AN ABSTRACT OF THE THESIS OF

Jingwen Li for the degree of Master of Science in Food Science and Technology presented on June 15, 2018.

Title: Composition of Produced from Different Crop Levels

Abstract approved:

______

Michael C. Qian

The production of high-quality is an important target for worldwide, and low crop level (yield) has been one management criterion long believed to achieve wine quality. Low yields are most often achieved by removing clusters from the grapevine. Some studies show that cluster thinning can enhance grape maturity and color intensity. The reduction of crop level also increased wine quality and intensity by raising the content of quality-important compounds like and anthocyanins. However, some other studies indicated that differences between wines made from crop- thinning vines and full crop vines were not always detected. Variations in vineyard climate and grape cultivar and rootstock may override the outcomes of cluster thinning practices. The impacts of cluster thinning on wine quality are still inconclusive.

In this study, Pinot noir wine composition was investigated over three growing seasons (2013 to 2015) in twelve commercial wineries in Oregon where crop level was adjusted using cluster thinning at lag-phase of berry development using a cluster number per shoot regime (e.g. 2, 1.5 and 1 cluster/shoot) and compared to a full crop control (non-thinned). Two sites used variable ton per hectare treatments (e.g. 1.31, 1.01, and 0.71 ton/hectare).

After , fruits from field replicates were combined to produce the wine. The phenolic compounds and volatile aroma compounds were analyzed by HPLC, UV- spectrometry and GC-MS techniques. Results showed that the effects of cluster thinning treatments on Pinot noir wine composition were dependent on vineyard site and year. For wine phenolic compounds, the influences of cluster thinning on major phenolic compounds were observed in six wineries but various with the and wineries. Results also showed that cluster thinning increased total monomeric anthocyanins (TMA) and total phenolic content (TP) in wines from five of the six wineries. Certain volatile compounds were mainly influenced by cluster thinning treatments, depending on wineries and vintages. It was demonstrated that wines from one were insensitive to cluster thinning treatments. Further research is underway to confirm the influence of cluster thinning practices on volatile compounds in Pinot noir wines.

©Copyright by Jingwen Li

June 15, 2018

All Rights Reserved

Composition of Pinot noir Wines Produced from Different Crop Levels

by

Jingwen Li

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Presented June 15, 2018 Commencement June 2019

Master of Science thesis of Jingwen Li presented on June 15, 2018

APPROVED:

Major Professor, representing Food Science and Technology

Head of the Department of Food Science and Technology

Dean of the Graduate School

I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request.

Jingwen Li, Author

ACKNOWLEDGEMENTS

I express sincere appreciation for the guidance, encouragement, and support given by my major advisor, Dr. Michael Qian. Also, thanks to Dr. Yanping Qian, who offered kind help and encouragement in my study and my life in Corvallis.

I offer sincere thanks to Dr. Patricia Skinkis and the Lab for providing the Pinot noir wine samples, and thanks for their great work in vineyard management research, field data collection. Thanks to Dr. Patricia Skinkis for data interpretation and guidance on my research.

Thanks to Dr. Patricia Skinkis, Dr. Elizabeth Tomasino and Dr. Alexandra Stone for serving as my committee members and giving me assistance and guidance on my research.

Thanks to Ludwig Ring, Quintin M Ferraris, Yueqi An, Mengying Fu, Estefania Abarca, Fei He and Xiaoxi Yuan and all the people who worked with me in the lab for their generous assistance and friendship. Thanks to my friends who have helped me through the years of study. Thanks to the staff at the Department of Food Science and Technology, Oregon State University, for their friendliness and assistance.

Finally, I want to thank my parents and my whole family for all their support, love and motivation.

CONTRIBUTION OF AUTHORS

Dr. Michael Qian contributed to the overall wine composition research concept, research design, data interpretation, and revision. Dr. Patricia Skinkis designed the larger study of which this work was conducted, designed the field and wine production experiments, coordinated sample collection, assisted the interpretation of the data and did manuscript revision for the overall research. Dr. Elizabeth

Tomasino contributed to manuscript revision for the overall research.

TABLE OF CONTENTS Page

Chapter 1 LITERATURE REVIEW ...... 1 The aroma compounds in wine ...... 1 1.1.1. Grape-derived aroma compounds ...... 1 1.1.2. Fermentation-derived aroma compounds ...... 17 Non-volatile phenolic compounds in wine ...... 23 Cluster thinning to reduce yields ...... 26 Justification of research ...... 28

Chapter 2 PHENOLIC COMPOSITION OF PINOT NOIR WINES PRODUCED FROM DIFFERENT CLUSTER THINNING LEVELS ...... 30 Abstract ...... 31 Introduction ...... 31 Materials and methods ...... 33 2.3.1. Chemicals ...... 33 2.3.2. Vineyard experimental design ...... 33 2.3.3. Determination of wine chemical composition ...... 36 Results ...... 38 2.4.1. Wine total phenols and anthocyanins ...... 38 2.4.2. Wine major phenolic compounds ...... 38 Discussion ...... 39 2.5.1. Wine total phenols and anthocyanins ...... 39 2.5.2. Wine major phenolic compounds ...... 40 Conclusions ...... 41

Chapter 3 VOLATILE COMPOSITION OF PINOT NOIR WINES PRODUCED FROM DIFFERENT CLUSTER THINNING LEVELS ...... 47 Abstract ...... 48 Introduction ...... 48 Materials and methods ...... 50

TABLES OF CONTENTS (Continued)

Page

3.3.1. Chemicals ...... 50 3.3.2. Quantitative analysis of wine volatile compounds ...... 51 3.3.3. Odor activity value ...... 55 3.3.4. Statistical analysis ...... 55 Results ...... 56 3.4.1. Volatile aroma compounds ...... 56 Discussion ...... 60 3.5.1. Grape-derived volatile compounds ...... 60 3.5.2. Fermentation-derived volatile compounds ...... 62 Conclusions ...... 63

Chapter 4 GENERAL CONCLUSION ...... 97

Bibliography ...... 99

APPENDIX ...... 121

LIST OF FIGURES

Figure Page

Figure 1.1 Scheme of plant isoprenoid biosynthetic pathways...... 3

Figure 1.2 Structure of some glycoconjugated terpenoid compounds...... 4

Figure 1.3 Chemical structures of important in grapes...... 5

Figure 1.4 Chemical structures of -derived norisoprenoids...... 6

Figure 1.5 Formation of norisoprenoids either via direct carotenoid degradation or via glycosylated intermediates...... 7

Figure 1.6 Formation of norisoprenoid compounds from β-carotene...... 7

Figure 1.7 Proposed formation of β-damascenone from a glycosidic precursor...... 9

Figure 1.8 Structures of norisoprenoids important to the aroma of wine...... 9

Figure 1.9 Stereoisomers of vitispirane...... 10

Figure 1.10 Biosynthesis of (a) short-chain acids, aldehydes, alcohols, esters and lactones, and (b) methyl ketones. AAT, acyl CoA transferase; MKS, methyl ketone synthases; ACP, acyl carrier protein...... 16

Figure 1.11 Some of the major classes of aroma compounds (shown in blocks) produced by yeast during alcoholic fermentation ...... 17

Figure 1.12 A simplified metabolic map of yeast production, indicating known metabolic linkages ...... 19

Figure 1.13 AATase enzyme biosynthetic pathway for synthesis ...... 20

Figure 1.14 The flavonoid ring system...... 24

LIST OF TABLES

Table Page

Table 1.1 Odors of some important aromatic volatile compounds in grapes...... 12

Table 2.1 The information of wine companies and and cluster thinning treatments in the fields with yields (Kg/meter) and percent thinned in 2013 to 2015 vintage years...... 34

Table 2.2 Total phenols and total monomeric anthocyanins in Pinot noir wine with different cluster thinning treatments from 2013 to 2015 vintage ...... 42

Table 2.3 Phenolic composition in Pinot noir wine with different cluster thinning treatments from 2013 to 2015 vintage ...... 44

Table 3.1. Selected quantification ions for isotope volatile compounds in Pinot noir wine ...... 64

Table 3.2 Composition of volatile compounds in Pinot noir wines from Winery A with cluster thinning treatments from 2013 to 2015 (ug/L) ...... 65

Table 3.3 Composition of volatile compounds in Pinot noir wines from Winery B with cluster thinning treatments from 2013 to 2015 (ug/L) ...... 68

Table 3.4 Composition of volatile compounds in Pinot noir wines from Winery C with cluster thinning treatments from 2013 to 2015 (ug/L) ...... 71

Table 3.5 Composition of volatile compounds in Pinot noir wines from Winery D with cluster thinning treatments from 2013 to 2015 (ug/L) ...... 74

Table 3.6 Composition of volatile compounds in Pinot noir wines from Winery E with cluster thinning treatments from 2013 to 2015 (ug/L) ...... 77

Table 3.7 Composition of volatile compounds in Pinot noir wines from Winery F with cluster thinning treatments from 2013 to 2015 (ug/L) ...... 80

Table 3.8 Thresholds and odors of some important aromatic volatile compounds in wines...... 83

Table 3.9 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery A...... 85

Table 3.10 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery B...... 87

LIST OF TABLES (Continued) Page

Table 3.11 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery C...... 89

Table 3.12 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery D...... 91

Table 3.13 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery E...... 93

Table 3.14 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery F...... 95

1

Chapter 1 LITERATURE REVIEW

Pinot noir wine is a light- to medium-bodied wine that is produced from grapes of vinifera cv. Pinot noir. Pinot noir grapes are grown around the world, mostly in the cooler climates (Tate, 2001). Many producers consider Pinot noir to be a fickle grape that requires optimum growing conditions, defined by warm days and cool evenings in order to produce its quality fruit and wines typical for the variety. It is also highly susceptible to wind, frost, rot and fungal diseases. In the winery, Pinot noir wine is highly reflective of its (e.g. growing conditions) and quality can be easily affected by fermentation methods and yeast strains (John Winthrop Haeger, 2004).

The Willamette Valley of Oregon has the largest concentration of vineyards and wineries in the state. It is an ideal growing region, as it is a cool climate typified by warm, dry summers and a cool late summer/early fall ripening period. Rainfall occurs primarily in the late autumn, winter, and early spring (John W Haeger & Storchmann, 2006).

The aroma compounds in wine

Wine is an ancient beverage and has been prized throughout time for its unique and pleasing flavor (Amerine & Singleton, 1977). The aroma of wine is complex due to the diversity of sources and mechanisms involved in its development, including 1) grape- derived aroma compounds such as , norisoprenoids, aliphatics, phenylpropanoids, and methoxypyrazines, 2) fermentation-derived compounds, including sugar and amino acid-derived volatile acids, esters, and higher alcohols, 3) aroma compounds generated during aging and storage, like -derived aroma compounds and aroma compounds from chemical changes associated with oxidative processes in wine. (Robinson, Boss, Solomon, Trengove, Heymann, & Ebeler, 2014).

1.1.1. Grape-derived aroma compounds

Many compounds in grape berries contribute to wine flavor and aroma, and most of them exist as odorless and glycosidically-bound forms which can be hydrolyzed into active aroma compounds during fermentation or aging (Hjelmeland & Ebeler, 2015). 2

1.1.1.1.

Many studies have investigated the chemistry, synthesis and systematic classification of terpenes (Marais, 2017). Terpenes are synthesized in berries and stored in the grape skin (Marais, 2017). Based on the number of isoprene units in the molecule, terpenes are classified into subgroups, including hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20) (Mahmoud & Croteau, 2002). Monoterpenes

(C10) and sesquiterpenes (C15) are biologically synthesized from isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) as shown in Figure 1.1 (Sapir-Mir, Mett, Belausov, Tal-Meshulam, Frydman, Gidoni, et al., 2008). These precursors are formed either through the cytosolic mevalonic-acid (MVA) pathway in the cytosol and endoplasmic reticulum (ER) compartment and from three molecules of acetyl-CoA (Newman & Chappell, 1999) or through the plastidial 2-C-methylerythritol-4-phosphate (MEP) pathway from pyruvate and glyceraldehyde-3-phosphate in plastid (Rohmer, 1999). Then monoterpenes are formed from 2E-geranyl diphosphate (GPP) and sesquiterpenes are formed from 6E-farnesyl diphosphate (FPP) (Sapir-Mir, et al., 2008). 3

Figure 1.1 Scheme of plant isoprenoid biosynthetic pathways. (Sapir-Mir, et al., 2008)

Considerable research has been done on identification and contribution of compounds to wine aroma, especially grapes and wines (, Avellone, Filizzola, Monte, Catanzaro, & Agozzino, 2013; Celik, Karaoğlan, Darıcı, Kelebek, İşçi, Kaçar, et al., 2015; Ribéreau-Gayon, Boidron, & Terrier, 1975). The majority of identified terpenes that contribute to grape and wine flavor and aroma are monoterpenes and only one sesquiterpene sub-family member (rotundone) is reported to be important for wine aroma (Dunlevy, Kalua, Keyzers, & Boss, 2009; Wood, Siebert, Parker, Capone, Elsey, Pollnitz, et al., 2008). In grapes, monoterpenes are present in both free and glycosidically-bound form. And some bound-form terpenes can be released either chemically (Michlmayr, Nauer, Brandes, Schümann, Kulbe, del Hierro, et al., 2012; Versini, Orriols, & Dalla Serra, 1994) or hydrolytically with glycosidases in grapes or from yeast and bacteria during the vinification phase (Michlmayr, et al., 2012; 4

Skouroumounis & Sefton, 2000). Only a small fraction of monoterpene alcohols in grapes exist as free volatile forms whereas most of them are non-volatile in the form of glycosides (Hjelmeland & Ebeler, 2015; Maicas & Mateo, 2005). Several glycosidically bound forms of monoterpenes have been identified in grapes, including O-β-d- glucopyranosides, 6-O-α-l-rhamnopyranosyl-β-d-glucopyranosides, 6-O-α-l- arabinofuranosyl-β-d-glucopyranosides, and 6-O-β-d-apiofuranosyl-β-d- glucopyranosides as shown in Figure 1.2 (Hjelmeland & Ebeler, 2015; Voirin, Brun, & Bayet, 1990; P. J. Williams, Strauss, Wilson, & Massy-Westropp, 1982). This high proportion of bound monoterpenes is recognized as a wine’s “hidden aromatic potential” since only free terpenes contribute to the aroma of a wine (Dimitriadis & Williams, 1984; Gunata, Bayonove, Baumes, & Cordonnier, 1985).

Figure 1.2 Structure of some glycoconjugated terpenoid compounds. (Maicas & Mateo, 2005) 5

Monoterpenes play important roles in many different wine grape varieties (Marais, 2017). The most important monoterpenes are monoterpene alcohols, including terpineol, , , nerol and citronellol (Figure 1.3), which are key aroma compounds for floral grape varieties (Barbera, Avellone, Filizzola, Monte, Catanzaro, & Agozzino, 2013; Celik, et al., 2015; Ebeler, 2001; Ribéreau-Gayon, Boidron, & Terrier, 1975; Skinkis, Bordelon, & Wood, 2008). However, monoterpenes in the non-floral varieties, such as Pinot noir, are usually below odor-active threshold values (Chatonnet, Dubourdie, Boidron, & Pons, 1992).

Figure 1.3 Chemical structures of important monoterpene alcohols in grapes.

It is known that terpenes play important roles in many different wine grape varieties. The concentration of terpenes in grapes and wine obviously depends on various factors, including cultivar, region, climate and wine-making techniques (Baron, Prusova, Tomaskova, Kumsta, & Sochor, 2017; Celik, et al., 2015; Ji & Dami, 2008). For instance, it was reported that terpene alcohols of wines from Cv. (Vitis vinifera L.) were affected by deficit irrigation (Ou, Du, Shellie, Ross, & Qian, 2010). Pinot noir, as a non- floral variety, also contain monoterpene alcohols, but generally at levels below the sensory thresholds (Fang & Qian, 2005). It was found that the monoterpenes in Pinot noir wine had increasing trends with grape maturation and postharvest dehydration. (Fang & Qian, 2006; Moreno, Cerpa-Calderón, Cohen, Fang, Qian, & Kennedy, 2008). Feng et al. also reported that leaf removal resulted in higher concentrations of some bound-form 6

terpenoids in Pinot noir grapes (Feng, Yuan, Skinkis, & Qian, 2015). Moreover, sunlight exposure and UV resulted in an increase of nerol, geraniol and citronellol but not linalool (Song, Smart, Wang, Dambergs, Sparrow, & Qian, 2015). Low grapevine vigor increased levels of linalool, nerol and geraniol (Song, Smart, Dambergs, Sparrow, Wells, Wang, et al., 2014). Since continuing discoveries of important new compounds, such as rotundone, the attention that is given to this group of compounds will keep increasing.

1.1.1.2. Norisoprenoids

Norisoprenoids are natural compounds that have attracted tremendous attention (P. Williams & Allen, 1996; Peter Winterhalter, 1992). They consist of a megastigma carbon skeleton and differ in the position of the oxygen functional group, being either absent (megastigmanes), attached to carbon 7 (damascones), or attached to carbon 9 (ionones) as shown in Figure 1.4 (Peter Winterhalter & Rouseff, 2002).

Figure 1.4 Chemical structures of carotenoid-derived norisoprenoids. (Peter Winterhalter & Rouseff, 2002)

The norisoprenoids are important aroma compounds that contribute to flower-like and fruity aromas of wine (Ebeler, 2001) and they have two main sources. (1) Norisoprenoids could arise from the direct degradation of carotenoid compounds, since are unstable due to typical highly conjugated double-bond structure (Figure 1.5), including β- carotene, lutein, neoxanthin and violaxanthin (Kanasawud & Crouzet, 1990; Mordi, Walton, Burton, Hughes, Ingold, & Lindsay, 1991; Peter Winterhalter & Rouseff, 2002). Among these carotenoids, the degradation of β-carotene is especially important to norisoprenoids and produce carbonyl compounds consist of 13, 11, 10 or 9 carbon atoms, retaining the terminal group of the β-carotene parent as illustrated in Figure 1.6. For 7

instance, the generation of β-ionone from β-carotene cleavage can be caused by cleavage dioxygenases, photo-oxygenation and thermal degradation (Isoe, Hyeon, & Sakan, 1969; Kanasawud & Crouzet, 1990). (2) Norisoprenoids could also come from the hydrolysis of glucoside molecules through glycosidases activity or acid hydrolysis as shown in Figure 1.5 (Di Stefano, Bottero, Pigello, Borsa, Bezzo, & Corino, 1998; Gunata, Bayonove, Baumes, & Cordonnier, 1985; Skouroumounis, Massy-Westropp, Sefton, & Williams, 1992). For example, β-damascenone could be generated from the glycosidic precursor as illustrated in Figure 1.7.

