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STRATEGIES FOR THE DEVELOPMENT OF MALATE SENSORS DEVOTED TO WINEMAKING MONITORING

STRATEGIES FOR THE DEVELOPMENT OF MALATE SENSORS DEVOTED TO WINEMAKING MONITORING. WHY TO DETECT MALIC ACID ? THE MALOLACTIC FERMENTATION (MLF). MLF = secondary fermentation, occurs after alcoholic fermentation, lasts from 2 weeks to several months (if T is too low).

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STRATEGIES FOR THE DEVELOPMENT OF MALATE SENSORS DEVOTED TO WINEMAKING MONITORING

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  1. STRATEGIES FOR THE DEVELOPMENT OF MALATE SENSORS DEVOTED TO WINEMAKING MONITORING

  2. WHY TO DETECT MALIC ACID ? THE MALOLACTIC FERMENTATION (MLF) MLF = secondary fermentation, occurs after alcoholic fermentation, lasts from 2 weeks to several months (if T is too low). -Transformation of malic acid (diacide) in lactic acid (monoacide) - Bacterial process (Oenococcus oeni) - Deacidification: decrease in titratable acidity and increase in pH - Wine stabilisation and flavour change MLF is usually encouraged for all dry red wines: [Malic acid] in musts: 1-5 g/L [Malic acid] in red wines : 0-0.1 g/L MLF is avoided or partially performed for white wines. MONITORING OF MLF IS FUNDAMENTAL FOR WINE PRODUCERS.

  3. + C O N A D H + H 2 C O O H C H O H 2 C H 3 C H Alcool dehydrogenase C H 3 3 Acetobacter Acetic acid C O 2 C O O H C O O H C H O H C H O H C H C H 3 2 C O O H SUBSTRATES OF INTEREST ALCOHOLIC FERMENTATION(Saccharomyces cerevisiae) + N A D H Sugars C O C O Pyruvate decarboxylase Ethanol Ethanal Pyruvate Lactic bacteria D-lactate L-malate L-lactate MALOLACTIC FERMENTATION(Oenococcus oeni)

  4. Suitable for any winery Low cost but low speed and accuracy. WIDELY USED METHOD : PAPER CHROMATOGRAPHY Legend : T - Tartaric acid L - Lactic acid M - Malic acid

  5. ENZYMATIC RECOMMENDED METHOD Malate dehydrogenase L-MDH (EC 1.1.1.37) Spectrophotometric determination at 340nm (e = 6300 M-1.cm-1) L-malate + NADH + H+ Oxaloacetate + NAD+ GOT* + L-glutamate * Glutamate-oxaloacetate transaminase (EC2.6.1.1) L-aspartate + 2-oxoglutarate Costly, not adapted to small wineries Laboratory analysis : delays between sampling and results. Need of easy and portable analytical devices as BIOSENSORS

  6. PRINCIPLE OF DH-BASED BIOSENSORS DEHYDROGENASE (DH) PRODUCT SUBSTRATE + NADH + H+ + NAD+ Optical detection (=340 nm) AMPEROMETRIC DETECTION Mediated oxidation (monoenzymaticsystem) Direct oxidation High Potential No selectivity Bienzymatic systems

  7. BI-ENZYMATIC SENSOR MONO-ENZYMATIC SENSOR THE MALATE DEHYDROGENASE-REACTION : DIFFERENT OPTIONS FOR SENSOR DEVELOPMENT Malate dehydrogenase L-MDH (EC 1.1.1.37) L-malate + NADH + H+ Oxaloacetate + NAD+ High concentrations of NAD+ + appropriate mediator Enzymatic consumption of NADH (regeneration of NAD+)

  8. NADH + H+ NAD+ Mandatory addition of ferricyanide Interferences with wine samples BI-ENZYMATIC SYSTEM BASED ON DIAPHORASE (EC 1.8.1.4) Diaphorase (Clostridium kluyverii) 2 Fe(CN)63- 2 Fe(CN)64- 250 mV vs. SCE 2 e- * Related papers : 1. J.-L. Marty and T. Noguer. Analusis, 21 (1993) 6-8. 2. T. Noguer and J.-L. Marty. Enzyme Microb. Technol., 17 (1995) 453-456. 3. T. Noguer and J.-L. Marty. Anal. Chim. Acta, 347 (1997) 63-70.

  9. NADH + H+ NAD+ High stability Dissolved in solution High overpotential for H2O2 oxidation : high interferences BI-ENZYMATIC SYSTEM BASED ON NADH OXIDASE (EC 1.6.99) NADH oxidase (Thermus thermophilus) H2 O2 O2 650 mV vs. Ag/AgCl 2 e- * Related papers : 1. T. Noguer and J.-L. Marty. Anal. Let., 30 (1997) 1069-1080. 2. T. Noguer, A. Gradinaru, A. Ciucu and J.-L. Marty. Anal. Let., 32 (9) (1999) 1723-1738.

