Kinetic determination of
l(−)malic acid in wines using
sequential injection analysis
Marcela A. Segundo
1, António O.S.S. Rangel
∗Escola Superior de Biotecnologia, Universidade Católica Portuguesa, R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal
Abstract
In the present work a sequential injection system for the enzymatic determination ofl(−)malic acid in wines is described. The determination was based on the detection of NADH, formed by oxidation ofl-malate to oxaloacetate, catalysed by l-malate dehydrogenase. A kinetic method was implemented by stopping the flow when a solution plug containing sample, NAD+and enzyme reached the detector. The rate of reaction was measured by monitoring absorbance changes at 340 nm during a fixed period of time. A linear calibration curve (rate of reaction versus concentration) was obtained for concentrations ofl(−)malic acid between 0.01 and 0.15 g l−1. The determination rate was about 22 h−1and samples were diluted 25 or 50 times before introduction to the system. Results obtained from 16 wine samples were comparable to those obtained by the reference method.
Keywords: Sequential injection analysis; Malic acid; Kinetic enzymatic determination; Wines
1. Introduction
l(−)Malic acid is one of the most important
or-ganic acids present in wine. Beyond its effect on sensory properties, its concentration can be regarded as an indicator of wine microbiological stability as sufficient high levels can enable malolactic fermen-tation to happen. If not controlled, this fermenfermen-tation can bring undesirable effects, including the excessive reduction in acidity of high pH wines leading to risk of spoilage, production of undesirable flavours, colour
∗Corresponding author. Tel.:+351-2-25580064; fax:+351-2-25090351.
E-mail address: [email protected] (A.O.S.S. Rangel).
1Present address: REQUIMTE/Departamento de
Qu´ımica-F´ısica, Faculdade de Farm´acia, Universidade do Porto, Rua An´ıbal Cunha, 164, 4050-047 Porto, Portugal.
changes and formation of amines [1]. Appropriate monitoring of l(−)malic acid concentration before bottling is considered good practice to assess wine microbiological stability[2].
The method for determining l(−)malic acid rec-ommended by Office International de la Vigne et du Vin (OIV) [3]is based on the enzymatic conver-sion of l(−)malate to oxaloacetate in the presence of NAD+ andl(−)malate dehydrogenase (l-MDH). The reaction product NADH is measured spectropho-tometrically at 340 nm; this procedure also includes another enzyme, glutamate-oxaloacetate transaminase (GOT), in order to favour the reaction towards the production of NADH.
For fast and automatic determination ofl(−)malic acid, several flow systems have been proposed in the past years resorting to segmented flow analysis (SFA) [4,5], flow injection analysis (FIA) [6–14]
and sequential injection analysis (SIA) [15]. Except for the chemometric FTIR-based system described by Schindler et al. [15], all of them were based on enzymatic reactions, using spectrophotometric
[4,5,8,9,13,14], fluorimetric [6,7,11,14], electro-chemical[10]or chemiluminescence-based detection
[12]. The enzyme l-MDH was used in solution
[4,5,8,9,13], immobilised on different supports[7,14]
or co-immobilised with other enzymes [6,10–12]. Nevertheless, interference associated to the complex wine matrix was reported and blank measurements
[4,6,7,9], sample treatment prior to its introduction
[10–12] or implementation of in-line dialysis in the flow system[4,5,7,9,13,14]were necessary.
In the present work the development of a spec-trophotometric SIA system to determine l(−)malic acid in wines is aimed. The methodology chosen is based on the detection of NADH formed in the reac-tion catalysed byl-MDH in the presence of NAD+. A kinetic approach is considered in order to avoid inter-ference due to intrinsic absorption of the wine matrix. SIA was regarded as the perfect tool for its implemen-tation as precise control of volume delivery and flow halting are inherent to this technique, which is essen-tial in kinetic-based procedures.