Figure 1.5 Formation of norisoprenoids either via direct carotenoid degradation or via glycosylated intermediates. (Mendes-Pinto, 2009)

Figure 1.6 Formation of norisoprenoid compounds from β-carotene. (Charalambous, 1990) 8

Apart from previously mentioned β-ionone and β-damascenone, several norisoprenoids are essential to the aroma of wine, including 2, 2, 6- trimethylcyclohexanone (TCH), vitispirane, actinidiol, 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN), acetal and 4-(2,3,6-trimethylphenyl)-buta-1,3-diene (TPB) (Mendes-Pinto, 2009). And their structures are shown in Figure 1.8. β-Damascenone and β-ionone belong to rose ketones, a diverse group of aroma compounds which first discovered from Bulgarian (Demole, 1970). These compounds are described as -like depending on the concentration (Baumes, Wirth, Bureau, Gunata, & Razungles, 2002; CAPONE, Skouroumounis, & SEFTON, 2002). β-Damascenone smells sweet and honey-like with an odor threshold of 0.05 µg/L in water/ethanol (90+10, w/w) (Guth, 1997). Studies have reported that when β-damascenone is present below its threshold in wines, it enhances the aroma intensity of fruity-type aromas and has the ability to mask the “herbaceous” aroma associated with 2-isobutyl-3-methoxypyrazine (Escudero, Campo, Fariña, Cacho, & Ferreira, 2007; Ferreira, Ortín, Escudero, López, & Cacho, 2002; Pineau, Barbe, Van Leeuwen, & Dubourdieu, 2007; M. A. Sefton, Skouroumounis, Elsey, & Taylor, 2011) . β-Ionone is often described as sweet or violet aroma with an odor threshold of 0.09 µg/L in water/ethanol (90+10, w/w) (Ferreira, Lopez, & Cacho, 2000). The concentrations of β-damascenone and β-ionone in grapes and wines are usually above their sensory thresholds (Bindon, Dry, & Loveys, 2007; Fang & Qian, 2005; Ristic, Bindon, Francis, Herderich, & Iland, 2010). Therefore, they are considered important aroma contributors to many wine varieties such as Merlot, and Pinot noir (Fang & Qian, 2005; Gürbüz, Rouseff, & Rouseff, 2006; Lopez, Ferreira, Hernandez, & Cacho, 1999). 9

Figure 1.7 Proposed formation of β-damascenone from a glycosidic precursor. (Kinoshita, Hirata, Yang, Baldermann, Kitayama, Matsumoto, et al., 2010)

Figure 1.8 Structures of norisoprenoids important to the aroma of wine. (1) TCH, (2) β-damascenone, (3) β-ionone, (4) vitispirane (5) actinidiol, (6) TDN, (7) Riesling acetal, (8) TPB (Mendes-Pinto, 2009)

TDN is characterized by “petrol” aromas with the sensory threshold of 2 µg/L in both model wine and neutral matrices (Sacks, Gates, Ferry, Lavin, Kurtz, & Acree, 10

2012). TDN concentrations of 1.3±0.8 µg/L in many , including Pinot noir, and are significantly lower than 6.4±3.8 µg/L in the Riesling wines (Eggers, Bohna, & Dooley, 2006; Sacks, Gates, Ferry, Lavin, Kurtz, & Acree, 2012). Vitispirane is also considered to be an important odorant in aged wine (Marais, Van Wyk, & Rapp, 1991; Simpson & Miller, 1983; P Winterhalter, Sefton, & Williams, 1990). Vitispirane has two chiral carbons and thus two pairs of diastereomers as shown in Figure 1.9. The odor of the diastereomers is distinctly different. The pair of cis enantiomers with green and flowery-fruity aroma were fresher and more intense than the trans pair, which was characterized by a heavy scent of exotic flowers with an earthy-woody undertone (Schulte-Elte, Muller, & Rautenstrauch, 1978). TDN and vitispirane are found at the concentration below their detection thresholds in many wine varieties (Eggers, Bohna, & Dooley, 2006; Sacks, Gates, Ferry, Lavin, Kurtz, & Acree, 2012). However, their levels can increase above its threshold during aging, which potentially imposes negative sensory attributes on the wine (Sacks, Gates, Ferry, Lavin, Kurtz, & Acree, 2012; Simpson, 1978; Peter Winterhalter & Rouseff, 2002).

Figure 1.9 Stereoisomers of vitispirane. (Eggers, Bohna, & Dooley, 2006)

Additional important aroma active norisoprenoids in wine include TPB, TCH, actinidiol and riesling acetal. TPB has a pleasant floral aroma at low concentrations while pungent or chemical odor at high concentrations (Cox, Capone, Elsey, Perkins, & Sefton, 2005). 11

TPB has been found above its sensory threshold in several white wine varieties, including Semillon, , and Riesling wines but not detected in red wines (Cox, Capone, Elsey, Perkins, & Sefton, 2005; Janusz, Capone, Puglisi, Perkins, Elsey, & Sefton, 2003)

It is reported that the presence of TCH was responsible for the “rock-rose-like” aroma in young Ports wines (Gunata, Bayonove, Baumes, & Cordonnier, 1985) at levels of 50-400 ng/L, which was higher than its sensory threshold in water of 44.3 ug/L (de Freitas, Ramalho, Azevedo, & Macedo, 1999). Actinidiol is known to be derived from actinidiolide (Sakan, Isoe, & Hyeon, 1967). Actinidiol and its isomers exist in some wines at levels higher than β-damascenone, vitispirane, and TDN, and are also found in many aged wines (Mendes-Pinto, 2009). Riesling acetal had a fruity odor while its enantiomers differ distinctly in their odor properties that (+)-Riesling acetal had a weak woody, fruity and flowery note, whereas the (-)-isomer exhibited a slight camphoraceous and flowery note (Dollmann, Full, Schreier, Winterhalter, Güntert, & Sommer, 1995).

Since norisoprenoids are considered to arise from carotenoid precursors found in the grape, factors that influence the composition of carotenoids will affect the level of norisoprenoids in grapes (Mendes-Pinto, 2009; Yuan & Qian, 2016). The profiles of carotenoids in grapes are affected by several factors including plant variety, climatic conditions, stage of maturity, terroir and viticulture practices (Mendes-Pinto, 2009). It is reported that carotenoid levels decreased progressively from the onset of fruit development to the end of maturation (A Razungles, Bayonove, Cordonnier, & Sapis, 1988). Sunlight exposure before resulted in increased carotenoid levels, while exposure after veraison increased norisoprenoid levels in (Vitis vinifera L.) grape berries (AJ Razungles, Baumes, Dufour, Sznaper, & Bayonove, 1998b). Moreover, it has been reported that warm regions produce grapes with the high level of carotenoids (Crupi, Mazzon, Marino, La Spada, Bramanti, Cuzzocrea, et al., 2010; Marais, Van Wyk, & Rapp, 1991).

1.1.1.3. Phenylpropanoids and benzenoids

Phenylpropanoids and benzenoids are a family of volatile molecules with the structure of 12

a planar, cyclic delocalized π-electron system (Dudareva, Negre, Nagegowda, & Orlova, 2006; Dunlevy, Kalua, Keyzers, & Boss, 2009). Volatile phenylpropanoids and benzenoids come from a range of sources and they can have a significant influence on wine aroma (Robinson, Boss, Solomon, Trengove, Heymann, & Ebeler, 2014). Some of the important and common phenylpropanoids and benzenoids are listed in Table 1.1 with their corresponding odor descriptions and sensory thresholds.

Table 1.1 Odors of some important aromatic volatile compounds in grapes.

Compound Sensory Threshold (ug/L) Odor description guaiacol 9.5[4] phenolic, spicy eugenol 6[2] honey, clove 2-methylphenol 31[2] woody, phenolic 3-methylphenol 68[1] leather 3-ethylphenol 250[2] musty 4-ethylguaiacol 33[1] caramellic 4-vinylguaiacol 1100[2] spice, anise 4-methylphenol 60[2] horse 4-ethylphenol 440[2] medicine, horse ethyl cinnamate 1.1[2] spice, sweet methyl anthranilate 3[2] grapes ethyl anthranilate 16[3] spice, sweet 2-phenylethyl alcohol 14000[5] pleasant floral ethyl phenylacetate 250[2] Sweetish solvent 2-phenylethyl acetate 250 [4] rose, honey, tobacco [1] Li, Tao, Wang, and Zhang (2008), [2] Genovese, Dimaggio, Lisanti, Piombino, and Moio (2005), [3] Jiang and Zhang (2010), [4] P. Zhao, Gao, Qian, and Li (2017) [5] (Feng, 2014)

Volatile phenylpropanoids, such as 2-phenylethyl alcohol, phenylacetaldehyde, benzaldehyde and benzyl acetate are generally considered to be derived from phenylalanine, which is formed through the Shikimic acid pathway in plastids (Dudareva, 13

Klempien, Muhlemann, & Kaplan, 2013). 2-Phenylethyl alcohol and 2-phenylethyl acetate, produced from substituted phenylalanine and tyrosine (Rossouw, Næs, & Bauer, 2008) are thought important to white wine aroma since they are typically found at concentrations above odor threshold (Guth, 1997; López, 2003). Methyl anthranilate is considered to be responsible for the distinctive “foxy” aroma of the Washington () (Wang & Luca, 2005) and may also contribute to the aroma of Pinot noir (L Moio & Etievant, 1995). Vinyl phenols and ethyl phenols are breaking- down products from hydroxycinnamic acids and the ethyl phenols with leather characters are considered detrimental wine aroma (Chatonnet, Dubourdie, Boidron, & Pons, 1992; Lattey, Bramley, & Francis, 2010; Wedral, Shewfelt, & Frank, 2010). Guaiacol, eugenol and 4-vinyl guaiacol were also found to have negative impacts on Pinot noir wine because of their smoky, spicy, woody, animal and medicinal aroma characteristics (Fang & Qian, 2005). However, it was reported they contributed to the complexity of wine aroma at lower concentration (< 4 mg/L) (Suárez, Suárez-Lepe, Morata, & Calderón, 2007).

Volatile phenylpropanoids and benzenoids deserve significant consideration because of their observed abundance in hydrolysates of glycoside isolates from juices and wines. In grapes, their non-volatile precursors can be converted to the odor-active compounds through enzyme or acid hydrolysis during fermentation (Chatonnet, Dubourdieu, Boidron, & Lavigne, 1993; Laforgue & Lonvaud-Funel, 2012). For example, they can constitute 10 to 20% of the total hydrolyzed volatile fraction in Chardonnay juice (M. Sefton, Francis, & Williams, 1993) and 51% of the total hydrolyzed fraction in wine (Boido, Lloret, Medina, Fariña, Carrau, Versini, et al., 2003).

The content of volatile phenylpropanoids and benzenoids in wine are affected by multiple factors. In grapes, levels of their precursors are influenced by grape variety, grape maturity, variations in water and nutrient availability, and sunlight and temperature conditions (Bell & Henschke, 2005; Fang & Qian, 2006; Koundouras, Marinos, Gkoulioti, Kotseridis, & Van Leeuwen, 2006). For instance, Pinot noir wine produced from deficit- irrigated vines had higher contents of guaiacol, 4-methylguaiacol, 4-ethylguaiacol, and 4- 14

vinylguaiacol relative to wine produced from well-watered vines (Qian, Fang, & Shellie, 2009). But there was no consistent effect of water deficit on Shikimic acid derivatives such as benzaldehyde, benzyl alcohol and 2-phenylethanol, and 2-methoxy-4-vinylphenol in Merlot grapes over the 2 years’ study (Song, Shellie, Wang, & Qian, 2012). Moreover, it was reported that grape maturation decreased ethyl cinnamate, ethyl dihydroxycinnamate, and ethyl anthranilate in Pinot noir wine (Fang & Qian, 2006). Benzyl alcohol and 2-phenylethyl alcohol in Pinot noir grapes dramatically increased over the whole growing season (Fang & Qian, 2012). The process, practices such as yeast strains and fermentation conditions have significant impacts on the release of free-form compounds from their precursors during fermentation (Ferreira, Lopez, & Cacho, 2000; Heresztyn, 1986). Oak barrels used for fermentation and wine aging process are also important sources (Prida & Chatonnet, 2010; Spillman, Sefton, & Gawel, 2004aa; 2004bb). Over 50 volatile phenylpropanoids have been identified in the smoke from pyrolyzed oak (Guillén & Manzanos, 2002).

1.1.1.4. Aliphatic volatile compounds

The major aroma compounds derived from fatty acids in grapes tend to be the C6- aldehydes and alcohols (Dunlevy, Kalua, Keyzers, & Boss, 2009; Ferreira, Fernndez,

Peña, Escudero, & Cacho, 1995; Iyer, Sacks, & PadillaZakour, 2010) and many of them are thought to be responsible for “green” aromas in wines (Kotseridis & Baumes, 2000;

Schwab, DavidovichRikanati, & Lewinsohn, 2008). The C6 compounds are generally formed by a series of enzymatic reactions when the grape is crushed (Hatanaka, 1993; Mwenda & Matsui, 2014; Prestage, Linforth, Taylor, Lee, Speirs, & Schuch, 1999; Sánchez & Harwood, 2002) (Figure 1.10). 9/13-Hydroperoxides are formed by the lipoxygenase (LOX) activity from linolenic acid in the presence of oxygen. These 9/13- hydroperoxides are then transformed to hexanal with the help of hydroperoxide lyase (HPL) and isomerase inter-converts the two hexenals. Finally, an alcohol dehydrogenase (ADH) reduces the aldehydes to the corresponding alcohols (Schwab, Davidovich Rikanati, & Lewinsohn, 2008). 15

The important C6 compounds in wine include 1-hexanol, (Z)-3-hexenol, (E)-3-hexenol, (Z)-2-hexenol, (E)-2-hexenol, hexanal and (E)-2-hexenal and they have been receiving special attention for a long time in wine production because of leafy and grassy odor (Joslin & Ough, 1978). The presence of 1-hexanol in wines arises from the 1-hexanol in the grapes and from the reduction of hexanal, (E)-2-hexenal, (E)-2-hexenol, and (Z)-2- hexenol during fermentation (Herraiz, Herraiz, Reglero, Martin-Alvarez, & Cabezudo, 1990; Joslin & Ough, 1978). 1-Hexanol and (E)-2-hexenol have been reported as precursors of esters (e.g., hexyl acetate) in the final wine(Dennis, Keyzers, Kalua, Maffei, Nicholson, & Boss, 2012). Even though at high concentration, 1-hexanol in wine is usually below its odor threshold (8000 µg/L) (Guth, 1997). (E)-3-hexenol and (Z)-3- hexenol have been referred as the most essential C6 compounds that seem to be sufficiently stable and remain unaffected by the metabolic activity of yeasts (Di Stefano & Ciolfi, 1982; Herraiz, Herraiz, Reglero, Martin-Alvarez, & Cabezudo, 1990). Some believe (E)-3-hexenol/(Z)-3-hexenol ratio can act as an indicator of wines (Boss, Keyzers, Kalua, Dennis, Williams, & Forde, 2010; Dennis, Keyzers, Kalua, Maffei, Nicholson, & Boss, 2012; Moret, Scarponi, & Cescon, 1984; José M Oliveira, Marta Faria, Filomena Sá, Filipa Barros, & Isabel M Araújo, 2006; Rapp, Volkmann, & Niebergall, 1993; Yuan & Qian, 2016). 16

Figure 1.10 Biosynthesis of (a) short-chain acids, aldehydes, alcohols, esters and lactones, and (b) methyl ketones. AAT, alcohol acyl CoA transferase; MKS, methyl ketone synthases; ACP, acyl carrier protein. (Schwab, DavidovichRikanati, & Lewinsohn, 2008)

The content of C6 compounds in wine depend on numerous factors, like the terroir, variety and maturity of grapes and winemaking procedures, such as must protection, skin and enzymatic , type of preservative used and moment of its application (Cabaroglu, Canbas, Baumes, Bayonove, Lepoutre, & Günata, 1997; Luigi Moio, Ugliano, Genovese, Gambuti, Pessina, & Piombino, 2004; Nicolini, Versini, & Amadei, 1996; Nicolini, Versini, Amadei, & Marchio, 1996; José M. Oliveira, Marta Faria, Filomena Sá, Filipa Barros, & Isabel M. Araújo, 2006). Reports have shown that higher hexanol contents during must incubation and higher (Z)-3-hexenol levels are found in musts that include grape leaves (Joslin & Ough, 1978). In Pinot noir grapes, C6 alcohols are accumulated during véraison but their contents continuously decrease during berry ripening (Fang & Qian, 2012). The levels of C6-aldehydes continue to increase after véraison until grapes reach harvest maturity and then start to decrease (Fang & Qian, 17

2012). In another hand, viticultural practices such as deficit irrigation, fertilization, and shoot thinning are shown to reduce levels of C6 compounds in grapes, leading to less herbaceous aroma in final wine (Mendez-Costabel, Wilkinson, Bastian, Jordans, McCarthy, Ford, et al., 2014; Song, Shellie, Wang, & Qian, 2012; S. Y. Sun, Jiang, & Zhao, 2012).

1.1.2. Fermentation-derived aroma compounds

Fermentation is the process of transforming grape musts (freshly pressed fruit juice that contains the skins, seeds, and stems of the fruit) into wine by yeasts in the absence of oxygen (Conde, Silva, Fontes, Dias, Tavares, Sousa, et al., 2007). Fermentation-derived volatiles make up the largest percentage of total aroma composition. The formation of these compounds is variable and yeast strain specific (Lambrecht & Pretorius, 2000). Through sugar and amino acid metabolism, higher alcohols, esters, volatile fatty acids and compounds are produced during fermentation (Swiegers, Bartowsky, Henschke, & Pretorius, 2005) ( Figure 1.11 ).

