Determination of Substrate and Product Concentrations in Lactic Acid

A 1H NMR method is described for the determination of the concentration of a chemically diverse group of metabolites over a wide concentration range i...
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Anal. Chem. 2001, 73, 1862-1868

Determination of Substrate and Product Concentrations in Lactic Acid Bacterial Fermentations by Proton NMR Using the ERETIC Method Virginie Silvestre, Ste´phane Goupry, Michel Trierweiler, Richard Robins, and Serge Akoka*

LAIEMsUMR CNRS 6006, Faculte´ des Sciences, Universite´ de Nantes, 2 rue de la Houssinie` re, BP 92208, 44322 Nantes Cedex 3, France

A 1H NMR method is described for the determination of the concentration of a chemically diverse group of metabolites over a wide concentration range in fermentation medium. The method employs the use of the ERETIC signal, which produces a pseudo-FID acquired with the FID derived from the sample components. It is shown that this signal, once calibrated against an external standard solution, can be used to determine accurately the concentration of all components present in the medium. The method is rapid, reliable, and applicable to a wide range of biological fluids. Problems such as the invisibility of certain components in the spectrum are overcome by a simple regulation of the pH. There is a growing interest in the use of 1H NMR to determine simultaneously the concentrations of numerous analytes in complex mixtures such as biological fluids.1,2 A major drawback to this approach, however, is the difficulty of calibrating the spectra with sufficient accuracy. The addition of an internal standard at a known concentration is possible, but this leads to intrinsic problems such as potential interference with analytes already present. Moreover, it is desirable that the T1 of the standard is close to or smaller than that of the analytes, since the recovery time, thus the duration of the experiment, is determined by the longer T1. Standard compounds are not consistent in their behavior and can even give nonlinear calibrations.3 In addition, in complex matrixes, it is often difficult to find a suitable standard compound that resonates in a transparent region of the spectrum. In a recent interlaboratory survey, up to 10-fold differences in concentration were recorded on the same blood sample.4 These authors discuss in detail the various sources of such large differences. A further difficulty in using 1H NMR to determine concentration is that species of interest in the matrix may interact, leading to an underestimation of their concentration. This effect can be * Corresponding author: (e-mail) [email protected]; (tel) (33)251125707; (fax) (33) 251125712. (1) Lindon, J. C.; Nicholson, J. K.; Holmes, E.; Everett, J. R. Magn. Reson. 2000, 12, 289-320. (2) Fan, T. W. Prog. Nucl. Magn. Reson. Spectrosc. 1996, 28, 161-219. (3) Kriat, M.; Confort-Gouny, S.; Vion-Dury J.; Sciaky, M.; Viout, P.; Cozzone, P. J. NMR Biomed. 1992, 5, 179-184. (4) Lindon, J. C.; Sweatman, B. C. J. Magn. Reson. Anal. 1996, 2, 66-74.

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serious, as has been demonstrated for lactic acid, which forms complexes with macromolecular components so that up to 60% of the analyte present becomes invisible.5,6 Indeed, a wide range of metabolites has been shown to bind to proteins.7 A role for 1H NMR has long been recognized in the analysis of fermentations, which are generally complex processes in which a number of metabolites show dynamic and inter-related changes in their concentrations over time.8 It is often desirable to follow closely the simultaneous metabolism of these components. However, to measure the dynamics of these changes by conventional methods requires a large number of individual analyses, each of which is subject to its own sources of error and uncertainty. In some cases, components present in the matrix interfere in the determination of similar species, such as the effect of diacetyl on the colorimetric determination of acetoin.9 It is hard to correct for this type of effect as the extent to which it occurs changes with time. Potentially, 1H NMR could alleviate such difficulties. However, the problems of accurate calibration for a chemically diverse range of species remain. One approach to avoiding the problems cited above is to use a chemically inert, species-independent reference. Recently, the ERETIC method has been reported as a means of providing such a reference. This method relies on the generation of a signal of a defined frequency and amplitude by a broad-band antenna in the magnet of the NMR spectrometer. This is recorded as a pseudo-FID acquired with the FID derived from the sample components. This signal, once calibrated against an external standard solution, can be used to determine accurately the concentration of analyte in unknown samples10 and to follow reaction kinetics.11 In this paper, the application of the ERETIC method for the simultaneous determination of all the principal components in the medium during the cofermentation of glucose and citric acid by (5) Bell, J. D.; Brown, J. C. C.; Kubal, G.; Sadler, P. J. FEBS Lett. 1988, 235, 81-86. (6) Chatham, J. C.; Forder, J. R. Biochim. Biophys. Acta 1999, 1426, 177-184. (7) Kragh-Hansen, U. Pharmacol. Rev. 1981, 33, 17-53. (8) Tran-Dinh, S.; Herve, M.; Wietzerbin, J. Eur. J. Biochem. 1991, 201, 715721 and references therein. (9) Xanthopoulos, V.; Picque, D.; Bassit, N.; Boquien, C.-Y.; Corrieu, G. J. Dairy Res. 1994, 61, 289-297. (10) Akoka, S.; Barantin, L.; Trierweiler, M. Anal. Chem. 1999, 71, 2554-2557. (11) Billault, I.; Akoka, S. Instrum. Sci. Technol. 1999, 28, 233-240. 10.1021/ac0013204 CCC: $20.00

