Influence of Chirality of Lactones on the Perception of Some Typical

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The Influence of Chirality of Lactones on the Perception of Some Typical Fruity Notes through Perceptual Interaction Phenomena in Bordeaux Dessert Wines Panagiotis Stamatopoulos, Eric Brohan, Celine Prevost, Tracey Ellen Siebert, Markus J. Herderich, and Philippe Darriet J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03117 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 9, 2016

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Journal of Agricultural and Food Chemistry

The Influence of Chirality of Lactones on the Perception of Some Typical Fruity Notes through Perceptual Interaction Phenomena in Bordeaux Dessert Wines Panagiotis Stamatopoulos*†,‡, Eric Brohan§, Celine Prevost§, Tracey E. Siebert┴, Markus Herderich┴ and Philippe Darriet†,‡ †

Univ. de Bordeaux, ISVV, EA4577 Œnologie, F-33140 Villenave d'Ornon, France.

‡

INRA, ISVV, USC 1366 Œnologie, F-33140 Villenave d’Ornon, France.

§

Sanofi, 13 quai Jules Guesde, 94400 Vitry, France.

┴

The Australian Wine Research Institute, PO Box 197, Glen Osmond (Adelaide) SA

5064, Australia. *corresponding

author

(telephone:

+33557575868;

fax:

[email protected])

ACS Paragon Plus Environment

+33557575813:

email:


1

ABSTRACT

2

Recent studies concerning the aroma of noble rot dessert wines revealed the importance of a

3

well-known phenomenon in perfumery, the perceptual blending, to create the perception of

4

“overripe orange” nuances. Thus, compounds associated with both oak wood aging (3-

5

methyl-4-octanolide, eugenol) and Botrytis cinerea development under the form of noble rot

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(2-nonen-4-olide, γ-nonalactone) are contribute to a specific aroma of great noble rot dessert

7

wines through perceptual interaction phenomena. This synthetic perception phenomenon was

8

established from reconstitution, addition and omission sensory experiments, using wine

9

extracts fractions supplemented with the volatile compounds previously mentioned. In order

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to better understand the sensory impact of these compounds, the goal of this research was to

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study the contributions of enantiomeric forms of 2-nonen-4-olide and γ-nonalactone and the

12

diastereoisomers of 3-methyl-4-octanolide. After multidimensional chiral chromatography

13

analysis, the relative proportions of enantiomers or diastereomeric forms were first

14

established, and then sensory experiments were carried out using the reference compounds

15

with isolated fractions from dessert wines. A dominance of R form was established for 2-

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nonen-4-olide which was correlated with wine aging while the S form is more dominant in

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young dessert wines. Furthermore, the reconstitution experiments confirmed perceptual

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interaction phenomena and revealed the sensory contribution of (R)-2-nonen-4-olide and cis-

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3-methyl-4-octanolide concerning the “overripe orange” nuances, whereas no sensory impact

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for the γ-nonalactone isomers was observed.

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KEYWORDS: Chirality, lactones, enantiomers, diastereoisomers, perceptual interactions,

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dessert wines aroma


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INTRODUCTION

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γ- and δ-Lactones are well known naturally occurring chiral flavor compounds. Whereas γ-

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lactones are important aroma components of many fruits, δ-lactones can also be detected in

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dairy products and other fermented foods.1 The γ-lactones contribute aromas reminiscent of

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stone fruits such as peach or apricot, while δ-lactones have characteristic milky notes that

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often evoke coconut.2 Perception threshold studies revealed that γ-lactones appear to be more

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aroma intense than the corresponding δ-lactones.3,4 Almost all lactones have stereocenters and

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the abundance of their individual enantiomers in nature varies greatly, but there is a general

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tendency for the (R)-configuration to dominate. Specific and characteristic enantiomeric ratios

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have been reported for lactones in apricots, peaches, raspberries, strawberries, plums,

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mangoes, passion fruit, and juices, wines, and spirits.5,6 In these studies (R)-γ-decalactone and

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(R)-γ-dodecalactone were prevalent in strawberries and a significant excess of (R)-γ-

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decalactone was also detected in raspberry juice. The sensory properties of the enantiomers of

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a series of γ-lactones were described by Mosandl and Günther.7 Identified for the first time in

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whiskey by Suomalainen & Nykanen,8 the “oak lactone” was cited as a specific aroma

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compound from beverages aging in oak barrels. Kepner et al.9 then correctly identified the cis

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isomer of “oak lactone” in a Cabernet Sauvignon wine. They concluded that this compound