Figure 1.11 Some of the major classes of aroma compounds (shown in blocks) produced by yeast during alcoholic fermentation (Styger, Prior, & Bauer, 2011) 18

1.1.2.1. Higher alcohols

Higher alcohols possess more than two carbon atoms with a higher molecular weight and boiling point than ethanol and have the characteristic pungent odor (Rapp & Mandery, 1986). Higher alcohols can be synthesized from intermediates of sugar metabolism through anabolic reactions, producing fatty acid- CoA precursors from pyruvate and acetyl-CoA via the tricarboxylic acid (TCA) cycle (Bell & Henschke, 2005; Crowell, Guymon, & Ingraham, 1961; Swiegers, Bartowsky, Henschke, & Pretorius, 2005). Alternatively, it can be synthesized from branched-chain amino acids (valine, leucine, isoleucine, threonine, and phenylalanine) through the Ehrlich reaction (S. J. Boulton & Jackson, 1996; Dickinson, Lanterman, Danner, Pearson, Sanz, Harrison, et al., 1997; Dickinson, Salgado, & Hewlins, 2003). The simple sketch of synthesis of higher alcohol is shown in Figure 1.11.

The branched-chain higher alcohols, including isoamyl alcohol and isobutyl alcohol, are synthesized from the branched-chain amino acids, leucine, and valine respectively (Figure 1.12). Isoamyl alcohol has whiskey, malt, and burnt odors and isobutyl alcohol has wine, solvent and bitter odors (Francis & Newton, 2005). Isoamyl alcohol is considered the important odorant in many wines, including Pinot noir, Merlot, and Cabernet Sauvignon wines (Fang & Qian, 2005; Gürbüz, Rouseff, & Rouseff, 2006; Kotseridis & Baumes, 2000). The aromatic amino acids, including phenylalanine and tyrosine, produce aromatic alcohols, such as 2-phenylethyl alcohol (Figure 1.12) (Rossouw & Bauer, 2009; Rossouw, Næs, & Bauer, 2008) which has a honey, dry rose and lilac aroma (Fang & Qian, 2005; Ferreira, Lopez, & Cacho, 2000; Francis & Newton, 2005a; Guth, 1997; López, 2003). Of aromatic higher alcohols, benzyl alcohol and 2- phenylethyl alcohol are described with positive characteristics such as floral, fruity, and dry rose in Pinot noir wines (Fang & Qian, 2005).

Amino acids together with ammonium constitute the yeast assimilable nitrogen (YAN) is important for yeast growth, successful fermentation, and the formation of wine higher alcohols (Bell & Henschke, 2005). Moreover, viticultural practices such as nitrogen fertilization, trellis style, and soil management techniques can influence grape amino acid 19

composition and concentration, subsequently impact higher alcohols in wine (Bell & Henschke, 2005; Schreiner, Scagel, & Lee, 2013).

Figure 1.12 A simplified metabolic map of yeast aroma compound production, indicating known metabolic linkages (Styger, Jacobson, & Bauer, 2011)

1.1.2.2. Esters

Volatile esters constitute one of the most important groups of aroma compounds and are largely associated with the fruity aromas in wine and other fermented beverages (Lilly, Lambrechts, & Pretorius, 2000). Although esters can be synthesized by enzyme-free equilibrium reaction between an alcohol and an acid at low pH, this manner of ester formation only accounts for a small part of esters in wine (Lambrecht & Pretorius, 2000). The majority of esters are produced through enzymatic reaction which is catalyzed by esterases, lipases and alcohol acetyltransferases (AATase) (Choi, Miguez, & Lee, 2004; Fenster, Parkin, & Steele, 2000, 2003a, 2003b; Gobbetti, Fox, & Stepaniak, 1997; Lilly, Lambrechts, & Pretorius, 2000; Liu, Holland, & Crow, 2004; Mason & Dufour, 2000; Nardi, FiezVandal, Tailliez, & Monnet, 2002). The acids are combined with coenzyme 20

A (CoA) to form acetyl-CoA or acyl-CoA at the presence of enzyme acyl-CoA synthetase and then react with the alcohol to form an ester (Lambrecht & Pretorius, 2000; Park, Shaffer, & Bennett, 2009). The biosynthetic pathway for ester synthesis catalyzed by AATase is illustrated in Figure 1.13.

Figure 1.13 AATase enzyme biosynthetic pathway for ester synthesis (Sumby, Grbin, & Jiranek, 2010).

Two important groups of esters in wine are ethyl esters and acetate. Ethyl esters (such as ethyl butanoate, ethyl hexanoate, ethyl octanoate) are formed from ethanolysis of acyl- CoA which is an intermediate metabolite of fatty acid metabolism (S.-J. Lee, Rathbone, Asimont, Adden, & Ebeler, 2004; Saerens, Delvaux, Verstrepen, Van Dijck, Thevelein, & Delvaux, 2008). Acetate esters (such as isoamyl acetate, propyl acetate, hexyl acetate, 2-phenylethyl acetate), are the result of the reaction of acetyl-CoA with alcohols that are formed from the degradation of amino acids and carbohydrates (S.-J. Lee, Rathbone, Asimont, Adden, & Ebeler, 2004; Saerens, Delvaux, Verstrepen, Van Dijck, Thevelein, & Delvaux, 2008). Ethyl acetate is recognized as the most common ester in wine because of its formation from ethanol and acetic acid (Ribéreau-Gayon, Glories, Maujean, & Dubourdieu, 2000). It is an important contributor to wine aroma and has a desirable and fruity character to the wine at concentrations below 8000 mg/L (Ribereau-Gayon, 1978). However, at higher concentrations, it can impart a solvent or nail varnish-like aroma (Bartowsky & Henschke, 2008).

The formation of esters is influenced by yeast strains and other external factors. As shown in Figure 1.11, the content of amino acids in grape must before fermentation could influence the concentration of esters. It is reported that isoamyl acetate, 2-phenylethyl 21

acetate, ethyl hexanoate, ethyl octanoate increased with additional amino acids in the grape juice before fermentation (Hernández-Orte, Ibarz, Cacho, & Ferreira, 2006). However, amino acid supplementation is not always positively correlated with higher ester concentrations in wine (Miller et al., 2007). Moreover, the fermentation temperature, nutrient availability, pH, unsaturated fatty acid levels, and oxygen levels also play an important part in determining the end levels of esters in a wine (S.-J. Lee, Rathbone, Asimont, Adden, & Ebeler, 2004; Quilter, Hurley, Lynch, & Murphy, 2003; Rojas, Gil, Piñaga, & Manzanares, 2003; Yoshimoto, Fukushige, Yonezawa, & Sone, 2002).

1.1.2.3. Volatile fatty acids

Fatty acids are essential constituents of the plasma membrane and precursors of more complex molecules, such as phospholipids (Demel & De Kruyff, 1976). They are synthesized through the repetitive condensation of acetyl-CoA, and catalyzed by the fatty acid synthetase complex (Lambrecht & Pretorius, 2000). Yeasts produce short-(<6 carbons), medium-(6 to 12 carbons), and long- chain fatty acids, with the short- and medium-chain fatty acids comprising the volatile fatty acids.

Acetic acid (C2) accounts for >90% of the volatile fatty and is formed from pyruvic acid as shown in Figure 1.11 (Bell & Henschke, 2005). Acetic acid interacted with ethyl acetate possesses a vinegar-like aroma (Pretorius & Lambrechts, 2000). Short- chain fatty acids including 2-methylpropanoic, 2-methylbutanoic, 3-methylbutanoic acids, butanoic and propanoic acids potentially contribute sweet, rancid and acid odors to wine flavor (Francis & Newton, 2005). It is reported that the short-chain fatty acids are potential important volatile compounds in Pinot noir wines, as reflected by their high flavor dilution values (FD) (Fang & Qian, 2005) with sweaty, cheesy-like aromas (Francis & Newton, 2005).

The medium-chain fatty acids, hexanoic (C6), octanoic (C8), and decanoic (C10) acid, also contribute to wine aroma (Francis & Newton, 2005), which are responsible for a fresh flavor in wine in low concentration but an unpleasant odors like sweaty or rancid at high levels (Lukić, Plavša, Sladonja, Radeka, & Peršurić, 2008). They can also modify the 22

perception of other taste sensations (Maujean, Dubourdieu, Ribérean-Gayon, & Glories, 2000). The concentrations of medium-chain fatty acids are dependent on anaerobic growth conditions, must composition, grape cultivar, yeast strain, fermentation temperature, and winemaking practices (Bardi, Cocito, & Marzona, 1999; Edwards, Beelman, Bartley, & McConnell, 1990). Moreover, medium-chain fatty acids are considered to responsible for sluggish fermentations, as they are inhibitory to S. cerevisiae and to some bacteria (Bisson, 1999). However, another study has suggested that cell growth is inhibited because fatty acid biosynthesis is prevented by the lack of oxygen that is one of the causes for sluggish fermentations (Bardi, Cocito, & Marzona, 1999).

1.1.2.4. Volatile sulfur compounds

Volatile sulfur compounds in wines have been extensively studied because of their negative effect on wine aroma (Mestres, Busto, & Guasch, 2000). Sulfur compounds originate from the degradation of sulfur-containing amino acids and the metabolism of some sulfur-containing pesticides (Mestres, Busto, & Guasch, 2000). The degradation process consists of enzymatic and non-enzymatic pathways (Mestres, Busto, & Guasch, 2000). The non-enzymatic pathways include photochemical, thermal and other chemical reactions of sulfur compounds during winemaking and storage (Mestres, Busto, & Guasch, 2000).

The most abundant volatile sulfur compounds in wines are hydrogen sulfide, methanethiol, dimethyl mercaptans, methyl thioesters and liberated glutathione and cysteine poly-functional (Dubourdieu & Tominaga, 2009; Roland, Schneider, Le Guernevé, Razungles, & Cavelier, 2010; Swiegers & Pretorius, 2007). Sulfur-containing compounds in wines have been extensively studied because of their effects on wine aroma (Mestres, Busto, & Guasch, 2000). In general, the aromatic contributions of these compounds are considered detrimental to wine quality, since the odor of these compounds can be characterized by cabbage, , or rubber odors (Mestres, Busto, & Guasch, 2000). Hydrogen sulfide is a very important sulfur compound with rotten egg aroma (Swiegers & Pretorius, 2007). The aroma of dimethyl sulfide was 23

described as asparagus, corn, and molasses. However, some believe sulfur compounds have a positive impact on the aroma and flavor of the wine. For example, compounds such as 3-mercaptohexanol can impart fruity flavors to a wine (Swiegers & Pretorius, 2007). In addition, these volatile sulfur compounds can enhance the fruity aromas at very low concentrations in wine (Escudero, Campo, Fariña, Cacho, & Ferreira, 2007; Segurel, Razungles, Riou, Salles, & Baumes, 2004).

Many parameters can influence the concentration of sulfur compounds in wine, such as deficiencies of nutrients, yeast strains, metal ions, and fermentation temperature (Franson, 2004) It has been found that the formation of hydrogen sulfide depends largely on the yeast strain and on the composition of the grape must (Butzke & Park, 2011; Winter, Henschke, Higgins, Ugliano, & Curtin, 2011). Aging process is also a very important factor that influences sulfur compounds in wine (J. He, Zhou, Peck, Soles, & Qian, 2013; Ugliano, Dieval, Siebert, Kwiatkowski, Aagaard, Vidal, et al., 2012). Dimethyl sulfide, methionol, diethyl sulfide, and diethyl disulfide increase in wine with aging and with increased temperature (Fedrizzi, Magno, Badocco, Nicolini, & Versini, 2007; MARA, 1979; Simpson, 1979). Ugliano also reported that hydrogen sulfide, methyl mercaptan, and dimethyl sulfide were found to increase during aging (Ugliano, et al., 2012). Lower postbottling oxygen exposure resulted in increased hydrogen sulfide and methyl mercaptan but dimethyl disulfide and dimethyl sulfide were not affected (Ugliano, et al., 2012). However, He et al. found that hydrogen sulfide, methanethiol, and thioacetates decreased during the aging process but the accumulation of dimethyl disulfide during storage was not obvious during storage for Pinot noir and Chardonnay wines. (J. He, Zhou, Peck, Soles, & Qian, 2013).

Non-volatile phenolic compounds in wine

Wine phenols include the non-flavonoids (hydroxycinnamates, hydroxybenzoates, and the stilbenes) and the flavonoids (anthocyanins, flavan-3-ols, and flavonols) (Waterhouse, 2002). The flavonoids comprise the majority of the phenols in and are derived from extraction of the skins and seeds of grapes during the fermentation process 24

(Waterhouse, 2002). They are all polyphenolic compounds with multiple aromatic rings possessing hydroxyl groups as shown in Figure 1.14.

Figure 1.14 The flavonoid ring system.

Flavonoid compounds, especially anthocyanins and flavan-3-ols, are major contributors to the intensity and stability of and wine astringency (Waterhouse, 2002). Anthocyanins provide the color in the skins of red or black grapes and in wines. The color is based on the fully conjugated 10 electron A–C ring π-system and is lost when the system is disrupted (Waterhouse, 2002). Anthocyanins are normally in the form of glycoside as shown in Fig. 1.15. Their corresponding flavonoid ring system is anthocyanidin which is unstable and only found in trace quantities in grapes or wines (Waterhouse, 2002). There are normally five main monomeric anthocyanins, including cyanidin-3-O-glucoside (Cy), peonidin-3-O-glucoside (Pn), delphinidin-3-O-glucoside (Dp), petudinidin-3-O-glucoside (Pt) and malvidin-3-O-glucoside (Mv) (Gomez-Miguez, Cacho, Ferreira, Vicario, & Heredia, 2007; Han, Zhang, Pan, Zheng, Chen, & Duan, 2008; Tang, Liu, Han, Xu, & Li, 2017; Waterhouse, 2002). The conditions of maceration, fermentation, and aging will affect the composition of wine anthocyanins while its total concentration and composition affect wine color (Mazza & Francis, 1995). For instance, wine aging causes intensive decrease of anthocyanins during the storage period and wines aged at higher temperature showes lower anthocyanin levels and less intense coloration (Ivanova, Vojnoski, & Stefova, 2012). In red wines, the anthocyanins not only provide appealing color, but also their higher concentration has been used as one of the indicators for high quality of berry and wine (Jensen, Demiray, Egebo, & Meyer, 2008; Vidal, Francis, Williams, Kwiatkowski, Gawel, Cheynier, et al., 2004). However, it is reported that high anthocyanin level in berries cannot ensure deep and stable color in wine and 25

tannins may be also essential for color and quality of wine (R. Boulton, 2001; Véronique Cheynier, Dueñas-Paton, Salas, Maury, Souquet, Sarni-Manchado, et al., 2006; Holt, Francis, Field, Herderich, & Iland, 2008; Pérez-Magariño & González-San José, 2006).

Figure 1.15 Anthocyanidin structures and conjugate forms (anthocyanins).

For Pinot noir, only non-acylated anthocyanins were detected from the grape berry skins (F. He, He, Pan, & Duan, 2010). The non-acylated anthocyanins are vulnerable to decoloring which means it can be hard to achieve and maintain good red color in Pinot noir wine (Carew, Sparrow, Curtin, Close, & Dambergs, 2014). Tannin is important to the mouthfeel, astringency and especially color stabilization of Pinot noir, but most tannin in Pinot noir fruit is in the seed that can be difficult to extract and can be bitter (Carew, Sparrow, Curtin, Close, & Dambergs, 2014). Apart from mentioned five anthocyanins, pelargonidin-3-O-glucoside was also found in Pinot noir wine, though it was only in trace amounts (F. He, He, Pan, & Duan, 2010).

Flavan-3-ols are the most abundant class of flavonoids in grapes and wines, and they are found in both the seed and skin of the grapes (Waterhouse, 2002). Catechin compromises the majority of monomeric flavan-3-ols (Cilliers & Singleton, 1990). The concentration of flavan-3-ols is influenced by many factors such as method, cultivar, pasteurization, and vintage (Fuleki & Ricardo-da-Silva, 2003). Flavonol is always found in a glycoside form and there are only three forms of the simple flavonoid aglycones in 26

grapes including quercetin, myricetin, and kaempferol. They are mostly shown as 3- glucosides or 3-glucuronides and small amounts of diglycosides (Véronique Cheynier & Rigaud, 1986). Since flavonols absorb UV light strongly at 360nm, and they appear mostly in the outermost layer of cells in the berry, indicating plant produces these compounds as a natural sunscreen. Meanwhile, it is reported that sunlight on the berry skin strongly enhanced the levels of the flavonols in Pinot noir grapes (Price, Breen, Valladao, & Watson, 1995).

Cluster thinning to reduce yields

It is perceived that grapes from low-yielding grapevines make higher quality wine. Recently, the cluster thinning practices to adjust yields receive increasing attention in the winery industry. The famous Burgundy, France Pinot noir production region has long controlled the grapevine yield to achieve wine quality (Pomerol, 1995). As a result, many other regions have adopted this practice in hopes of achieving quality of the famed wine regions of France. A study of the Oregon winegrape industry found that yields were reduced primarily to achieve quality fruit and wine (Uzes & Skinkis, 2016).