© 2001 American Chemical Society Published on Web 03/15/2001

lactic acid bacteria is reported. The quantitative fluxes as determined by the ERETIC method correlate well with previous data obtained by classical methods.12 Thus, the method is shown to provide a rapid, reliable, and accurate determination of all the compounds involved. Furthermore, it is shown that, by a simple adjustment of the pH, reported NMR invisibility is avoided. EXPERIMENTAL SECTION Sample Preparation for NMR. Biological Samples. Lactococcus lactis subsp. lactis biovar diacetylactis strain B7/2147, obtained from the national collection of lactic acid bacteria (Institute of Food Research, Norwich, U.K.), was stored at -80 °C in M17 medium13 with 15% (v/v) glycerol. Routine culture was in sterile (30 min, 121 °C, 1 bar) M17 broth. The bacteria were precultured for 6 h in M17 with glucose (Sigma-Aldrich, L’Isle d’Abreau Chesnes, France) and sodium citrate‚2H2O (Merck S. A., Nogent-sur-Marne, France) at, respectively, 27.8 and 13.9 mM, added by sterile filtration. The culture was started by adding 200 µL of preculture to 200 mL of sterile M17 (glucose 27.8 mM, citrate 13.9 mM). The conditions of fermentation were as follows: static, anaerobic (closed Duran bottle), 30 °C, initial pH 6.3, and left to evolve freely for 10 h. The dynamics of the fermentation were followed by stopping the culture after different periods of incubation. Fermentation was arrested by centrifugation (4472g, 10 min, +4 °C), and the recovered supernatant was sealed and stored at -20 °C until required for analysis. The NMR sample was prepared as follows: 1.0 mL of supernatant, 70 µL of 3.19 M sodium phosphate buffer (pH 2.0), and 50 µL of 1,4-dioxane-d8 (Eurisotope, Saclay, France) were mixed and filtered with a syringe filter unit (Millipore, 0.22 µm, CA), and 950 µL of the filtrate was pipetted into the NMR tube (5-mm i.d.; Wilmad). Model Samples. Model mixtures of commercial compounds for validation and calibration of the NMR method were prepared either in distilled water or in M17 broth. Standard solutions of glucose (Sigma-Aldrich), sodium citrate‚2H2O (Merck S. A.), lithium lactate (ICN Biomedicals, Parc Club d’Orsay, France), sodium acetate (Prolabo, Gradignan, France), acetoin (Fluka, L’Isle d’Abreau Chesnes, France), and diacetyl (Fluka) were prepared by weighing in volumetric flasks. To test the effect of phosphate buffer, solutions of all compounds were prepared in M17 + phosphate buffer and in distilled water + phosphate buffer (0.4 M). For the NMR sample, 1140 µL of the relevant solution was mixed with 60 µL of dioxan-d8, the solution was filtered, and 950 µL of the filtrate was placed in the NMR tube. The solution in M17 + phosphate buffer is known as solution A. The concentrations of these solutions were as follows (in mM): 26.10 (glucose), 19.26 (citric acid), 2.45 (diacetyl), 6.11 (acetoin), 26.02 (acetic acid), and 76.96 (lactic acid). Three other solutions were prepared from solution A by dilution in M17 + phosphate buffer. Concentrations were 75, 50, and 25% of those of solution A for solutions B-D, respectively. Determination of the Concentrations of Substrates and Fermentation Products by Classical Methods. Diacetyl concentration was determined by measuring at 366 nm the yellow (12) Goupry, S.; Croguennec, T.; Gentil, E.; Robins, R. J. FEMS Microbiol. Lett. 2000, 182, 207-211. (13) Terzaghi, B. E.; Sandine, W. E. Appl. Microbiol. 1975, 29, 807-813.

