identification of dialkylpyrazines off flavors in oak wood

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Chemistry and Biology of Aroma and Taste

IDENTIFICATION OF DIALKYLPYRAZINES OFF FLAVORS IN OAK WOOD Svitlana Shinkaruk, Morgan Floch, Andrei Prida, Philippe Darriet, and alexandre pons J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03185 • Publication Date (Web): 18 Aug 2019 Downloaded from pubs.acs.org on August 19, 2019

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Journal of Agricultural and Food Chemistry

IDENTIFICATION OF DIALKYLPYRAZINES OFF FLAVORS IN OAK WOOD

Svitlana Shinkaruk‡§, Morgan Floch‡, Andréi PRIDA†, Philippe DARRIET‡, and Alexandre PONS ‡†

‡

Univ. Bordeaux, Unité de recherche Œnologie, EA 4577, USC 1366 INRA, ISVV, 33882

Villenave d’Ornon cedex, France §

Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, 33400, Talence, France

† Seguin

Moreau France, Z.I. Merpins, B.P. 94, F-16103 Cognac, France

1 ACS Paragon Plus Environment


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Abstract :

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Volatile extractive compounds from high quality oak wood (Quercus sp.) are responsible for

3

important pleasant olfactory notes, such as coconut, wood, vanilla, caramel, and spice.

4

Recently, a new off-flavor reminiscent of rancid butter was detected in oak wood. Using gas

5

chromatography-olfactometry (GC-O) coupled to several detection modes, as nitrogen-

6

phosphorus detection (GC-O-NPD) or mass spectrometry (GC-O-MS), and multidimensional

7

GC-O coupled to time-of-flight mass spectrometry (MDGC-O-TOF-MS), six compounds

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containing nitrogen atoms were identified. The volatiles were suggested to belong to 2,5-

9

disubstituted pyrazines family, which was confirmed by comparison with synthetic reference

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compounds. For this purpose, the symmetric and dissymmetric 2,5-dialkylpyrazines were

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prepared from methyl esters of corresponding aliphatic amino acids (Val, Leu, and Ile) by a

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three step - one pot reaction under mild reducing conditions. Organoleptic descriptors and

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odor detection thresholds were also determined, whereas a bacterial origin explaining these

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off flavors was hypothesized.

15 16 17

Keywords: aroma, oak wood, dialkylpyrazine, off flavor, wine, barrel aging


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INTRODUCTION

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Nowadays, it is well recognized by connoisseurs and winemakers that the use of oak wood

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offers a valuable contribution to the richness and complexity of the wine aroma. Contact with

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wood can occur during winemaking and ageing or only during ageing or maturation in

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barrels. Oak (Quercus robur, Quercus petraea, Quercus alba) is currently the most

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widespread wood used by coopers for the maturation of high quality wines.

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The main steps of barrel production are selection and cutting of oak trees from a forest,

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transformation of logs into staves, seasoning of wood and then assembling of barrels from

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staves. This last step contains operations of bending and toasting, during both of them various

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chemical reactions take place leading to the production of odorant compounds from neutral

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wood biopolymers.

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Volatile extractive compounds from oak wood are responsible for pleasant olfactory notes,

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such as coconut, wood, vanilla, caramel, and spice. That is why the wine aging in oak casks

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plays an essential role in the development of a rich aromatic palette. For many years, the

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research has been focused on the identification and quantitation of aromatic compounds

33

released during barrel ageing. Vanillin (vanilla), β-methyl-γ-octalactone (coconut), volatile

34

phenols (spicy) and 2-furanemethanethiol (roasted coffee) are considered to be the key

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molecules associated with oak ageing, including ageing in barrels1-2 and treatment with oak

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chips.3 Many other odorants are also associated directly or indirectly with oak wood aging,

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increasing the complexity of wines aged in oak wood barrels.4-5 These molecules of different

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chemical natures can be native in oak heartwood or appear during the cooperage process (oak

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seasoning and toasting). Their occurrence and concentrations result from different factors

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such as the availability and structure of starting components (lignin, (poly)saccharides, amino

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acids, extraneous components, etc) and processing parameters (temperature, duration, water

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activity, oxygen level, etc).6-7 The quality of the oak wood is a crucial point as well. It can 3 ACS Paragon Plus Environment


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negatively affect the odor transfer from oak to wine and thus, wine quality. Therefore,

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detailed knowledge about the origins of flavor and off-flavor generation is required if

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producers want to precisely control the impact on wine of the wood they use to make barrels.

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Off-flavor formation in oak wood can sporadically occur due to microbiological spoilage and

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non-controlled auto-oxidative processes during manufacturing and storage. Indeed, in the

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past, it was not unusual for wines aged specifically in new barrels to present an odor of fresh

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sawdust or sap. These odors were grouped under the general heading of “sawdust” and

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“planky“ aroma. Some unsaturated aldehydes and more specifically (E)-2-nonenal was

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associated with this off-flavor.8 Poorly selected wood that has been insufficiently seasoned or

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toasted, may increase the risk to obtain these odors.

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There are also other sources of contamination of the wood and barrels, directly or indirectly

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responsible for the spoilage of the products stored in them. Concerning the moldy off-odors,

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several compounds belonging to the haloanisole or halophenol families have been identified.9-

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10

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cork, on the wood and in the wine cellars.11-12 In 2010, Chatonnet identified a bacterial

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contamination of cork and wood responsible for the presence in wine of 2-methoxy-3,5-

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dimethylpyrazine, reminiscent of “fungal” and “corky” aroma.13 This pyrazine is thermally

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unstable, the off-flavor contamination can be prevented by the suitable thermal treatment of

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wood (toasting).

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The present study aimed at the molecular identification of a new off-flavor, reminiscent of

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rancid butter, recently detected by coopers in oak wood staves used to make barrels. The

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volatiles were identified as dialkylpyrazines by comparing to synthetic compounds. Their full

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structural analysis and organoleptic characteristics were described for the first time. We also

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considered the influence of some technological parameters on the presence of these pyrazines

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in oak wood.

Their occurrence was correlated with the presence of different kind of microbiota on the


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Materials and Methods

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Chemicals. Diisobutyl aluminium hydride solution in tetrahydrofuran (THF, 1M) and α-

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amino acid methyl ester hydrochlorides (H-Val-OMe, H-Leu-OMe, H-Ile-OMe), 2-octanol

71

(99%), (E)-2-nonenal (97%), butyric acid (99 %), trimethylamine (+99%), and ammonium

72

sulfate were purchased from Sigma-Aldrich Chemicals (Saint Quentin Fallavier, France).

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THF was dried by refluxing a solution containing sodium wires and benzophenone under

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nitrogen and distilled immediately before use. All moisture-sensitive reactions were carried

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out under an argon atmosphere.

76

General features. Nuclear Magnetic Resonance Spectroscopy (NMR): 1H, and

77

spectra were recorded on a Bruker Avance I (1H: 300 MHz, 13C: 75 MHz), and on Avance III

78

(1H: 600 MHz,

79

solvent. Chemical shifts () and coupling constants (J) are expressed in ppm and Hz,

80

respectively. Thin-layer chromatography (TLC) was performed on 60F TLC plates: thickness

81

0.25mm, particle size 10 µm, pore size 60 Å. Merck silica gel 60 (70–230 mesh and 0.063–

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0.200 mm) was used for flash chromatography. Spots were revealed with UV at 254 nm.