Reducing yields by way of cluster thinning is a viticultural management practice that is used to achieve grape yield and quality (Kliewer & Dokoozlian, 2005). The practice is conducted by removing the grape clusters from the grapevine to modify vine fruit to vegetative growth ratio (vine balance) (Kliewer & Dokoozlian, 2005). The intrinsic self- regulatory mechanism of the grapevines controls the vine balance between vegetative growth and fruit growth at a particular yield (McDonnell, Dry, Wample, & Bastian, 2008). Studies showed that if the factors that influence the crop load was changed, such as pruning levels, irrigation and microclimates, the grapevine will compensate for such changes due to yield compensation (Coombe & Dry, 1992; Freeman, Lee, & Turkington, 1979; Smart, Shaulis, & Lemon, 1982). Ough (1984) suggested that cluster thinning will benefit wine quality because of accelerated grape maturity (Ough & Nagaoka, 1984). It is reported that cluster thinning increased the berry weight, soluble solids, improved color and flavor at harvest (Bravdo, Hepner, Loinger, Cohen, & Tabacman, 1985; Bureau, Baumes, & Razungles, 2000; Gil, Esteruelas, González, Kontoudakis, Jiménez, Fort, et 27

al., 2013; Jackson & Lombard, 1993; Q. Sun, Sacks, Lerch, & Heuvel, 2011). Moreover, it is also reported that cluster thinning increased anthocyanin and polysaccharide contents in Syrah wine (Gil, et al., 2013). Guidoni has reported that cluster thinning treatment increased the concentrations of cyanidin-3-O-glucoside (Cy), peonidin-3-O-glucoside (Pn) and petudinidin-3-O-glucoside (Pt), but malvidin-3-O-glucoside (Mv) was not affected by cluster thinning treatments (Guidoni, Allara, & Schubert, 2002). For the volatile compounds, it was reported that cluster thinning decreased C6-alcohols in grape berries while no influence on the content of total C13-norisoprenoids in Syrah berries. (Bureau, Baumes, & Razungles, 2000). Mechanical thinning influenced wines’ aroma, taste, and mouthfeel of and varieties (Vitis vinifera L.) (Diago, Vilanova, Blanco, & Tardaguila, 2010). For Pinot noir, cluster thinning led to less yield per vine, but higher cluster weights, berries per cluster, and berry weights (Reynolds, Price, Wardle, & Watson, 1994). It also increased soluble solids, pH, and juice color (Reynolds, Price, Wardle, & Watson, 1994). Moreover, cluster thinning increased ethanol, color (anthocyanins), astringency and aroma of Pinot noir wine (Reynolds, Yerle, Watson, Price, & Wardle, 1996).

However, cluster thinning is not always able to accelerate ripening and improve fruit composition (Keller, Mills, Wample, & Spayd, 2005; Keller, Smithyman, & Mills, 2008; Ridomi, Pezza, Intrieri, & Silvestroni, 1995). For example, Keller et al. (2005) reported that despite a large yield drop, only a slight increase in soluble solid content but no effect on acidity and color parameters was observed. Some studies also reported that higher crop levels are not always responsible for lower quality wine (Chapman, Matthews, & Guinard, 2004; Kliewer, Freeman, & Hosssom, 1983; McCarthy, Cirami, & Furkaliev, 1987). For instance, Chapman et al. (2004) found the grape from higher yielding vines have positive effects on flavor and aroma of wines (Chapman, Matthews, & Guinard, 2004).

In addition, the timing of cluster thinning has an integral role in affecting the physiology of the grapevine (McDonnell, Dry, Wample, & Bastian, 2008). Generally, cluster thinning can be done at any time from pre-bloom through just prior to harvest. Vance and Skinkis (2013) suggested cluster thinning to be conducted at either lag phase (when 28

harvest yields can be more accurately estimated) or at véraison (to remove clusters that are visibly green and lagging in development). Jackson and Lombard (1993) also reported that lag-phase cluster thinning is appropriate in order to affect maturity (Jackson & Lombard, 1993). But Keller et al. found that the lag-phase cluster thinning showed no effect on vegetative growth, cluster yield components or advanced fruit maturity on Cabernet Sauvignon (Keller, Smithyman, & Mills, 2008). Moreover, other reports show different conclusions in the timing of cluster thinning. It was reported that pre-bloom cluster thinning were more effective to improve grape quality by elevating total soluble solids and color properties, but that is risky when berry set and cluster shape were unknown (Dokoozlian & Hirschfelt, 1995). Ough and Nagaoka (1984) reported the removal of one-third, two-thirds or none of the clusters two weeks after bloom had no effect on wine aroma intensity (Ough & Nagaoka, 1984). Chapman et al. (2004) showed cluster thinning only at early fruit development period influenced aroma and flavor of Cabernet Sauvignon (Chapman, Matthews, & Guinard, 2004). Whereas Reynolds et al. (2007) found that more delay in thinning time increased total soluble solids and monoterpene alcohols in Chardonnay Musqué (Reynolds, Schlosser, Sorokowsky, Roberts, Willwerth, & de Savigny, 2007).

Justification of research

Pinot noir wine has been increasing in popularity in the United States. Oregon’s Willamette Valley is considered one of the best places in the US for producing high- quality Pinot noir grapes and wine. Cluster thinning is a crop management that commonly used in Oregon vineyards (Uzes & Skinkis, 2016). Most commercial vineyard managers in this region currently remove ~25 to 40% of the vine’s total fruit each year (Uzes & Skinkis, 2016). Moreover, cluster thinning is one of the most expensive management practices in vineyards for Pinot noir produced in Oregon. It is necessary to investigate the most efficient cluster thinning practice, reducing vineyard costs without losing grape quality. Furthermore, it is very important to understand how grapes respond to cluster thinning in Oregon to optimize grape and wine quality. Owing to the inconclusive results of the impacts of cluster thinning on grape and wine composition, it is supposed that the 29

various responses of vine growth and berry development to cluster thinning may be due to the specific region and grape cultivar (Feng, 2014). On the other hand, it could also be due to that Pinot noir is generally a low-yielding cultivar. The cluster thinning may not alter source to sink relationships or to yield ratios enough to overcome ripening limitations (Reeve, Skinkis, Vance, McLaughlin, Tomasino, Lee, et al., 2018). Thus, studies for finding balance points of crop level that are suitable for individual site characteristics of Oregon vineyards are necessary to re-adjust current viticulture practices.

The overall hypothesis of this dissertation is that the grape compositions could be modified by altering cluster thinning, thus consequently affecting the important wine compositions. This hypothesis will be tested herein through determination of whether there exists a correlation between crop level and the quality-important compositions in Pinot noir wines.

30

Chapter 2 PHENOLIC COMPOSITION OF PINOT NOIR WINES PRODUCED FROM DIFFERENT CLUSTER THINNING LEVELS

Jingwen Li, Patricia A. Skinkis, and Michael C. Qian,

31

Abstract

Phenolic compounds are formed in the grape berry and affect bitterness, astringency, and red wine color. Crop level (yield), controlled by cluster thinning, can influence phenolic composition in grape berries and final wine quality. In this study, cluster thinning Pinot noir was investigated. Crop level was reduced by cluster thinning using a cluster per shoot regime (e.g. 2, 1 and 0.5 cluster/shoot) and compared to a full crop control (non- thinned), whereas two experimental sites used ton per hectare measurements (e.g. 1.31, 1.01 and 0.71 ton/hectare). Total phenol content (TP), total monomeric anthocyanins (TMA) and major phenolic compounds, including catechin, epicatechin, caffeoyltartaric acid and anthocyanins were quantified by UV-spectrometry and HPLC. Results showed no consistent influence of cluster thinning on phenolic compounds across six wineries (A, B, C, D, E and F). Cluster thinning increased TMA or TP in wines from Winery A, B, C, D and E in some vintages. The complicated impacts of cluster thinning practices on major phenolic compounds were dependent on specific wineries and vintage years. But cluster thinning had no impacts on the phenolic compounds for Winery F except the increase of Pt and Mv with cluster thinning in 2013 and 2014 vintage.

Keywords: Pinot noir wine, cluster thinning, TP, anthocyanins, UV-spectrometry, HPLC

Introduction

Vine balance is referred to as the balance between vegetative growth (canopy) and fruit yield. The goal is to maintain healthy canopy growth and achieve adequate fruit production with optimal fruit quality (Vance & Skinkis, 2013). Cluster thinning is a yield management practice that often used in Oregon vineyards to adjust crop level (yield) to achieve the quality goal (Uzes & Skinkis, 2016). Studies have shown cluster thinning practice affects the vine size (weight of cane prunings), yield, cluster weight, berries per cluster and berry weight (Bravdo, Hepner, Loinger, Cohen, & Tabacman, 1985; Bureau, Baumes, & Razungles, 2000; Gil, et al., 2013; Jackson & Lombard, 1993; Q. Sun, Sacks, Lerch, & Heuvel, 2011). The chemical prosperities, such as Brix, titratable acidity, anthocyanins, phenolics and monoterpenes were also impacted (Reynolds, 2009). 32

However, recent studies in Oregon Pinot noir have shown vine size or fruit composition changes to be due primarily to vineyard management practices other than cluster thinning (Reeve, Skinkis, Vance, Lee, & Tarara, 2016).

Wine contains many phenolic substances, most of which originate in the grape berries. The phenolics have a number of important functions in wine, affecting bitterness and astringency, especially in red wine (Waterhouse, 2002). Wine phenolics are grouped into two categories, the flavonoids and non-flavonoids (Waterhouse, 2002). The chromophoric nature of the flavonoid ring structure results in the absorption of light in both the ultraviolet and visible spectra, which protects plants from sunlight (Koes, Quattrocchio, & Mol, 1994; Shirley, 1996; Smith & Markham, 1998). Anthocyanins are a group of water-soluble flavonoids widely distributed in grapes and are responsible for wine color and can act as antioxidants (Pojer, Mattivi, Johnson, & Stockley, 2013). For Pinot noir, only non-acylated anthocyanins were detected from the grape berry skins (F. He, He, Pan, & Duan, 2010). The non-acylated anthocyanins are vulnerable to decoloring which means it can be hard to achieve and maintain good red color in Pinot noir wine (Carew, Sparrow, Curtin, Close, & Dambergs, 2014). Flavan-3-ols constitute the most abundant class and include simple monomeric catechins, oligomeric and polymeric proanthocyanidin forms in the grape skins and seeds (Cortell & Kennedy, 2006). These compounds contribute to the bitterness and astringency of wines (Kallithraka, Bakker, & Clifford, 1997). Flavonols are present in the berry skin as glycosylated forms of kaempferol, quercetin, myricetin, and isorhamnetin, which function as photoprotectant, and are increased by high sunlight exposure (Haselgrove, Botting, Heeswijck, Høj, Dry, Ford, et al., 2000; Price, Breen, Valladao, & Watson, 1995).

Wine phenolic compounds, especially anthocyanins, are key components for the wine quality (Jackson & Lombard, 1993; Mazza & Francis, 1995; Somers, 1971). Grape phenolic compounds can be modified by altering crop load, thus consequently affecting the wine phenolic compositions. Reynolds reported that cluster thinning increased anthocyanins and phenolics in wine (Reynolds, Yerle, Watson, Price, & Wardle, 1996). However, the results of cluster thinning on Oregon Pinot noir grape and wine 33

composition are inconclusive. It is supposed that the specific region and grape cultivar are responsible for the various responses of vine growth and berry development to cluster thinning. Moreover, cluster thinning is labor-intensive and comes at a high cost for Oregon Pinot noir producers. It is necessary to investigate the most efficient cluster thinning practice to reduce vineyard costs without losing grape quality. The goal of this study was to determine wine phenolic composition of Pinot noir wines produced from acluster thinning ptrial conducted across multiple vineyards over a three year period.

Materials and methods

2.3.1. Chemicals

Folin-Ciocalteu reagent, sodium carbonate, sodium acetate, potassium chloride and gallic acid were purchased from EM Science (Darmstadt, Germany), citric acid and tartaric acid were purchased from Avantor (Center Valley, PA). GC grade of methanol was obtained from EMD (Gibbstown, NJ) and ethanol was purchased from Aaper Alcohol and Chemical Co. (Shelbyville, KY). Formic acid was purchased from J.T.Baker (88%, Center Valley, PA). Milli-Q quality water was obtained from a Milli-Q purification system (Millipore, North Ryde, NSW, Australia).

2.3.2. Vineyard experimental design

From 2013 to 2015, a cluster thinning experiment was conducted in 12 commercial vineyards using protocols outlined by the Oregon State University Viticulture Program. All collaborators developed their vineyard experiment using the same randomized complete block design with whole row plots and at least three field replicates. The fruit yield was reduced by cluster thinning using two or more cluster per shoot thinning regime (e.g. 2, 1.5 and 1 cluster/shoot), compared to a full crop control (non-thinned). Two sites used yield per hectare measurements (e.g. 1.31, 1.01 and 0.71 ton/hectare). After harvest, fruit from field replicates were combined to produce one wine per crop level. Wines were produced to winery collaborator’s commercial standard but using the same method for all their treatment wines in the study. Wines were stored under temperature-controlled 34

conditions at each winery for two years until they were picked up by OSU and stored on campus under-temperature controlled conditions until analysis.

According to companies that produced the wine, the wine samples were sorted alphabetically and labeled as Winery A to Winery L. The cluster thinning treatments with yields and percent of clusters thinned (the percentage of clusters removed from the grapevines) from 2013 to 2015 vintage year is shown (Table 2.1). In this study, the results and discussion will be based on the wines from six wineries (Winery A to Winery F). The results for the other six wineries (Winery G to Winery L) that only provided wines for one or two years were shown in appendix.

Table 2.1 The information of wine companies and vineyards and cluster thinning treatments in the fields with yields (Kg/meter) and percent thinned in 2013 to 2015 vintage years.

Yield Year Treatment Percent Thinned Winery (cluster/shoot or ton/hectare) Kg/meter 1 cls 1.20 44.3% 2013 1.5 cls 1.44 21.5% No Thin 2.21 0.0% 1 cls 1.53 42.7% A 2014 1.5 cls 2.00 23.7% No Thin 2.65 0.0% 1 cls 2.37 45.4% 2015 1.5 cls 2.96 25.9% No Thin 3.57 0.0% 1 cls 0.82 28.4% 2013 No Thin 1.13 0.0% 1 cls 1.47 44.5% B 2014 No Thin 2.51 0.0% 1 cls 1.16 35.9% 2015 No Thin 1.75 0.0% 0.71 T 1.12 42.8% C 2013 1.01 T 1.19 28.3% 1.31T 1.51 15.2% 35

(Table 2.1 continued) 0.71 T 0.95 66.7% 2014 1.01 T 1.37 46.2% 1.31T 1.89 37.1% 0.71 T 1.13 46.6% 2015 1.01 T 1.34 20.8% 1.31T 1.68 18.1% 1 cls 1.16 37.9% 2013 1.5 cls 1.41 14.9% No Thin 1.54 0.0% 1 cls 1.20 35.3% D 2014 1.5 cls 1.68 18.6% No Thin 2.25 0.0% 1 cls 1.75 45.7% 2015 No Thin 4.01 0.0% 1 cls 0.51 36.0% 2013 1.5 cls 0.85 12.0% 2 cls 0.84 8.0% 1 cls 0.81 44.1% E 2014 2 cls 1.54 0.0% No Thin 1.19 0.0% 1 cls 0.94 41.6% 2015 No Thin 2.08 0.0% 0.81 T 1.77 26.9% 2013 1.22 T 1.96 0.0% 0.81 T 1.51 35.9% F 2014 1.22 T 2.35 4.0% 0.81 T 1.88 24.8% 2015 1.22 T 2.42 0.8% Cls: clusters per shoot. No Thin: no clusters were removed. T: ton/hectare 36

2.3.3. Determination of wine chemical composition

2.3.3.1. Total monomeric anthocyanin analysis

The spectrophotometric method based upon absorbance change under different pH was used to assay total monomeric anthocyanins (TMA) (Giusti & Wrolstad, 2005). Before analysis, wine sample was diluted 10 times with milli-Q water. An aliquot of 0.5 mL of the diluted sample was placed into two disposable cuvettes, diluted with either 2 mL of 0.4 M sodium acetate buffer (pH 4.5) or 2 mL of 0.025 M potassium chloride buffer (adjust to pH 1.0 with concentrated hydrochloric acid) and allowed to equilibrate for at least 15 min at room temperature. Optical absorbance was measured at both 520 nm and 700 nm. The concentration of monomeric anthocyanins (expressed as mg/L malvidin-3- glucoside equivalent) was calculated based on the absorbance difference of two pH reaction solutions. Assays were performed in triplicates.

TMA calculations should be made as follows: TMA (mg/L) = (A×MW×DF×1000)/(ε×p) Where:

A = (ABS6789: − ABS<889:)=> ?.8 − (ABS6789: − ABS<889:)=> A.6 MW: molecular weight of malvidin-3-glucoside (528.89g/mol) DF: dilution factor p: pathlength in cm ε : molar extinction coefficient for malvidin-3-glucoside

2.3.3.2. Total phenolic content analysis

Total phenolic content (TP) was determined using the Folin-Ciocalteu colorimetric method (V. L. Singleton, Orthofer, & Lamuela-Raventós, 1999). Briefly, wine samples were diluted 10 times with milli-Q water and 1 mL of aliquots of the diluted sample was added to a test tube containing 5 mL of diluted Folin-Ciocalteu (FC) Reagent (1:10 diluted with Milli-Q water). After mixing, solutions were allowed to react for 5 min before the addition of 4 mL of 7.5% sodium carbonate solution (w/v). The resultant 37

mixture was mixed homogeneously and then allowed to react for 2 hours at room temperature and in dark environment. The standard curve was prepared the same day using solutions of gallic acid (40, 80, 120, 160 and 200 ppm). The absorbance of samples was measured at 740nm using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The absorbance of samples was measured in a 1cm disposable cuvette against a reagent blank carried through the same procedure. Assays were performed in triplicates, and values were reported as mg/L gallic acid equivalents.