2. Experimental
2.1. Reagents and solutions
All chemicals used were of analytical reagent grade with no further purification and water from a MilliQ plus system was used throughout.
l-MDH (1200 U mg−1, EC 1.1.1.37, ref. 127914,
5 mg ml−1) and NAD+(grade III, 90%, ref. 710113) were purchased from Boehringer.
The buffer carrier solution was prepared by dissolv-ing 37.5 g of glycine and 1 g of EDTA in 25 ml of a 2 mol l−1 sodium hydroxide solution. After that, the volume was made up to 500 ml with water and pH was adjusted to 9.5 by dropwise addition of concentrated sodium hydroxide solution. The enzyme solution was prepared daily by dissolving 10l of commercial so-lution in 20 ml of buffer soso-lution. The aqueous solu-tion of 75 mmol l−1NAD+was prepared daily.
Working standard solutions ofl(−)malic acid in the concentration range 0.010–0.150 g l−1were prepared
by rigorous dilution of 5 g l−1 stock solution using buffer solution. The wine samples were diluted 50- or 25-fold in buffer solution before introduction into the system.
2.2. Apparatus
The solutions were propelled by a Gilson Minipuls 3 peristaltic pump, equipped with Gilson PVC pumping tubes. This pump was connected to the central channel of an eight-port electrically actuated selection valve (Valco VICI C25-3118E).
A Unicam 8625 UV–visible spectrophotometer equipped with a thermostatic cell holder and a flow through cell from Hellma (80l, ref. 178.710QS) was used as detection system and the wavelength was set at 340 nm. The cell holder was connected to an I. S. Co GTR 190 thermostatic bath.
Omnifit PTFE tubing (0.8 mm i.d.) and Gilson end-fittings and connectors were used to assemble the different parts of the manifold.
Signal acquisition (3 Hz) and device control were achieved using a 386 personal computer equipped with an Advantec PCL-818L interface card and a PCLD-8115 wiring board terminal, running software written in QuickBasic 4.5 (Microsoft).
For the reference procedure, the spectrophotometric measurements were carried out at 340 nm using the referred Unicam spectrophotometer.
2.3. Manifold and procedure
System components were arranged as shown schematically inFig. 1. The holding coil (HC) length was 100 cm while the tubing connecting the selection valve to the detector was 46 cm long. Other tubing connected to the valve were 30 cm long. The protocol of flow and timing sequence for the determination of
l(−)malic acid in wines is listed inTable 1.
First, sample, NAD+ and enzyme solution were sequentially aspirated into the HC; after flow reversal, the stacked zones were sent through the connection be-tween the valve and the detector towards the flow cell. After a certain period of time, the flow was stopped and signal acquisition was performed during a pre-set time interval. Subsequently, the pump was re-activated and the content of the flow cell was washed out.
Fig. 1. Manifold for the determination ofl(−)malic acid in wines. SV: selection valve; P: peristaltic pump; HC: holding coil; D: detection system; S: sample or standard; NAD+ = 75 mmol l−1 NAD+solution; E= 2.5 mg l−1enzyme solution; C: glycine buffer solution(pH = 9.5); W: waste.
2.4. Reference method
The reference method for l(−)malic acid in wine was performed using the test kit “UV method for the determination ofl(−)malic acid in foodstuffs and other materials” from Boehringer (ref. 139068). The measurements were done after the procedure “Deter-mination of l(−)malic acid in wine” which is de-scribed in test kit package. Prior to the analysis, the samples were diluted 20-fold with water.
3. Results and discussion
3.1. Development of the sequential injection system
The determination ofl(−)malic acid was based on the rate of formation of NADH from the reaction
catal-Table 1
Protocol sequence for determination ofl(−)malic acid in wines
Step Valve position Operation time (s) Flow rate (ml min−1) Volume (l) Description
a 5 8 0.90 120 Aspirate sample/standard solution to HC b 6 4 0.45 30 Aspirate NAD+solution to HC c 7 6 0.90 90 Aspirate enzyme solution to HC
d 8 7.5 2.70 337 Dispense HC content towards the flow cell e 8 120 – – Stop period, signal acquisition
f 8 17 3.55 1006 Dispense carrier to wash flow cell
ysed byl-MDH. The production of NADH was mon-itored by measuring absorbance at 340 nm during a pre-set time interval. The rate of reaction was deter-mined by plotting absorbance values versus time and calculating the slope when the relation between them was linear. This relationship was considered linear af-ter visual inspection of the graphs (number of points >50) and when correlation coefficient was superior to 0.995. Calibration curves were established by plotting the rate of reaction versusl(−)malic acid concentra-tion; linear relation was expected for concentration values lower than the Michaelis constant of l-MDH
[16].