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Material. Non-toasted oak wood (Q. petraea) material originated from France was provided

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by Seguin Moreau cooperage. The raw material served for identification of dialkylpyrazines

85

was chosen by sensory assessment of several staves, according to its untypical and strong

86

“rancid butter-like” flavor.

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For studying effect of seasoning on the presence of dialkylpyrazines in oak staves, two

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different batches of fresh oak wood staves were selected for their strong characteristic off

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flavor. This experiment was conducted in 2013. Tainted oak staves were kept in open air, as

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others staves used for the barrel production, undergoing the microclimate of Cognac, in terms

91

of rain fall, wind and temperature at Seguin Moreau cooperage.

13C:

13C

NMR

151 MHz), spectra referenced using the lock frequency of deuterated



92

The dialkypyrazine content was then monitored during the experiment. Sampling was

93

performed by shaving the stave: 20 g were collected from each stave. The samples were

94

stored under normal storage conditions at 20 °C in the dark until use.

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In addition, the study of dialkylpyrazine occurrence was done on the seasoned wood material

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randomly selected from Seguin Moreau seasoning yard (250 different samples coming from

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individual staves).

98

Isolation of Volatile Compounds. Oak wood (10 g) was ground to obtain a homogenous

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powder and moistened with distilled hydroalcoholic solution (100 mL, 12% EtOH) at room

100

temperature for 24 h. After filtration, the hydroalcoholic solution was extracted in triplicate

101

with 5 mL of dichloromethane for 5 min at 750 rpm). The extract was dried with Na2SO4 and

102

concentrated at ambient temperature under nitrogen gas to a final volume of 0.5 mL.

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High-Resolution Gas Chromatography (GC). Analyses were carried out using a Trace GC

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Ultra (Thermo Fisher, Waltham, MA, USA) gas chromatograph equipped with a PTV injector

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and an ODO-1 sniffing port (SGE) (GC-O), and coupled to a nitrogen-phosphorus detector

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(GC-O-NPD), or a mass spectrometer DSQ II (Thermo Fisher, Waltham, MA, USA)

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functioning in EI mode (GC-O-MS).

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The oak wood extracts were analyzed on a polar BP20 capillary column (SGE, 50 m, 0.25

109

mm i.d., 0.25 µm film thickness) or an HP5-MS type fused silica nonpolar capillary column

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(Agilent J&W, 50 m, 0.22 mm i.d., 0.25 µm). A sample extract (1 µL) was injected in a PTV

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injector in splitless mode (injection temperature 150 °C for 10 s, 14 °C/sec to 230 °C, purge

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time: 1 min, purge flow: 50 mL/min). The compounds eluting at the end of the capillaries

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were split with a Y-splitter (GlasSeal, Sigma, St Quentin Fallavier, France) with a ratio 1:1,

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v/v and transferred via two deactivated capillaries (SGE, Ringwood, Australia) to the sniffing

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port and the NPD or the MS detector. The program temperature was as follows: 45 °C for 1


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min, increasing by 3 °C/min to 250 °C, followed to a 20 min isotherm. The carrier gas was

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helium N 60 (Linde gas, France) with a constant flow rate of 1 mL/min.

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The mass spectra source parameters (EI mode) such as source temperature, electron energy,

119

and emission current were set at 210 °C, 70 eV, and 30 µA, respectively. Ions were detected

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within a range of m/z 40 – 250. Transfer line temperature was set at 250 °C.

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Linear retention indices (LRI) were obtained by simultaneous injection of samples and a

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series of alkanes (C7-C23).14

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High-Resolution Gas Chromatography-Olfactometry (GC-O). GC-O analysis was carried

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out by four operators in triplicate. The assessors were familiar with the GC-O technique and

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performed GC-O analysis of different types of oak wood and wine samples at least one month

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before the GC-O analysis of tainted oak samples. In the true experiment of GC-O analysis, the

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odor characteristics and aroma intensities of the separated odorants were evaluated by

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assessors, and the aroma intensities were defined using a four level intensity scale from - to

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+++, rate as follows “-” weak, “+” moderate, “++” strong, and “+++” very strong. The

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odoriferous zones with same retention times and common olfactory descriptors reminiscent of

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the odor perceived in oak wood samples by the four assessors were selected for further

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identification by MDGC-O-TOF-MS.

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Heart-cut Multidimensional Gas Chromatography- Olfactometry – Time of flight Mass

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Spectrometry (MDGC-O-TOF-MS). The MDGC separations were performed on two

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capillary columns with different stationary phases (BP20, HP5) on two GC ovens: oven I was

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a Hewlett Packard 5890 series II, while oven II was an Agilent 6890 coupled with a JEOL-

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Accutof JMS T100 (Jeol, France). The two chromatographs were connected with a

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temperature-controlled transfer line set at 230 °C. The outlet of the first column was

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connected to a sniff port (ODO I; SGE France) and to the second column, via a Gerstel MCS

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2 multicolumn switching system. The end of the second column was split (1:1) via a



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crosspiece (Gerstel) between TOF-MS detection (JEOL) and the olfactory detection port

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(ODP 3, Gerstel, Germany). For oven I, only 10 % of the total flow was transferred to the

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deactivated fused silica column connected to ODO I, whereas 50 % of the flow was

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transferred to ODP 3 in oven II. The MDGC system was operated under constant pressure to

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maintain the balance between the two columns throughout the oven temperature program.

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Concerning the operating conditions of the JEOL AccuTOF mass spectrometer, it operated in

147

positive-ion mode (70 eV) or in chemical ionization mode (MeOH). The source temperatures

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were set at 220 °C and 160 °C for EI and CI experiments, respectively. The couples

149

voltage/current were set at 70 V/300 µA and 200 V/300 µA for EI and CI, respectively. This

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system was controlled by ‘‘Mass Center ’’ software (version 1.3.4 m; JEOL, Inc.) associated

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with the NIST mass spectral library (National Institute of Standards and Technology, NIST,

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Gaithersburg, MD, USA) and the Flavors and Fragrances database of Natural and Synthetic

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Compounds (FFNSC, 2nd edition, Wiley).

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The AccuTOF was tuned by infusion of FC 43 (Sigma-Aldrich, Inc., St. Louis, MO) to meet

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the manufacturer’s recommendations for resolution (>6000 FWHM). The recording intervals

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were set to 0.4 s with a wait time of 0.003 s and a sampling interval of 0.5 ns over a mass

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range of 50-250 Da.

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Identification of Odorants. The oak wood extracts were analyzed by MDGC-O-TOF-MS as

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described in the previous section. Specific odorant zones reminiscent of the tainted oak wood

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were detected by sniffing the effluent after the second dimension on the ODP. So, to validate

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the identification of volatile compounds, the synthesized reference compounds dissolved in

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dichloromethane were analyzed by MDGC-O-TOF-MS under the same conditions. The

163

compounds were identified by comparison of the LRI on BP20 and HP5 capillaries, their odor

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qualities, and their mass spectra (TOF-MS-EI) with those of the respective reference

165

compounds.


