# Development of a purification protocol guided by bitter taste receptor activation and sensory analysis to isolate new tasteactive compounds in barrel-aged wines

Delphine Winstel, Christine Belloir, Pierre Waffo-Téguo, Loïc Briand, Axel Marchal

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Delphine Winstel, Christine Belloir, Pierre Waffo-Téguo, Loïc Briand, Axel Marchal. Development of a purification protocol guided by bitter taste receptor activation and sensory analysis to isolate new tasteactive compounds in barrel-aged wines. 2021, Proceedings of the 16. Weurman Flavour Research Symposium, 10.5281/zenodo.5347016. hal-03368011

HAL Id: hal-03368011

https://hal.inrae.fr/hal-03368011

Submitted on 6 Oct 2021

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

# Development of a purification protocol guided by bitter taste receptor activation and sensory analysis to isolate new taste-active compounds in barrel-aged wines

DELPHINE WINSTEL<fnref n="1" />, Christine Belloir<fnref n="2" />, Pierre Waffo-Téguo<fnref n="1" />, Loïc Briand<fnref n="2" /> and Axel Marchal<fnref n="1" />

## Abstract

Wine sensory quality is strongly dependent on oak wood ageing, as it can occasionally increase the perception of bitterness. This sensory phenomenon is due to the release of non-volatile molecules from wood. The search for such bitter compounds, requires both reliable purification tools and powerful identification techniques. The aim of this work was to propose the development of an original inductive approach, using separative techniques, sensory analysis and human bitter taste receptor activation, to discover new taste-active molecules. This new methodology was implemented on oak wood extracts to isolate and purify bitter compounds. By using High Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR), a new galloyl glucopyranosyl compound, named GG-DMC, was elucidated for the first time. Its bitter properties were validated by receptor activation and sensory analysis in two matrices. These findings demonstrated the relevance and the effectiveness of the developed strategy.

Keywords: bitterness, taste-guided purification, taste, TAS2R, receptors

## Introduction

During barrel ageing, wines and spirits undergo organoleptic changes caused by the release of aroma and taste molecules. While the key aromatic compounds released from oak wood have been identified, the bitter and sweet molecular determinants remain largely unknown. A gain in sweetness is frequently observed during ageing [1], that can be explained by the release of sweet triterpenoids from oak wood [2, 3]. Ageing in barrels can sometimes increase the perception of bitterness in wines, which has a negative impact on their value. This phenomenon has been widely attributed to ellagitannins, whose bitter characteristics have been suggested [4]. However, results showed that the detection thresholds of the main ellagitannins were significantly higher than their concentrations in wines, suggesting their low influence on wine bitterness [5]. More recently, the bitter properties of lyoniresinol, a lignan extracted from oak wood, have been demonstrated [6]. Despite these significant acquisitions, these compounds cannot explain the gustatory importance of oak ageing. Consequently, a large number of non-volatile molecules released by the oak remain to be identified, as well as their sensory contribution. To isolate these bitter molecules, an inductive approach with a fractionation protocol guided by gustatometry has been implemented in our laboratory for over 10 years [2, 7-8]. The success of this approach is based on the choice of raw extract, the reliability of the sensory test, and on the access to various complementary analytical techniques. In order to improve the search for taste-active compounds, new methodologies could be used. In an attempt to overcome the masking phenomena occurring during tasting, the fractions, obtained with the fractionation protocol, were screened by measuring the activation of human bitter taste receptors (TAS2Rs). Therefore, this work aims at proposing a complementary strategy for the discovery of taste-active compounds.

## Experimental

### Samples

Oak wood used for this study was supplied by the cooperage company Seguin-Moreau (Merpins, France). It was sampled in April 2017 from a batch of staves used to make barrels. The three levels of toasting corresponded to untoasted wood (UW), to toasted wood (TW) under the same conditions as those for "bousinage" of the barrels (around 180° C for a few minutes) and to heavy toasted wood (HTW) (200°C for 4 hours). The botanical species was assigned to *Quercus petraea* according to the method described by Marchal et al [9].

