Effect of vine water and nitrogen status, as well as temperature, on

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Chemistry and Biology of Aroma and Taste

Effect of vine water and nitrogen status, as well as temperature, on some aroma compounds of aged red Bordeaux wines Nicolas Le Menn, Cornelis van Leeuwen, Magali Picard, laurent riquier, Gilles de Revel, and Stéphanie MARCHAND J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00591 • Publication Date (Web): 02 Jun 2019 Downloaded from http://pubs.acs.org on June 3, 2019

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Journal of Agricultural and Food Chemistry

Effect of Vine Water and Nitrogen Status, as well as Temperature, on some Aroma Compounds of Aged Red Bordeaux Wines

Nicolas LE MENN1,2, Cornelis VAN LEEUWEN3, Magali PICARD1,2, Laurent RIQUIER1,2, Gilles de REVEL1,2, Stephanie MARCHAND1,2 1

University of Bordeaux, ISVV, EA 4577, Unité de recherche OENOLOGIE, F-33882 Villenave d'Ornon, France 2 INRA, ISVV, USC 1366 OENOLOGIE, F-33882 Villenave d'Ornon, France 3 EGFV, Bordeaux Sciences Agro, INRA, Univ. Bordeaux, ISVV, F-33882 Villenave d’Ornon, France

Corresponding autor : Stephanie MARCHAND [email protected] (05 57 57 58 41) University of Bordeaux, ISVV, 210 chemin de Leysotte F-33882 Villenave d'Ornon, France

1 ACS Paragon Plus Environment


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1

ABSTRACT

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Wine aging bouquet is defined as a positive, complex evolution of aromas during bottle aging.

3

The aim of this study was to look for the link between some of the vine status parameters and

4

the development, during wine aging, of volatile compounds, such as DMS, tabanones and some

5

wine aromatic heterocycles. The potential influence of air temperature was investigated, as well

6

as vine nitrogen and water status. Wines were obtained by microvinification from plots of Vitis

7

vinifera L. cv. Merlot, Cabernet-Sauvignon and Cabernet franc, over vintages from 1996 to

8

2007, and cellar-aged until 2014. Wine aging aromas, were quantified using GC/MS. The effect

9

of the vintage and vine water and nitrogen status were greater than the varietal effects. The nine

10

aroma compounds measured showed very high levels in the 2003 vintage. The results revealed

11

a positive link between vine nitrogen status and dimethyl-sulfide and N,S,O- heterocycle levels

12

measured in the aged wines. Levels of 4-[2-butylidene]-3,5,5-trimethyl-2-cyclohexen-1-one

13

and

14

tabanone) isomers are upper when the vines were affected by a water deficit.

4-[(3E)-1-butylidene]-3,5,5-trimethyl-2-cyclohexen-1-one

(megastigmatrienones;

15 16

KEYWORDS

17

Vine water status, vine nitrogen status, red wine aging aromas, Vitis vinifera, dimethyl

18

sulfide, odorous heterocycles, tabanone.


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INTRODUCTION

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Wine quality is closely related to its aromatic expression, itself influenced by some vine

21

parameters including the grape variety, viticultural management techniques and environmental

22

factors, like soil and climate.1 The influence of environmental factors on wine quality and

23

typicity is referred to as the “terroir effect”.2 Terroir has a spatial (variability of soil,

24

topography, and climatic conditions among locations where wine is produced) and temporal

25

(year-to-year variability of climatic conditions: the so-called “vintage effect”) dimension.3,4 It

26

has been shown that the soil effect in terroir expression is largely mediated by the availability

27

of water and nitrogen5 and the climate effect is mediated by air temperature and water balance.6

28

More recently, and in connection with the two dimensions of terroir cited, it has been observed

29

that microorganisms are also distributed according to the terroir.7 Vintage characteristics are

30

perceptible during the tasting of the young wines and modified by bottle aging, when wines

31

develop their specific “aging bouquet”.8 The aging bouquet is considered one of the most

32

important quality attributes of premium wines. In the early 1980’s, the aging bouquet of wine

33

was described as a qualitative complexity of aging aromas. Recent studies have highlighted the

34

fact that the mental representation of the wine aging bouquet concept by wine professionals

35

include the terroir dimension, as well as vineyard characteristics. The sensory definition of the

36

aging bouquet of red Bordeaux wines has been shown to be structured around seven main

37

aromatic nuances: “undergrowth”, “spicy” “truffle”, “fresh red- and black-berry fruits”,

38

“liquorice”, “mint”, and “toasted”.8 It is a common observation by wine experts that the quality

39

of the aging bouquet varies with the precise origin of the wine (including vineyard soil and

40

microclimate) and vintage (reflecting the climatic conditions of the year of production).