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originated from oak wood, since this isomer was not detected in the same wine aged in

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stainless steel vats. It was later shown that there were actually two diastereoisomers of “oak

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lactone”10 and not one as was originally reported. These two diastereoisomers were isolated

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from the oak and also identified from three species; Q. mongolica, Q. serrata and Q. alba

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(North America). Otsuka et al.11 also identified the “oak lactones” directly from wood and

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they showed an increase in the concentration of “oak lactone” in beverages aged in oak wood

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barrels. In wine, the importance of enantiomeric and diastereoisomeric forms of volatile

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compounds on the aroma perception of wines has been much documented including research



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works on monoterpenes12,13, thiols14, esters15and lactones.6,16 Regarding the lactones, the R

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isomer for both γ-octalactone and γ-nonalactone was shown to be the most abundant

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compound in wines.6 Most lactones are present at levels below their olfactory detection

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thresholds but some authors17 have demonstrated synergistic effects between these

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compounds. These interactions could explain the involvement of lactones in the aroma of the

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wines.

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Bordeaux dessert wines are produced by the action of Botrytis cinerea mold under the form of

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“noble rot” on perfectly ripe grapes. This action changes the composition of the berries by

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producing and concentrating odoriferous compounds, and also by increasing the cysteinylated

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and glutathionylated precursors of volatile thiols.18–20 Other studies21–24 have demonstrated the

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key role of volatile compounds belonging to various chemical families such as thiols,

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furanones, Strecker aldehydes and lactones, in the aromas of noble rot dessert wines. Some

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studies revealed higher concentrations of lactones compared to dry white wines from the same

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grape variety.21,25 Moreover, massoia lactone was identified in noble rot dessert wines21 but

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only the (R) isomer is present in wines.26 The most abundant lactones in dessert wines are γ-

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nonalactone and cis- and trans-3-methyl-4-octanolide (for wines with oak barrels aging).

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Along with other volatile compounds investigated in Bordeaux dessert wines, lactones were

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significantly correlated with the typicality rating.27 Further studies28,29 on the noble rot dessert

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wines aroma, revealed the importance of a well-known phenomenon in perfumery,

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specifically, the perceptual blending in order to attain the “overripe orange” aroma nuances in

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these wines. Thus, compounds associated with both oak (3-methyl-4-octanolide, eugenol) and

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B. cinerea over maturation (2-nonen-4-olide, γ-nonalactone) were shown to contribute to the

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specific aromatic nuance of great noble rot dessert wines. This demonstration of the

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perceptual blending phenomenon, was established from sensory reconstruction and omission


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tests, with wine extract fractions supplemented with the volatile compounds previously

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mentioned.

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To better understand the involvement of the compounds of interest on the sensory perception

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of the perceptual blending the enantiomeric distribution of lactones, associated to the

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“overripe orange” nuances in Bordeaux dessert wines, associated with aging such as 2-nonen-

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4-olide, 1, γ-nonalactone, 2, and 3-methyl-4-octanolide, 3, (Figure 1) were determined.

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Subsequently, the sensory impact of each enantiomer or diastereoisomers was then studied by

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means of reconstitution experiments.

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MATERIALS AND METHODS

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Reagents and Reference Compounds: Dichloromethane (99.99%) was supplied by Fischer

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Scientific (Illkirch, France) and absolute ethanol (99.9%) by Merck (Semoy, France). Micro

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filtered water was obtained using Milli-Q Plus water system (resistivity = 18.2 MΩ,

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Millipore, Saint-Quentin-en-Yvelines, France). The racemic forms of the compounds were

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provided by Sigma Aldrich (Saint-Quentin-Fallavier, France) [3-methyl-4-octanolide (98%),

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γ-nonalactone (98%) and eugenol, 4 (99%)]. Racemic 2-nonen-4-olide (99.5%) and the

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diastereoisomers of 3-methyl-4-octanolide [trans (99%) and cis (99%)] were provided by

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Firmenich SA (Geneva, Switzerland). The enantiomeric forms (R)-γ-nonalactone (99%) and

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(S)-γ-nonalactone (98%) were kindly donated by the AWRI (Urrbrae, Australia).

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Separation of (R)-2-nonen-4-olide and (S)-2-nonen-4-olide from a Racemic Mixture by

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Preparative HPLC: HPLC was performed on a Knauer preparative 1800 pump connected to

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a Knauer K-2600 UV with a variable mono wavelength detector (Knauer, Berlin, Germany).