complex formed by the reaction of 3,3′-diaminobenzidine (SigmaAldrich, L’Isle d’Abreau Chesnes, France) and diacetyl in acidic conditions.14 Lactic acid concentration was determined spectrophotometrically by following the increase in absorption at 340 nm using L-lactate dehydrogenase, the pyruvate produced being removed using an alanine:2-oxo-glutarate aminotransferase/excess glutamate couple.15 Citric acid concentration was measured by following the decrease in absorption at 340 nm using citrate lyase, the oxaloacetate produced and its decarboxylation product pyruvate being removed by malate dehydrogenase and lactate dehydrogenase, respectively.16 Acetic acid was determined spectrophotometrically using the Boeringer Manheim, Food Analysis kit (Boeringer Manheim S. A., Meylan, France), in which the NADH produced during the formation of citrate from acetate and oxaloacetate by citrate lyase is determined at 340 nm. The oxaloacetate required was synthesized in situ by the reaction of malate and NAD+ catalyzed by malate dehydrogenase.17 Glucose concentration was determined by following the NADPH formed during the reaction of glucose with ATP and NADP+ catalyzed by hexose kinase and glucose-6-phosphate dehydrogenase.18 Determination of the Concentrations of Substrates and Fermentation Products by the ERETIC Method. An electronic signal was synthesized by multiplication between two components: an exponentially decreasing low-frequency component and a sinusoidal high-frequency component. As a result, a pseudoFID that has all the characteristics of a real NMR signal and whose parameters (frequency, magnitude, phase, and T2) can be freely controlled from the spectrometer console is obtained. The second rf channel of the spectrometer produced the high-frequency component. An electronic device produced the low-frequency component. After multiplication, the pseudo-FID was transmitted through the carbon coil and received by the proton coil at the same time as the NMR signal. After Fourier transformation, an additional peak was obtained in the NMR spectrum (Figure 1) which was used as a quantitative reference. A complete description of the ERETIC method has been given in previous papers.10,19 Calibration of the equivalent concentration of the ERETIC peak was performed using eq 1, where [Lact] and ALact are respectively

[ERETIC] ) (AERETIC/ALact)[Lact]

(1)

concentration of lactic acid and peak area of the lactic acid methyl group in the most concentrated solution in M17 + 0.4 M phosphate buffer (pH 2.0) and AERETIC is the area of the ERETIC peak. Because of the stability over time previously observed,10 this calibration was performed once a week. (14) Pien, J.; Baisse, J.; Martin, R. Lait 1937, 17, 673-698. (15) Noll, F. In Methods of Enzymatic Analysis, 3rd ed.; Bergmeyer, H. U., Ed.; VCH: Weinheim: 1988; Vol. 6, pp 582-588. (16) Mo ¨llering, H. In Methods of Enzymatic Analysis, 3rd ed.; Bergmeyer, H. U., Ed.; VCH: Weinheim, 1989; Vol. 7, pp 2-12, (17) Beutler, H.-O. In Methods of Enzymatic Analysis, 3rd ed.; Bergmeyer, H. U., Ed.; VCH: Weinheim, 1988; Vol. 6, pp 639-645. (18) Kunst, A.; Draeger, B.; Ziegenhorn, J. In Methods of Enzymatic Analysis, 3rd ed.; Bergmeyer, H. U., Ed.; VCH: Weinheim, 1988; Vol. 6, pp 163-172. (19) Barantin, L.; Akoka, S.; LePape, A. French Patent CNRS 95 07651, 1995.

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Table 1. Chemical Shifts and T1 Relaxation Times for the Chemical Groups Used To Quantify Concentrationsa δ (ppm) attribution

pH 6.2

pH 2.0

glucose C2 (CH) citric acid (CH2) diacetyl (CH3) diacetyl (CH3) Hyd. acetoin (CH3) acetic acid (CH3) lactic acid (CH3)

3.24 2.67; 2.53 2.34 2.33 2.22 1.91 1.32

3.24 2.96; 2.81 2.34 2.33 2.22 2.09 1.40

T1 (s) pH 6.2

pH 2.0

1.70 0.68 3.36 3.54 4.42 4.58 1.74

1.84 0.77 2.28 2.33 2.23 3.89 1.65

a Measurements were performed in M17 medium with 0.4 M phosphate buffer (pH 2.0) and without phosphate buffer (pH 6.2).