2.3.3.3. Major phenolic compounds analysis

An aliquot (1 mL) of wine sample was transferred into a 1.5 mL centrifuge tube and centrifuged in a microcentrifuge (Minispin plus, Eppendorf, Hamburg, Germany) at 11,000 rpm for 5 min. Twenty microliters of the supernatant were injected into HPLC system. The HPLC system was equipped with an Agilent 1100 series diode array detector (Palo Alto, CA). It consisted of a G1311A quaternary pump, a G1313A autosampler, G1316A thermostat column and G1315B photo-diode array detector, controlled by ChemStation software (Agilent, v.10.02). The separation was carried out on a Prodigy C18 column (100 Å, 5 µm, 250×4.6 mm, Phenomenex, Torrance, CA). The mobile phase consisted of two solvents: solvent A, 5% formic acid in milli-Q water; solvent B, 100% methanol (HPLC grade), with a flow rate of 1 mL/min. The following gradient was employed, 0-34 min (3-36% B); 34-45 min (36% B); 45-55 min (36-100% B); 55-60 min (100%-3% B); 60-70 min (3% B). The absorbance at 280 nm was used to measure the content of caffeoyltartaric acid and flavan-3-ols. The absorbance at 520 nm and 280 nm was used to measure the content of anthocyanins and flavonols, respectively. All compounds were identified through comparison with UV spectra and by comparing their retention times and UV spectra with previous data (Yuan, 2013). External calibration was performed using malvidin-3-glucoside. Anthocyanin compounds were quantified using this calibration curve and reported as malvidin-3-glucoside equivalent. 38

2.3.3.4. Statistical analysis

Mean values were calculated. Treatment differences were analyzed using analysis of variance (ANOVA) using SPSS version 20 (IBM, Chicago, IL). Statistical differences between two treatment levels were identified using Student t-test at the p<0.05 level. Statistical differences among three or more than three treatment levels were identified using Tukey’s HSD at the p<0.05 level.

Results

In this study, total phenols (TP) and total monomeric anthocyanins (TMA), and major phenolic compounds in Pinot noir wines were measured from six wineries (Winery A, B, C, D, E and F) from 2013 to 2015 vintage.

2.4.1. Wine total phenols and anthocyanins

TP and TMA contents in Pinot noir wines were shown in Table 2.2. A significant increase (p<0.05) of TP with cluster thinning treatments was observed in wines from most wineries (A, B, C, D and E) in some vintage, like Winery A in 2014 vintage, Winery B in 2013 vintage, Winery C in 2013 vintage while cluster thinning had no impacts on TP in other vintages for these wineries. Cluster thinning treatments had no impacts on TP in the wines from Winery F.

Cluster thinning treatments had no significant influence on TMA in Pinot noir wines from Winery B, C and F for all three vintages. For Winery A, 1 cls treatment resulted in the lowest (p<0.05) TMA in 2105 vintage. For Winery D, TMA content was significantly higher (p<0.05) in 1 cls treatment in 2014 and 2015 vintages. For Winery E, 1 cls treatment resulted in a higher (p<0.05) TMA in 2013 vintage but lower (p<0.05) TMA in 2014 vintage.

2.4.2. Wine major phenolic compounds

In this study, major phenolics in Pinot noir wines were measured to understand the effect of cluster thinning practices on wine phenolic composition. The contents of major 39

phenolic compounds in Pinot noir wines were shown in Table 2.3. Previous studies showed that five anthocyanins (Dp, Cy, Pt, Pn, and Mv) were normally identified in Pinot noir grapes and wines (Carew, Sparrow, Curtin, Close, & Dambergs, 2014; F. He, He, Pan, & Duan, 2010). However, only four anthocyanins (Dp, Pt, Pn, and Mv) were detected while Pt and Dp could only be detected in some wine samples. Quercetin glycosides could not be identified in our study.

Results showed that there was no consistent difference in major phenolic compounds among treatments, although some variations were observed. Divergent impacts of cluster thinning on major phenolic compounds were observed in different vintages. For instance, in Winery A, cluster thinning significantly decreased (p<0.05) caffeoyltartaric acid in 2013 vintage but increased (p<0.05) it in both 2014 and 2015 vintage. For Winery D, cluster thinning practice significantly increased (p<0.05) caffeoyltartaric acid in 2013 vintage but decreased (p<0.05) it in 2014 vintage, while no difference was observed in 2015 vintage. Moreover, complex impacts of cluster thinning on major phenolic compounds were also observed in different wineries. Cluster thinning significantly increased (p<0.05) Pn and Pt in wines from Winery A but significantly decreased (p<0.05) Pn and Pt in wines from Winery B in 2013 vintage. Thus, the influence of cluster thinning practices was various with vintages and wineries. On the contrary, cluster thinning practice had limited influence on wine major phenolic compounds for Winery F. Only increases (p<0.05) of Pt in 2104 vintage and Mv in 2013 vintage were observed for all three vintages in Winery F.

Discussion

2.5.1. Wine total phenols and anthocyanins

Several studies have shown that low crop level can increase anthocyanins and total phenolics of the fruit and wine of , , Merlot, and Pinot noir (Guidoni, Allara, & Schubert, 2002; Mazza & Francis, 1995; Reynolds, Yerle, Watson, Price, & Wardle, 1996). Prajitna et al. also reported that cluster thinning increased linearly the polyphenolic composition of Chambourcin wines as indicated by increases in 40

total anthocyanins and total phenolics (Prajitna, Dami, Steiner, Ferree, Scheerens, & Schwartz, 2007). In our study, cluster thinning practices increased TMA or TP in wines from Winery A, B, C, D and E in some vintages, which is in agreement with previous studies. Kennedy reported that a positive relationship between grape berry development and anthocyanin concentration has been found (Kennedy, Matthews, & Waterhouse, 2002). Cluster thinning may increase polyphenols indirectly by advancing fruit maturity or directly by increasing the substrate levels necessary for polyphenol synthesis. The impacts of cluster thinning practices on TP and TMA content were also various in different vintages might be due to year differences (e.g. weather, temperature and sunlight). There was no consistent difference observed across the wineries. The location (e.g. viticultural practices, terroir and microclimate) of each winery had a general effect on Pinot noir wine which could be related to composition changes (Jackson & Lombard, 1993; Ough & Nagaoka, 1984).

2.5.2. Wine major phenolic compounds

Caffeoyltartaric acid (hydroxycinnamic acid esters) as one of the major phenols of white grape juices is the best substrates for grape polyphenol oxidase (Veronique Cheynier & Ricardo da Silva, 1991). It was significantly increased by 0.71 T and 1.01 T treatments in Winery C while not influenced by cluster thinning practices in Winery E and F for all three years. Catechin and epicatechin are responsible for the bitterness of wines (v. Singleton, 1974). They are influenced by cluster thinning in different ways. Zhao et al. also found that the effect of cluster thinning on catechins (catechin, epicatechin and epigallocatechin) concentration in Cabernet Sauvignon (Vitis vinifera L.) was complex and involved many factors (X. Zhao, Wang, Liu, Li, Sun, & Shu, 2006). But Peña-Neira reported that the cluster thinning resulted in higher concentrations of catechin in berry skins of Syrah (Peña-Neira, Cáceres, & Pastenes, 2007).

As mentioned before, some studies reported that cluster thinning can increase anthocyanins of the fruit and wine of Pinot noir. Peña-Neira reported that the cluster thinning treatments resulted in higher concentrations of the phenolic compounds including Mv, Cy and catechin in Syrah (Peña-Neira, Cáceres, & Pastenes, 2007). 41

Guidoni reported that cluster thinning treatment increased the concentrations of Cy, Pn, and Pt, but Mv was not affected (Guidoni, Allara, & Schubert, 2002). However, our results were not in agreement with these earlier studies, as cluster thinning can positively or negatively influence each anthocyanin, depending on vintage and winery.

Major phenolic compounds differed by winery and vintage and was not consistent for any cluster thinning level. The 2013 to 2015 vintages varied by climate (e.g. weather, temperature and sunlight) and base crop levels with 2013 being a wet harvest with low base yields and 2014 and 2015 being a warm dry season with a warm harvest and high yields. Hydroxycinnamic acid composition in noir grapes (Vitis vinifera L.) was related to the average maximum temperatures of the warmest month (MATWM). Sunlight exposure has a positive impact on the synthesis of anthocyanins in Pinot noir grapes (J. Lee & Skinkis, 2013; Song, Smart, Wang, Dambergs, Sparrow, & Qian, 2015). Moreover, the location (e.g. viticultural practices, terroir and microclimate) of each winery also played a role in the effects of cluster thinning practices on wine phenolics (Ough & Nagaoka, 1984).

Conclusions

The results of the six wineries (Winery A, B, C, D, E and F) that provided the wine samples from 2013 to 2015 vintage were discussed in this chapter. Cluster thinning practices increased TMA or TP in wines from Winery A, B, C, D and E in some vintages. However, the positive influence was dependent on vintage and wineries. Cluster thinning practice had no impacts on phenolic compounds for Winery F from 2013 to 2015 vintage except the increase (p<0.05) of Pt and Mv with cluster thinning in 2014 and 2013 vintage. Cluster thinning impacts on major phenolic compounds were also various with vintages and wineries. There were no consistent impacts of cluster thinning practices on wine phenolics across the vintages and wineries, which had greater effects on wine phenolics than cluster thinning levels. 42

Table 2.2 Total phenols and total monomeric anthocyanins in Pinot noir wine with different cluster thinning treatments from 2013 to 2015 vintage

Winery Year Treatments Total Phenolsa Total Monomeric Anthocyaninsb 1 cls 5950±297 7.25±0.49 2013 1.5 cls 5959±73 6.08±1.19 No Thin 5869±212 4.40±2.85 1 cls 4673±127 a 9.93±2.76 A 2014 1.5 cls 4766±219 a 9.56±2.08 No Thin 4180±132 b 13.1±1.8 1 cls 6264±518 10.9±0.1 a 2015 1.5 cls 6722±356 16.9±0.6 b No Thin 6542±603 16.1±0.4 b 1 cls 6984±128 a 16.6±2.6 2013 No Thin 5844±367 b 15.2±1.6 1 cls 5750±164 19.3±3.6 B 2014 No Thin 6096±212 19.2±4.9 1 cls 8651±482 17.9±0.0 2015 No Thin 8479±563 21.4±3.9 0.71 T 5293±122 a 7.30±1.55 2013 1.01 T 5710±79 b 9.80±1.70 1.31T 5402±124 a 7.76±0.85 0.71 T 7875±237 13.6±3.3 C 2014 1.01 T 7168±167 15.2±4.5 1.31T 7713±240 9.46±3.35 0.71 T 8258±229 21.0±7.0 2015 1.01 T 8028±629 24.4±2.2 1.31T 7744±579 26.9±0.8 1 cls 5601±110 7.55±1.35 2013 1.5 cls 5847±51 8.43±0.89 No Thin 5679±226 5.40±2.08 1 cls 7349±249 a 7349±249 a D 2014 1.5 cls 6165±234 b 6165±234 b No Thin 6726±191 b 6726±191 b 1 cls 5140±332 a 22.4±1.1 a 2015 No Thin 4059±301 b 14.4±2.2 b E 2013 1 cls 5430±155 10.5±1.6 a 43

(Table 2.2 continued) 1.5 cls 5314±108 5.17±1.46 b 2 cls 5492±96 9.55±0.50 a 1 cls 4763±205 a 7.30±1.84 a 2014 2 cls 5680±139 b 11.9±2.5 b No Thin 4878±375 a 11.7±0.6 b 1 cls 5187±253 17.7±4.1 2015 No Thin 4744±294 11.7±1.5 0.81 T 4423±187 11.6±1.4 2013 1.22 T 4489±150 10.9±0.1 0.81 T 5280±353 19.8±0.7 F 2014 1.22 T 5408±277 18.2±2.4 0.81 T 5355±503 23.3±3.8 2015 1.22 T 5180±390 25.9±1.7 Mean ± SD presented (n=2). Different letters indicating differences in means (Tukey HSD and student t-test at α=0.05). estate: unknown; aas peak area; bas mg/kg malvidin-3-monoglucoside equivalent.

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Table 2.3 Phenolic composition in Pinot noir wine with different cluster thinning treatments from 2013 to 2015 vintage Hydroxycinnamic Flavan-3-olsa Anthocyaninsb acid estersa Delphinidin-3- Petunidin-3- Peonidin-3- Malvidin-3- Winery Year Treatments Caffeoyltartaric acid Catechin Epicatechin monoglucoside monoglucoside monoglucoside monoglucoside 1 cls 472±11 a 1102±2 a 445±28 nd 2.0±0.1 2.2±0.1 a 5.3±0.2 a 2013 1.5 cls 526±11 b 1020±50 a 421±9 nd nd 2.0±0.0 b 3.7±0.1 b No Thin 604±14 c 1245±1 b 460±3 nd nd 2.0±0.0 b 3.6±0.1 b 1 cls 1325±1 a 849±5 a 488±23 nd 2.0±0.0 a 2.6±0.1 9.1±0.7 A 2014 1.5 cls 1302±118 a 838±52 ab 602±34 nd 1.9±0.0 b 2.2±0.1 7.4±1.8 No Thin 1008±10 b 690±38 b 619±36 nd 1.9±0.0 b 2.4±0.1 8.7±1.6 1 cls 1347±45 a 876±4 a 456±0 a nd 2.4±0.3 3.3±0.4 10.4±0.9 a 2015 1.5 cls 1708±9 b 941±5 b 526±4 b nd 2.6±0.1 3.5±0.4 14.9±1.0 b No Thin 1532±23 b 897±2 a 545±1 b nd 2.1±0.2 3.3±0.2 11.4±0.7 b 1 cls 2112±356 687±7 350±3 2.0±0.0 2.3±0.2 a 2.0±0.0 a 7.6±0.0 a 2013 No Thin 1508±44 679±8 335±7 2.2±0.1 3.7±0.5 b 3.4±0.4 b 10.5±0.5 b 1 cls 1747±10 a 839±2 a 489±20 2.2±0.1 3.0±0.1 3.1±0.0 a 18.1±0.2 a B 2014 No Thin 2033±44 b 1000±49 b 513±42 2.3±0.1 3.4±0.6 3.4±0.0 b 21.3±0.1 b 1 cls 2987±36 1280±0 387±4 3.0±0.1 3.5±0.3 4.4±0.9 23.1±3.8 2015 No Thin 3174±43 1315±6 332±12 3.1±0.1 4.5±0.6 5.4±0.2 25.3±0.6 0.71 T 1667±42 a 890±133 419±11 a 1.9±0.1 1.9±0.0 ab 2.2±0.0 5.3±0.2 2013 1.01 T 1246±6 b 1024±7 375±25 b nd 2.1±2.0 a 2.1±2.1 5.4±0.1 1.31T 514±2 c 1044±20 565±19 c nd 1.8 ±0.2 b 2.2±0.0 5.1±0.5 C 0.71 T 909±32 a 1843±25 a 464±22 2.0±0.0 a 2.3±0.0 2.4±0.0 8.7±0.2 a 2014 1.01 T 1251±48 b 1485±21 b 461±15 2.1±0.0 b 2.6±0.1 2.7±0.4 12.1±1.2 b 1.31T 880±14 a 1655±49 c 435±11 1.9±0.0 a 2.2±0.1 2.3±0.0 6.4±0.3 c 45

(Table 2.3 continued) 0.71 T 970±37 a 1514±0 364±7 a 2.8±0.1 a 3.5±0.1 a 4.1±0.3 a 20.7±0.1 a 2015 1.01 T 559±5 b 1512±0 303±0 b 3.1±0.1 ab 3.7±0.1 a 4.1±0.3 a 23.3±1.2 a 1.31T 524±11 b 1533±5 290±1 b 3.4±0.2 b 5.3±0.3 b 4.8±0.1 b 31.3±1.0 b 1 cls 536±11 a 426±92 754±34 nd nd nd 3.2±0.0 a 2013 1.5 cls 834±19 b 408±109 662±11 nd nd 1.8±1.3 2.9±0.2 b No Thin 690±3 c 436±11 666±36 nd nd 1.9±0.0 2.7±0.0 b 1 cls 1486±2 a 1036±3 a 648±3 nd nd 2.0±0.0 5.9±0.9 D 2014 1.5 cls 947±14 b 730±2 b 655±24 nd nd 1.9±0.0 4.2±0.0 No Thin 2119±67 c 942±25 c 607±33 nd nd 1.9±0.1 4.7±0.5 1 cls 1157±159 300±19 a 506±12 2.6±1.5 3.1±1.8 3.7±1.0 22.6±5.3 2015 No Thin 1638±241 506±45 b 466±12 2.4±1.4 3.7±1.3 5.4±2.0 27.3±6.1 1 cls 1030±306 423±13 760±36 nd nd 1.8±0.0 3.0±0.0 2013 1.5 cls 1025±26 428±6 735±54 nd nd 1.7±0.0 2.8±0.2 2 cls 811±190 440±8 693±45 nd nd 1.8±0.0 2.8±0.1 1 cls 947±29 832±27 703±12 ab nd nd 2.0±0.0 a 6.0±0.0 a E 2014 2 cls 864±27 976±2 730±19 a nd nd 2.1±0.0 a 6.1±0.0 b No Thin 949±3 839±87 654±0 b nd nd 2.3±0.1 b 8.2±0.0 c 1 cls 520±4 a 562±5 a 574±0 a 2.1±0.0 2.6±0.1 3.4±0.1 16.7±0.5 a 2015 No Thin 1242±41 b 452±4 b 465±4 b 2.0±0.1 2.7±0.4 3.3±0.6 11.2±0.7 b 0.81 T 378±3 710±4 436±27 1.9±0.0 2.3±0.0 2.2±0.0 10.1±0.1 a 2013 1.22 T 383±9 663±64 400±27 1.9±0.1 2.2±0.0 2.1±0.0 8.2±0.1 b 0.81 T 1448±65 861±29 596±1 2.1±0.0 3.2±0.0 a 3.0±0.0 24.3±0.8 F 2014 1.22 T 1245±19 939±3 651±27 2.1±0.0 2.9±0.1 b 2.9±0.1 24.3±0.1 0.81 T 1617±286 772±47 503±8 2.4±0.0 3.3±0.4 3.2±0.5 24.1±2.2 2015 1.22 T 1390±173 620±189 494±15 2.3±0.0 3.5±0.1 3.9±0.1 28.8±0.7

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(Table 2.3 continued) Mean ± SD presented (n=2). nd: not detected. Different letters indicating differences in means (Tukey HSD and student t-test at α=0.05). estate: unknown; aas peak area; bas mg/kg malvidin-3-monoglucoside equivalent.