<footnote n="1">¹ Unité de recherche Œnologie, EA 4577, USC 1366 INRAE, ISVV, Université de Bordeaux, F33882 Villenave d'Ornon, France, delphine.winstel@u-bordeaux.fr</footnote>

<footnote n="2">² Centre des Sciences du Goût et de l'Alimentation, AgroSup Dijon, CNRS, INRAE, Université de Bourgogne Franche-Comté, 21000 Dijon, France</footnote>
Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS) analysis

The LC-HRMS platform consisted of a Vanquish system (Thermo Fisher Scientific, Les Ulis, France) with binary pumps, an autosampler and a heated column compartment and with an Exactive Orbitrap mass spectrometer, equipped with a heated electrospray ionization (HESI-II) probe (both from Thermo Fisher Scientific). Liquid chromatography separation was performed on a C18 column (Hypersil Gold 2.1mm × 100 mm, 1.9 µm particle size, Thermo Fisher Scientific) with water containing 0.1% of formic acid (A) and acetonitrile with 0.1% of formic acid (B) as mobile phases. The flow rate was 600 µL/min and eluent B varied as follows: 0 min, 10%; 1.0 min, 10%; 5.0 min, 50%; 5.3 min, 98%; 6.0 min, 98%; 6.15 min, 10%; 7 min, 10%. Optimization of voltages, gas values, and temperatures applied for ion transfer and ionization was performed in negative mode.

Extraction of oak wood

The three modalities of sessile oak wood (600 g) were macerated in 6 L of H₂O/EtOH solution (50:50; v/v), for 2 weeks and under an inert atmosphere. Filtration (0.45 µm) was used to remove wood sawdust and particles. The three solutions containing soluble wood compounds were evaporated *in vacuo*, suspended in water, and freeze-dried to obtain brownish powders.

Liquid-liquid extractions

The powder of untoasted wood extract was dissolved in 450 mL of water and was washed twice with 450 mL of *n*-heptane. This aqueous layer was then extracted successively with methyl *tert*-butyl ether (MTBE) (6×500 mL), ethyl acetate (EtOAc) (5×800 mL) and with water-saturated butan-1-ol (BuOH) (4×800 mL). The combined organic layers were evaporated *in vacuo*, suspended in water, and freeze-dried to get powders of MTBE (1.5 g), EtOAc (1.6 g), BuOH (3.6 g) and aqueous (14.6 g) pre-purified extracts.

Centrifugal partition chromatography (CPC)

CPC was performed on a Spot prep II LC coupled with a SCPC-100+1000 (Armen Instrument, Saint-Avé, France), both controlled by Armen Glider Prep V5.0 software. A 1 L rotor was employed. The choice of an appropriate biphasic system of solvents was based on the study of the partition of extract compounds in both phases following the procedure described by Marchal et al [2]. The partition coefficient, *Kd*, was calculated as the ratio of the solute area in each phase. On this basis, various systems were tested, and the BuOH extract was fractionated using the ternary biphasic system EtOAc/propan-2-ol/H₂O (5:1:5, v/v). Separation was performed by one CPC run of 3.6 g injection. Six fractions F1 to F6 were constituted on the basis of their similar chromatographic profile, after being combined, evaporated *in vacuo*, suspended in water and freeze-dried.

Preparative liquid chromatography

Preparative HPLC analyses were performed using a Waters Prep 150 LC including a 2545 Quaternary Gradient Module, a 2489 UV/Visible detector and a 2424 ELSD detector (Waters, Guyancourt, France). Separations were performed using a SunFire Prep C18 OBD (19 mm × 250 mm, 5 µm particle size, Waters) equipped with a SunFire preparative C18 guard cartridge (20 × 19 mm, 5 µm particle size, Waters). The mobile phase was a mixture of ultrapure water and acetonitrile, both containing 0.1% of formic acid. UV detection was carried out at 280 nm and chromatographic peaks were collected manually just after the detector. Samples obtained after successive injections were pooled, evaporated *in vacuo* to remove acetonitrile and freeze-dried twice.

Nuclear Magnetic Resonance (NMR) experiments

NMR experiments were conducted on a Bruker UltraShield® System Avance 600 NMR, spectrometer (¹H at 600.27 MHz and ¹³C at 150.95 MHz) equipped with a 5 mm TXI probe. All 1D (proton) and 2D (¹H-¹H COSY, ¹H-¹H ROESY, ¹H-¹³C HSQC, and ¹H-¹³C HMBC) spectra were acquired at 300 K in methanol-*d*₄.