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Preparation of dialkylpyrazines. A 1M solution of diisobutyl aluminium hydride in THF

167

(30 mmol, 30 mL) was added dropwise to a suspension of α-amino acid methyl ester

168

hydrochloride (6 mmol) and freshly distilled trimethylamine (6 mmol) in anhydrous THF (20

169

mL) at -78 °C under argon and stirred for 2 h. For the synthesis of dissymmetric pyrazines, a

170

mixture of two corresponding amino acid esters in molar ratio 1:1 was used. The reaction was

171

then quenched with MeOH (5 mL) and allowed to warm to room temperature. Then EtOAc

172

(90 mL) and water (50 mL) were added, the pH was adjusted to 4 with 1M HCl, and the well

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stirred mixture was kept exposed to the air overnight. After the separation of organic layer,

174

the aqueous solution was extracted with EtOAc (3 x 30 mL). The combined organic phases

175

were subsequently washed with 1% HCl aqueous solution (60 mL) and then with saturated

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sodium chloride aqueous solution (2 x 40 mL), dried over magnesium sulfate, filtered, and

177

concentrated under reduced pressure to give yellow oil. The residue was then purified by

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silica gel column chromatography using diethyl ether and pentane as an eluent in a volume

179

ratio of 2:98. When needed, the fraction, containing the dissymmetric pyrazine, was isolated

180

and concentrated for further purification by semi-preparative HPLC. For HPLC purification,

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three different phases were used, namely a normal phase (Varian Dynamax Microsorb 100–5

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Si column, 250*21.4 mm) and a chiral phase (Chiralpak IA, 250*20 mm) with different

183

organic solvent systems, as well as a nitrile bonded phase under both reversed phase or

184

normal phased conditions (Phenomenex Luna CN 5µm, 100A, 250*10 mm) in order to

185

achieve the best separation for each pyrazine mixture (data non shown). For example, for the

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pre-purified 3 slightly contaminated by 1 and 6, HPLC purification was performed on Luna

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CN column (250x10 mm, 5 µm particles; Phenomenex, Le Pecq, France) using UV detection

188

at 274 nm. The injection volume was 250 µL at a concentration of 40 mg/L of pre-purified

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pyrazines in acetonitrile (HPLC grade). The separation was performed in isocratic mode with

190

mobile phase containing 0.7% of acetonitrile in hexane, flow rate was 1.8 mL/min in the first



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4 min, then 2.2 mL/min in the following 25 min. The elution order and retention times were

192

as follows: 1 at 13.25 min, 3 at 13.95 min, 6 at 14.45 min.

193 194

2,5-diisopropylpyrazine (1). Yellow liquid, isolated yield: 38%. TLC (diethyl ether/pentane:

195

70/30): Rf = 0.80. 1H NMR (300 MHz, CDCl3): δ 8.37 (s, 2H, CHar), 3.07 (septet, J = 6.9 Hz,

196

2H, CH), 1.31 (d, J = 6.9 Hz, 12H, CH3). 13C NMR (75 MHz, CDCl3): δ 159.45 (C), 141.96

197

(CH), 33.67 (CH), 22.36 (CH3). Positive HREIMS m/z 164.1313 [M]+ (th. mass for C10H16N2:

198

164.1314). MS (EI, 70 eV) m/z (%) 164 (30), 163 (15), 149 (100), 150 (12), 136 (50), 134

199

(20), 121 (25), 107 (6). LRIpolar= 1495.

200 201

2-(sec-butyl)-5-isopropylpyrazine (2). Yellow liquid, isolated yield: 8%. TLC (diethyl

202

ether/pentane: 80/20): R f= 0.75. 1H NMR (600 MHz, CDCl3): δ 8.40 (d, J = 1.5 Hz, 1H,

203

CHar-6), 8.34 (d, J = 1.5 Hz, 1H, CHar-3), 3.08 (septet, J = 6.9 Hz, 1H, CH(CH3)2), 2.81

204

(apparent sextet, J = 6.9 Hz, 1H, CH(CH3)CH2), 1.79 (ddq, J = 14.4, 7.4, 6.6 Hz, 1H, CH2),

205

1.68 (apparent dp, J = 14.4, 7.4 Hz, 1H, CH2), 1.33 (d, J = 6.9 Hz, 6H, CH(CH3)2), 1.29 (d, J

206

= 6.9 Hz, 3H, CHCH3), 0.85 (t, J = 7.4 Hz, 3H, CH2CH3).

207

159.44 (Cq-5), 158.70 (Cq-2), 142.75 (CHar-3), 142.16 (CHar-6), 40.84 (CH(CH3)2), 33.70

208

(CH), 29.82 (CH2), 22.38, 22.36 (CH(CH3)2), 20.16 (CHCH3), 12.20 (CH2CH3). Positive

209

HREIMS m/z 178.1454 [M]+ (th. mass for C11H18N2: 178.1470). MS (EI, 70 eV) m/z (%) 178

210

(20), 163 (45), 150 (100), 149 (18), 136 (33), 135 (68), 121 (12), 107 (5). LRIpolar= 1568.

211

2-isobutyl-5-isopropylpyrazine (3). Yellow liquid, isolated yield: 7%. TLC (diethyl

212

ether/pentane: 80/20): Rf = 0.73. 1H NMR (300 MHz, CDCl3) δ 8.39 (d, J = 1.5 Hz, 1H,

213

CHar-6), 8.32 (d, J = 1.5 Hz, 1H, CHar-3), 3.07 (septet, J = 6.9 Hz, 1H, CH(CH3)2), 2.63 (d, J

214

= 7.2 Hz, 2H, CH2), 2.08 (tsep, J = 7.2, 6.6 Hz, 1H, CH2CH), 1.32 (d, J = 6.9 Hz, 6H,

215

CH(CH3)2), 0.94 (d, J = 6.6 Hz, 6H, CH2CH(CH3)2).

13C

13C

NMR (75 MHz, CDCl3): δ

NMR (75 MHz, CDCl3): δ 159.35


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(Cq-5), 154.04 (Cq-2), 143.90 (CHar-3), 142.08 (CHar-6), 44.22 (CH2), 33.69 (CH(CH3)2),

217

29.21 (CH2CH(CH3)2), 22.50, 22.37 (CH3). Positive HREIMS m/z 178.1460 [M]+ (th. mass

218

for C11H18N2: 178.1470). MS (EI,70 eV) m/z (%) 178 (18), 163 (19), 150 (8), 137 (11), 136

219

(100), 135 (10), 121 (75), 122 (8) LRIpolar= 1581.

220

2,5-di-sec-butylpyrazine (4). Yellow liquid, isolated yield: 37%. TLC (diethyl ether/pentane:

221

80/20): Rf = 0.71. 1H NMR (600 MHz, CDCl3): δ 8.36 (s, 2H, CHar), 2.82 (apparent sextet, J

222

= 6.9 Hz, 2H, CH), 1.76 (ddq, J = 14.4, 7.4, 6.6 Hz, 2H, CH2a), 1.65 (apparent dp, J = 14.4,

223

7.4 Hz, 2H, CH2b), 1.30 (d, J = 6.9 Hz, 6H, CHCH3), 0.85 (t, J = 7.4 Hz, 6H, CH2CH3). 13C

224

NMR (151 MHz, CDCl3) δ 158.69 (Cq), 142.76 (CHar), 40.77 (CH) , 29.83 (CH2), 20.10

225

(CHCH3), 12.19 (CH2CH3). MS (EI,70 eV) m/z (%) 192 (15), 177 (42), 164 (100), 150 (28),

226

149 (13), 135 (100), 121 (15), 107 (5). Positive HREIMS m/z 192.1621 [M]+ (th. mass for

227

C12H20N2: 192.1626). LRIpolar= 1640.