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Chapter 3 VOLATILE COMPOSITION OF PINOT NOIR WINES PRODUCED FROM DIFFERENT CLUSTER THINNING LEVELS

Jingwen Li, Patricia A. Skinkis, and Michael C. Qian,

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Abstract

To determine the impacts of crop level on Pinot noir wine quality, a cluster thinning experiment was conducted in Oregon wineries over a 3-year period (2013-2015). Crop level was reduced by cluster thinning at the lag phase of berry development using a cluster per shoot regime (e.g. 2, 1 and 0.5 cluster/shoot) and compared to a full crop load control (non- thinned), whereas two experimental sites used ton per hectare measurements (e.g. 1.31, 1.01 and 0.71 ton/hectare). The volatile composition of Pinot noir wines with different crop levels were analyzed by GC-MS and GC-FID in this study. The results showed that certain wine volatiles were affected by the cluster thinning treatments. However, specific volatile compounds varied with wineries and vintages more than crop level. Low thinning (1.5 cls or non-thinned treatments) in Winery A but 1 cls treatment for Winery E resulted in significantly higher free-from or total β-damascenone. Cluster thinning introduced significantly lower 2- phenylethyl alcohol in wines from Winery A, C, D and F, although the influences were varied with vintages. It was also found that cluster thinning practices had limited influence on the wine volatile compositions for Winery B across all three years.

Keywords: Cluster thinning, volatile composition, GC-MS, GC-FID, odor activity value

Introduction

Cluster thinning is a vineyard management practice that removes grape clusters from the grapevine to achieve target yields assumed to allow for vine balance or achieve quality. It is commonly used in vineyards to adjust crop level and achieve desirable vine balance (Kliewer & Dokoozlian, 2005). It was reported that cluster thinning practices improves yield, fruit composition, vine size, bud hardiness, and wine quality (Kok, 2011; Stergios & Howell, 1977). However, some studies reported that cluster thinning practices (or the higher yields) are not always responsible for lower quality wine (Chapman, Matthews, & Guinard, 2004; Kliewer, Freeman, & Hosssom, 1983; McCarthy, Cirami, & Furkaliev, 1987). For instance, Chapman et al. (2004) found the grape from higher yielding vines have positive effects on flavor and aroma of wines (Chapman, Matthews, & Guinard, 2004). Cluster thinning increased soluble solids, pH, and juice color (Reynolds, Price, Wardle, & Watson, 1994). It also increased

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ethanol, color (anthocyanins), astringency and aroma of Pinot noir wine (Reynolds, Yerle, Watson, Price, & Wardle, 1996). However, Vance found that crop thinning reduced yields but had no effect on Pinot noir berry weight or cluster size in 2010 and 2011 studying seasons (Vance, 2012). Reeve et al. reported that cluster thinning had limited impact on vegetative growth but increased total soluble solids and pH, in two of three studying seasons (Reeve, Skinkis, Vance, Lee, & Tarara, 2016). Reeve et al. also proposed that cluster thinning may not alter source to sink relationships or canopy to yield ratios enough to overcome ripening limitations in Oregon (Reeve, Skinkis, Vance, McLaughlin, Tomasino, Lee, et al., 2018).

Aroma is one of the most important attributes of wine quality. Wine aroma can be classified by their origins into three groups: grape-derived aroma compounds, fermentation-derived aroma compounds, and aging-derived aroma compounds (Rapp & Mandery, 1986; Robinson, Boss, Solomon, Trengove, Heymann, & Ebeler, 2014). The amount of the aroma compounds can be influenced by various environmental factors such as climate and soil type, cultivar, the conditions of the fruit, the conditions during the fermentation stage and different post- fermentation treatments (Rapp & Mandery, 1986). Cluster thinning can influence the synthesis of primary and secondary metabolites in grapes such terpenoids, C13-norisoprenoids,

C6 compounds and methoxypyrazines (MendezCostabel, et al., 2014; Romero, Gil-Muñoz, del Amor, Valdés, Fernández, & Martinez-Cutillas, 2013; Song, Shellie, Wang, & Qian, 2012),

In Oregon vineyards, cluster thinning is conducted annually to achieve a narrow target yield range and is conducted manually at a considerable expense to the grower. It is important to know how cluster thinning influences wine quality. Most studies conducted in Oregon to date have focused on vine growth and fruit composition effects. Results of these cluster thinning studies show limited benefit to fruit ripening or quality (Reeve, Skinkis, Vance, Lee, & Tarara, 2016; Reeve, et al., 2018) compared to other practices being used, and a considerable amount of work is in progress on yield management (Skinkis, in progress). This work investigates the wine composition impacts of cluster thinning to determine if there are appreciable wine quality benefits. The objective of this study is to determine the volatile aroma compounds present in Pinot noir wines produce with different cluster thinning levels.

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Materials and methods

3.3.1. Chemicals

All chemical standards used for identification and quantitation in this study was of analytical reagent grade unless otherwise stated. Ethyl acetate (99%), ethyl butanoate (98%), ethyl hexanoate (98%), ethyl octanoate (98%), ethyl decanoate (98%), hexyl acetate (

98%), octyl acetate ( 99%), octyl butyrate ( 98%), diethyl succinate ( 99%), 2- phenylethyl acetate (98%), ethyl phenylacetate (98%), isoamyl acetate (99%), isobutyl acetate (98%), ethyl 2-methylpropanoate (98%), ethyl 2-methylbutanoate (98%), ethyl

3-methylbutanoate (98%), acetaldehyde (99%), propanol (99%), isobutanol (99%), isoamyl alcohol (98%), methyl propionate (99+%),1-hexanol (99%),2-ethyl-1-hexanol (≥96%), 1- octanol (≥99.5%), 1-octen-3-ol (98%), benzyl alcohol (99.8%), benzeneethanol (99%), linalool (97%), nerol (98%), geraniol (98%), α-terpineol (90%), β-citronellol (95%), β- damascenone (≥90%), β-ionone (95%), hexanoic acid (99%), octanoic acid (≥98%), decanoic acid (≥98.0%), 3-methylphenol (99%) and 4-methylphenol (99%) were purchased from Sigma-Aldrich (St. Louis, MO). Eugenol (98%), guaiacol (≥98%), 2-methylphenol (99%), 3- ethylphenol (95%), 4-ethylphenol (98%), 4-ethylguaiacol (≥98%), 4-methylguaiacol (99%), ethyl thioacetate (≥98%), dimethyl sulfide (99%), dimethyl disulfide (≥98%), dimethyl trisulfide (≥98%), diisopropyl disulfide (≥98%) and ethyl methyl sulfide (96%) were purchased from TCI America (Cambridge, MA). GC grade of methanol was obtained from EMD (Gibbstown, NJ) and ethanol was purchased from Aaper Alcohol and Chemical Co.

(Shelbyville, KY). Isotope compounds: ethyl butanoate-4,4,4-d3 (99.8%), ethyl hexanoate-d11

(98.7%), ethyl octanoate-d15 (98.5%), hexanoic-d11 acid (98.5%), decanoic-d19 acid (98.6%), octanoic-d15 acid (99.2%), (±)-linalool-d3(vinyl-d3) (99.2%), 2-phenyl-d5-ethan-1,1,2,2-d4-ol

98.5%), ethyl (±)-2-methylbutanoate-d9 (99.1%) were purchased from CDN Isotopes Inc.

(Canada). Ethyl decanoate-d19 was synthesized by our lab. A synthetic wine solution was made by dissolving 3.5 g of L-tartaric acid in 1 L of 12% ethanol solution and adjusting pH to 3.5 with 1 M NaOH. Milli-Q quality water was obtained from a Milli-Q purification system

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(Millipore, North Ryde, NSW, Australia). Standard stock solutions were prepared in methanol individually.

3.3.2. Quantitative analysis of wine volatile compounds

3.3.2.1. Highly volatile compound analysis

Headspace (HS)-GC-FID method was used for analyzing acetaldehyde, ethyl acetate, isoamyl acetate, propanol, isobutyl, and isoamyl alcohol. One mL of wine was pipetted into a 20 mL auto-sampler vial and 20µL of the internal standard (2.5 mg/mL of methyl propionate) was added to the sample. Then the vial was tightly capped with Teflon-faced silicone septa. The sample was incubated at 50 °C for 15 min with 500 rpm agitation to reach the equilibrium between the sample and headspace. The extraction and injection were conducted by an autosampler (CTC Analytics, Inc., Zwingen, Switzerland) that equipped with a 2.5 mL syringe. The syringe temperature was kept at 70 °C. An aliquot (1000µL) of headspace gas was taken by syringe at a rate of 100µL/S and then injected into the GC injection port in split mode 10:1.

The analysis of the extracted volatile compounds was carried out by using a Varian CP 3800 gas chromatograph equipped with a flame ionization detector (Varian, Inc., Palo Alto, CA). Separation was performed by a DB-wax capillary column (30 m × 0.25 mm i.d., 0.5m film thickness, Agilent Technologies, Inc., Santa Clara, CA). The carrier gas was nitrogen at 2 mL/min. The initial oven temperature was 35°C, and held for 4 minutes, then increased to 150°C at a rate of 10°C /min, finally held for 5 minutes. The inlet temperature was 250 °C and the FID temperature was 250 °C. Identifications were conducted by comparing retention time with authentic pure standards. All analyses were carried out in triplicates.

The standard calibration curve was prepared by spiking known amount of pure chemical standards into 1 mL of 5% ethanol solution in a 20 mL of autosampler vial, and then follow the same procedures as described above.

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3.3.2.2. Major volatile compound analysis

Solid phase microextraction (SPME)-GC-MS method was used to quantitate major volatile compounds in wine. Two mL of wine was diluted with 8 mL of saturated sodium chloride citric buffer (0.25 g/L, pH 3.5) in a 20mL auto-sampler vial with a 1cm Teflon stir bar. Then

10µL of isotope internal standard mixture (ethyl octanoate-d15, 50 mg/L; ethyl butanoate-

4,4,4-d3, 500 mg/L; ethyl (±)-2-methylbutyrate-d9, 200mg/L; 2-phenyl-d5-ethan-1,1,2,2-d4-ol,

1500 mg/L; ethyl decanoate-d19, 50 mg/L; (±)-linallol-d3(vinyl-d3), 100 mg/L; a-terpineol-d3,

200 mg/L; hexanoic-d11 acid, 1000 mg/L; octanoic-d15 acid, 500 mg/L; decanoic-d19 acid 200 mg/L) was added into the diluted sample. The sample vials were tightly capped with Teflon- faced silicone septa. The volatiles in headspace were extracted by a preconditioned 2cm- 50/30µm DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA). The extraction and injection were conducted by autosampler (Gerstel, Linthicum, MD). Samples were first equilibrated at 50 °C in a thermostatic bath for 30 min and then extracted by SPME fiber for 30 min at the same temperature with stirring (500 rpm). After extraction, the fiber was inserted into the GC injection port in 250 to desorb the analytes for 5 minutes in a splitless mode.

The analysis was performed on an Agilent 6890 gas chromatograph equipped with an Agilent 5973 mass selective detector (Agilent Technologies). Compound separation was achieved using a ZB-WAX-PLUS column (30 m × 0.25 mm i.d., 0.5 µm film thickness, Phenomenex, Torrance, CA). The column initial temperature was 40 °C, held for 4 min and then raised to 210°C at 4 °C/min, and held at 210 °C for 10 min. A constant helium column flow rate of 1.5 mL/min was used. MS transfer line and ion source temperature were 280 and 230 °C, respectively. Electron ionization mass spectrometric data from m/z 40~350 were collected using a scan mode with an ionization voltage of 70 eV. Compound quantification was achieved by selective mass ions via Chemstation (Agilent Technologies) software, and the SIMs of compounds were listed in Table 3.1.

Standard calibration curve was prepared by spiking known amount of pure chemical standards into 2 mL of synthetic wine (3.5g/L tartaric acid, ethanol 12% V/V, pH 3.5) diluted with 8 mL of saturated sodium chloride citric buffer in a 20 mL of autosampler vial, and then follow the

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same procedures as described previously. Results were calculated through Chemstation software (Agilent Technologies).

3.3.2.3. C13-norisoprenoids potential

C13-norisoprenoids exist in wines as both free and bound forms or precursors. The bound form and the precursors are aroma potentials that can be released during aging. Since acid hydrolysis could accelerate this aroma releasing process, it can be used to evaluate the C13- norisoprenoid potential of the wine. Two mL of the wine sample was diluted with 8 mL of saturated sodium chloride citric buffer (0.2 M, pH 2.5) and a 1cm Teflon stir bar in a 20ml autosampler vial. Ten µL of internal standard (2-phenyl-d5-ethan-1,1,2,2-d4-ol, 1500 mg/L) was added in and then the vial was tightly capped with baked Teflon-faced silicone septa. The sample vials then were incubated in a 99°C water bath for 1 hour. After incubation, the vial was cooled to room temperature for analysis. The samples analyzed using the same SPME- GC-MS procedure as free-form major volatile compound analysis. All analyses were conducted in triplicates.

3.3.2.4. Volatile sulfur compound analysis

A solid-phase microextraction and gas chromatography-pulsed flame photometric detection technique (SPME-GC-PFPD) was used to quantify volatile sulfur compounds in Pinot noir wines. An aliquot (1 mL) of wine was diluted with 9 mL of saturated sodium chloride water with 2% ethanol in a 20 mL auto-sampler glass vial, in which 20µL of internal standard solution (0.5 mg/L of ethyl methyl sulfide and 0.004 mg/L of diisopropyl disulfide) was added.

The extraction and injection were conducted by a Combipal autosampler (CTC Analytics, Inc.,) that equipped with a Carboxen-PDMS fiber (85µm, Supelco). The samples were equilibrated for 15 min at 30 °C, and extracted at the same temperature for 20 min with agitation of the SPME fiber. After extraction, the SPME fiber was injected directly into GC injection port with the splitless mode at 300 °C.

The analysis was achieved on a Varian CP-3800 gas chromatography equipped with a pulsed flame photometric detector (PFPD) (Varian). The separation was performed using a DB-

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FFAP capillary column (30 m×0.32 mm i.d., 1 m film thickness, Agilent Techonologies, Inc.). The chromatographic program was set at 35 °C for 3 min, raised to 150 °C at a rate of 10 °C /min, held for 5min, and then raised to 220 °C at a rate of 20 °C /min and held at the final temperature (220 °C) for 3 min. The carrier gas was nitrogen with a constant flow rate of 2 mL/min. The temperature of the detector was 300 °C. The detector voltage was 500V, the gate delay for sulfur compounds was 6ms, and the gate width is 20ms. The peak identification was achieved by comparing the retention time with the pure standard. The sulfur responses of specific compounds were calculated by taking the square root of the peak area. All analyses were conducted in triplicates.

The standard calibration curve was prepared by spiking known amount of pure chemical standards into 10 mL of saturated sodium chloride water with 2% ethanol in a 20 mL autosampler glass vial, and then follow the same procedures as described above.

3.3.2.5. Volatile phenolic compound analysis

Volatile phenols and methyl anthranilate were analyzed by SBSE-GC-MS method with an ethylene glycol−silicone (EG) coated stir bar (0.5 mm film thickness, 10 mm length, Gerstel Inc.,) (Zhou et al., 2015). Ten mL of wine sample was mixed with 10 mL of phosphate buffer (1M, pH =7.2). An aliquot of 20µL internal standard (50 mg/L 3,4-dimethylphenol) was added. EG coated stir bar was placed into each vial and stirred for 3 hours at room temperature at 1000 rpm for extraction. After extraction, the EG stir bar was removed from the sample, rinsed with milli-Q water, dried with Kimtech wipers (Kimberly-Clark Professional Inc., Roswell, GA), then transferred into a thermal desorption tube for GC-MS analysis.

Analysis of the extracted volatile compounds was carried out using an Agilent 7890 gas chromatograph coupled with a 5975 mass selective detector (Agilent Techonologies, Inc.) and a Gerstel MPS-2 multipurpose TDU autosampler with a CIS-4 cooling injection system (Gerstel Inc.). The analytes were thermally desorbed at the TDU in splitless mode, ramped from 25 to 220 °C at a rate of 100 °C/min, and held at 220 °C for 2 min. The CIS-4 was cooled to -80 °C during the sample injection, and then heated at 10 °C/s to 250 °C for 10 min.

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Solvent vent mode was used during the injection with a split vent flow of 50 mL/min. Separation was achieved by using a ZB-WAX-PLUS capillary column (60m × 0.25mm i.d., 0.5µm film thickness, Agilent Technologies, Inc.). The oven program was set at 35 °C for 4 min, raised to 150 °C at a rate of 20 °C/min, and then raised to 230 °C at a rate of 4 °C/min, and held for 10 min. Helium served as the carrier gas with a constant flow of 1.5 mL/min. The MS transfer line and ion source temperature were 280°C and 230°C, respectively. The mass selective detector in full scan mode was used to collect the data. Electron ionization mass spectrometric data from m/z 35 to 300 were collected, with an ionization voltage of 70 eV.

Standard calibration curve was prepared by spiking known amount of pure chemical standards into 10 mL of synthetic wine (3.5g/L tartaric acid, ethanol 12% V/V, pH 3.5) diluted with 10 mL of saturated sodium chloride buffer in a 40 mL of sample vial, and then follow the same procedures as described previously. Results were calculated through Chemstation software (Agilent Technologies, Inc.).

3.3.3. Odor activity value

To evaluate the contribution of each volatile compound to the wine aroma profile, the odor activity value (OAV) was determined. OAV is an indicator of the importance of a specific compound to the odor of a sample. It was calculated as the ratio between the concentration of an individual compound and the sensory threshold value found in the literature.

3.3.4. Statistical analysis

Mean values were calculated. Treatment differences were analyzed using analysis of variance (ANOVA) using SPSS version 20 (IBM, Chicago, IL). Statistical differences between two treatment levels were identified using Student t-test at the p<0.05 level. Statistical differences among three or more than three treatment levels were identified using Tukey’s HSD at the p<0.05 level.

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Results

3.4.1. Volatile aroma compounds

Volatile aroma compounds in Pinot noir wines with different cluster thinning treatments in twelve wineries were analyzed in this study. The results and discussion will be based on the wines from six wineries (A, B, C, D, E and F) that provided the wine samples from 2013 to 2015 vintage. The contents of volatile aroma compounds in Pinot noir wines in these wineries were shown in Table 3.2 (Winery A), Table 3.3 (Winery B), Table 3.4 (Winery C), Table 3.5 (Winery D), Table 3.6 (Winery E) and Table 3.7(Winery F). The impacts of cluster thinning treatments on volatile compounds are discussed based on the statistical difference at α=0.05. The odor active value (OAV) of each compound in Pinot noir wine for each winery was calculated and showed in Table 3.9 (Winery A), Table 3.10 (Winery B), Table 3.11 (Winery C), Table 3.12 (Winery D), Table 3.13 (Winery E) and Table 3.14 (Winery F).