Studies concerning the influence of several parame-ters in the analytical performance were carried out us-ing the univariate method; the conditions used and the results obtained are discussed in the following para-graphs.
3.1.1. Aspiration order
Zone overlapping is essential to attain mixture of reagents and sample in SIA. Therefore, the order in which these solutions were aspirated was of utmost importance. In this particular application the solutions were drawn into the HC by the following order: sam-ple/standard, NAD+and enzyme. This order was cho-sen for two reasons. First, as kinetic measurements were carried out, enzyme should be the last reagent to be aspirated into the HC to minimise its dilution. Fur-ther, the plug of solution placed between the other two should be kept as small as possible to allow suitable overlapping of the three zones. Hence, NAD+solution was preferred as its concentration can be raised if nec-essary; this procedure could not be applied to samples.
3.1.2. Volume of solution sent to detector
Preliminary studies were performed to assess if the volume of solution sent to detector (Table 1,
Fig. 2. Absorbance values obtained for 0.20 g l−1l(−)malic acid standard during the stop period (Table 1, step e) for different time intervals (s) during which the HC content was sent to detector prior to signal acquisition: (a) 6.5, (b) 7.0, (c) 7.5, (d) 8.0 and (e) 8.5.
step d) influenced the rate of reaction. Hence, 90l of 0.20 g l−1 l(−)malic acid standard, 30 l of 50 mmol l−1NAD+solution and 90l of 1.25 mg l−1 enzyme solution were sequentially aspirated into the HC containing carrier buffer at pH = 9.5. These stacked zones were sent during different time in-tervals through the flow cell before flow halting, corresponding to different volumes containing differ-ent proportions of the species involved. The results obtained for intervals between 6.5 and 8.5 s were depicted in Fig. 2. For the data acquisition interval from 20 to 45 s after flow stop, absorbance values increased 0.096, 0.115, 0.128, 0.115 and 0.087 after sending HC content towards the flow cell for 6.5, 7.0, 7.5, 8.0 and 8.5 s, respectively. For further studies, the HC content was sent during 7.5 s through the flow cell before flow halting as this value gave the highest change in absorbance.
3.1.3. Temperature
Temperature increase has a drastic effect in enzyme kinetics as it increases the rate of reaction as well as deactivates the enzyme. In the proposed system, temperature studies were carried out by changing the temperature of the cell holder between 25 and 60◦C. Two aspects were examined: linear relation between absorbance and time during the period of signal ac-quisition and temperature influence on rate of reaction and calibration curve. For these experiments, 90l of
l(−)malic acid standards between 0.05 and 0.20 g l−1,
30l of 50 mmol l−1 NAD+ solution and 90l of
Fig. 3. Absorbance values obtained for 0.10 g l−1 l(−)malic acid standard during the stop period (Table 1, step e) at different temperatures: (a) 25◦C, (b) 30◦C, (c) 35◦C, (d) 40◦C, (e) 45◦C, (f) 50◦C, (g) 55◦C and (h) 60◦C.
1.25 mg l−1 enzyme solution were sequentially aspi-rated; pH of carrier buffer was 9.5. The results ob-tained for the 0.10 g l−1standard are shown inFig. 3. Considering the interval of signal acquisition between 15 and 45 s, linear relation between absorbance and time was verified for temperatures between 30 and 55◦C. At 25◦C, linear relation was obtained between 20 and 45 s while linear response was not verified at 60◦C at any time interval.