228

2-sec-butyl-5-isobutylpyrazine (5). Yellow liquid, isolated yield: 7%. TLC (diethyl

229

ether/pentane: 80/20): Rf = 0.70. 1H NMR (300 MHz, CDCl3): δ 8.30 (d, J = 1.5 Hz, 1H), 8.28

230

(d, J = 1.5 Hz, 1H), 2.75 (apparent sextet, J = 6.9 Hz, 1H, CH(CH3)CH2), 2.59 (d, J = 7.2 Hz,

231

2H, CH2CH(CH3)2), 2.11 – 1.98 (m, 1H, CH2CH(CH3)2), 1.83 – 1.76 (m, 1H, CHCH2CH3),

232

1.72 – 1.65 (m, 1H, CHCH2CH3), 1.25 (d, J = 6.9 Hz, 3H, CHCH3), 0.89 (d, J = 6.6 Hz, 6H,

233

CH2CH(CH3)2), 0.79 (t, J = 7.4 Hz, 3H, CHCH2CH3). 13C NMR (75 MHz, CDCl3): δ 158.48

234

(Cq-2), 154.00 (Cq-5), 144.03 (CHar-3), 142.88 (CHar-6), 44.20 (CH2CH(CH3)2), 40.76

235

(CH(CH3)CH2), 29.77 (CHCH2CH3), 29.12 (CH2CH(CH3)2), 22.44 (CH2CH(CH3)2), 20.08

236

(CH(CH3)CH2), 12.11 (CHCH2CH3). MS (EI, 70 eV) m/z (%) 192 (15), 177 (25), 164 (30),

237

150 (70), 149 (10), 135 (10), 121 (100), 107 (5). Positive HREIMS m/z 192.1620 [M]+ (th.

238

mass for C12H20N2: 192.1626). LRIpolar= 1656.

239

2,5-diisobutylpyrazine (6). Yellow liquid, isolated yield: 21%. TLC (diethyl ether/pentane:

240

80/20): Rf = 0.69. 1H NMR (300 MHz, CDCl3): δ 8.32 (s, 2H), 2.63 (d, J = 7.2 Hz, 4H, CH2), 11 ACS Paragon Plus Environment


241

2.09 (apparent nonet, J = 6.9 Hz, 2H, CH2CH(CH3)2), 0.93 (d, J = 6.7 Hz, 12H, CH3). 13C

242

NMR (75 MHz, CDCl3): δ 153.95 (Cq), 144.08 (CHar), 44.24 (CH2), 29.20 (CH), 22.49

243

(CH3). Positive HREIMS m/z 192.1629 [M]+ (th. mass for C12H20N2: 192.1626). MS (EI,70

244

eV) m/z (%) 192 (13), 177 (15), 150 (85), 149 (10), 135 (10), 107 (100). LRIpolar= 1673.

245

Purity check of reference substances. Synthesized substances were dissolved in ethanol in

246

defined concentrations (approximatively 100 µg/mL) and were stepwise diluted with CH2Cl2

247

(1:10, by vol.). The solutions (1 µL) were subjected to GC-O and were checked for odor-

248

active compounds. Thus, all reference substances were checked for purity by GC-MS and

249

GC-O before used for threshold determinations.

250

Determination of olfactory detection thresholds. Odor detection thresholds (ODT),

251

corresponding to the perception of a stimulus without recognizing the odor quality were

252

determined in two sessions, using a model solution of wines (12% double-distilled ethanol, 5

253

g/L L(+)-tartaric acid, pH 3.5). The pH of model solutions was adjusted with NaOH (5 M).

254

For ODT measurements of a given odorant, experiments were performed in two steps. The

255

first test consisted of selecting the best range of concentration for ODT evaluation for a given

256

compound. During the second session, the same assessors individually evaluated the ODT of

257

selected volatile compound. The sensory panel consisted of 9 experienced assessors; students

258

and researchers (four male, five female) from the institute, between 20 and 40 years old

259

working specifically on wine flavors.

260

Before the sensory evaluation and the determination of odor detection thresholds, purity of the

261

synthesized compounds was determined by GC-MS. The presence of highly odorant

262

compounds possibly found at trace level was also checked by the analysis of each samples of

263

dialkylpyrazine by GC-O.


Page 12 of 36

Page 13 of 36


264

Samples were evaluated at controlled room temperature (20°C), in individual booths, using

265

AFNOR (Association Française des Normes) standard glasses containing about 50 mL of

266

liquid. The solutions were presented in an ascending procedure with a three-alternative forced

267

choice (3-AFC) presentation. Reference substances were first dissolved in ethanol (1 g/L) and

268

were stepwise diluted with ethanol before used to spike the wine model solution. All samples

269

were prepared shortly before the sensory analysis. For each compound, eight concentrations

270

were selected, and the subjects received a set of three glasses labeled with three-digit random

271

codes: two blank samples and one containing the odorant. The odor detection threshold was

272

defined as the lowest concentration perceived by 50% of tasters. 15

273 274

Quantitative analysis of dialkylpyrazines by SPME-GC-MS. Solid Phase Microextraction-

275

Gas chromatography-mass spectrometry (GC-MS). Five grams of ammonium sulfate, 9 mL

276

of reverse osmosis-purified water (Milli-Q, Millipore, Bedford, MA, USA), 10 µL of internal

277

standard (octan-2-ol in ethanol, 100 mg/L), and 1 mL of sample were added to 20 mL amber

278

vial successively. The vial was placed in the thermostatic enclosure of the autosampler

279

(Combipal, CTC Analytics) at 50 °C for 5 min, and the needle fiber (PDMS/DVB) was

280

inserted into the headspace of the sample for 25 min. The extracted chemicals were desorbed

281

thermally into the GC-MS injector for 5 min at 240 °C.

282

A Varian 3400CX GC coupled with 4000MS ion trap MS/MS system (Varian, Agilent

283

Technologies, Palo Alto, CA, USA) controlled by a computer using Varian Saturn

284

Workstation software (ver. 5.52) was used for the analysis of dialkylpyrazines. Separation

285

was performed on a BP20 capillary column (60 m, 0.25 i.d, 0.5 µm film thickness) from SGE.

286

The carrier gas was helium N 60 (Air Liquide) with a constant flow rate of 1 mL/min. The

287

1177 injector was set at 240 °C. The GC split valve was set to open after 4 min delay of the

288

initially closed valve. The column temperature was set at 40 °C for 1 min and then 13 ACS Paragon Plus Environment


289

programmed at 3 °C/min to 190 °C then ramped at a rate of 15 °C/min to 240 °C and held at

290

this temperature for 20 min. The total run time was about 54 min. The MS system was

291

operated in the electron impact (EI) mode, and tuned with perfluorotributylamine (PFTBA).

292

Temperatures of the trap, manifold and transfer line were set at 150 °C, 70 °C and 240 °C,

293

respectively. Quantitation of pyrazines was performed with the following ions: m/z 149 for 1,

294

m/z 150 for 2, 5, and 6, m/z 136 for 3 and m/z 164 for compound 4 and m/z 59 for octan-2-ol.

295

Qualifier ions were m/z 164 for 1, m/z 178 for 2 and 3, m/z 192 for 4, 5, and 6.

296

Calibration. The method was validated with respect to linearity, LOD, and LOQ

297

requirements. Stock standard solutions of each dialkylpyrazine were prepared in ethanol at 1

298

g/L and kept at 6 °C before use. Concerning the calibration curves, increasing concentrations

299

of each dialkylpyrazine were spiked to an oak wood maceration solution, reaching the levels

300

found in tainted oak wood samples. This last solution was prepared according to the following

301

protocol: (1) 2 g of non-toasted oak wood were stirred in 100 mL of hydroalcoholic solution

302

(EtOH 12 % vol.) for 24 h at room temperature; (2) the solution was filtered to remove oak

303

wood particles and used as is for method validation experiments. Finally, spiked solutions

304

were extracted according to the SPME protocol previously described. The linearity of the

305

calibration curves was higher than R²>0.995 whatever the dialkylpyrazine, all LODs values

306

were below 2 µg/L (Table 1S in Supplementary Data).