  10. Solution Sensing layer Mediator 4 K+ 4 e- 2 NADH + 2 H+ 2NADOX (FMN) Fe4 [Fe(CN)6]3 Prussian Blue 2H2O2 -150 mV vs Ag/AgCl Fe4K4[Fe(CN)6]3Prussian White 2NADOX (FMNH2) 2 NAD+ 1/2 O2 + H2O Working electrode 4 OH- + 4K+ Solution Sensing layer Mediator 4 H+ + 4K+ 4 K+ 4 e- 2 NADH + 2 H+ 2NADOX (FMN) Fe4 [Fe(CN)6]3 Prussian Blue -150 mV vs Ag/AgCl Fe4K4[Fe(CN)6]3Prussian White 2NADOX (FMNH2) 2 NAD+ Working electrode BI-ENZYMATIC SYSTEM INVOLVING PRUSSIAN BLUE AS MEDIATOR NAD and FMN must be added in solution Precipitated on WE surface

  11. MONO-ENZYMATIC SYSTEM INVOLVING MELDOLA’S BLUE AS MEDIATOR Malic acid Oxaloacetic acid In solution L-MDH Incorporated in the electrode material NADH NAD+ MB+ MBH H+ + 2e- - 150 mV vs Ag/AgCl MB : FAST EXCHANGE OF ELECTRONS WITH NADH

  12. MELDOLA’S BLUE-MODIFIED ELECTRODES : EVALUATION OF INTERFERENCES DUE TO WINE PHENOLIC COMPOUNDS 10% MBRS-modified SPE,pyrophosphate buffer 0.1 M, pH 9.3 Gallic acid 1 mM, 50 µL red wine (Caramany) WORKING AT -150 MV VS Ag/AgCl ALLOWS REDUCING INTERFERENCES

  13. PB AND MB-BASED SENSORS : COMPARATIVE TABLE (Batch measurements in stirred buffered solution)

  14. GOOD CORRELATIONS BUT NAD (and FMN) MUST BE ADDED IN REACTIONAL MEDIUM RESEARCHS FOCUS ON OBTENTION OF A FAD-BOUND NADH OXIDASE (GTP Technology, Labège, France) REAL SAMPLES ANALYSIS COMPARISON WITH COMMERCIALLY AVAILABLE KITS

  15. AN ALTERNATIVE TO THE CLASSICAL MDH : THE MALATE:QUINONE OXIDOREDUCTASE(MQO,EC 1.1.99.16). MQO from Corynebacterium glutamicum is a FAD-dependent peripheral membrane enzyme (FAD tightly bound). Involved in citric acid cycle. - Natural aceptor : ubiquinone (ménaquinone) Alternative metabolic pathway (PEP shunt) for the conversion of malate to oxaloacetate in E. coli. Van der Rest et al., J Bacteriol. 182(24) (2000) 6892-6899

  16. L-Malic acid Oxaloacetic acid MQO-FAD MQO-FADH2 Medox Medred e- THE PRINCIPLE OF MQO-BASED BIOSENSOR MQO used in this work in a recombinant enzyme (E. coli) produced by GTP Technology, Labège (France). NO COENZYME NEEDED, MONOENZYMATIC SYSTEM REACTION ESSENTIALLY IRREVERSIBLE BUT : APPROPRIATE MEDIATORS MUST BE FOUND

  17. High Interferences High Interferences SELECTION OF MEDIATOR(S) FOR MQO Analytical responses of the sensors to 1 mM malic acid (0,134g/L) (Working potentials were selected by cyclic voltammetry)

  18. MQO-BASED SENSORS PERFORMANCES

  19. HIGH INTERFERENCES USING DPIP AS MEDIATOR Evaluation of interferences AS : Analytical signal (to 1mM malate), IS = Interference signal (to 100-fold diluted red wine or 0.05 mM gallic acid)

  20. REAL SAMPLES ANALYSIS Wine analysis using MQO biosensors using DPIP or PMS as mediators. Average of triplicate measurements, spiked wine samples.

  21. Advantages & Drawbacks of MQO-sensor MQO : cofactorless enzyme, irreversible conversion of malate BUT : Poor stability, supplied in (NH4)2SO4 by GTP technology, must be desalted before immobilization : loss of activity Mediators : DPIP and PMS were used in solution, high interferences with DPIP,low stability of PMS (light sensitive). Appropriated mediators still must be found : * Efficient electronic transfert with FADH2, * Low detection potential, reduced interactions with polyphenolic compounds * Incorporable in screen-printed electrodes

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