Results related to temperature influence on rate of reaction are shown inFig. 4 for 0.15 and 0.20 g l−1 standards. Maximum activity was obtained for 50◦C, followed by similar values at 45 and 55◦C. Calibra-tion curves (reacCalibra-tion rate versusl(−)malic acid con-centration) were established; sensitivity was similar at 50 and 55◦C. Its value decreased for lower temper-atures; sensitivity at 30◦C was 60% lower than that found at 50◦C.
Fig. 4. Rate of reaction (absorbance units s−1) values obtained for 0.15 (䊐) and 0.20 (䊉) g l−1L(–)malic acid standards at different temperatures.
3.1.4. Sample volume
Different values of sample volume were tested be-tween 90 and 250l. The conditions used were the same described for temperature studies; in this case, temperature was fixed at 40◦C and absorbance val-ues acquired between 20 and 45 s were considered for the determination of rate of reaction. Slope values ob-tained from the calibration curves were between 1.9× 10−2and 2.1×10−2absorbance units s−1for volumes between 120 and 250l. In this case the lowest value (120l) was chosen for further experiments in order to avoid high blank signals from the wine samples.
3.1.5. Composition of carrier buffer
The carrier buffer was prepared as described by Lima et al.[13], except for the presence of hydrazine. Glycine and EDTA concentrations were not changed and influence of pH value was assessed between 8.5 and 10.5 using the conditions described in the pre-vious section. The results are summarised inFig. 5. For 0.20 g l−1l(−)malic acid standard, linear relation between absorbance and time was obtained for carrier solution with pH between 9.0 and 10.0, considering the interval of signal acquisition between 15 and 45 s. For these values, calibration curves were established (Fig. 5B) and maximum sensitivity occurred for pH value of 9.5.
3.1.6. NAD+concentration
The NAD+ concentration was studied between 12.5 and 100 mmol l−1using the conditions described
Fig. 5. (A) Absorbance values obtained for 0.20 g l−1 L(–)malic acid standard during the stop period for carrier solution with different pH. (a) 8.5, (b) 9.0, (c) 9.5, (d) 10.0 and (e) 10.5. (B) Rate of reaction (absorbance units s−1) values obtained forl(−)malic acid standard solutions between 0.05 and 0.20 g l−1 using carrier solution at different pH values: 9.0 (䊐), 9.5 (䊊) and 10.0 (䉱).
in sample volume experiment. Calibration curves (re-action rate versusl(−)malic acid concentration) were established; sensitivity was similar for concentration values between 50 and 100 mmol l−1, but reaction rate values were higher for increasing concentrations. For 12.5 and 25 mmol l−1 the slope was about 50 and 75% lower than the value obtained for the other concentration values. The concentration chosen was 75 mmol l−1 in order to have high values of reaction rate and low reagent consumption.
3.1.7. Enzyme concentration
The enzyme concentration was studied between 0.62 and 10 mg l−1using the conditions described in sample volume experiment, except for concentration of NAD+that was 75 mmol l−1. Results obtained for
Fig. 6. Absorbance values obtained for 0.10 g l−1l(−)malic acid standard during the stop period using different concentrations of enzyme (mg l−1): (a) 10, (b) 5.0, (c) 2.5, (d) 1.25 and (e) 0.62.
0.10 g l−1 l(−)malic acid standard using different enzyme concentrations were shown in Fig. 6. Ab-sorbance and time were linearly related when enzyme concentrations were 1.25 or 2.5 mg l−1, considering signal acquisition between 15 and 45 s. Calibration curves were established for these concentration val-ues; the sensitivity obtained for the more diluted so-lution was about 55% of that achieved for 2.5 mg l−1.