307

Results and discussion

308

Identification of volatile compounds associated with rancid flavors in oak wood (OW).

309

Two batches of non-toasted oak wood (Q. petraea) were selected on the basis of sensorial

310

descriptors. The control sample (OW-C) had an odor reminiscent of a mixture of fresh

311

coconut, hay, vanilla aromas whereas the tainted sample (OW-R) was strongly marked by an

312

off-flavor reminiscent of rancid butter. The GC-O analysis performed by four assessors led to

313

the detection of about 50 odor-active compounds. Among them, eight odorant zones were


Page 14 of 36

Page 15 of 36


314

reminiscent of this rancid odor. Two of them were found in both batches and only six of them

315

were systematically associated with the OW-R batch (Table 1). In order to elucidate the

316

chemical structure of odorous molecules, additional GC analyses coupled with different

317

detectors were performed.

318

Two aroma compounds present in both batches were identified by GC-O-MS by comparing

319

their retention indices and mass spectrum with that of pure commercial products (Table 1).

320

These were well described in oak wood and were (E)-2-nonenal (rancid, sawdust, LRI 1521)

321

and butyric acid (rancid cheese, LRI 1619).8 Others six odoriferous zones could not be

322

identified in the one-dimensional approach as their mass spectra were covered by diverse-co-

323

eluting substances making the interpretation of the mass spectrum difficult. To achieve higher

324

resolution, a two-dimensional approach was next applied.

325

Using serially connected columns, a polar column as the first dimension and an apolar column

326

as the second dimension, the six odoriferous zones were successfully associated with six

327

chromatographic peaks and their respective mass spectra were obtained by TOF-MS (Table 2,

328

Figures 1S-6S in Supplementary Data). The mass spectra obtained did not match with

329

compounds available in libraries such as NIST and FFNSC. To achieve the identification of

330

these compounds we followed the procedure described hereafter.

331

Identification of unknown compounds

332

For structure identification, the oak wood extracts marked or not with rancid odors were

333

analyzed by GC-O coupled to NPD or MS detectors. NPD detection revealed that six

334

odoriferous zones of interest contained nitrogen (Figure 1). The mass spectra obtained for

335

these peaks by MDGC-O-TOF-MS(EI) showed common characteristic ion clusters with low

336

intensity at m/z 107, 94, 80, 67, and 53 (Figure 1S-6S in Supplementary Data). This pattern is

337

typical for alkylsubstituted pyrazine derivatives.16 For all compounds, the hypothesis of alkyl



338

substitution on the pyrazine ring is also supported by the presence of a peak at m/z (M-15)

339

produced by methyl loss.

340

Pyrazine identification is challenging due to substitutions that can occur at different positions

341

of the heterocycle. While naturally occurring methylpyrazines can carry up to four alkyl

342

groups on various locations,17 those carrying larger alkyl side chains usually favor a 2,5-

343

dialkyl arrangement. Nevertheless, 2,6-isomers also occur, although in smaller quantities

344

because of their biosynthetic origin.18

345

The MS spectrum of pyrazine 1 showed a molecular ion of m/z = 164 and a series of peaks

346

(164→149→121; 164→136→121) corresponding to the loss of a ·CH3 fragment (-15) and of

347

a neutral C2H4 (-28) (Figure 1S in Supplementary Data). This fragment pattern is

348

characteristic to pyrazines bearing an isopropyl moiety. Moreover, as shown in Table 2, high-

349

resolution GC-TOF-MS data indicated C10H16N2 (164.1313, -0.04 Da) as the molecular

350

formula for 1, therefore containing four double-bond equivalents and two nitrogen atoms.

351

Deeper analysis of MS profile for the compound 1 demonstrated that the fragmentation and

352

the intensity of ions was very close to that of 2,5-isopropylpyrazine already found as by

353

product of certain bacteria16, 19 and beetle.20

354

The MS spectra of pyrazines 2 and 3 showed a molecular ion of m/z = 178, suggesting that

355

one of the alkyl substituents contains an additional methylene moiety (Figures 2S and 3S in

356

Supplementary Data). Both spectra presented a fragment pattern of isopropyl moiety (M-15,

357

M-28) and the peaks corresponded to the fragmentation of butyl fragment C4H9 (M-57, M-

358

42). The loss of neutral propene CH2=CHCH3 (-42) produced by McLafferty rearrangement

359

can occur in both iBu and secBu fragments. However, the loss of a neutral C2H4 (-28) was

360

observed only for sec-butylpyrazines.16-17 By further comparison of fragment pattern and peak


Page 16 of 36

Page 17 of 36


361

intensity, we proposed for 2 and 3 a structure bearing a sec-butyl and an isobutyl group,

362

respectively.

363

In mass spectra of compounds 4-6 (Figures 4S-6S in Supplementary Data), the additional shift

364

of 14 Da compared to 2 and 3 was observed for the molecular ions (m/z 192) and principal

365

fragments, that indicated the presence of two C4H9 groups. The mass spectrum of 4 presents

366

two principal fragments m/z = 164 and m/z = 135 of high intensity, that corresponds to the

367

loss of a neutral C2H4 (-28) and of a butyl fragment (-57), respectively. The fragmentation

368

pattern of 4 was tentatively assigned to 2,5-di-sec-butylpyrazine, whose MS data were not yet

369

reported in literature. The interpretation of MS spectrum of 6 was facilitated by the presence

370

of a pronounced peak at m/z = 150 (M-42) and the absence of peaks corresponded to the loss

371

of C2H4 (-28) typical for sec-butyl branching.16 Based on these observations, we hypothesized

372

this compound to be diisobutylpyrazine. Finally, the MS spectrum of 5 was suggested to be 2-

373

sec-butyl-5-isobutylpyrazine as it showed the fragmentation patterns typical for both iso- and

374

sec-butyl moieties and matched to fragment ions description already published for this

375

compound.16

376

All pyrazines corresponding to odoriferous zones 1-6 found only in OW-R samples were

377

proposed to bear 2,5-dialkyl substitution (Figure 2) and thus to be likely generated from

378

aliphatic amino acids (valine, leucine, and isoleucine) by dipeptide intramolecular cyclisation.

379

Some of them were already described and synthesized but at that time no consolidated

380

spectral data validating their structure were published.

381

Chemical synthesis

382

Symmetrical 2,5-dialkylpyrazines are classically synthesized by cycloamination reaction

383

from alkanolamines in the presence of dehydrogenation catalysts21-23 and by the self-

384

condensation of in situ formed -aminoketones.17,

24-25


The last ones usually prepared


Page 18 of 36

385

from corresponding -azidoketones using the mild azide reduction or the catalytic

386

reduction.17,

387

obtained in two steps by electrolytic oxidation of ketones in ammoniacal methanol, was

388

also achieved.26 Using oximes as starting material for pyrazine cycle formation was

389

demonstrated in the synthesis of 2,5-dimethylpyrazine, which was prepared from

390

oximinoacetone

391

alkylchloropyrazines mixtures, derived from corresponding aliphatic amino acid

392

anhydrides, was largely exploited by Ohta et al.28-29 Using Grignard reagent for direct

393

alkylation of pyrazine ring led to the mixture of three regiomers with the major alkylation

394

in 2,6-positions.19 Recently, an excellent regioselectivity was achieved in the synthesis of

395

symmetrical 2,5-disubstituted pyrazines from (Z)--haloenol acetates, but only one

396

example concerned the linear alkyl-substituted substrate.30 However, these methods have

397

some disadvantages, such as using expensive reagents or not commercially available

398

starting materials (which often requires the multi-step preparation), low selectivity,

399

unsatisfactory product yields and tedious experimental or purification procedures.

