© The Authors, published by EDP Sciences, 2019

1. Introduction

Current oenological practices are focused in searching strategies for reducing alcohol content in wine, as climate changes has provoked an important increase of sugar amounts in must. With the simplest Saccharomyces yeast strains anaerobic conditions, fermentation of grapes with increased sugar content will produce wine with high alcohol by volume percentage (%ABV) and thus a product with penalized mouth-feel and/or taint and/or flavour [1, 2]. In consequence, an important reduce of market consumption or severe tax government policies, could be expected. Amongst the broad spectrum of viticultural, physical or microbiological processes suggested for alcohol reduction, the concatenated use of non-Saccharomyces yeast strains to first aerobically sequester excess of sugar content by respiration, followed by the use of anaerobic Saccharomyces strains for final fermentation, has gained attention in the last years [3, 4, 5]. Methods comprising the use of several non-Saccharomyces yeast strains for assimilating must sugar to produce wine with reduced alcohol content and appropriate organoleptic properties has been extensively reported at lab- medium- and pilot-scales [6, 7, 8], but scarcely reported at industrial scale [9]. Finally, analysis of relevant wine parameters of reduced alcohol wines is carried out currently by a set of different enzymatic colorimetric trials and chromatographic schemes. Basic wine analysis comprises the quantification of primary metabolites such as ethanol, acetic acid, D-glucose, D-fructose, glycerol, lactic acid, malic acid and/or isovaleric acid -each with a particular enzymatic test kit-, that present some disadvantages such as time consuming; they present certain complexity in terms of sample preparation and chemical manipulations, they are costly and require some level of analytical expertise.

The present work proposes a “one-shot” evaluation of basic dealcoholized wine parameters with one- and two-dimensional Nuclear Magnetic Resonance (NMR) fingerprint and profiling [10, 11, 12]. Mono-varietal Mexican Cabernet Sauvignon wines with reduced alcohol content, were prepared in a large-scale regime (3.0 Ton grape, final volume of c.a. 2900 L) with non-Saccharomyces Candida zeplina strain, in co-inoculation with S. cerevisiae for final fermentation. Said reducing alcohol content procedure, was compared with at least two alcoholic fermentations at the same scale, carried out respectively with S. bayanux or S. cerevisiae (CM) strains. Ethanol reduction with simultaneous profiling and quantification of key metabolites such as acetic acid, malic acid, sorbic acid, fumaric acid and shikimic acid were carried out with a recent OIV procedure [10]. Finally, the use of 2D-NMR acquisition schemes are proposed in order to increase the number of observables for quantifying efficiency of alcohol reduction, with a set of additional parameters so far not explored.

2. Materials and methods

2.1. Large scale fermentations

Large scale production of wine with reduced Alcohol content (%alc. v/v) was carried out in two different regions: Baja California, Mexico (Monte Xanic, hereinafter called as MX) and Parras, Coahuila, Mexico (Casa Madero, hereinafter called asCM), using the variety Cabernet Sauvignon, obtaining yields of c.a. 2950 L of wine, using as raw material 3.0 Ton Cabernet Sauvignon grapes. Prior to vinification, used grapes presented the following chemical properties, resumed in Table 1.

Large-scale alcohol reduction were done with the following non-conventional strains (Tables 2–4), whereas results will be compared against a large-scale control process (Saccharomyces bayanus, Strain 10N45).

Large scale fermentations herein developed, can be resumed as:

• A.

  Standard Saccharomyces bayanus, Strain 10N45.

• B.

  Co-inoculation Non-Saccharomyces Candida zemplinina (Enartis Ferm) +S. cerevisiae 10N45

• C.

  Inoculation w/Saccharomyces Bayanus ex uvarumES-U42 (Enartis Ferm)

• D.

  Inoculation w/Saccharomyces cerevisiae (IONYS).