41

However, this link is not easy to establish on a scientific basis, as it requires precise data on the

42

soil type and climatic conditions of the vintage, to understand how these environmental factors

43

influence grape composition, especially through vine water and nitrogen status, as well as



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temperature, during grape ripening. It is very rare that the precise water and nitrogen status of

45

the vines that produced the grapes used for a particular bottle-aged wine are known. Another

46

difficulty is that soil composition varies considerably in commercial vineyards.9 This point was

47

addressed by micro-vinification of grapes harvested from a limited number of vines, where

48

pedological soil composition was considered as homogeneous.3,10 The other major parameters

49

that affect berry composition are vine water and nitrogen status,5 quantified using several

50

indicators.6,11 High nitrogen status in vines favors high nitrogen levels in grape berries. This

51

results in high total nitrogen and amino acid concentrations (particularly arginine, proline, and

52

ammonium). Consequently high yeast-available nitrogen values (YAN) provide a reliable

53

indicator of vine nitrogen status12. Several studies have reported that the amount of yeast-

54

available nitrogen influences fermentation dynamics, as well as the generation of by-products,

55

impacting wine aromas.13 At the end of fermentation, yeast autolysis returns free amino acids

56

to the wine.14 One of these, cysteine, is involved in the synthesis of odorous compounds via a

57

Maillard-like reaction including -dicarbonyl compounds.15 Consequently, nitrogen, sulfur and

58

oxygen heterocycles are generated, intensifying “nutty”, “toasty”, and “spicy” notes that

59

contribute to the aroma of aged wines.15–17 Even when aromatic heterocycles are not apparently

60

involved in the organoleptic expression of the wine aging bouquet18, they are linked to the

61

nitrogenous compounds in wines.15,17 A recent study highlighted the positive correlation

62

between 7 aromatic heterocycles (5-methylfurfural (4), 2-acetylfuran (3), thiazole (1), 2-

63

ethylthiazole, 2-methylpyrazine, 2-acetyl-3-methylpyrazine (6), and 2-acetylthiophene (5) ;

64

Figure 1) and the age of Champagne reserve wines, as well as their amino acid content.19 Many

65

wine aromas result from the chemical or biochemical conversion of non-volatile molecular

66

compounds detectable in grapes, consisting of a glycoside or amino acid linked to an aroma

67

precursor.1,20–22 For example, the “truffle” note is due to the presence of dimethyl sulfide (DMS

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(8)).18 The DMS (8) has recently been highlighted to contribute significantly and positively to


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red Bordeaux wine bouquet aroma and affect fruity aroma perception.18,23 The main precursor

70

of DMS (8) is S-methyl methionine, a derivative of methionine synthesized in vines and

71

accumulated in the berries.24 Although some DMS (8) is released during alcoholic

72

fermentation,25 DMS (8) levels increase significantly during bottle aging, and the DMS (8)

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release is highly dependent on wine storage conditions, particularly temperature and redox

74

status.26,27 The pDMS is the quantity of DMS (8) that may be released during vinification and

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aging, depending on numerous factors, including vine water deficit and nitrogen status,

76

assessed by the Yeast Available Nitrogen content in must.24 Vine water status influences the

77

production of other aroma-precursor compounds. The C13-norisoprenoids content in grape

78

berries increases with the severity of vine water deficit.20,28,29 For example, tabanones (9 to 13)

79

are a set of compounds derived from the C13-norisoprenoid metabolism in vines and oak trees,

80

including all 5 isomers (megastigma-4,6Z,8E-trien-3-one (9), megastigma-4,7E,9-trien-3-one

81

(10), megastigma-4,6Z,8Z-trien-3-one (11), megastigma-4,6E,8E-trien- 3-one (12), and

82

megastigma-4,6E,8Z-trien-3-one (13); Figure 1). Four of the five isomers are present in grape

83

juice ((9)(11)(12)(13)) and the last one is transferred to wine by contact with oak wood.