400

Therefore, in order to get easy and rapid access to target pyrazines, special attention was

401

paid to recently published syntheses via biomimetic dimerization of amino acid

402

derivatives.31-32 Symmetrical 2,5-disubstituted pyrazines (1, 4, and 6) were synthesized

403

from corresponding commercially available amino acid methyl esters in a three-step one-

404

pot reaction (Figure 3) by the improved procedure already described by Rojas et al.31 The

405

same approach was applied for the preparation of dissymmetric 2,5-dialkylpyrazines (2,

406

3, and 5). Using the mixture of two aliphatic amino acid derivatives led to the formation

407

of three pyrazines, as illustrated in Figure 3, the challenge was their careful

408

chromatographic separation by HPLC.

25

The dimerization of stable -aminoketone hydrochlorides, which were

in

satisfactory

yield.27

The


catalytic

hydrogenation

of

Page 19 of 36


409

The identification of unknown odoriferous zones was validated by comparison with

410

synthetic chemical standards in terms of retention time and TOF-MS matches as well as

411

by co-injecting pure compounds and a tainted oak wood sample extract on polar (BP20)

412

and apolar (HP5) column.

413

Odor detection threshold

414

The odor detection threshold of pyrazines identified in oak wood is depicted in Table 3. These

415

values are quite high compared to those obtained for others pyrazines such as 2-alkyl-3-

416

methoxypyrazines (some ng/L), for example, it exceeds 1 mg/L for the pyrazine 5 having a

417

different bulky alkyl group in positions 2- and 5-. However, as already demonstrated for

418

others compounds, these compounds having similar structure and descriptors may contribute

419

to the rancid butter off flavor of oak wood by additive effects.33 For instance, in the case of

420

oak chips, at 10 g/L (average level in the winemaking process), oak wood containing high

421

dialkylpyrazine levels (maximum level) can release its content into the wine and contaminate

422 437 439 427 431 429 432 428 423 433 438 436 424 425 430 434 435 426 440 441

methoxypyrazines often like initially to odor methylation amino followed are potatoes of These aroma 2-Alkyl-3compounds amide condensation be different characteristic peas threshold formed have and components acid proposed [10] or byglyoxal, afoods boiled were Olow by [16]. ofand the an

442

250 samples of oak wood (Q. petraea) from several batches were analyzed and extracted in

443

enological conditions (wine model solution). Based on these observation and sampling, most

444

of the samples did not contain dialkylpyrazines, we asses that less than 1 % of oak wood

445

samples were pyrazine-contaminated. The occurrence of these compounds in oak wood

446

sample is quite low. However, it is important to note that some of them presented in high

447

level. The highest concentrations were found for 4 and 5, reaching 94.9 and 80.1 µg/g,

448

respectively, whereas pyrazine 1 was found at the lowest level.

449

Pyrazines are widely distributed in nature and are utilized by bacteria, plants, vertebrate and

450

invertebrate animals and also by humans.34 Alkylated pyrazines have frequently been

451

described in processed food including coffee,35-36 beer,37 cocoa and chocolate.38 Their

it by reaching their ODT.

Occurrence of dialkylpyrazines in oak wood.



452

formation has been related to dry-roasting processes in which they are formed in the Maillard-

453

type reaction by different chemical pathways. Thus, thirty-six alkylpyrazines were identified

454

from the model reactions of amino acids and ascorbic acid.39 The alkylpyrazine pattern on real

455

food matrixes can be modulated by competing their formation pathways using amino acid

456

additives.40 Toasting of oak wood is also responsible for the formation of some pleasant

457

pyrazines such as 2-acetyl-pyrazine or 2-acetyl-3-methylpyrazine, both reminiscent of roasted

458

flavors.41 The family of 3-alkyl-2-methoxypyrazines is the most abundant in grapes and wine

459

and makes a large contribution to very characteristic green and herbaceous varietal aromas.42

460

Some alkylated pyrazines not bearing O-containing substituents were recently identified in

461

famous Chinese liquors43 and Cocoa liquors44. To our knowledge, the type of pyrazines

462

identified in this study has not previously been described in wine or other alcoholic

463

beverages.


Page 20 of 36

Page 21 of 36


464

Different bacteria have been reported to synthesize pyrazines and especially pyrazines bearing

465

two alkyl groups. Some myxobacteria seem to be specifically able to produce 2,5-

466

dialkylpyrazines from branched amino acids via reduction and dimerization reactions.18 For

467

example, the mycobacterium Chondromyces crocatus is able to produce pyrazines 1, 2, 3, and

468

6.17 Bacillus species also release some alkylated pyrazines, e.g. Paenibacillus polymyxa

469

produces 2,5-diisopropylpyrazine 1 as a main compound, and in a lesser extent 2, 3, 5, and

470

6.16 Moreover, five of 23 Pseudomonas strains isolated from wine cork sample are able to

471

produce up to 14 pyrazines alkylated at different positions; among which, 2,5-

472

dialkylpyrazines were represented by 1, 2, and 3.45 So, to our knowledge, some of the

473

pyrazines identified in this study were produced by bacteria isolates on agar plates and have

474

not been reported to be detected in any food products or in nature. Only pyrazine 1 is an

475

exception to this observation, as it can be produced by unknown microorganisms living on

476

rotting pineapples acting as an attractant of the pineapple beetle Carpophilus humeralis.20

477

Based on the known biochemical origins of these pyrazines in nature, we can hypothesize that

478

pyrazines are not so widely distributed in oak wood; it is likely to consider their occurrence

479

due to processing and/or bacterial contamination. During stave storage and manufacturing, it

480

is not excluded that they were in contact with the soil or close to it, so the bacterial

481

contamination of the oak wood from the soil and the participation of bacterial metabolism in

482

the development of off-flavors on the wood can be suggested.

483

Impact of seasoning of the level of alkylpyrazines in oak wood

484

Outdoor seasoning is a traditional technique that cooper use to decrease, first and foremost,

485

the oak’s humidity in order to ensure the barrel’s mechanical strength. Natural seasoning is an

486

operation that takes several years. This length of time is necessary to obtain high quality wood

487

that is properly suited to the aging and improvement of wine.46 The dehydration takes place

488

during the first 10 months and followed by a period when the wood actually ‘matures’, thus 21 ACS Paragon Plus Environment


489

improving its physical and organoleptic qualities. Enzymatic reactions are also involved,

490

caused by enzymes secreted by the fungal microflora that develop on the wood. Several

491

species have been identified including A. pullulans, which present on wood during seasoning,

492

represents 80% of the total microflora.47-48

493

In this experiment, two batches of non-seasoned oak wood were selected due to the presence

494

of dialkylpyrazines. Before seasoning, batch B contained more pyrazines than batch A (Figure

495

4). The concentration of the various pyrazines assayed in samples by SPME-GC/MS was also

496

different: in batch A, pyrazines 4 and 5 were at least four times more abundant than the

497

others, whereas in batch B, pyrazines 2 and 3 were found at higher concentration. In both

498

samples, compounds 1 and 6 were detected at lower level compared to the others.