2.2. Nuclear magnetic resonance (NMR) spectroscopy

Sample preparation for NMR studies comprised the addition of 100 μL of a mixture of D₂O and chemical-shift reference sodium 3-(trimethylsilyl)-propionate-2, 2, 3, 3-d₄ (TSP), phosphonate buffer KH₂PO₄ 0.1% and 2% NaN₃ to 900 uL of wine sample, whereas pH was finally adjusted to a value of 3.9 for all samples. Samples were finally versed in standard 5 mm NMR tubes.

All spectra were recorded on a Bruker 600 AVANCE III HD equipped with a 5 mm ¹H/D TXI probehead with z-gradient. The following set of NMR experiments were conducted at a temperature of 298 K, stabilizing the temperature with a Bruker VCU flow unit:

• a)

  Quantitative one-dimensional proton nuclear magnetic resonance spectra (q-1D-¹H-NMR) used to measure the reduction of alcoholic content in wines were carried out by recording a total of 64 transients, that were collected into 28,844 complex data points, with a spectral width of 20 ppm (12019 Hz), an optimized recovery delay of 5.6 seconds to obtain quantitative signal integration and acquisition times of 1.2 s, produced experimental times of 7 minutes per experiment. No apodization function was used prior to Fourier Transformation.

• b)

  1D-¹H experiments with water-to-ethanol solvent presaturation were carried out as elsewhere reported [12].

• c)

  Two-dimensional ¹H-¹³C Heternouclear Single Quantum Coherence experiment [13] with a home-made water-to-ethanol multipresaturation scheme [12], prior to INEPT polarization transfer [14] were recorded by acquiring 7810 × 256 points with 32 scans per transient. Heteronuclear ¹H-¹³C spectral widths were setup respectively at 20 (¹H), 180 (¹³C) ppm. With acquisition times for the direct F2 dimension of 325 ms and a recovery delay of 1 s. produced experimental times of 3 h 18min per HSQC experiment.

Figure 1. Quantitative one-dimensional proton nuclear magnetic resonance (q-1D-1H NMR) spectra of large-scale fermented wine samples of MX (top) and CM (bottom) obtained with: standard S.bayanus as control (black); Non-Saccharomyces Candida zemplinina + S. cerevisiae 10N45 (blue); Saccharomyces Bayanus ex uvarum ES-U42 (pink) and Saccharomyces cerevisiae (IONYS, brown).

Figure 2. Stacked proton one-dimensional Nuclear Magnetic Resonance spectra (1H-NMR) with improved water-to-ethanol multipresat. Scheme [12] of Mexican monovarietal Cabernet Sauvignon wines, with different large-scale fermentations (A to D fermentation processes, see Materials and Methods, see as well Figure legends). Top: stacked 1D-1H NMR from Monte Xanic; Bottom: stacked 1D-1H NMR from Casa Madero. NMR signature of Mexican Cabernet Sauvignon shows the signal assignment of relevant metabolites prone to be quantified by the PULCON/NMR-OIV method [10, 15].

Figure 3. Alcohol reduction (with respect standard S.bayanus controls), wines' final pH, Total Acidity (T.A.), Volatile Acidity (V.A.) and free sulphites of large-scale fermentation trials.

Figure 4. Two-dimensional 1H-13C Heternouclear Single Quantum Coherence (HSQC) experiments, applying a home-made water-to-ethanol multipresaturation scheme [12] used to detect novel metabolites with the addition of a 13C NMR dimension. Magenta: 1H-13C HSQC of large-scaled fermented wines with Saccharomyces Bayanus ex uvarum ES-U42 strain; Black: 1H-13C HSQC of large-scaled fermented wines with standard Saccharomyces Bayanus control; Blue: 1H-13C HSQC of large-scaled fermented wines with Co-inoculation of Non-Saccharomyces Candida zemplinina (Enartis Ferm) with S. cerevisiae 10N45 strain. Bottom: 1H-13C HSQC pulse program, with the addition of a multipresat module, prior to the INEPT polarization transfer module.

Figure 4. Continued.