3.4.1.1. Grape-derived volatile compounds

Terpenoids: Five terpenoids including a-terpineol, β-citronellol, linalool, nerol and geraniol were analyzed in this study. Wineries including A, B and C were relatively insensitive to cluster thinning practices. Cluster thinning had no influence on terpenoids for these wineries except a few terpenoids were significantly influenced. For instance, only nerol and geraniol increased significantly (p<0.05) in 1.5 cls in 2014 vintage in Winery A. However, the other wineries including D, E and F were relatively sensitive to cluster thinning practices. Take Winery D for an example, treatment of l cls had the highest (p<0.05) linalool, a-terpineol, nerol and geraniol but lowest (p<0.05) β-citronellol in 2013 vintage. In 2014 vintage, linalool was not detected. β-Citronellol was not detected in 1 cls. No significant difference was observed for other terpenoids. In 2015 vintage, 1 cls had significantly higher (p<0.05) a- terpineol but the lower (p<0.05) linalool, β-citronellol, nerol and geraniol. Results showed that there was no consistent difference in terpenoids across wineries and vintages, although some variations were observed.

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C13-norisoprenoids: C13-norisoprenoids have been identified as potential impact odorants in wines, contributing berry, honey and fruity notes in many red wines (Qian, Fang, & Shellie, 2009), especially β-damascenone and β-ionone (Ferreira, Lopez, & Cacho, 2000; Gomez- Miguez, Cacho, Ferreira, Vicario, & Heredia, 2007). TDN might have a negative influence on grape and wine flavor for its kerosene-like aroma. Vitispirane was another important odorant in wines. After acid hydrolysis, the total concentration of β-damascenone, vitispirane and TDN was obtained.

Cluster thinning has no impacts on C13-norisoprenoid compounds for Winery B except that 1 cls had significantly higher (p<0.05) free-form β-damascenone and total TDN in 2014 vintage but significantly lower (p<0.05) free-form TDN in 2015 vintage. However, other wineries

(Winery A, C, D, E, and F) were more sensitive to cluster thinning practices since most C13- norisoprenoid compounds were influenced by cluster thinning treatments in each vintage. Take Winery A as an instance, 1.5 cls or non-thinned treatments had significantly higher (p<0.05) free-form β-damascenone, β-ionone, vitispirane, total β-damascenone and total TDN in both 2013 and 2014 vintage. In 2015 vintage, only free β-damascenone and free vitispirane were significantly higher (p<0.05) in 1.5 cls or non-thinned treatment. The complicated impacts of cluster thinning practices on C13-norisoprenoids were also observed in Winery C,

D, E and F. There was no consistent trend of C13-norisoprenoid compositions with cluster thinning across wineries and vintages.

Volatile phenolic compounds: Guaiacol, 2-methylphenol, 3-ethylphenol, 4-ethylguaiacol and 4-ethylphenol were analyzed in this study. The impact of cluster thinning treatments on volatile phenolic compounds was various from the wineries and vintages. For Winery A, cluster thinning treatments (1 cls and 1.5 cls) significantly decreased (p<0.05) guaiacol and 4- ethylguaiacol while increased (p<0.05) 3-ethylphenol in 2013 vintage. It also resulted in significantly lower (p<0.05) 4-ethylguaiacol in 2014 vintage. In 2015 vintage, 1 cls had significantly lower (p<0.05) 4-ethylguaiacol but significantly higher (p<0.05) guaiacol. However, volatile phenolic compounds in wines for the other wineries (Winery B, C, D, E and F) were rarely impacted by cluster thinning practices, since only a few significant differences were observed among treatments. For example, cluster thinning treatments had no influence

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on volatile phenolic compounds for Winery B except that 1 cls had significantly lower (p<0.05) 4-ethylphenol in 2014 vintage.

3.4.1.2. Fermentation-derived volatile compounds

Higher alcohols: Higher alcohols such as propanol, isobutyl alcohol, isoamyl alcohol, benzyl alcohol and 2-phenylethyl alcohol were analyzed in this study. Cluster thinning practices had limited influence on higher alcohols in wines from some wineries, including Winery B and E. Only a few higher alcohols were significantly influenced by cluster thinning practices for these wineries. For instance, cluster thinning only significantly increased (p<0.05) benzyl alcohol in 2014 vintage for Winery B.

However, in the other wineries (Winery A, C, D and F), the influence of cluster thinning on higher alcohols was more complicated. Such as in Winery A, propanol, isobutyl alcohol and isoamyl alcohol was significantly increased (p<0.05) but 1-hexanol, 1-octanol and 1-octen-3- ol were significantly decreased (p<0.05) by cluster thinning (1 cls or 1.5 cls) treatments in 2013 vintage. In 2014 vintage, 1 cls had significantly higher (p<0.05) propanol but lower 1- hexanol 1-octanol, 1-octen-3-ol and benzyl alcohol, while 1.5 cls had the lowest (p<0.05) 2- phenylethyl alcohol. In 2015 vintage, cluster thinning practices had no impacts on most higher alcohols. The complicated impacts of cluster thinning practices on higher alcohols were also observed in Winery C, D and F. There was no consistent trend of higher alcohols with cluster thinning across wineries and vintages, although some statistical differences were observed among cluster thinning treatments.

Esters: Esters were the major class of aroma-active compounds analyzed in this study, contributing fruity and floral aromas to wines. Cluster thinning had limited influence on esters in Pinot noir wines from some wineries, including Winery B and C with very few esters influenced. Take Winery B for instance, 1 cls had significantly lower (p<0.05) hexyl acetate and ethyl 2-methylbutanoate in 2013 vintage. In 2014 vintage, 1 cls had significantly higher (p<0.05) ethyl isobutyrate. In 2015 vintage, 1 cls had significantly higher (p<0.05) ethyl acetate but lower (p<0.05) hexyl acetate, ethyl isobutyrate and ethyl 2-methylbutanoate.

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However, for other wineries including Winery A, D, E and F cluster thinning had the significant influence on ester compounds in different ways, depending on the wineries and vintages. Take Winery A for example, in 2013 vintage, cluster thinning treatments (1 cls and 1.5 cls) resulted in significantly lower (p<0.05) ethyl acetate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, hexyl acetate, octyl acetate and isobutyl acetate but significantly higher (p<0.05) diethyl succinate, ethyl isobutyrate, and ethyl 2-methylbutanoate compared to non-thinned treatment. Cluster thinning practices had no significant impacts on ethyl phenylacetate, octyl butyrate, 2-phenylethyl acetate, isoamyl acetate in 2013 vintage. But in 2014 vintage, the concentrations of ethyl acetate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, octyl butyrate, isobutyl acetate and isoamyl acetate significantly increased (p<0.05) in 1.5 cls while other esters were not influenced. In 2015 vintage, there had no significant difference was seen for all esters except that 1 cls had significantly higher (p<0.05) ethyl acetate. Based on subtotal OAV of esters in wines form Winery A, 1.5 cls had the lowest subtotal OAV in 2013 but the highest 2014 and 2015 vintage. The complex impacts of cluster thinning practices on esters were also observed in Winery D, E and F, indicating that there was no consistent difference in these esters across wineries and vintages.

Fatty acids: Hexanoic acid, octanoic acid and decanoic acid were identified. At high concentrations, fatty acids are associated with rancid, cheesy and vinegar-like aromas, but they are usually present below their detection thresholds in wines (Louw, Tredoux, Van Rensburg, Kidd, Naes, & Nieuwoudt, 2010).

Cluster thinning practice had limited influence on fatty acids in Pinot noir wines from some wineries including Winery B and F. Take Winery F as an example, crop thing treatment had no influence on fatty acids except 0.81 T had significantly lower (p<0.05) hexanoic acid and octanoic acid in 2015 vintage. However, the different impacts of cluster thinning on fatty acids were shown in the other wineries (Winery A, C, D and E). Take Winery A as an example, in 2013 vintage cluster thinning treatments (1 cls and 1.5 cls) resulted in significantly lower (p<0.05) octanoic and hexanoic acids. In 2014 vintage, three fatty acids had the highest (p<0.05) concentration in 1.5 cls. In vintage 2015, no difference was observed

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for all three fatty acids. The complex impacts of cluster thinning practice on fatty acids were also observed in Winery C, D and E. There was no consistent difference in these middle-chain acids across wineries and vintages.

Sulfur compounds: The aromatic contributions of sulfur compounds are widely considered detrimental to wine quality, which has garlic, onion or rubber odors, although someone reported that it also has a positive impact on the aroma and flavor of the wine. In this study, methyl thioacetate (MeSOAc), ethyl thioacetate (EtSOAc), dimethyl sulfide (DMS), dimethyl disulfide (DMDS), and dimethyl trisulfide (DMTS) were analyzed with cluster thinning practices. The impacts of cluster thinning on sulfur compounds were different from wineries, vintages, and individual compound. Generally, significant differences in sulfur compounds among cluster thinning treatments were observed for all six wineries. Like in Winery A, 1.5 cls had the lowest (p<0.05) MeSOAc, DMS and DMDS in 2013 vintage. In 2014 vintage, 1.5 cls had the lowest (p<0.05) MeSOAc but the highest (p<0.05) DMS. In 2015 vintage, there had no significant difference with cluster thinning treatments. The complicated impacts of cluster thinning practices on sulfur compounds were also observed in the other wineries. The impacts of cluster thinning practices on sulfur compounds of Pinot noir wines were varied with wineries and vintages.

Discussion

The major groups of compound affected by the treatments were terpenoids, C13- norisoprenoids, esters and alcohols which will be further discussed below.

3.5.1. Grape-derived volatile compounds

Terpenoids: Monoterpene alcohols are primarily responsible for wine floral aromas. Pinot noir contains monoterpene alcohols generally at levels below the sensory thresholds (Fang & Qian, 2005). But these compounds may still contribute to wine aroma by interacting with other compounds (Loscos, Hernandez-Orte, Cacho, & Ferreira, 2007). The impacts of cluster thinning practices on monoterpene alcohols of Pinot noir wines were varied with wineries and vintages. Cluster thinning practices had limited influence on terpenoids in the wines from

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some wineries, including Winery A, B and C. Cluster thinning practices could introduce the significant difference of terpenoid compounds among treatment wines from Winery D, E and F in some vintages. The variations among wineries may due to microclimates, sites, and viticultural practices in each winery (Jackson & Lombard, 1993). Nevertheless, the OAVs of terpenoids in Pinot noir wine were very low, indicating that the concentration of terpenoids in the wine was far below their sensory thresholds. The subtotal OAV for terpenoids were mainly less than one, suggesting that the impact of terpenoids on the aroma of Pinot noir wine is very small.

C13-norisoprenoids: Impacts of cluster thinning practices on C13-norisoprenoids depended on the winery and vintage. Although cluster thinning treatments indeed increased the content of each C13-norisoprenoid for those six wineries in some vintages, no consistent trend was observed for C13-norisoprenoids across the wineries or vintages. In this study, β-damascenone had very high OAVs, indicating they are important contributors to the wine aroma. For Winery A, low thinning (1.5 cls or non-thinned treatments) resulted in significantly higher (p<0.05) free-from β-damascenone. For Winery E, cluster thinning (1 cls) had significantly higher (p<0.05) free-from or total β-damascenone. But for Winery B, C13-norisoprenoids were insensitive to cluster thinning practices. Similarly, Bureau et al. reported that cluster thinning was independent of the biosynthesis of the C13-norisoprenoid glycosides in Syrah grapes (Bureau, Baumes, & Razungles, 2000). For other wineries (Winery C, D, and F), the influence of cluster thinning practices on β-damascenone was varied with vintages. Limited information is available to explain the observation in this study at this point. Factors that influence the composition of carotenoids (precursor of C13-norisoprenoids) will affect C13-norisoprenoids in grapes (Mendes-Pinto, 2009; Yuan & Qian, 2016). The factors such as terroir and viticulture practices in these six wineries could play a role in the C13-norisoprenoid synthesis in the grape berries and thus affect the C13-norisoprenoid concentration in wine (Jackson & Lombard, 1993; Mendes-Pinto, 2009).

In addition, cluster thinning had no evident impact on grape-derived volatile compositions with high variation over three studied seasons. As we know that grape-derived volatile compounds (terpenoids and C13-norisoprenoids) are associated with sunlight exposure and

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canopy density (Belancic, Agosin, Ibacache, Bordeu, Baumes, Razungles, et al., 1997; Feng, Yuan, Skinkis, & Qian, 2015; AJ Razungles, Baumes, Dufour, Sznaper, & Bayonove, 1998a; Song, Smart, Wang, Dambergs, Sparrow, & Qian, 2015). Effects of cluster thinning on grape- derived volatile compounds are possibly owing to the secondary changes in cluster-zone sunlight exposure and canopy density. However, in the current study, cluster-zone sunlight exposure and canopy density were rarely affected by cluster thinning and hence it is reasonable to expect little changes in grape-derived volatile composition.

3.5.2. Fermentation-derived volatile compounds

Higher alcohols: The impacts of cluster thinning practices on higher alcohols of Pinot noir wines were varied with wineries and vintages. Cluster thinning practices had limited influence on some wineries, including Winery B and E. 2-Phenylethyl alcohol and isoamyl alcohol were reported as important odor-active higher alcohols in the AEDA study of Pinot noir wine (Fang & Qian, 2005). 2-Phenylethyl alcohol and isoamyl alcohol also had OAVs higher than one. Cluster thinning introduced significantly lower (p<0.05) 2-phenylethyl alcohol in wines from Winery A, C, D and F, although the influences were varied with vintages. Isoamyl alcohol was influenced by cluster thinning in different ways, depending on wineries and vintages.

Esters: Condurso et al. found that the majority of the identified esters were present in quantities statistically higher in thinned samples than in the control ones for Syrah wines (Condurso, Cincotta, Tripodi, Sparacio, Giglio, Sparla, et al., 2016). However, in our study cluster thinning is not always able to increase the concentrations of esters. The impacts of cluster thinning practices on esters of Pinot noir wines were varied with wineries and vintages. Cluster thinning practices had limited influence on some wineries, including Winery B and C. Ethyl acetate, ethyl hexanoate, ethyl octanoate, isoamyl acetate and ethyl isobutyrate had higher OAVs (>10) in Pinot noir wines, which contributed a lot to wine aroma. However, cluster thinning had divergent influences on these esters. Only in Winery D, ethyl acetate was significantly decreased by cluster thinning treatments (1 cls and 1.5 cls) from 2013 to 2015 vintage.

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The impact of cluster thinning practice on middle-chain fatty acids and sulfur compounds of Pinot noir wines were still varied with wineries and vintages. The variation among wineries may due to terroir and viticulture practices in each vineyard site (Jackson & Lombard, 1993). Higher alcohols, esters, fatty acids and sulfur compounds are yeast metabolites. Their formation can be affected by many factors such as yeast strain, fermentation conditions, oxygen availability, grape nutrient status and nitrogen level (Bell & Henschke, 2005; J. He, Zhou, Peck, Soles, & Qian, 2013; Ugliano & Moio, 2005). Therefore, the changes of those compound compositions may also attribute to the alteration in grape nutrient status or other winemaking parameters.

Conclusions

The results of the analysis of three vintages in six wineries demonstrated the role of cluster thinning practices on the volatile compounds of Pinot noir wine in Western Oregon. Certain wine volatile compounds were affected by the cluster thinning treatments, but no consistent trend was found. Because the impacts of cluster thinning on wine volatiles are highly varied over the three vintages and the six wineries, it is conclusive that cluster thinning has limited impact on wine volatile compositions of Pinot noir under our regional conditions. Pinot noir is generally a low-yielding cultivar and vine sizes are often large due to sufficient water resources in Oregon’s Willamette Valley. It is rare that grapevines are unable to ripen fruit due to a limited canopy. In other words, when vine yields are already low, cluster thinning only serves to reduce yields, rather than increase fruit and wine quality. Based on work in the larger study, the cluster thinning is not shifting the source-sink relationship to a significant level to alter grape volatile composition. Thus, cluster thinning may not influence the source- sink relationship and affect the grape volatile composition.

