfate(IV) (SO32-), sorbate (C6H7O2-), putrescyne (C4H12N2) and glucose (C6H12O6). Reduction of signal in presence of mentioned above substances could be explained by their inhibiting influence on catalytic activity on tyrosinase. On the other hand, because of difference in substrate specificity of enzymes, they did not affect the signal of laccase sensor.
ANALYSIS OF WINE SAMPLES35
The results obtained based on tyrosinase were, apart from white wine Michal Schneider Riesling, significantly higher than those obtained with the use of laccase. This phenomenon can be probably attributed to difference in enzyme’s activity at selected pH of supporting electrolyte, and to different substrate specificity.
The considerably higher values of phenols content observed for electrochemical method comparing with spectrophotometric method suggest significant influence of additive type interferences.
REFERENCES
1. H. Claus, D. Hein, Systematic and Applied Microbiology 29 (2006) 3.
2. S. Garavagia, M.T. Cambria, M. Miglio, S. Ragusa, V. Lacobazzi, F. Palmeri, C. D’Ambrrosio, M. Rizzi, Journal of Molecular Biology 342 (2004) 1519.
3. J. Adamski, P. Nowak, J. Kochana, Electrochimica Acta 55 (2010) 2363.
4. A. L. Waterhouse (red), R. E. Wrolstad, Current Protocols In Food Analytical Chemistry, Wiley 2001, I, 1.1–1.8.