The T1 values of each compound were determined by using an inversion recovery sequence21 with 12 inversion-time values ranging from 5 ms to 25 s and by using the T1 calculation software of the spectrometer. As for concentration measurements, spectra were measured with water suppression using the Dante method20 during recuperation and inversion times and with parameters as given above. Determination of the Concentrations. Determination of the concentration of each compound was done for each experiment using eq 2:

Figure 1. Time evolution of a 500-MHz NMR spectrum of supernatant from a fermentation of lactic acid bacteria in M17 medium with 0.4 M phosphate buffer (pH 2). (A) t ) 0 h, glucose (Gluc), citric acid (Cit), and M17 medium components are visible. Note that the unfermented medium contains trace amounts of lactic acid (Lact) and acetic acid (Acet). The * marks a broad peak that interferes with the baseline for citric acid determination; (B) t ) 6 h; (C) t ) 10 h, glucose and citric acid have disappeared and lactic acid, acetic acid, acetoin, and diacetyl (Diacet) have reached their maximum concentrations (see Table 4).

NMR Measurements. All experiments were performed on a DRX 500 Bruker spectrometer. A dual probe (1H/13C) was used. The proton channel was used for transmitting rf pulses and receiving. Fully relaxed spectra were acquired with the following parameters: flip angle 90° (12 µs), repetition time 22 s, SW ) 6000 Hz, SI ) 32 K, NS ) 20, DS ) 4, RG ) 40. Spectra were measured with water suppression using the Dante method.20 A pulse train was applied during all of the recovery period with the following parameters: pulse duration 5 µs, delay between two pulses 95 µs, and pulse power 5 mW (attenuation of 34 dB). During NMR experiments, the probe and sample temperature was controlled and kept at 303 K. Five acquisitions were performed for each measurement in order to estimate the precision. An exponential multiplication was applied to the FID inducing a line broadening of 1 Hz. A baseline correction was performed using a cubic spline algorithm included in the software of the spectrometer. Baseline points were carefully chosen in order to obtain a flat baseline. Spectra were processed in the frequency domain using the curve-fitting package of the spectrometer; lines were fitted by pure Lorentzian curves. (20) Morris, G. A.; Freeman, R. J. Magn. Reson. 1978, 29, 433-437.

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[Comp] ) NH[ERETIC](AComp/AERETIC)

(2)

where

NH )

number of protons under the peak to be quantified number of protons under the calibrating peak

(in the present case, for calibration against lactate, the number of protons under the calibrating peak is 3). Acomp is the area of the peak to be quantified, AERETIC is the area of the ERETIC peak, and [Comp] is the concentration of analyte. Resonance Assignments. Values found in the literature2 were used for an initial assignment of the resonances of the compounds studied. However, care should be taken in using these values, as many of them are sensitive to solution conditions, notably pH and ionic strength. Therefore, chemical shifts were precisely determined for standard solutions in M17 at the two values of pH used. As reference, the methyl resonance of the lactate was set to be at 1.32 ppm at pH 6.2, which is equivalent to sodium 3-trimethylsilyl2,2,3,3-tetradeuteriopropionate (TSP) at 0.0 ppm at neutral pH. RESULTS AND DISCUSSION Measured Resonance Lines. For each compound, one chemical shift position was selected for quantification. Two criteria were used for this selection: a minimum overlap with other lines in the spectrum and a minimum number of lines due to spinspin coupling in order to obtain the best signal-to-noise ratio and precision. The selected chemical shifts are indicated in Figure 1 and Table 1 with the corresponding attributions. (21) Freeman, R.; Hill, H. D. W. J. Chem. Phys. 1969, 54, 3140-3143.