Sensory analysis

Taste evaluation was performed in a dedicated room, under normal daylight, at room temperature (around 20°C) and with normalized glass. Fractions or pure compounds were tasted by five experts in wine tasting. They were asked to give their written consent to participate and instructed to spit out the samples after tasting. They described the gustatory perception (bitterness, sourness, sweetness, astringency) of each glass using the vocabulary of wine tasting and were asked in particular to evaluate the bitterness intensity on a scale from 0 (not detectable) to 5 (strongly detectable). The newly identified compounds, named GG-DMC, was tasted at 5 mg/L in water (eau de source de Montagne, Laqueuille, France), as well as in a non-oaked white wine (Bordeaux 2013).
Measurement of the activation of the TAS2Rs by calcium-mobilization assays

To measure the activation of 25 human TAS2Rs, two preliminary steps were necessary. Seeding on 96-well microtiter plates, lasting 24 hours, on HEK293 cell lines stably transfected with the Ga16-gust44 protein (gustducin) was first carried out. The next step, transient transfection with one of the TAS2R expression plasmid was performed and promoted by a common transfection reagent, lipofectamine 2000TM (Thermo Fisher Scientific). Mock cells were also transfected with empty expression vector. Subsequently, transfected cells were loaded with the calcium fluorescent probe, Fluo4-AM (Molecular Probes). Cells were then stimulated with oak wood extracts at increasing concentrations: from 0.1 to 10 mg/mL for the sessile oak wood extract, from 0.0003 to 1 mg/mL for the UW, TW and HTW extracts, from 0.0033 to 3.3 mg/mL for the MTBE, EtOAc, BuOH and aqueous phase extracts of UW, and from 0.001 to 1 mg/mL for the first CPC fractionation of the UW_extract. The changes of fluorescence of TAS2R-expressing cells were recorded using a fluorometric imaging plate reader (Flexstation®, Molecular Devices) at 510 nm following excitation at 488 nm. All the measurements were carried out in duplicate.

**Results and discussion**

To isolate new bitter molecules, an inductive approach with a fractionation protocol guided by gustatometry has been implemented in our laboratory. This approach involves an extraction step followed by the development of a fractionation protocol implemented using various separation techniques such as liquid/liquid extractions, centrifugal partition chromatography (CPC), and preparative-HPLC. After each fractionation step, sensory analysis was used to select relevant fractions. However, masking phenomena between compounds of a same fraction can occur during tasting and could affect the efficiency of this approach. Consequently, a complementary strategy was implemented by coupling the sensory analysis to measurement of the activation of the human bitter taste receptors. After each fractionation step, the extracts obtained were tested for their ability to activate functionally expressed TAS2Rs using *in vitro* calcium imaging assay on the 25 human TAS2Rs (Figure 1).

<figure><img src="image_4.png" /><figcaption>**Figure 1: Fraction screening strategies during the fractionation protocol.**</figcaption></figure>

TAS2Rs activation test by an extract of sessile oak wood

To begin this new approach, a preliminary test was performed on a sessile oak wood extract. A screening of the different bitter taste receptors was carried out in order to identify the best activated TAS2R receptor by the oak wood. The receptor activations following stimulation with increasing concentrations of extract were measured. Fluorescence changes were compared to a control well, i.e. a well without addition of extract, and responses of mock-transfected cells were subtracted. The results showed that cells expressing five of the twenty-five TAS2Rs responded to an oak wood extract (Figure 2). The best response was obtained for TAS2R7, followed by TAS2R4, TAS2R16, TAS2R38, and TAS2R43 receptors. For TAS2R7, fluorescence increased with concentration then decreased from 3 g/L. Indeed, at too high concentrations, the environment could become toxic to the cells, which would lead to a decrease in the cell response. To continue this approach, we focused on the TAS2R7 receptor, which induced robust cellular response.
<figure><img src="image_5.png" /><figcaption>**Figure 2:** Activation of TAS2R receptors by an extract of sessile oak wood.</figcaption></figure>

### TAS2R7 activation test by UW, TW, HTW extracts

The same protocol was performed on three different extracts, which corresponded to different toasting levels of sessile oak wood extracts: heavy toasted wood (HTW), toasted wood (TW) and untoasted wood (UW). In order to analyse the ability of the fractions to activate bitter taste receptors, a dose-response curve was established, which allowed the calculation of the half maximal effective concentration (EC₅₀ value). This value reflects the taste potency of a substance: the lower the EC₅₀ value obtained, the greater the potency of the substance. Solutions of increasing concentrations of these extracts were added to the cells, then the activation of the TAS2R7 receptor was measured (Figure 3).