3.1.8. l(–)Malic acid determination
First trials were performed using the conditions listed in Table 2, except for temperature and time period considered for rate of reaction assessment; the values initially used were 40◦C and 20–60 s, respec-tively. Using these conditions, a calibration curve was established between 0.025 and 0.25 g l−1ofl(−)malic acid. This range was appropriate for determination in wines after 1:20 dilution. This situation caused high
Table 2
Range of values used in the study of system variables and chosen conditions for its operation
Parameter Range Chosen value Time interval for propelling HC
content to detector (s)
6.5–8.5 7.5 Temperature (◦C) 25–60 50 Sample volume (l) 90–250 120 pH of carrier buffer 8.5–10.5 9.5 NAD+ concentration (mmol l−1) 12.5–100 75 Enzyme concentration (mg l−1) 0.62–10 2.5 Time period for signal acquisition (s) – 40–120
blank values for some samples(absorbance > 0.800) before measurement of enzyme activity. Hence, at-tempts were made to change the calibration range to lower concentration values in order to increase wine dilution. This was achieved by increasing temperature to 50◦C and by extending signal acquisition to 120 s and considering values between 40 and 120 s for de-termination of rate of reaction. The final conditions used for determination in wines were summarised in
Table 2.
3.2. Evaluation of the method and its application to wine samples
The performance of the proposed system for the de-termination ofl(−)malic acid in wines was evaluated regarding to application range, detection limit, sam-pling frequency, accuracy and repeatability.
The standards concentration varied between 0.010 and 0.150 g l−1 and the calibration curve was estab-lished by plotting rate of reaction versus concentra-tion of l(−)malic acid. This range was appropriate for determination in wines containing between 0.5 and 7.5 g l−1 of l(−)malic acid when diluted 50 times. Wines containing less than 0.5 g l−1 were analysed after 1:25 dilution. Samples were diluted in carrier buffer; for red wines, sample dilution should be per-formed some time before determination to allow sta-bilisation of wine colour. For the samples analysed, the maximum time necessary was 2 h.
3.2.1. Detection limit
The detection limit was calculated as the concen-tration corresponding to the intercept value plus three times sy/x[17], which is given by:
sy/x=
i(yi− ˆyi)2
n − 2
1/2
For seven different calibration curves(n = 10), the calculated detection limit was about 0.009 g l−1.
3.2.2. Sample throughput
The time required for a complete analytical cycle is not merely the addition of the time required for each step performance. As the time spent for proper port selection in the selection valve must also be accounted, it took 110.5 s to complete an analytical cycle. Hence,
Table 3
Results obtained by the proposed methodology (Cp) and by the
reference method (Cr) for the determination of l(−)malic acid
(g l−1) and values of absolute deviation (D) and relative deviation (RD) between the two methods
Samplea Crb Cpc D (g l−1) RD (%) 1 1.85 (±0.01) 2.01 (±0.13) +0.16 +8.6 2 0.78 (±0.05) 0.79 (±0.08) +0.01 +1.3 3 5.42 (±0.03) 5.29 (±0.13) −0.13 −2.4 4 3.03 (±0.01) 3.00 (±0.13) −0.03 −1.0 5 0.54 (±0.03) 0.44 (±0.02) −0.10 −18.5 6 0.30 (±0.01) 0.22 (±0.02) −0.08 −26.6 7 1.36 (±0.02) 1.42 (±0.04) 0.06 +4.4 8 2.59 (±0.01) 2.47 (±0.10) −0.12 −4.6 9 1.58 (±0.01) 1.66 (±0.04) +0.08 +5.1 10 2.07 (±0.01) 2.05 (±0.05) −0.02 −1.0 11 2.19 (±0.02) 2.21 (±0.12) +0.02 +0.9 12 2.77 (±0.01) 2.98 (±0.04) +0.21 +7.6 13 1.13 (±0.04) 0.80 (±0.11) −0.33 −29.2 14 1.01 (±0.01) 0.85 (±0.07) −0.16 −15.8 15 0.81 (±0.02) 0.97 (±0.06) 0.16 +19.6 16 1.07 (±0.01) 1.04 (±0.05) −0.03 −2.8
a1–9: white wines; 10–14: red wines; 15–16: Port wines. bS.D. (n = 2).
cS.D. (n = 5).
the determination rate was about 22 determinations per hour.
