Presentation on theme: "STRATEGIES FOR THE DEVELOPMENT OF MALATE SENSORS DEVOTED TO WINEMAKING MONITORING."— Presentation transcript:
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). -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.
SUBSTRATES OF INTEREST CO 2 COOH CHOH CH 3 L-malate L-lactate MALOLACTIC FERMENTATION (Oenococcus oeni) COOH CHOH CH 2 COOH ALCOHOLIC FERMENTATION ( Saccharomyces cerevisiae) O COOH C CH 3 CO 2 H C CH 3 O NADH + H + NAD + CH 2 OH CH 3 Pyruvate decarboxylase Alcool dehydrogenase Sugars Ethanal Ethanol Pyruvate Acetobacter Acetic acid Lactic bacteria D-lactate
Legend : T - Tartaric acid L - Lactic acid M - Malic acid WIDELY USED METHOD : PAPER CHROMATOGRAPHY Suitable for any winery Low cost but low speed and accuracy.
ENZYMATIC RECOMMENDED METHOD L-malate Oxaloacetate + NAD + + NADH + H + Spectrophotometric determination at 340nm ( = 6300 M -1.cm -1 ) GOT* + L-glutamate L-aspartate + 2-oxoglutarate * Glutamate-oxaloacetate transaminase (EC18.104.22.168) Costly, not adapted to small wineries Laboratory analysis : delays between sampling and results. Malate dehydrogenase L-MDH (EC 22.214.171.124) Need of easy and portable analytical devices as BIOSENSORS
+ NAD + PRINCIPLE OF DH-BASED BIOSENSORS DEHYDROGENASE (DH) SUBSTRATE PRODUCT + NADH + H + Optical detection ( =340 nm ) AMPEROMETRIC DETECTION Direct oxidation High Potential No selectivity Bienzymatic systems Mediated oxidation (monoenzymatic system)
+ NAD + THE MALATE DEHYDROGENASE-REACTION : DIFFERENT OPTIONS FOR SENSOR DEVELOPMENT Malate dehydrogenase L-MDH (EC 126.96.36.199) L-malate Oxaloacetate + NADH + H + High concentrations of NAD + + appropriate mediator Enzymatic consumption of NADH (regeneration of NAD + ) MONO-ENZYMATIC SENSOR BI-ENZYMATIC SENSOR
Diaphorase (Clostridium kluyverii) 2 Fe(CN) 6 3- 250 mV vs. SCE 2 e- 2 Fe(CN) 6 4- NADH + H + NAD + BI-ENZYMATIC SYSTEM BASED ON DIAPHORASE ( EC 188.8.131.52) Mandatory addition of ferricyanide Interferences with wine samples * 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.
NADH oxidase ( Thermus thermophilus) O2O2 650 mV vs. Ag/AgCl H 2 O 2 NADH + H + NAD + 2 e- Dissolved in solution High stability BI-ENZYMATIC SYSTEM BASED ON NADH OXIDASE ( EC 1.6.99) High overpotential for H 2 O 2 oxidation : high interferences * 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.
Fe 4 [Fe(CN) 6 ] 3 Prussian Blue Fe 4 K 4 [Fe(CN) 6 ] 3 Prussian White 2NADOX (FMNH 2 ) 2 NADH + 2 H + 2 NAD + Mediator Working electrode Sensing layer 4 e - Solution -150 mV vs Ag/AgCl 2NADOX (FMN) 4 K + 4 H + + 4K + BI-ENZYMATIC SYSTEM INVOLVING PRUSSIAN BLUE AS MEDIATOR 1/2 O 2 + H 2 O Fe 4 [Fe(CN) 6 ] 3 Prussian Blue Fe 4 K 4 [Fe(CN) 6 ] 3 Prussian White 2NADOX (FMNH 2 ) 2 NADH + 2 H + 2 NAD + 2H 2 O 2 4 OH - + 4K + Mediator 4 e - Solution 2NADOX (FMN) 4 K + Working electrode Sensing layer -150 mV vs Ag/AgCl Precipitated on WE surface NAD and FMN must be added in solution
NADH NAD + MB + MBH H + + 2e - Malic acid Oxaloacetic acid - 150 mV vs Ag/AgCl L-MDH MONO-ENZYMATIC SYSTEM INVOLVING MELDOLA’S BLUE AS MEDIATOR In solution Incorporated in the electrode material MB : FAST EXCHANGE OF ELECTRONS WITH NADH
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 MELDOLA’S BLUE-MODIFIED ELECTRODES : EVALUATION OF INTERFERENCES DUE TO WINE PHENOLIC COMPOUNDS
(Batch measurements in stirred buffered solution) PB AND MB-BASED SENSORS : COMPARATIVE TABLE
REAL SAMPLES ANALYSIS COMPARISON WITH COMMERCIALLY AVAILABLE KITS 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)
MQO from Corynebacterium glutamicum is a FAD-dependent peripheral membrane enzyme (FAD tightly bound). Involved in citric acid cycle.- Natural aceptor : ubiquinone (ménaquinone) AN ALTERNATIVE TO THE CLASSICAL MDH : THE MALATE:QUINONE OXIDOREDUCTASE ( MQO,EC 184.108.40.206). 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
MQO-FAD MQO-FADH 2 Med ox Med red e-e- L-Malic acidOxaloacetic acid 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 THE PRINCIPLE OF MQO-BASED BIOSENSOR
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) High Interferences
Evaluation of interferences HIGH INTERFERENCES USING DPIP AS MEDIATOR AS : Analytical signal (to 1mM malate), IS = Interference signal (to 100-fold diluted red wine or 0.05 mM gallic acid)
REAL SAMPLES ANALYSIS Wine analysis using MQO biosensors using DPIP or PMS as mediators. Average of triplicate measurements, spiked wine samples.
MQO : cofactorless enzyme, irreversible conversion of malate BUT :Poor stability, supplied in (NH 4 ) 2 SO 4 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 FADH 2, * Low detection potential, reduced interactions with polyphenolic compounds * Incorporable in screen-printed electrodes Advantages & Drawbacks of MQO-sensor