84

Tabanone levels in wines and spirits increase during aging and it could contribute to the spicy

85

and toasty notes mentioned in the Bordeaux red wines bouquet aroma description.30,31 Vine

86

water status depends on the climatic conditions of the vintage (especially rainfall and reference

87

evapotranspiration) and soil water-retention capacity. It is also influenced by plant material

88

(particularly rootstocks), as well as the training system and planting density.32 In some regions,

89

winegrowers modify vine water status through irrigation, but this is not permitted in the

90

Bordeaux area. Vine water status can be assessed by several techniques, among which the

91

measurement of stem water potential during the season or the measurement of K13C on grape

92

must or wine are accurate indicators.6 Stem water potential is measured by means of a pressure

93

chamber in the field6 and K13C by stable isotope ratio mass spectrometry (EA/IRMS).33 Results



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of both approaches to assess vine water status are well correlated.34 Vine nitrogen status

95

depends on soil type, climatic conditions of the vintage, vineyard floor management, and

96

fertilization practices and is influenced by water availability. According to White et al. (2007)35,

97

high soil water availability may increase soil nitrogen content, but this is not always the case.

98

The effect of vine water status on aroma precursors in grapes has already been

99

investigated.20,21,24 The influence of aroma precursors is also dependent on training systems,1

100

cluster thinning, and leaf removal,22 but these practices were homogeneous over the plots

101

considered in the scope of this study. Although both nitrogen and water status can influence the

102

levels of aroma precursors in wines, their role in the molecular composition of wines during

103

aging had not previously been investigated in depth. The aim of this study was to profile some

104

key aging aroma compounds (DMS, N,S,O-heterocycles, and tabanones) in a set of aged red

105

Bordeaux wines produced from Cabernet Sauvignon, Cabernet-franc, and Merlot grapes. The

106

wines were obtained by micro-vinification in several vintages (1996 to 2007). Soil composition,

107

as well as vine water and nitrogen status, were precisely quantified for each variety on each

108

type of soil in each vintage. Molecular compositions were interpreted in relation to the recorded

109

indicators of nitrogen and water status in the corresponding vines and juices.

110

MATERIALS AND METHODS

111

Vines and vineyards

112

The wines collected for analysis were produced from grape berries harvested from vines

113

cultivated on nine plots in the Saint-Emilion appellation in the Bordeaux area in the 1996 to

114

2007 vintages. For each vintages, date of harvest was the same as for the other vineyard vine

115

plots used for commercial winemaking. Each plot had approximately 100 vines and was small

116

enough to consider that the soil was homogeneous. Grape yields were reduced to be similar

117

between each plot at 35 hL/ha, as well as to be able to be get in each vintage. They were located

118

on three soil types: sandy soil with a water table accessible to the root system, resulting in little


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or no water deficit; clay soil with over 50% clay beyond 50-cm depth, inducing moderate water

120

deficits, and gravelly soil with over 50% coarse elements, inducing moderate to severe water

121

deficit. Soil composition is described in.36 On each soil, the following Vitis vinifera L. varieties

122

were planted: Merlot (clone 181), Cabernet-Sauvignon (clone 191), and Cabernet franc (clone

123

326), all grafted on 3309C rootstock.

124

Vine water status

125

Vine water status was assessed in each block and each vintage using several indicators: stem

126

water potential, pre-dawn leaf water potential, and carbon isotope discrimination measured on

127

grape sugar at ripeness ;K13C).6 These data were presented Table S2 (Supporting information).

128

In order to measure K13C on grape must, 2 mL of juice are introduced into an Eppendorf tube

129

and centrifuged at 10 000 RPM. Tin capsules (TIN 6×4 mm) are delicately introduced into a

130

96-well (8 mm) microplate (SARSTEDT n° 83.1835). Five O of grape juice is introduced in

131

each tin capsule by means of a micropipette P10. The location of the samples must be carefully

132

registered. The microplate is placed in a non-ventilated stove at 60°C during 24 hours. Tin

133

capsules are compressed and turned into small balls without any remaining air and are then

134

analysed by an elemental analyser (EA, VarioMicroCube, Elementar, F-69623 Villeurbanne,

135

France) coupled to isotope ratio monitoring by mass spectrometry (IRM-EA/MS,

136

Isoprime/Elementar, F-69623 Villeurbanne, France). The tin capsules are injected in the

137

oxidation tube (950°C) under helium flux (200 mL.min-1) and oxygen flux (30 mL.min-1),

138

reduction furnace temperature being fixed at 550°C. Combustion gases were dried and eluted

139

to a specific column that physically retains the CO2 (60°C) and then releases it with an increase

140

in temperature (210°C). An open split system allowed regulation of gas withdrawing to the

141

IRM-MS, current trap is fixed at 200 µA. The overall measurement duration was 600 s.