3.2.3. Comparison with the recommended procedure
In order to evaluate the accuracy of the proposed system, 16 samples of table and Port wine were anal-ysed. The results (Cp) were compared with those
ob-tained by the reference procedure (Cr)[17]; they are
presented inTable 3.
For comparison purposes, a linear relationship
(Cp= C0+SCr) was established. The equation found
wasCp= −0.02(±0.13)+1.00(±0.06)Cr, where the
values in parentheses are the limits of the 95% confi-dence intervals. From these figures it is clear that the estimated intercept and slope do not differ significantly from the values 0 and 1, respectively. Thus, there is no evidence for systematic differences between the two sets of results[17]obtained by the proposed methodol-ogy and by the reference method. Furthermore, when paired t-test was performed on the data obtained for white and red wines separately, t values of 0.564(n = 10) and 0.619 (n = 5) were obtained for each type of wine, respectively. The comparison between those values with thet(P = 0.05, d.f. = 9) = 2.26 and the
t(P = 0.05, d.f. = 4) = 2.78 indicates no significant
difference for the mean concentrations obtained by the two methods for both white and red wines.
When looking at the results for individual samples, absolute deviations between the reference procedure and the proposed methodology were only higher than 0.16 g l−1for two samples (Table 3). The concentra-tions obtained for these two samples corresponded to low activity values, which were conditioned by the ap-plied sample dilution; therefore, more accurate results could be expected if a lower sample dilution was used. Nevertheless, these two results do not compromise the usefulness of this method for the wine industry, as variations in thel(−)malic acid concentration during malolactic fermentation can be efficiently monitored with the proposed flow system.
3.2.4. Repeatability
It was estimated by calculating the relative stan-dard deviation from five consecutive injections of wine samples. Standard deviations up to 0.13 g l−1 were obtained for 16 analysed samples (Table 3). Consid-ering concentration values obtained by the proposed methodology, relative standard deviations between 4.5 and 13.8% were found for samples containing less than 1.0 g l−1 of l(−)malic acid; values between 1.3 and 6.5% were determined for samples containing more than 1.0 g l−1.
4. Conclusions
The proposed system was able to perform determi-nation ofl(−)malic acid in wines, with results compa-rable to those obtained by the reference method, with minimum sample treatment (dilution) and low enzyme consumption. In fact, only 10l of commercial en-zyme solution were necessary to prepare the diluted solution consumed daily.
Compared to other previously described flow sys-tems, neither blank measurements [4,6,7,9]nor sam-ple treatment using gelatin [10] or nylon cartridge
[11,12]and in-line dialysis[4,5,7,9,13,14]were nec-essary in the proposed system. In fact, the absence of these features in the present system was possible since the analytical signal was based on absorbance change due to production of NADH. Hence, the contribution of intrinsic absorption of wine matrix did not affect
the determination of concentration as the absolute value of absorbance was not considered.
Moreover, enzyme consumption in the present sys-tem was about 10 times lower than that obtained in the merging zones system described by Garc´ıa de Mar´ıa et al. [8]. Besides that, use of hydrazine was also avoided in the proposed system as it is considered a carcinogenic reagent.
Compared to the reference method, less NAD+was necessary per assay and only one enzyme (l-MDH) was used. Nevertheless, repeatability was lower in the SIA system. This feature is not important if it is considered as a screening tool to follow or assess the extension of malolactic fermentation during wine production.
Finally, as several ports of selection valve were not used, it would be possible to implement other determi-nations using the proposed manifold. Determination of ethanol and glycerol could be performed after mi-nor adjustments by connecting immobilised enzyme reactors in the available ports[18]. The same detec-tor could be used as these determinations were also carried out spectrophotometrically at 340 nm.
Acknowledgements
The authors acknowledge the financial support from IFADAP through Project AGRO 273 and from PRAXIS XXI/Agˆencia de Inovação through Project P076-P31B-09/97-INSIA. M.A. Segundo thanks FCT for the grant PRAXIS XXI BD/13648/97.
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