Figure 5. Quantification of acetic acid, malic acid, sorbic acid, fumaric acid and shikimic acid according to the PULCON/NMR method [15]. In all cases, an external reference of each metabolite, with known concentration, has been used for calibration. External standards were prepared in concentrations close to the OIV reference mean value (dotted red line) as follows: [acetic acid] = 650 mg/L; [malic acid] = 1436 mg/L; [sorbic acid] = 152 mg/L; [fumaric acid] = 49 mg/L; [shikimic acid] = 51 mg/L.

3. Results and discussion

Q-1D-¹H-NMR spectra of full set of samples (Fig. 1) were obtained in order to calculate %alc. (v/v) of each fermented product, by means of signal integration of methyl (δ = 1.026 ppm, triplet)-to-methylene (δ = 3.497 ppm, quartet) signals, with respect water signal (δ = 4.7 ppm, singlet). Direct percentage of integrated methylene signal (δ = 3.497 ppm, quartet), with respect integrated H₂O signal (δ = 4.7 ppm, singlet), immediately provides the alcohol percentage of fermented samples, as methylene and water signals present both the same number of observed spins (I = 2). Alcohol reduction of each large scale trial, computed by q-1D-¹H-NMR was of around 1% in all cases (see Fig. 3), as expected and verified by cross-check methods. In the other hand, multipresat 1D-¹H-NMR experiments will serve to detect and quantify appropriate metabolites such as acetate (1.9–1.92 ppm), malate (2.45–2.48 ppm), sorbate (5.84–5.87 ppm), fumaric (6.65–6.61 ppm) and shikimic (6.68–6.69 ppm) by the PUlse Length based CONcentration (PULCON) method [15].

Detection and quantification of acetate, malate, sorbate, fumaric and shikimic moieties from the NMR signature is related to the easiness for signal selection of isolated-intense resonances from above mentioned metabolites, even exposed within the q-1D-¹H-NMR spectra, without multipresat scheme (Fig. 1), but at low signal-to-noise ratio. However, novel schemes involving solvent elimination and addition of a second dimension for dispersing encumbered resonances from the 1D-¹H fingerprint will shed light in novel resonances or exposed regions, potentially ready to be quantified with PULCON/NMR procedures. For that it is presented the use of a two-dimensional ¹H-¹³C Heternouclear Single Quantum Coherence (HSQC) experiments, applying a home-made water-to-ethanol multipresaturation scheme [12] for assigning novel resonances involving relevant metabolites associated to oenological practices or quality control parameters.

Set of novel assigned resonances with the use of two-dimensional ¹H-¹³C HSQC are: tyrosine, phenylalanine, fructose, glycerol, glutamine, lactic acid and quercetin. Assignments were done by confirming a ¹H-resonance with its correlation to a specific ¹³C chemical shift. It is worth noting to highlight as example that quercetin proton resonance (6.68 to 6.77 ppm) present a shift as a function of large-scale fermentation scheme. Despite the last, the easiness to identify quercetin relies on the unambiguous carbon correlations (δ¹³C = 116 and 137 ppm) with shifted protons, proving though the advantages of using additional NMR dimensions.

Quantification with PULCON/NMR method consists in referring the signal integral of an unambiguously assigned resonance (or set of them), with respect signal intensity and line width of a known external reference, prepared at chemical conditions close to the sample of unknown concentration. Set method has been proposed for proteins [15] and recently to wine targeting [10], whereas for the later study, 20 mg/L of citric acid was used as external reference [10]. For the present study a set of external references were used for cross-check validation: 650 mg/L of acetic acid; 1436 mg/L of malic acid; 152 mg/L of sorbic acid; 49 mg/L of fumaric acid and 51 mg/L of shikimic acid. All external reference solutions were prepared at similar pH and buffer conditions with respect wine samples (see Materials and Methods). PULCON/NMR targeted analysis is stressed in Fig. 5 and Table 5.