142

Measured masses by IRM-MS are m/z 44 and 45 corresponding to CO2 without and with a 13C,

143

respectively. Isotope ratio is expressed as a relative deviation, K13C in per mil (‰) against the



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144

international standard, V-PDB (Vienna-Pee Dee Belemnite) according to K13C (‰) =

145

1000×[(Rs / Rst)-1] where R corresponds to the carbon 13 isotope ratio of the sample (s) and

146

the standard (st). Results given in this study are an average of two measurements validated if

147

the gap between the two values is lower than 0.3‰. Otherwise, the analysis is repeated.37 Pre-

148

dawn water potential was measured using a pressure chamber, every second week from the end

149

of June until the harvest. Stem water potential was measured every week between 2 and 4 pm

150

local time over the same period.38 The lowest value for both pre-dawn and stem water potential

151

recorded over the season was selected as an indicator of the intensity of the vine water deficit

152

over the season. For each plot, K C was measured using mass spectrometry on four individual

153

samples of grapes collected prior to harvest. K C is expressed compared to a standard and

154

values ranged from -27 p. 1000 (no water deficit) to -20 p. 1000 (severe water deficit).6

155

Vine nitrogen status

156

Vine nitrogen status was assessed on all plots during all the vintages studied (1996 to 2007) by

157

quantitation of nitrogen compounds in the must. Yeast available nitrogen (YAN) was measured

158

in the must using the Sörensen method (quantitation of protons measured after derivation of

159

primary and secondary amines by formaldehyde).39 Total nitrogen was measured using the

160

Kjeldahl method.40 Petiole and leaf blade total nitrogen content was also measured at veraison.11

161

These data were presented Table S2 (Supporting information). As some wines were no longer

162

available when the aromatic compounds were analyzed and some indicators were not measured

163

in all vintages, the experimental design was incomplete. The wines inventory and available

164

nitrogen and water status indicators are listed in Table 1.

165

Microvinification

166

The wines were made by microvinification in 50 L tanks. Grapes were hand-harvested at the

167

same time as the commercial vineyards where the plots were located. After crushing and

168

destemming, 5 g/hL SO2 was added to the must, which was then placed in 50 L tanks in a

13

13


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temperature-controlled room at 28°C. Reactivated commercial yeast was added at 20 g/hL

170

using the 522 Davis strain (Laffort, Floirac, France). During alcoholic fermentation, the cap

171

was punched down daily. When alcoholic fermentation was completed, cap punching was

172

reduced to alternate days. The must was aerated when it reached a density of 1.050, 20 g/hL of

173

the fermentation activator thiazote® (Laffort, Floirac, France) was added, together with the

174

amount of ammonium sulfate required to reach 180 mg/L yeast-available nitrogen. When the

175

level of must yeast available nitrogen was higher than 180 mg/L at harvest, no ammonium

176

sulfate was added. After 15 days' skin contact, the liquid phase was transferred to 30 L tanks

177

and the solids were pressed. All press wines were added to the free run and represent 12% of

178

the total volume. Lactic bacteria (Oenococcus oeni Vitilactic F, Martin Vialatte, Magenta,

179

France) were added at 1 g/hL to start malolactic fermentation. They were all quick and occurred

180

in homogeneous conditions and 5 g/hL SO2 were added when it was completed. After 2 months,

181

the wine was sterile-filtered and bottled using natural cork stoppers. The bottles were stored at

182

20°C until opening. All bottles were stored in the same room, ensuring homogeneous conditions

183

for wines from different varieties, soils, and vintages.

184

Chemicals and standards

185

The chemical structures of the 13 studied compounds are presented in Figure 1. All solvents

186

were HPLC grade. Absolute ethanol and methanol (purity > 99%) were obtained from Merck

187

(Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q Plus water system

188

(Millipore, Saint-Quentin-en-Yvelines, France). Sodium chloride (purity > 99 %), boric acid,

189

and hydrochloric acid were supplied by VWR-Prolabo (Fontenay-sous-bois, France). Thiazole

190

(1), 4-methylthiazole (2), 2-ethylthiazole, 2-acetylthiazole, 2-methylthiazole, 2,4,5-

191

trimethyloxazole, 3-acetyl-2,5-dimethylfuran, 2-acetylfuran (3), 5-methylfurfural (4), 3-

192

acetylthiophene, 2-acetylthiophene (5), 2,3-dimethylthiophene, 2,5-dimethylthiophene,

193

acetylpyrazine, 2,3-diethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-3-methylpyrazine, 2-acetyl-



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194

3-methylpyrazine (6), tetramethylpyrazine (7), dimethyl sulfide (DMS) (8), and thiophene