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The preparative column was a Chiralpack (Chiral Technologies®, Illkirch, France) AS 10µm

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model (250 × 30 mm). The eluent consisted of heptane/ethanol (95:5, v/v) with a flow rate of

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45 mL/min at a constant temperature of 25 °C. The injection was 2 mL of 30 mg of racemic



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2-nonen-4-olide dissolved in mobile phase. A total of 3 injections were performed (90 mg of

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racemic 2-nonen-4-olide). After HPLC purification, (R)-2-nonen-4-olide (23.8 mg) and (S)-2-

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nonen-4-olide (23.4 mg) were obtained. The purity of each enantiomer was assessed by HPLC

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using a Chiralpak AS 10 µm (250 × 4.6 mm). The eluent consisted of 5% of ethanol in

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heptane with a flow rate of 1 mL/min. Detection wavelength was 215 nm. Chiral purity was

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99.5% and 98% (by area) for the first and second eluting peaks, respectively.

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Wine Samples: The wines selected are listed in Table 1 and they were used for the various

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quantitative and sensory analyses, including gas and liquid chromatography.

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Wine Extraction for Semi-Preparative HPLC: A 750 mL wine sample was extracted

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successively using 60, 40 and 40 mL dichloromethane with magnetic stirring (700 rpm) for 10

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min each and separated in a separatory funnel for 10 min. The organic phases were collected,

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combined dried over sodium sulfate and concentrated to approximately 2 mL using a Buchi

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R-114 rotary evaporator (Buchi, Rugis, France) and then further concentrated under nitrogen

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flow (100 mL/min) to obtain 750 µL of raw wine extract.

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Semi-Preparative HPLC on Wine Extracts: The method used is that proposed by Ferreira

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et al.,30 and later adapted by Pineau et al.31 A volume of 250 µL of raw wine extract was

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injected on a reversed phase (RP) Novapak® C18 column (300 × 7.8 mm, 6 µm internal

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diameter (i.d), Waters, Saint Quentin, France) with a guard column of the same phase. An

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Ultimate 3000 semi-preparative HPLC system (Dionex, Courtaboeuf, France) was utilized for

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this procedure. Chromatographic conditions were as follows: Flow rate 1 mL/min; gradient,

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eluent A, micro filtered water, eluent B, ethanol; 0-2 min, 0%B, 2-50 min, 0-100% B linear.

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Fifty 1 mL effluent fractions were collected.


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Quantitation

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Spectrometry: The method was applied as described by Ferreira et al.32 and concerning 2-

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nonen-4-olide and eugenol as described by Stamatopoulos et al.28

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Enantiomeric Separation of Lactones by Chiral Multidimensional Gas Chromatography

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Mass Spectrometry: All analyses were performed using a multidimensional gas

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chromatograph (MD-GC) consisting of two independent model 7890A GCs (Agilent, Palo

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Alto, CA), interconnected by means of a thermo-regulated transfer line kept at 230 °C. For the

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first GC (GC-1), was equipped with a flame ionization detector (FID) and an ODP sniffing

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port (Gerstel, Mülheim an der Ruhr, Germany). GC-1 was retrofitted with a pressure-driven

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switching valve, multi column switching system (MCS), (Gerstel, Mülheim an der Ruhr,

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Germany) to transfer selected fractions eluted from the first capillary column directly into the

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analytical column of the second GC (GC-2). The carrier gas, helium N 60 (Air Liquide,

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Floirac, France), was delivered in constant flow mode (1 mL/min). The inlet pressure was at

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215 kPa. GC-1 was fitted with a 50 m × 0.22 mm internal diameter (i.d) × 0.25 µm film BP20

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(SGE, Ringwood, Australia). The oven temperature program was as follows: 45 °C for 1 min,

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then increasing by 4 °C/min up to 230 °C, and held at 230 °C for 20 min. The injector was

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held at 250 °C. GC-2 (Agilent 7890A) was coupled with a TOF (Time of Flight) Mass

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Spectrometer (JEOL, Croissy sur Seine, France) and fitted with a chiral column, 25 m × 0.25

136

mm internal diameter (i.d) × 0.25 µm DAC Beta Dex Mega (Mega, Legnano, Italy). In GC-1,

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the column was connected to the MCS (situated inside the oven of GC-1) and routed via a

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transfer line thermostatically controlled at 230 °C. The oven temperature program was the

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same as the first oven (45°C initially then 4 °C/min increase up to 230 °C). The MSTOF was

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used in full scan mode (m/z 40–300). MS data were recorded and processed using Mass

141

Center software (version 2.4.0) (JEOL, Croissy sur Seine, France) equipped with an NIST

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2008 MS library (US National Institute of Standards and Technology, Gaithersburg, MD).

of

Higher

Aliphatic

Lactones

by

Gas


Chromatography

Mass


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General Conditions for Sensory Analyses: Sensory analyses were performed as described

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by Martin and de Revel.33 Samples were evaluated in individual booths, at controlled room

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temperature (20 °C), using covered black AFNOR (Association Française des Normes)

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glasses containing 50 mL liquid, coded with random three-digit numbers. Sessions lasted

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approximately 10 min.