499

After a 6 months seasoning period, 2,5-dialkylpyrazines had completely and systematically

500

disappeared, excepted for pyrazine 1 which was detected at trace level after the seasoning. To

501

explain these results several hypotheses can be formulated. First, pyrazines located at the

502

surface of the stave could be removed by the mechanical action of rainfall. Nevertheless, it is

503

not excluded that some fungus developing on the surface of the wood may metabolize these

504

pyrazines, as was already described in other matrices.34, 49

505

Finally, from an industrial point of view, these results reveal that shortening the seasoning

506

time can lead to off-flavor in oak wood, which leads to the contamination of wines aged in

507

barrels.

508

In conclusion, the oak wood used in wine making releases into the wine a variety of

509

compounds which usually positively contribute to the sensorial complexity of wines. In this

510

work, we describe for the first time a new off flavor reminiscent of rancid butter in oak wood.

511

Thanks to traditional GC-O-MS supported by multidimensional strategies, we bring new

512

chemical data to interpret this odor: six newly identified dialkylpyrazines were associated 22 ACS Paragon Plus Environment

Page 22 of 36

Page 23 of 36


513

with the rancid butter aroma detected in certain oak staves. Whereas the impact of seasoning

514

had been clearly demonstrated, their origin was quite unclear and needs further investigation.

515

In view of the sporadic nature of the contamination, the study of these new compounds is

516

worth pursuing in order to better the quality management of wood containers and wood pieces

517

used in enology.



518

References

519 520

(1) Chatonnet, P.; Dubourdieu, D.; Boidron, J.-N. Effects of Fermentation and Maturation in Oak Barrels on the Composition and Quality of White Wines. Aust. N. Z. Wine Ind. J. 1991, 6, 73-84.

521 522 523

(2) Tominaga, T.; Blanchard, L.; Darriet, P.; Dubourdieu, D. A Powerful Aromatic Volatile Thiol, 2Furanmethanethiol, Exhibiting Roast Coffee Aroma in Wines Made from several Vitis vinifera Grape Varieties. J. Agric. Food Chem. 2000, 48 (5), 1799-802.

524 525

(3) de Simón, B. F.; Cadahía, E.; Muiño, I.; del Álamo, M.; Nevares, I. Volatile Composition of Toasted Oak Chips and Staves and of Red Wine Aged with Them. Am.J. Enol. Vitic. 2010, 61 (2), 157-165.

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(4) Chatonnet, P.; Boidron, J. N.; Pons, M. Maturation of Red Wines in Oak Barrels: Evolution of Some Volatile Compounds and Their Aromatic Impact. Sci. Aliment. 1990, 10, 565-587.

528 529

(5) Cutzach, I.; Chatonnet, P.; Henry, R.; Dubourdieu, D. Identification of Volatile Compounds with a “Toasty” Aroma in Heated Oak Used in Barrelmaking. J. Agric. Food Chem. 1997, 45 (6), 2217-2224.

530 531 532

(6) Spillman, P. J.; Sefton, M. A.; Gawel, R. The Effect of Oak Wood Source, Location of Seasoning and Coopering on the Composition of Volatile Compounds in Oak-matured Wines. Aust. J. Grape Wine R. 2004, 10 (3), 216-226.

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(7) Chatonnet, P. Incidences du Bois de Chêne sur la Composition Chimique et les Qualités Organoleptiques des Vins: Applications Technologiques. University of Bordeaux II, Bordeaux, 1991.

535 536

(8) Chatonnet, P.; Dubourdieu, D. Identification of Substances Responsible for the 'Sawdust' Aroma in Oak Wood. J. Sci. Food Agric. 1998, 76 (2), 179-188.

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(9) Chatonnet, P.; Fleury, A.; Boutou, S. Identification of a New Source of Contamination of Quercus sp. Oak Wood by 2,4,6-Trichloroanisole and its impact on the contamination of Barrel-aged Wines. J. Agric. Food Chem. 2010, 58 (19), 10528-10538.

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(10) Cacho, J. I.; Campillo, N.; Viñas, P.; Hernández-Córdoba, M. Stir Bar Sorptive Extraction Polar Coatings for the Determination of Chlorophenols and Chloroanisoles in Wines Using Gas Chromatography and Mass Spectrometry. Talanta 2014, 118, 30-36.

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(11) Álvarez-Rodríguez, M. L.; López-Ocaña, L.; López-Coronado, J. M.; Rodríguez, E.; Martínez, M. J.; Larriba, G.; Coque, J. J. R. Cork taint of Wines: Role of the Filamentous Fungi Isolated from Cork in the Formation of 2,4,6-Trichloroanisole by O-methylation of 2,4,6-Trichlorophenol. Appl. Environ. Microbiol. 2002, 68 (12), 5860-5869.

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(12) Haas, D.; Galler, H.; Habib, J.; Melkes, A.; Schlacher, R.; Buzina, W.; Friedl, H.; Marth, E.; Reinthaler, F. F. Concentrations of Viable Airborne Fungal Spores and Trichloroanisole in Wine Cellars. Int. J. Food Microbiol. 2010, 144 (1), 126-132.

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(13) Chatonnet, P.; Fleury, A.; Boutou, S. Origin and Incidence of 2-Methoxy-3,5-dimethylpyrazine, a Compound with a “Fungal” and “Corky” Aroma Found in Cork Stoppers and Oak Chips in Contact with Wines. J. Agric. Food Chem. 2010, 58 (23), 12481-12490.


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(14) Van Del Dool, H.; Kratz, P. A Generalization of the retention Index System Including Linear Temperature Programmed Gas—liquid Partition Chromatography. J. Chromatogr. 1963, 11, 463-471.

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(15) AFNOR, Sensory analysis − Methodology − General Guidance for Measuring Odour, Flavour and Test Detection Thresholds by a Three Alternative Forced-Choice (3-AFC) Procedure − ISO 13301. In Analyse Sensorielle 7éme Edition, AFNOR: La plaine Saint Denis, 2007.

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(16) Beck, H. C.; Hansen, A. M.; Lauritsen, F. R. Novel Pyrazine Metabolites Found in Polymyxin Biosynthesis by Paenibacillus polymyxa. FEMS Microbiol. Lett. 2003, 220 (1), 67-73.

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(17) Dickschat, J. S.; Reichenbach, H.; Wagner-Döbler, I.; Schulz, S. Novel Pyrazines from the Myxobacterium Chondromyces crocatus and Marine Bacteria. Eur. J. Org. Chem. 2005, 19, 41414153.

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(18) Nawrath, T., Dickschat, J. S., Kunze, B., Schulz, S. The Biosynthesis of Branched Dialkylpyrazines in Myxobacteria. Chem. Biodiv. 2010, 7 (9), 2129-2144.

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(19) Schulz, S.; Fuhlendorff, J.; Reichenbach, H. Identification and synthesis of Volatiles Released by the Myxobacterium Chondromyces crocatus. Tetrahedron 2004, 60 (17), 3863-3872.

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(20) Zilkowski, B. W.; Bartelt, R. J.; Blumberg, D.; James, D. G.; Weaver, D. K. Identification of HostRelated Volatiles Attractive to Pineapple Beetle Carpophilus humeralis. J. Chem. Ecol. 1999, 25 (1), 229-252.