In the case of diacetyl, two chemical shifts were quantified (2.33 and 2.34 ppm at pH 2.0). This is because, in aqueous solution, the hydrated molecule (H3C-CO-C(OH)2-CH3 is formed which is in slow exchange with the nonhydrated molecule (H3C-COCO-CH3). This interpretation has been confirmed by 13C NMR on solutions of pure diacetyl in water or M17 medium (results not shown). Both lines were independently quantified, and the determination is given in equivalents to the nonhydrated form. Optimization of NMR Acquisition Conditions. In this study, we have chosen to use 1,4-dioxane-d8 to provide a field-frequency lock. Another choice could be D2O. However, it has previously been shown that acetoin or diacetyl exchanges deuterium with D2O.22 Similarly, due to the hydration/dehydration described above, diacetyl will exchange with D2O. Therefore, it is not possible to obtain accurate and precise measurements of the concentration of compounds containing labile hydrogens in the presence of D2O because the isotopic substitution induces a loss of intensity of the measured singlet and a multiplet (protondeuterium coupling) at virtually the same chemical shift. Furthermore, the kinetics of the isotopic substitution depend on pH, ionic strength, and concentration of the analyte. The measured T1 values for the different chemical groups used for quantification are given in Table 1 for the two pH values (2.0 and 6.2). They ranged from 0.68 to 4.58 s at pH 6.2 and from 0.77 to 3.89 s at pH 2. It appears that, for each chemical group, T1 was either virtually the same at the two pH values (glucose, citric acid, and lactic acid) or significantly lower at pH 2.0 (diacetyl, acetoin, and acetic acid). The highest value was 4.58 s: the ratio repetition time/T1 was therefore equal to 4.8 in the worst case (acetic acid). In such conditions, partial saturation due to longitudinal relaxation during the repetition time cannot induce intensity distortion greater than 0.82%. It has previously been shown that, for high accuracy, optimal signal-to-noise ratio is achieved by using large pulse angles of 70-90° and a repetition time higher than 4.6-fold the maximum T1.23 Therefore, we used a flip angle of 90°. It must be noted that the repetition time used was chosen by taking into account the T1 values obtained at the two values of pH. In the case of routine measurements performed at pH 2.0, the repetition time could be lower; 18.7s would induce intensity distortion lower than 0.8% at this pH. The total time required for acquisition of data would be significantly decreased. Figure 2 shows the relative evolution of measured line intensities versus the saturation power used for water suppression (expressed in attenuation of the maximum power). Experiments performed with different irradiation frequencies during the Dante pulse train (results not shown) showed that these evolutions were strongly related to the difference between the quantified line and the irradiation frequency. Such variations in intensity can be explained by a lack in selectivity of the water saturation. For attenuations higher than 34 dB (saturation power lower than 5 mW), no significant differences were observed, the difference between 34 and 40 dB being lower than 0.5% for all the measured lines. Hence, at this power, the direct saturation effect can be considered as negligible. We therefore chose an attenuation of 34 dB in order to be sure that water suppression did not have a significant influence on the determination of concentration. (22) Marsman, J. W.; Lubach, J.; Drenth, W. Recl. Trav. Chim. Pays Bas 1969, 88, 193-194. (23) Cookson, D. J.; Smith, B. E. Anal. Chem. 1982, 54, 2591-2593.

Figure 2. Evolution of measured line intensities (expressed as percentage deviation of the intensity measured at 40 dB) versus the saturation power used for water suppression (expressed in attenuation of the maximum power 12.5W): (b) glucose, (9) citric acid, (4) diacetyl, (O) acetoin, (0) acetic acid, and (2) lactic acid. Table 2. Accuracy, Given in Percentage Deviation from the True Value, for Concentrations Measured in Four Sample Preparations: H2O without Buffer; 0.4 M Phosphate Buffer (pH 2.0); M17 Medium (pH 6.2); M17 Medium with 0.4 M Phosphate Buffer (pH 2.0)a

glucose citric acid diacetyl acetoin acetic acid lactic acid

M17/PO4

M17

H2O/PO4

H2O

0.25 (-) 0.15 (+) 0.45 (-) 0.22 (-) 0.59 (-) 0.06 (-)

9.37 (-) 8.27 (-) 27.68 (-) 9.31 (-) 5.91 (-) 20.17 (-)

0.36 (+) 0.11 (-) 0.11 (+) 0.76 (-) 0.58 (-) 0.57 (-)

0.43 (+) 0.31 (+) 0.78 (+) 0.51 (-) 0.68 (-) 0.39 (+)

a (+), positive deviation (value overestimated); (-), negative deviation (value underestimated).

NMR Visibility of Compounds. Interactions between small metabolites and macromolecules in the matrix have been described previously.24,25 For example, 23% of the diacetyl present is retained by a 61% sodium caseinate aqueous solution.26 Such flavor-protein interactions involve nonspecific binding and depend on the nature and the hydrophobicity of the aroma compound. Lactate similarly interacts with proteins, and it has recently been shown that this interaction strongly influences the concentration determined by NMR.6 The addition of bovine serum albumin (0.2 mM) to a solution of lactate and alanine resulted in the diminution by ∼60% of the 1H NMR resonances due to lactate while those from alanine were unaffected. A similar effect was reported for acetate. This 1H NMR invisibility is considered to be due to the immobilization by nonspecific binding of the interacting compound. Consequently, the concentrations of lactate, acetate, diacetyl, and, probably, acetoin will tend to be underestimated in the 1H NMR spectrum, as fermentation broth contains excreted proteins and polypeptides. Table 2 shows the accuracy, given in percentage deviation from the true value, for concentrations measured in four sample preparations. As anticipated, measurements performed in M17 broth at pH 6.2 produce erroneous values, the underestimation ranging between 6% for acetate and 27% for diacetyl. When, (24) Sostmann, K.; Guichard, E. Food Chem. 1998, 62, 509-513. (25) Guichard, E.; Langourieux, S. Food Chem. 2000, 71, 301-308. (26) Landy, P.; Draux, C.; Voilley, A. Food Chem. 1995, 54, 387-392.