<figure><img src="image_6.png" /><figcaption>**Figure 3:** Activation of TAS2R7 by UW, TW, HTW extracts.</figcaption></figure>

For all three extracts, we found that the TAS2R7 receptor was activated. Nevertheless, a better dose-response was observed for UW extract leading to an EC₅₀ value of 3.04 ± 0.65 µg/mL. Conversely, the dose-response of HTW was half that of UW, with an EC₅₀ value of 7.36 ± 1.52 µg/mL. These results are consistent with the empirical observations of cooper and winemakers, and confirmed that untoasted wood brings more bitterness than heavy toasted wood. So, this UW extract was selected for further fractionation.

### TAS2R7 activation test by extracts of MTBE, EtOAc, BuOH and aqueous phase of UW

To begin the purification protocol, successive liquid-liquid extractions of UW have been carried out with solvents of increasing polarity. Four extracts were subsequently obtained and corresponded to MTBE, EtOAc, BuOH extracts and the aqueous phase. Increasing concentrations of these fractions were added to the cells expressing the bitter taste receptors and the activation of the TAS2R7 receptor was measured (Figure 4). The best responses were obtained for the aqueous phase and the butanol extract.
<figure><img src="image_7.png" /><figcaption>**Figure 4:** Activation of TAS2R7 by extracts of MTBE, EtOAc, BuOH and aqueous phase of UW.</figcaption></figure>

LC-HRMS analysis of the aqueous phase showed that this extract mainly contained ellagitannins. However, a previous study showed that the TAS2R7 receptor was activated by these hydrolysable tannins [10]. Thereby, it was possible that the measured signal for the aqueous phase was due to these compounds. Further studies will aim at validating this hypothesis. In order to search for new compounds, we only focused on butanol extract. The chemical complexity of the enriched BuOH extract required a further fractionation by CPC.

### Activation of TAS2R7 by CPC fractions of UW_BuOH extract

A ternary biphasic system EtOAc/propan-2-ol/H₂O (5:1:5, v/v) allowed the best partition of the BuOH extract between the two phases. At the end of this experiment, the tubes were analysed by LC-HRMS and pooled according to their composition to give fractions. After solvent evaporation and freeze-drying, 6 fractions (noted F1 to F6) were obtained as powder in variable quantities. Increasing concentrations of these fractions were added to the cells and the activation of the TAS2R7 receptor was measured (Figure 5).

<figure><img src="image_8.png" /><figcaption>**Figure 5:** Activation of TAS2R7 by six CPC fractions (F1 to F6) of UW_BuOH extract</figcaption></figure>

No signal was detected for F1, suggesting that TAS2R7 was not activated. Fraction F2 had the highest EC₅₀ of the other fractions. Fraction 6 obtained the best dose response with an EC₅₀ of 28 ± 7 µg/mL, followed by fraction F4 then F5 and F3. Then, tasting of the CPC fractions made it possible to distinguish a bitter fraction, the F5, which was in agreement with the positive response of the receptor to its contact. So, to continue this inductive approach, fraction F5 was selected. Its simplified chromatographic profile allowed us to perform a Preparative-LC.

### Activation of TAS2R7 by compound of m/z 539

To see which molecules were responsible for activating TAS2R7, fraction F5 was submitted to preparative HPLC with UV detection. A preliminary injection showed that the chromatograms presented a refined profile with only a few peaks detected both in ELSD and in UV a 280 nm. Thus, an appropriate gradient was chosen and the entire was fractionated by successive injections. The main components of fraction F5 were purified, including one compound of *m/z* 539. Its HRMS spectrum exhibited a deprotonated [M – H]⁻ ion at *m/z* 539.1411. Given the isotopic ratio and the experimental mass of the deprotonated ion, the empirical formula C₂₄H₂₈O₁₄ was assigned to
this compound. Before proceeding to its structural characterization, increasing concentrations of this unknown compound were added to the cells expressing the bitter receptor TAS2R7 (Figure 6). Results showed a robust dose-response of *m/z* 539 on TAS2R7 leading to an EC₅₀ value of 8 µg/mL or approximately of 15 µM. By comparison, magnesium sulphate, which is known as a bitter agonist of TAS2R7, presents an EC₅₀ on the order of 15 mM [11]. Measuring the activation of TAS2R7 helped to guide the fractionation to the isolation of a new bitter compound. Then, its structure was elucidated by using HRMS and NMR. To our knowledge, this compound, belonging to the galloyl glucopyranosyl family, had never been described in the literature previously, so we named it GG-DMC. This molecule was also tasted at 5 mg/L in water and in a unoaked white wine. It was perceived as bitter in these two matrices, which confirmed the results obtained during the activation of the bitter TAS2R7 receptor. By LC-HRMS screening, GG-DMC was also identified in several spirits.