195

(used as an internal standard), were purchased from Sigma–Aldrich (Saint Quentin-Fallavier,

196

France). Alfa Aesar (Johnson Mattey Company, Bischheim, France) supplied 2-

197

methylpyrazine. Acros organics (Geel, Belgium) supplied 2,3,5-trimethylpyrazine, 2,5-

198

dimethylthiophene and 2-ethylpyrazine. Megastigmatrienones (tabanones; 4-[butenylidene]-

199

3,5,5-trimethylcyclo-2-hexen-1-one) provided by Symrise AG (Holzminden, Germany) as a

200

mix of 5 isomers (m/m): 11% megastigma-4,6Z,8E-trien-3-one (9), 32% megastigma-4,7E,9-

201

trien-3-one (10), 35% megastigma-4,6Z,8Z-trien-3-one (11), 4% megastigma-4,6E,8E-trien-3-

202

one (12), and 18% megastigma-4,6E,8Z-trien-3-one (13). Dodecan-1-ol, used as an internal

203

standard, was supplied by Acros organic (Geel, Belgium). CDN Isotopes (Quebec, Canada)

204

supplied. 2-methylpyrazine-d6, also used as an internal standard

205

Quantitation procedures

206

Heterocycle quantitation

207

Heterocyclic compounds were quantified using the method proposed by Burin et al.16 Sample

208

preparation and chromatographic conditions were optimized and validated using an HS-SPME-

209

GC/MS device. For the quantitative study, 10 µL stock solution (about 330 mg/L in

210

ethanol/water 50% v/v) of 2-methylpyrazine-d6 was added to a 10 mL wine sample as an

211

internal standard. The spiked sample was placed in a 20 mL vial, 3 g sodium chloride were

212

added, and the vial was tightly sealed with a PTFE-lined cap. The spiked wine was

213

homogenized in a vortex shaker and then loaded onto a Gerstel MPS2 auto-sampling device

214

(Mülheim an der Ruhr, Germany). The program consisted of swirling the vial at 250 rpm at

215

40°C for 5 min, then inserting the SPME fiber into the headspace at 40°C for 55 min as the

216

solution was swirled again, and transferring the fiber to the injector for desorption at 250°C for

217

5 min. The SPME fiber used (Supelco, Bellefonte, PA, USA) was coated with 85 O* stationary-

218

phase carboxen/polydimethylsiloxane (Carboxen/PDMS). Gas chromatography analyses were


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219

carried out on an Agilent technologies 6890N gas chromatography device, coupled to an

220

Agilent technologies 5973 inert mass spectrometer. The capillary column was an HP-5MS

221

(50 m × 0.25 mm; film thickness 0.2 O* -21.5

< -1.4

< -0.8

Moderate to severe water deficit

-21.5 to -23

-1.1 to -1.4

-0.5 to -0.8

Moderate to weak water deficit

-23 to -24.5

-0.9 to -1.1

-0.3 to -0.5

Weak water deficit

-24.5 to -26

-0.6 to -0.9

-0.2 to -0.3

No water deficit

< -26

> -0.6

> -0.2

?

13C


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Table 2b: Levels of vine nitrogen status according to three indicators such as yeast available content of must at harvest, nitrogen leaf blade and nitrogen petiole (Van Leeuven, 2000)14. *N%MS : percentage of nitrogen masse compared of mass of dry matter

Level of vine nitrogen status

Yeast available nitrogen content of must at harvest (mg/L)

Nitrogen leaf blade (N%MS*)

Nitrogen petiole (N%MS*)

Very low

< 50

-

-

Low

50 to 100

< 0.4

< 1.8

Low to medium

100 to 150

0.4 to 0.6

1.8 to 2.4

Medium to high

150 to 200

0.4 to 0.6

1.8 to 2.4

High

200 to 250

> 0.6

> 2.4

Very high

>250

-

-



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Table 3: Correlations between aroma compound levels and wine ages. Spearman coefficient

Spearman p-value

R2

(1)

0.125

0.439

0.016

(2)

0.643

< 0.0001

0.413

(4)

0.341

0.032

0.116

(3)

0.719

< 0.0001

0.517

(5)

0.042

0.797

0.002

(6)

-0.056

0.731

0.003

(7)

0.415

0.008

0.173

DMS (8)

-0.251

0.119

0.063

(9)

0.749

< 0.0001

0.561

(11)

0.719

< 0.0001

0.517

(12)

0.637

< 0.0001

0.406

(13)

0.707

< 0.0001

0.500


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Effect of vine water and nitrogen status, as well as temperature, on

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