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Table 3. Concentrations (mM) of Metabolites Measured Using the ERETIC Method on Four Standard Solutions A-D in M17 with 0.4 M Phosphate Buffer (pH 2.0) ( SD Calculated from Five Measurements Performed on Each Tubea

glucose citric acid diacetyl acetoin acetic acid lactic acid a

A

B

C

D

slope

intercept

r2

26.39 ( 0.07 19.90 ( 0.04 2.52 ( 0.01 6.26 ( 0.03 26.40 ( 0.18 77.47 ( 0.21

19.47 ( 0.12 14.60 ( 0.04 1.79 ( 0.01 4.50 ( 0.01 19.82 ( 0.09 57.53 ( 0.24

13.67 ( 0.03 10.11 ( 0.04 1.24 ( 0.02 3.05 ( 0.01 13.25 ( 0.03 38.37 ( 0.09

7.57 ( 0.02 5.57 ( 0.04 0.65 ( 0.002 1.60 ( 0.01 6.99 ( 0.01 20.56 ( 0.06

0.996 1.007 1.009 1.009 1.004 0.999

0.118 0.270 0.013 0.013 0.029 0.014

0.9986 0.9984 0.9979 0.9975 0.9999 0.9994

Slopes, intercepts, and r2 coefficients were obtained by linear regression.

however, measurements were performed in water or in 0.4 M phosphate buffer at pH 2.0, the percentage deviation from the true value was lower than 0.78% for all compounds. Crucially, it was found that buffering the M17 fermentation broth to pH 2.0 with 0.4 M phosphate buffer alleviated the problem of binding, the percentage deviation from the true value being lower than 0.6% for all analytes. Hence, it is demonstrated that, for measurement of concentrations of small analytes by 1H NMR in complex media, conditions can be established in which interactions between bacterial metabolites and macromolecules are eliminated. If this precaution is not taken, as is frequently the case in the literature, measured concentrations are likely to be underestimated by up to 30%. While this can partially be avoided by preparing a calibration curve with a matrix identical to that of the sample, this approach becomes impossible or too fastidious when the composition of the sample matrix changes with time. Alternative methods, such as heat treatment (60 °C; 30 min) to try to denature the proteins or high ionic strength to minimize ionic interactions, were not sufficient to reveal all the lactate present (data not shown). In contrast, the use of a low pH proved efficient (Table 2) and is simply achieved by adding a small volume of concentrated phosphate buffer with the deuterium lock. Dilution is minimal (7%) and full visibility in the 1H NMR spectrum of all the analytes of interest in this study is achieved. Accuracy and Precision of the Concentration Measurements. The concentrations of the six analytes of interest were determined by the 1H NMR ERETIC method at four dilutions in M17 with phosphate buffer (pH 2.0). The values determined are summarized in Table 3 with standard deviations calculated from five replicate measurements on each tube. The coefficient of variation, calculated from these data for each measured concentration, ranged from 0.14 (acetic acid 6.99 mM) to 0.68% (acetic acid 26.40 mM), and the mean value was 0.36%. A linear regression analysis of the true values against the measured values was performed for each compound. Slope and intercept values are given in Table 3 with the corresponding r2 coefficients. In all cases, the fit for a linear correlation gave r2 > 0.997. The highest slope was 1.009 (diacetyl and acetoin) and the lowest was 0.996 (glucose). Intercepts ranged from 0.013 (diacetyl and acetoin) to 0.270 mM (citric acid). As can be seen from Table 3, the concentration measured by the ERETIC method is highly significantly correlated to the true values. The intercepts of regression give a measure of the systematic error of the method. For the metabolites produced by the fermentation, this is consistently below 0.03 mM. At the lowest 1866 Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

Figure 3. Evolution in time (h) of concentrations (mM) measured in the supernatant by the ERETIC method during the fermentation of lactic acid bacteria in M17 medium: (b) glucose, (9) citric acid, (4) diacetyl, (O) acetoin, (0) acetic acid, and (2) lactic acid.