<figure><img src="image_9.png" /><figcaption>**Figure 6:** *Activation of TAS2R7 by compound of m/z 539 (GG-DMC).*</figcaption></figure>

## Conclusion

The aim of this work was to propose an efficient methodology for discovering new taste-active compounds in oak wood. The results demonstrated the relevance and the effectiveness of combining separative techniques, sensory analysis and receptor activation to identify and purify bitter compounds. This purification protocol guided by *in vitro* and *in vivo* analysis led to the isolation of a new bitter compound, a galloyl glucopyranosyl derivative called GG-DMC. Its structural elucidation was carried out by high resolution mass spectrometry and nuclear magnetic resonance, and will be detailed in a forthcoming publication. Its bitter taste was validated by sensory analysis and receptor activation. These studies provide promising perspectives for a better understanding of the molecular markers responsible for the taste of foods and beverages such as wine.

## References

1. Marchal, A.; Pons, A.; Lavigne, V.; Dubourdieu, D. Contribution of Oak Wood Ageing to the Sweet Perception of Dry Wines: Effect of Oak Ageing on Wine Sweetness. Aust J Grape Wine Res. 2013;19:11–19.

2. Marchal, A.; Waffo-Téguo, P.; Génin, E.; Mérillon, J.-M.; Dubourdieu, D. Identification of New Natural Sweet Compounds in Wine Using Centrifugal Partition Chromatography–Gustatometry and Fourier Transform Mass Spectrometry. Anal Chem. 2011;83:9629–9637.

3. Gammacurta, M.; Waffo-Teguo, P.; Winstel, D.; Cretin, B.N.; Sindt, L.; Dubourdieu, D.; Marchal, A. Triterpenoids from Quercus Petraea: Identification in Wines and Spirits and Sensory Assessment. J Nat Prod. 2019;82:265–275.

4. Quinn, M.K.; Singleton, V.L. Isolation and Identification of Ellagitannins from White Oak Wood and an Estimation of Their Roles in Wine. Am J Enol Vitic. 1985;36:8.

5. Glabasnia, A.; Hofmann, T. Sensory-Directed Identification of Taste-Active Ellagitannins in American (Quercus Alba L.) and European Oak Wood (Quercus Robur L.) and Quantitative Analysis in Bourbon Whiskey and Oak-Matured Red Wines. J Agric Food Chem. 2006;54:3380–3390.

6. Marchal, A.; Cretin, B.N.; Sindt, L.; Waffo-Téguo, P.; Dubourdieu, D. Contribution of Oak Lignans to Wine Taste: Chemical Identification, Sensory Characterization and Quantification. Tetrahedron 2015;71:3148–3156.

7. Cretin, B.N.; Waffo-Teguo, P.; Dubourdieu, D.; Marchal, A. Taste-Guided Isolation of Sweet-Tasting Compounds from Grape Seeds, Structural Elucidation and Identification in Wines. Food Chem. 2019;272:388–395.

8. Sindt, L.; Gammacurta, M.; Waffo-Teguo, P.; Dubourdieu, D.; Marchal, A. Taste-Guided Isolation of Bitter Lignans from Quercus Petraea and Their Identification in Wine. J Nat Prod. 2016;79:2432–2438.

9. Marchal, A.; Prida, A.; Dubourdieu, D. New Approach for Differentiating Sessile and Pedunculate Oak: Development of a LC-HRMS Method To Quantitate Triterpenoids in Wood. J Agric Food Chem. 2016;64:618–626.

10. Soares, S.; Silva, M.S.; García-Estevez, I.; Großmann, P.; Brás, N.; Brandão, E.; Mateus, N.; de Freitas, V.; Behrens, M.; Meyerhof, W. Human Bitter Taste Receptors Are Activated by Different Classes of Polyphenols. J Agric Food Chem. 2018;66(33):8814–8823.

11. Behrens, M.; Redel, U.; Blank, K.; Meyerhof, W. The Human Bitter Taste Receptor TAS2R7 Facilitates the Detection of Bitter Salts. Biochem Biophys Res Commun. 2019;512:877–881.