From PULCON/NMR quantifications the following observations arise: At selected large-scale fermentation conditions, control and dealcoholized products present normal mean values of both acetic and shikimic acids. Normal mean values of malic acid were detected for both control and dealcoholized products of only Monte Xanic stocks. Acceptable mean values of fumaric acid were only obtained for dealcoholized wines from Casa Madero, fermented with co-inoculations S. cerevisiae 10N45 +C. zeplinia. Controls and dealcoholized wines in all cases present low values of sorbic acid (8–15 mg/L for CM samples; 64–100 mg/L for MX samples) with respect OIV accepted mean values (150mg/L), suggesting a strong susceptibility of all samples to be unprotected against fungi or bacteria attacks [16].

4. Conclusions

A multitask NMR strategy is herein proposed to analyse alcohol reduction and oenological practice in wines produced with the first large-scale Non-Saccharomyces or non-conventional Saccharomyces fermentation processes. Standard metabolites were detected and quantified with the PULCON/NMR procedure and quality recommendations can be done as a function of selected fermentation scheme for reducing %alc. (v/v). High-resolution two-dimensional ¹H-¹³C HSQC with solvent multipresat have revealed novel resonances ready to be targeted and quantified with PULCON/NMR method, towards a not known accuracy in wines' quality control.

Acknowledgments

Authors acknowledge the Mexican Ministry of Science and Technology (CONACyT) for funding: CONACyT program No. 682 “Cátedras CONACyT”, CONACyT program No. LN295321 “Laboratorios Nacionales” and CONACyT-México infrastructure grant: INFRA-2016 (269012) for funding Nuclear Magnetic Resonance experimental time.

References

All Tables

All Figures

	Figure 1. Quantitative one-dimensional proton nuclear magnetic resonance (q-1D-1H NMR) spectra of large-scale fermented wine samples of MX (top) and CM (bottom) obtained with: standard S.bayanus as control (black); Non-Saccharomyces Candida zemplinina + S. cerevisiae 10N45 (blue); Saccharomyces Bayanus ex uvarum ES-U42 (pink) and Saccharomyces cerevisiae (IONYS, brown).
In the text

Figure 2. Stacked proton one-dimensional Nuclear Magnetic Resonance spectra (1H-NMR) with improved water-to-ethanol multipresat. Scheme [12] of Mexican monovarietal Cabernet Sauvignon wines, with different large-scale fermentations (A to D fermentation processes, see Materials and Methods, see as well Figure legends). Top: stacked 1D-1H NMR from Monte Xanic; Bottom: stacked 1D-1H NMR from Casa Madero. NMR signature of Mexican Cabernet Sauvignon shows the signal assignment of relevant metabolites prone to be quantified by the PULCON/NMR-OIV method [10, 15].

In the text

	Figure 3. Alcohol reduction (with respect standard S.bayanus controls), wines' final pH, Total Acidity (T.A.), Volatile Acidity (V.A.) and free sulphites of large-scale fermentation trials.
In the text

Figure 4. Two-dimensional 1H-13C Heternouclear Single Quantum Coherence (HSQC) experiments, applying a home-made water-to-ethanol multipresaturation scheme [12] used to detect novel metabolites with the addition of a 13C NMR dimension. Magenta: 1H-13C HSQC of large-scaled fermented wines with Saccharomyces Bayanus ex uvarum ES-U42 strain; Black: 1H-13C HSQC of large-scaled fermented wines with standard Saccharomyces Bayanus control; Blue: 1H-13C HSQC of large-scaled fermented wines with Co-inoculation of Non-Saccharomyces Candida zemplinina (Enartis Ferm) with S. cerevisiae 10N45 strain. Bottom: 1H-13C HSQC pulse program, with the addition of a multipresat module, prior to the INEPT polarization transfer module.

In the text

	Figure 4. Continued.
In the text

Figure 5. Quantification of acetic acid, malic acid, sorbic acid, fumaric acid and shikimic acid according to the PULCON/NMR method [15]. In all cases, an external reference of each metabolite, with known concentration, has been used for calibration. External standards were prepared in concentrations close to the OIV reference mean value (dotted red line) as follows: [acetic acid] = 650 mg/L; [malic acid] = 1436 mg/L; [sorbic acid] = 152 mg/L; [fumaric acid] = 49 mg/L; [shikimic acid] = 51 mg/L.

In the text