concentrations determined (∼0.4 mM), this represents a maximal error of ∼7%. For the substrates, the error in the intercept is higher, notably 0.27 mM for citrate, representing an error of ∼4% in the lowest values determined. This systematic error is due primarily to imperfect smoothing of the baseline and could be improved by more rigorous baseline fitting. This explanation is consistent with the fact that the maximum intercept was found for citric acid, for which a broad line appears under the measured lines (Figure 1). It also explains why this parameter is different for each compound. When the intercept can be neglected (i.e., 1 mM for diacetyl, acetoin, or lactic acid, 10 mM for glucose, and 20 mM for citric acid concentration), the accuracy of the method can be measured by the slope of the linear regression at better than 1%. The variation coefficients obtained from the five measurements on the same tube demonstrate the short-term stability over time of the method and can be used to evaluate the precision. The worst value was found for citric acid, again consistent with a contribution of baseline correction introducing variability in the measurement. This step of the data processing is operatordependent. Nevertheless, the precision was 0.7% in the worst case. Kinetics of Fermentation. The evolution of the concentrations of all compounds measured by the 1H NMR ERETIC method during a typical lactic acid fermentation is plotted in Figure 3. The shapes of the curves reflect bacterial growth. At the beginning of the fermentation (0-4 h), the utilization of substrates and the appearance of products are minimal because of the low bacterial concentration. Between 4 and 8 h of culture, the growth of lactic acid bacteria is in exponential phase and all the concentrations

Table 4. Concentrations of Metabolites (mM) Measured by the ERETIC Method (Boldface Type) and by Enzyme Assay in Supernatant at Different Times after the Onset of Lactic Acid Fermentation in M17 Mediuma time (h)

glucose

citric acid

0

30.7 ( 0.4 30.2 ( 0.4 30.0 ( 0.8 30.0 ( 0.7 27.4 ( 0.8 28.5 ( 0.4 25.3 ( 0.6 25.6 ( 0.5 17.7 ( 0.5 17.5 ( 0.3 8.1 ( 0.3 7.0 ( 0.1

15.3(0.3 15.7 ( 0.3 15.7 ( 0.4 14.6 ( 0.1 14.9 ( 0.4 14.6 ( 0.3 13.9 ( 0.3 13.6 ( 0.1 7.7 ( 0.2 6.9 ( 0.07

2 4 5 6 7 8 10

diacetyl

acetoinb

0.0c

0.59 ( 0.01 0.68 ( 0.01 0.69 ( 0.01 0.41 ( 0.03 0.84 ( 0.01 1.37 ( 0.04 1.08 ( 0.02 1.55 ( 0.04 1.97 ( 0.03 2.05 ( 0.05 2.03 ( 0.02 2.02 ( 0.04 1.92 ( 0.05

acetic acid

1.38 ( 0.04 4.4 ( 0.1 3.53 ( 0.09 4.1 ( 0.2

0.75(0.04 0.91 ( 0.02 1.10 ( 0.06 0.96 ( 0.04 2.9 ( 0.1 3.27 ( 0.04 10.2 ( 0.3 10.6 ( 0.15 21.0 ( 0.6 18.93 ( 0.08 22.3 ( 0.6 18.97 ( 0.54 24.8 ( 0.8 24.5 ( 1.5

lactic acid 0.0c 1.3 ( 0.1 0.5 ( 0.1 3.2 ( 0.2 2.6 ( 0.1 8.8 ( 0.3 8.9 ( 0.2 22.0 ( 0.8 29.4 ( 0.6 43 ( 1 42.7 ( 0.7 54 ( 2 53.7 ( 0.3 59 ( 2 58 ( 2

a Standard deviations were calculated from five (ERETIC) or three (assay) measurements on the same sample. b The determination of the concentration of acetoin by classical methods is subject to interference by diacetyl (see Results and Discussion). cBy 1H NMR, low concentrations of acetic acid (0.54 ( 0.02 mM) and lactic acid (0.85 ( 0.06 mM) are detected in the M17 medium prior to fermentation (see Figure 1). This is taken into account in calibrating the ERETIC signal, as the reference solution is prepared in M17 medium.