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Sensory Panel: The panel consisted of 15 judges, 5 males and 10 females, aged 30.5 ± 4.6

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(mean ± SD). All panelists belong to the enology laboratory staff at ISVV, Bordeaux

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University. The judges were selected for their experience for assessing fruity aromas on wine.

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Olfactory Thresholds: The olfactory thresholds of the isomers of the 2-nonen-4-olide were

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performed in model solution consisting of twice-distilled ethanol (12%, v/v) in micro filtered

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water and tartaric acid (5 g/L) at pH 3.5 (adjusted using NaOH). The detection threshold of

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the aroma compound was determined with an ascending procedure (0.25, 0.5, 1, 2, 4, 8, 16,

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32, 64, 128 µg/L), using the three alternative force choice presentation method (3-AFC) (ISO

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13301 2001).34 The aroma detection threshold was defined as the concentration at which the

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probability of detection was 50%. This statistical value was determined according to the

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adaptation of the ASTM-E1432 method as described by Stamatopoulos et al.28

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Reconstitution and Omission Experiments: Two different types of dessert wines were

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considered for reconstitution and omission tests, which have been previously selected by

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experienced assessors.28 One was a typical example of a Bordeaux dessert wine, with a

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desirable odor, reminiscent of overripe fruit, particularly orange peel (typicality rating at 7.2 ±

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0.8), while the other was a non-typical Bordeaux dessert wine, with a fresh fruity aroma

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(typicality rating at 0.9 ± 0.3) (Table 1). After fractionation of each raw extract by reversed-

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phase HPLC, the four volatile compounds (i.e. 2-nonen-4-olide, 1, γ-nonalactone, 2, 3-

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methyl-4-octanolide, 3, and eugenol, 4) were added in several reconstitution tests at


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concentrations assayed in HPLC fractions 37 and 38 of the typical and the non-typical dessert

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wine, as previously described by Stamatopoulos et al.28 Names and composition of the

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samples are indicated in Table 2 and 3. In detail, the total aromatic reconstitution (TAR)

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samples corresponded to the 50 fractions of a Bordeaux dessert wine raw extract blended

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together (1 to 50). Partial aromatic reconstitution (PAR) samples corresponded to the

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combined 50 fractions except fractions 37 + 38. Reconstitution and omission samples (R

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samples) were prepared by supplementing PAR samples with eugenol and two of the

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stereoisomers in racemic form plus one of the third compound stereoisomer at 100% or at the

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natural ratio (R/S or cis/trans) for all twelve possible combinations (Table 2). Before the

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samples were presented to the panel, in order to reproduce wine-like conditions, they were

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supplemented with double-distilled ethanol and micro-filtered water in to obtain an ethanol

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level of 12 % (v/v) and pH value at 3.5.

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In the first sensory analysis session, the fractions corresponded to those of a “typical”

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Bordeaux dessert wine and the four volatile compounds (1-4) were supplemented at

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concentrations, enantiomeric and diastereoisomeric distributions assayed in that type of wine

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(Table 2). In the second session, the fractions corresponded to those of a “non-typical”

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Bordeaux dessert wine the concentrations and enantiomeric and diastereoisomeric

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distributions of the compounds added were related to those assayed in that type of wine

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(Table 3). Each sensory session was performed in duplicate on different days. The “overripe

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orange” aroma intensity, and typicality rating relatively to Bordeaux dessert wines, were

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determined by the panel on a 0-10 point, non-structured scale, where 0 = no odor perceived

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and 10 = high intensity. Concerning typicality experiments, the scale was 0 for a bad example

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of Bordeaux dessert wine and 10 for a good example of Bordeaux dessert wine as previously

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described.28



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Statistical data were analyzed using the R software version 2.15.1 (R Foundation for

192

Statistical Computing, Vienne Austria, 2011). Analysis of variance (ANOVA) was

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performed, the homogeneity of variance was tested with Levene’s test, while the normality of

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residuals was tested using the Shapiro-Wilk test. Statistical significance was set at 5%

195

(p

Influence of Chirality of Lactones on the Perception of Some Typical

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