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(21) Langdon, W. K.; Levis, W. W., Jr.; Jackson, D. R.; Cenker, M.; Baxter, G. E. Synthesis of Pyrazines. Cycloamination of Alkanolamines. Ind. Eng. Chem. Prod. Res. Dev. 1964, 3 (1), 8-11.

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(22) Gnanaprakasam, B.; Balaraman, E.; Ben-David, Y.; Milstein, D. Synthesis of Peptides and Pyrazines from -Amino Alcohols through Extrusion of H2 Catalyzed by Ruthenium Pincer Complexes: Ligand-Controlled Selectivity. Angew. Chem., Int. Ed. 2011, 50 (51), 12240-12244.

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(23) Daw, P.; Kumar, A.; Espinosa-Jalapa, N. A.; Diskin-Posner, Y.; Ben-David, Y.; Milstein, D. Synthesis of Pyrazines and Quinoxalines via Acceptorless Dehydrogenative Coupling Routes Catalyzed by Manganese Pincer Complexes. ACS Catalysis 2018, 8 (9), 7734-7741.

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(24) Suzuki, H.; Kawaguchi, T.; Takaoka, K. Reactions of Sodium Hydrogen Telluride with -Azido Ketones and -Azido Bromides. Bull. Chem. Soc. Jpn. 1986, 59 (2), 665-666.

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(25) Nakajima, M.; Loeschorn, C. A.; Cimbrelo, W. E.; Anselme, J. P. Substituted Pyrazines from the Catalytic Reduction of -Azido Ketones. Org. Prep. Proced. Int. 1980, 12 (5), 265-8.

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(26) Chiba, T.; Sakagami, H.; Murata, M.; Okimoto, M. Electrolytic Oxidation of Ketones in Ammoniacal Methanol in the Presence of Catalytic Amounts of KI. J. Org. Chem. 1995, 60 (21), 676470.

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(27) Zhang, C. Y.; Tour, J. M. Synthesis of Highly Functionalized Pyrazines by Ortho-Lithiation Reactions. Pyrazine Ladder Polymers. J. Am. Chem. Soc. 1999, 121 (38), 8783-8790.

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(28) Ohta, A.; Akita, Y.; Hara, M. Syntheses and reactions of Some 2,5-Disubstituted Pyrazine Monoxides. Chem. Pharm. Bull. 1979, 27 (9), 2027-41. 25 ACS Paragon Plus Environment


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(32) Badrinarayanan, S.; Sperry, J. Pyrazine Alkaloids via Dimerization of Amino Acid-Derived -Amino Aldehydes: Biomimetic Synthesis of 2,5-Diisopropylpyrazine, 2,5-Bis(3-indolylmethyl)pyrazine and Actinopolymorphol C. Org. Biomol. Chem. 2012, 10 (10), 2126-2132.

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642

Figure captions

643

Figure 1: GC-NPD chromatograms of extracts from wood marked (OW-R) or not (OW-C) by

644

rancid off flavors.

645

Figure 2: Chemical structures of dialkylpyrazines detected in non-toasted oak wood : 2,5-di-

646

isopropylpyrazine (1), 2-(sec-butyl)-5-isopropylpyrazine (2), 2-isobutyl-5-isopropylpyrazine

647

(3), 2,5-di-sec-butylpyrazine (4), 2-(sec-butyl)-5-isobutylpyrazine (5), 2,5-diisobutylpyrazine

648

(6).

649

Figure 3: Biomimetic synthesis of target pyrazines by spontaneous condensation of α-amino

650

aldehydes, formed in situ by mild reduction of corresponding amino acid esters.

651 652

Figure 4: Effect of seasoning length on the evolution of the dialkylpyrazine contents in the

653

oak wood in two batches A and B (results are expressed as µg/g of dry wood, Dw).

654


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Page 29 of 36


TABLES Table 1: Main odoriferous zones reminiscent of the oak wood marked (OW-R) or not (OW-C) by rancid odors. Identification of compounds by GC-O-MS. Odoriferous Occurrence in oak wood zones / Compounds identified a OW-C OW-R unknown (1)

a

-

++

Odor descriptorsb

LRIc

Buttery

1495

(E)-2-nonenal ++

++

Sawdust, rancid, 1521

unknown (2)

-

++

Cigarette smoke, 1568 rancid

unknown (3)

-

+

Cigarette smoke, 1581 rancid

butyric acid

+

+++

Rancid

1619

unknown (4)

-

++

Rancid, sweat

1640

unknown (5)

-

++

Smoke, rancid

1656

unknown (6)

-

++

Buttery

1667

Compounds were identified by comparing their mass spectra and retention indices (LRI)

with reference spectral databases and their odors with commercially available reference compounds. b Odor descriptors generated by the four assessors during GC-O. c Retention index (LRI) of odor peak on a BP20 (50 m x 0.25 mm, 0.25µm).



Page 30 of 36

Table 2: Linear Retention Indices (LRI) and mass data of the unknown odoriferous zones in oak wood marked by rancid odors obtained by MDGC-O-TOF-MS analysis.

a

Odoriferous zones

LRIa

LRIb

M+ experimental mass

M+ theoretical mass

Empirical formula

unknown 1

1495

1202

164.1313

164.1314

C10H16N2

unknown 2

1568

1285

178.1454

178.1470

C11H18N2

unknown 3

1581

1293

178.1460

178.1470

C11H18N2

unknown 4

1640

1366

192.1621

192.1626

C12H20N2

unknown 5

1656

1375

192.1620

192.1626

C12H20N2

unknown 6

1667

1383

192.1629

192.1626

C12H20N2

LRI of odor peak on a BP20 (50 m x 0.25 mm, 0.25µm).

nonpolar capillary column (50 m x 0.22 mm, 0.25 µm).


b

HP5-MS type fused silica

Page 31 of 36


Table 3: Odor detection thresholds (ODT) and occurrence of dialkylpyrazines in oak wood quantified by SPME-GC-MS analysis.

Compounds (odoriferous zone)

ODT (µg/L)a

Concentrations found in oak wood min – max (µg/g)b

2,5-diisopropylpyrazine (1) 220 nd – 3.5 2-(sec-butyl)-5-isopropylpyrazine (2) 920 nd – 40.2 2-isobutyl-5-isopropylpyrazine (3) 900 nd – 25.8 2,5-di-sec-butylpyrazine (4) 890 nd – 94.9 2-(sec-butyl)-5-isobutylpyrazine (5) 1050 nd – 80.1 2,5-diisobutylpyrazine (6) 980 nd - 4.2 a wine model solution (12% vol. EtOH, tartaric acid 5 g/L, pH 3.5). b data obtained from 250 samples coming from different individual staves, results expressed as µg/g dry wood; nd: not detected.



Page 32 of 36

FIGURES

(1)

(2)

(3)

(4) (5)(6)

OW-R

OW-C

Figure 1.


Page 33 of 36


N

N

N

N

1

2

N

N

N

N

3

4

N

N

N

N

5

6

Figure 2.



O R

DIBAL-H O

NH2

O R

N

dimerization H

R

NH2

N

R

Page 34 of 36

N

[O] R

N 1 4 6 2 3 5

H-Val-OMe H-Ile-OMe H-Leu-OMe H-Val-OMe + H-Ile-OMe H-Val-OMe + H-Leu-OMe H-Ile-OMe + H-Leu-OMe

Figure 3.


R

Page 35 of 36


Figure 4.



TOC graphic

Rancid Butter OFF-FLAVOR

N

N

N N N

N

N

N

N

N

N

N


Page 36 of 36

identification of dialkylpyrazines off flavors in oak wood

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