evolve quickly. By 8 h, the substrate concentrations are diminished, metabolism is slowing down, and the products, lactic and acetic acids, diacetyl, and acetoin, are approaching their maximal concentrations. The profiles observed using the 1H NMR ERETIC method closely follow those reported previously using classical methods to determine the metabolite concentrations.12 To validate the 1H NMR determinations of the concentrations by the ERETIC method, the NMR-derived data have been compared to the results obtained on the same samples by classical methods (Table 4). For the substrates, glucose and citric acid, the results obtained by 1H NMR ERETIC and classical enzymatic methods are identical. However, due to line splitting in the spectrum, the determination of these compounds by NMR becomes difficult at low concentrations. For the major products, lactic and acetic acids, the concentrations determined by 1H NMR ERETIC are in good agreement with those determined enzymatically. In contrast, the NMR-determined values for diacetyl are consistently lower than those determined by colorimetry,14 notably during the first 6 h of the fermentation. At the beginning of the fermentation (0, 2, and 4 h), when substrate concentrations are maximal, ∼0.6 mM diacetyl is found by the colorimetric test whereas no diacetyl peak is visible in the 1H NMR spectrum. This demonstrates that the colorimetric method is less accurate due to interference in the test method by one of the substrates present. This is confirmed by the closer agreement found between the two determination methods later in the fermentation when the substrates are totally consumed. Although the concentration of acetoin is commonly determined by a colorimetric method,27 in which R-naphthol and creatine react with acetoin to give a red complex, this method is widely recognized as prone to severe interference from diacetyl.9 The hydration of diacetyl in solution, as observed by 13C NMR (see above), could explain this crossreaction. The determination of the acetoin concentration by 1H NMR under appropriate conditions overcomes this problem of interference. (27) Walsh, J.; Cogan, T. M. J. Dairy Res. 1974, 41, 25-30.

Overall Efficiency of the Method. The 1H NMR ERETIC method allows the determination of numerous analytes simultaneously. It has the advantage over methods using external calibrations for each analyte in that only a single calibration is required. The precision of the method is shown to be comparable or better than that of classical methods of determining the concentration of each analyte separately (Table 4). In contrast to this time-consuming and tedious approach, the concentrations of six components of the medium can be determined in less than 30 min. No complex sample preparation is required, alleviating the risk that partial purification or concentration might alter the relative concentrations measured. Interference between compounds is avoided, provided they have distinct lines in the NMR spectrum. A major advantage of the ERETIC method is that dynamic measurements can be made. CONCLUSION ERETIC provides a generic method for the determination of concentration, which avoids contaminating the sample, thus retaining it in a suitable state for further analyses. The reference signal can be tuned in terms of both chemical displacement and amplitude to suit the particular constraints of the experiment. It is demonstrated that ERETIC provides accurate and precise determinations of a wide range of concentrations of chemically diverse species in a mixture without having to calibrate each analyte individually. The use of the ERETIC reference can be carried out on a commercial spectrometer with minimal modification. For ERETIC implementation, a second rf channel is needed but this is always present on the NMR spectrometer. Furthermore, the electronic device used to generate the ERETIC signal is easy to make.19 Indeed, many modern spectrometers are equipped with an electronic card able to synthesize a shaped rf pulse, a facility that can replace the electronic device for generating the ERETIC signal. This approach is presently under investigation in our laboratory. We therefore propose that the method described can be used routinely to measure metabolite concentrations in fermentation supernatants or other biological fluids. Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

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NMR has been widely exploited for the determination of the concentration of small molecular weight analytes in complex biological fluids.1,2 It has the major advantage of permitting the determination of the concentration of a number of metabolites simultaneously but has always suffered from problems of accurately calibrating the signal. While a classical internal reference can be used in this type of study, there are a number of disadvantages to this approach. First, in such media, chemical interactions can induce a lack of NMR visibility, which varies depending on the compounds measured. The use of the phosphate buffer at pH 2.0 has restored the visibility in our case, but low pH cannot be considered as a universal solution. Furthermore, the chemical reference may induce an increase in the duration of the experiment if it has a T1 significantly higher than that of the analytes present in the sample. ERETIC overcomes this problem, providing a method, that is at least as accurate and precise as that of classical techniques and considerably less time-consuming. In terms of the system studied, the cofermentation of glucose and citric acid by a lactic acid bacterial culture, ERETIC NMR

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offers considerable advantages over conventional analysis.12 Because of the speed of analysis, it is potentially possible to follow the kinetics of the fermentation much more closely. Furthermore, because the entire medium content of small molecular weight analytes is observed, unexpected metabolites will be detected. This is particularly important where comparisons between fermentation conditions and strains are being made. ACKNOWLEDGMENT The contribution of the Scientific Council of the Region of the Pays-de-la-Loire to the purchase of a 500-MHz spectrometer is gratefully acknowledged. S.G. acknowledges the financial support of the MERST for a doctoral bursary.

Received for review November 10, 2000. Accepted February 4, 2001. AC0013204