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Table 3.1. Selected quantification ions for isotope volatile compounds in Pinot noir wine Compounds Quantify ion Compounds Quantify ion ethyl butyrate-4,4,4-d3 (IS) 74 hexanoic-d11 acid (IS) 77 ethyl butanoate 71 hexanoic acid 73 ethyl isobutyrate 71 octanoic-d15 acid (IS) 77 isobutyl acetate 56 octanoic acid 73 ethyl octanoate-d15 (IS) 91 decanoic-d19 acid (IS) 77 methyl octanoate 87 decanoic acid 73 ethyl octanoate 88 ethyl decanoate-d19 (IS) 105 octyl acetate 70 ethyl decanoate 101 octyl butyrate 112 diethyl succinate 129 ethyl hexanoate-d11 (IS) 91 2-phenylethyl acetate 91 ethyl hexanoate 88 ethyl phenylacetate 164 hexyl acetate 84 ethyl±-2-methylbutyrate- 107 d9 (IS) 2-phenyl-d5-ethan-1,1,2,2-d4- 131 methyl 2-methylbutanoate 101 ol (IS) 2-phenylethyl alcohol 122 ethyl 2-methylbutanoate 102 benzyl alcohol 108 butyl 2-methylbutanoate 103 hexanol 69 (±)-linallol-d3(vinyl-d3) (IS) 124 1-octanol 84 linalool 121 1-octen-3-ol 85 nerol 93 β-damascenone 121 geraniol 123

β-ionone 177 α-terpineol-d3 (IS) 124 vitispirane 192 α-terpineol 121 TDN 157 β-citronellol 123

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Table 3.2 Composition of volatile compounds in Pinot noir wines from Winery A with cluster thinning treatments from 2013 to 2015 (ug/L)

Year 2013 2014 2015

Cluster Thinning 1 cls 1.5 cls No Thin 1 cls 1.5 cls No Thin 1 cls 1.5 cls No Thin Treatment Straight-Chain Esters a ethyl acetate 161±3 a 182±6 b 166±3 b 184±7 a 209±4 b 174±5 a 99.5±1.9 a 92.0±2.5 b 92.1±2.3 b ethyl butanoate 141±7 a 122±5 b 164±8 c 173±9 a 258±22 b 227±13 b 128±1 158±8 170±12 ethyl hexanoate 190±7 a 138±8 b 198±11 c 147±9 a 309±28 b 239±8 c 138±1 185±24 194±6 ethyl octanoate 145±2 a 110±3 b 156±3 c 136±15 a 274±14 b 220±20 c 140±1 153±14 172±9 ethyl decanoate 17.3±0.6 a 17.0±0.5 b 22.6±2.4 c 20.8±1.6 a 40.0±1.5 b 27.2±2.2 c 20.0±0.7 40.8±1.2 42.3±2.9 hexyl acetate 1.52±0.13 ab 1.35±0.07 a 2.13±0.32 b 1.55±0.00 1.62±0.73 1.15±0.14 4.13±0.39 nd 3.48±1.33 octyl acetate 0.55±0.07 a 0.65±0.07 b 1.83±0.04 a nd 2.65±0.38 nd nd nd 1.6±0.5 octyl butyrate 4.32±0.20 4.12±0.13 4.75±0.07 6.65±0.21 a 10.27±0.70 b 5.95±0.14 a 4.38±0.18 7.80±0.14 8.52±0.30 diethyl succinate 1230±68 a 1233±99 a 1195±90 b 4668±615 5478±213 7495±687 6042±394 6121±339 6061±825

2-phenylethyl acetate 8.58±0.81 8.15±0.85 8.33±1.24 16.23±1.94 17.17±0.91 19.05±0.78 18.88±1.38 17.80±1.48 19.05±2.00 ethyl phenylacetate 11.7±0.9 9.7±0.7 10.7±1.3 13.9±1.7 15.5±0.5 17.3±1.4 9.0±0.6 16.0±0.1 15.3±2.2 Branched-Chain Esters isoamyl acetate 455±44 466±8 435±15 529±38 a 721±62 b 597±80 ab 245±32 281±29 284±26 isobutyl acetate 46.3±4.0 a 51.3±0.4 a 56.0±3.3 b 50.9±1.0 a 80.8±8.1 b 67.9±7.2 b 80.0±3.6 75.6±1.1 59.7±4.6 ethyl isobutyrate 349±16 a 292±16 b 329±11 a 449±37 399±39 430±16 214±1 a 518±24 b 363±23 c ethyl 2-methylbutanoate 44.4±2.0 a 27.7±1.2 b 37.1±3.7 c 36.7±3.1 39.7±4.0 42.2±2.6 40.9±3.7 41.3±1.6 32.8±2.6 Alcohols propanola 39.4±2.2 a 36.3±0.6 a 34.6±2.8 b 56.1±3.7 a 41.2±5.9 b 47.7±1.0 ab 17.2±1.2 16.6±1.4 17.2±1.2 isobutyl alcohola 65.9±3.3 a 78.8±1.4 a 58.0±0.9 b 88.7±11.4 99.5±11.1 98.6±1.6 58.1±8.4 64.5±2.2 64.9±2.7 isoamyl alcohola 374±32 a 361±6 a 170±2 b 377±45 449±28 446±10 222±1 220±19 218±9

1-hexanol 1752±171 a 1796±73 2060±135 b 856±92 a 1040±108 ab 1098±40 b 1648±242 1841±121 1760±80 2-ethyl-1-hexanol 4.33±0.26 a 5.78±0.42 b 17.85±0.98 c 2.48±0.28 a 10.00±0.70 b 2.08±0.15 a 2.73±0.25 ab 2.38±0.88 a 3.73±0.53 b 1-octanol 277±30 a 259±13 b 337±25 a 183±7 a 217±12 b 216±2 b 205±8 110±75 203±6 66

(Table 3.2 continued) 1-octen-3-ol 3.27±0.28 a 3.60±0.18 b 4.85±0.21 ab 2.30±0.33 a 3.75±0.50 b 3.60±0.15 b 3.17±0.47 3.63±0.04 4.28±0.29 benzyl alcohol 726±38 802±79 934±63 620±58 a 681±26 ab 736±28 b 810±44 676±50 629±26 2-phenylethyl alcohol 54076±2160 a 49276±3803 a 52476±3225 b 43326±1567 a 39488±1310 b 43437±815 a 31280±11033 41562±578 39308±1413 Terpenoids linalool 7.85±0.14 8.68±0.38 8.30±0.35 9.30±0.63 8.18±0.11 8.13±0.88 7.83±0.28 7.2511±0.14 7.30±0.42 a-terpineol 25.9±2.2 25.2±1.9 23.4±4.0 19.5±0.6 19.2±1.6 20.4±0.8 20.1±2.3 22.5±0.1 23.8±1.4 β-citronellol 5.73±0.34 5.45±0.07 5.72±0.23 3.38±0.25 3.13±0.42 3.88±0.58 3.30±0.21 4.10±0.64 3.00±0.30 nerol 5.92±0.13 7.03±0.18 5.98±0.42 16.82±1.37 a 23.63±2.31 b 19.68±0.60 ab 4.95±0.28 10.15±0.42 14.23±0.26 geraniol 2.53±0.04 2.30±0.07 2.45±0.21 4.83±0.32 a 7.62±0.71 b 5.95±0.57 ab 7.13±1.17 2.78±0.32 5.18±0.60

C13-Norisoprenoids β-damascenone 2.28±0.21 a 3.08±0.31 b 2.38±0.19 a 3.07±0.08 a 4.00±0.05 b 3.63±0.30 b 1.20±0.00 2.50±0.07 2.65±0.23 β-ionone nd 0.40±0.05 nd 0.20±0.00 0.25±0.00 0.27±0.03 0.40±0.00 0.33±0.04 0.42±0.03 vitispiraneb 85.9±8.1 a 85.9±8.1 a 99.1±12.8 b 60.4±2.7 a 78.2±6.7 b 62.9±3.1 a 46.2±5.5 a 47.3±1.9 a 63.7±3.5 b

TDNb 30.8±1.4 30.8±1.4 31.1±2.9 25.9±0.6 a 27.8±0.5 b 27.3±0.5 ab 21.5±0.9 20.3±0.0 22.0±1.3 β-damascenone* 7.63±0.60 a 9.52±0.42 b 9.00±0.35 b 13.8±0.3 a 7.80±0.05 b 13.4±1.6 a 23.2±0.2 21.8±2.3 22.3±0.5 vitispiraneb* 453±19 372±77 472±20 392±18 412±118 413±22 719±163 575±93 572±90

TDNb* 149±13 a 158±19 a 171±11 b 234±10 a 192±18 b 255±11 a 476±59 489±79 443±84 Aldehydes acetaldehydea 3.50±0.26 a 9.92±0.67 b 4.16±0.34 c 19.2±0.3 21.0±0.3 21.1±0.2 2.76±0.16 2.43±0.22 2.47±0.03 Acids hexanoic acid 912±5 a 865±60 b 951±49 a 961±46 a 1362±15 b 1077±51 c 755±39 852±76 1097±35 octanoic acid 744±52 a 758±27 b 831±34 c 790±21 a 1164±39 b 812±16 a 809±72 933±132 901±5 decanoic acid 39.4±2.5 39.5±1.6 38.7±5.4 41.4±3.0 a 66.9±5.2 b 44.3±1.3 a 41.7±7.3 67.6±4.0 77.9±8.2 Volatile phenolic compounds guaiacol 17.2±7.3 a 26.6±5.7 a 32.5±2.9 b 14.9±11.7 12.7±6.2 18.9±9.6 27.8±7.0 a 11.4±3.4 b 16.7±3.6 ab

2-methylphenol 1.26±0.05 3.11±0.59 2.64±0.11 2.36±0.88 1.01±0.79 0.75±0.09 1.48±1.50 1.01±0.74 1.63±1.27 3-ethylphenol 0.92±0.13 a 1.49±0.45 b 0.62±0.10 b 1.09±0.46 0.61±0.37 0.76±0.13 0.83±0.61 0.39±0.41 1.26±1.02

4-ethylphenol 3.19±1.32 11.6±0.9 6.50±0.75 4.35±3.91 11.9±2.7 11.6±4.5 0.81±0.50 1.48±1.06 1.54±0.34

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(Table 3.2 continued) 4-ethylguaiacol 3.15±0.45 a 6.64±1.21 b 22.2±1.6 c 2.60±1.48 a 2.01±1.19 a 12.6±2.6 b 0.33±0.02 a 0.36±0.00 a 0.76±0.23 b Sulfur Containing Compounds methyl thioacetate 5.50±0.24 a 4.00±0.15 b 5.46±0.13 a 16.9±2.3 a 11.4±2.0 b 11.7±0.3 b 4.35±0.23 5.57±0.40 4.29±0.10 ethyl thioacetate nd nd nd 1.36±0.19 1.12±0.19 1.18±0.03 nd 0.33±0.04 nd dimethyl sulfide 126±7 a 78.4±0.7 b 119±2 c 85.9±2.9 a 133±12 b 119±5 b 129±5 117±2 135±6 dimethyl disulfide 0.25±0.00 a 0.16±0.01 b 0.32±0.02 a 0.20±0.06 0.26±0.06 0.21±0.01 6.40±0.55 6.27±0.44 5.86±1.12 dimethyl trisulfide 0.15±0.00 0.13±0.00 0.15±0.01 0.22±0.07 0.25±0.06 0.19±0.01 2.63±0.30 2.13±0.04 2.58±0.01

Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (Tukey HSD at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis.

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Table 3.3 Composition of volatile compounds in Pinot noir wines from Winery B with cluster thinning treatments from 2013 to 2015 (ug/L)

Year 2013 2014 2015 Cluster Thinning Treatment 1 cls No Thin 1 cls No Thin 1 cls No Thin Straight-Chain Esters ethyl acetatea 160±5 157±2 168±5 166±8 96.5±2.7 a 91.1±0.4 b ethyl butanoate 274±13 304±27 205±18 220±22 175±9 175±4 ethyl hexanoate 275±22 303±16 237±18 230±18 193±8 174±10 ethyl octanoate 259±15 285±24 199±6 222±27 181±17 186±9 ethyl decanoate 32.4±2.6 35.5±2.1 31.4±0.6 32.5±0.5 35.8±5.1 37.4±2.0 hexyl acetate 1.38±0.04 a 3.58±0.11 b 2.48±0.18 2.18±0.18 1.15±0.00 a 2.48±0.25 b octyl acetate nd 4.83±4.49 nd nd 1.03±0.25 ND octyl butyrate 6.15±0.21 9.48±1.23 8.50±0.88 8.20±0.14 8.18±0.81 8.23±0.82 diethyl succinate 2120±63 5869±186 1933±42 2089±207 1919±448 1804±23 2-phenylethyl acetate 6.13±0.25 23.58±2.37 10.60±0.21 12.72±0.43 17.00±4.45 12.78±0.39 ethyl phenylacetate 7.4±0.4 25.9±1.1 8.8±0.2 9.4±0.7 13.6±3.2 12.4±0.2 Branched-Chain Esters isoamyl acetate 564±45 397±13 798±63 793±39 325±17 334±31 isobutyl acetate 92.9±2.1 97.8±12.8 142.9±13.6 131.0±11.0 151.7±3.6 145.5±4.5 ethyl isobutyrate 588±26 618±72 550±31 a 444±13 b 466±18 a 532±15 b ethyl 2-methylbutanoate 43.5±1.6 a 51.5±3.9 b 35.4±4.9 29.7±2.8 24.3±1.6 a 28.1±0.3 b Alcohols propanola 55.6±6.6 51.1±6.8 41.3±1.9 41.9±2.6 13.7±0.6 14.3±0.5 isobutyl alcohola 157±10 157±4 211±7 204±12 108±4 a 119±2 b isoamyl alcohola 406±41 510±64 481±34 486±31 220±9 240±11 1-hexanol 1051±45 1241±168 1045±63 1021±58 1483±20 1484±162 2-ethyl-1-hexanol 3.00±0.22 4.75±1.13 2.82±0.31 3.53±0.18 1.68±0.20 2.13±0.11 1-octanol 276±20 309±34 236±19 240±14 187±7 170±14

69

(Table 3.3 continued) 1-octen-3-ol 4.47±0.23 5.23±0.25 3.32±0.25 3.23±0.28 4.03±0.39 4.80±0.35 benzyl alcohol 942±17 879±89 758±27 a 707±9 b 460±22 436±3 2-phenylethyl alcohol 35492±1403 36298±3787 34411±945 35099±459 25915±472 26378±405 Terpenoids linalool 8.37±0.18 nd 12.47±0.25 13.68±0.90 9.75±0.00 9.83±1.24 a-terpineol 32.6±4.0 37.3±3.3 23.3±1.2 22.0±0.6 14.5±1.8 16.9±1.1 β-citronellol 2.00±0.21 nd 2.83±0.04 2.95±0.40 3.00±0.28 2.00±0.35 nerol 7.73±0.51 8.57±2.33 7.55±0.66 7.48±0.86 9.05±1.20 9.95±0.78 geraniol 3.00±0.21 a 10.55±0.54 b 4.35±0.00 3.10±1.84 8.68±1.66 3.73±0.15

C13-Norisoprenoids β-damascenone 2.40±0.13 1.98±0.18 1.47±0.03 a 1.33±0.03 b 1.60±0.21 1.57±0.08 β-ionone 0.35±0.00 1.65±0.92 0.32±0.03 0.32±0.03 0.38±0.04 0.38±0.03 vitispiraneb 130±4 121±19 68.1±4.5 64.8±8.5 65.0±9.8 71.8±1.9 TDNb 32.1±1.5 32.5±2.4 26.5±1.1 25.6±1.1 20.3±0.1 a 20.6±0.0 b β-damascenone* 6.48±0.21 6.88±0.20 7.70±1.52 8.20±0.83 20.7±1.3 19.4±1.8 vitispiraneb* 551±155 466±10 573±94 623±101 744±223 760±163 TDNb* 257±25 256±5 232±23 a 162±31 b 523±127 524±188 Aldehydes acetaldehydea 7.16±0.43 7.04±0.24 5.60±0.34 a 4.93±0.07 b 2.48±0.09 a 2.12±0.06 b Acids hexanoic acid 1368±48 1364±23 948±34 887±22 976±75 996±17 octanoic acid 1424±3 a 1342±30 b 894±24 897±32 834±16 858±19 decanoic acid 72.5±9.7 75.2±4.6 73.6±4.1 76.6±4.3 68.5±4.7 74.7±4.4 Volatile phenolic compounds guaiacol 15.0±0.3 13.0±3.3 19.8±2.3 19.0±7.2 15.0±3.6 14.5±4.4 2-methylphenol 2.52±0.28 2.15±0.27 2.43±0.34 1.65±1.26 1.18±0.00 1.22±0.14 3-ethylphenol 0.29±0.07 0.21±0.03 0.43±0.18 0.42±0.24 0.76±0.92 0.62±0.75 4-ethylphenol 15.4±12.9 19.2±2.1 10.1±6.2 a 20.4±0.9 b 9.33±11.97 9.73±8.19

70

(Table 3.3 continued) 4-ethylguaiacol 4.49±0.39 5.76±1.27 8.62±5.67 8.52±0.97 13.1±3.9 14.3±0.4 Sulfur Containing Compounds methyl thioacetate 9.14±1.49 7.18±0.45 7.98±0.13 a 1.76±0.09 b 3.81±0.16 3.68±0.23 ethyl thioacetate 0.45±0.10 0.43±0.04 nd nd nd nd dimethyl sulfide 235±19 a 131±3 b 144±7 132±4 104±4 97.3±2.1 dimethyl disulfide 0.16±0.02 0.16±0.01 0.16±0.01 a 0.23±0.01 b 3.09±0.13 3.11±0.79 dimethyl trisulfide 0.17±0.03 0.14±0.00 0.17±0.03 0.16±0.01 1.65±0.04 a 1.11±0.00 b Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (student t-test at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis

71

Table 3.4 Composition of volatile compounds in Pinot noir wines from Winery C with cluster thinning treatments from 2013 to 2015 (ug/L)

Year 2013 2014 2015

Cluster Thinning 0.71 T 1.01 T 1.31 T 0.71 T 1.01 T 1.31 T 0.71 T 1.01 T 1.31 T Treatment Straight-Chain Esters a ethyl acetate 101±3 165±3 b 176±5 b 109±4 a 117±2 b 112±3 ab 114±5 a 132±1 b 110±1 a ethyl butanoate 181±6 a 182±4 a 146±4 b 183±16 183±16 176±3 118±7 a 112±10 a 143±5 b ethyl hexanoate 191±12 a 154±14 b 147±6 b 230±22 212±13 215±1 169±28 138±29 181±3 ethyl octanoate 204±7 201±18 141±3 210±20 237±2 231±4 158±19 122±1 184±16 ethyl decanoate 37.7±1.1 a 29.6±3.9 b 21.1±0.4 c 29.0±3.5 47.9±3.5 47.9±5.1 27.3±2.5 23.6±2.7 34.1±3.4 hexyl acetate 1.68±0.04 2.10±0.21 1.48±0.04 11.23±1.38 21.03±1.31 5.28±0.81 4.67±1.20 a 2.40±0.00 b 3.35±0.30 ab octyl acetate 2.13±0.11 1.08±0.18 1.00±0.13 1.15±0.21 1.58±1.38 1.98±1.52 0.80±0.07 0.73±0.11 0.88±0.53 octyl butyrate 9.00±1.05 7.28±1.87 4.95±0.17 6.82±0.98 11.73±5.98 10.70±5.87 6.93±0.67 5.85±1.71 6.75±1.00 diethyl succinate 3320±476 2649±769 1251±151 7230±559 10205±465 7983±801 1475±93 1524±102 2168±204

2-phenylethyl acetate 28.05±3.04 17.40±5.59 9.38±1.40 13.4±0.4 20.8±0.3 20.1±2.9 18.63±1.29 21.65±1.27 18.60±1.88 ethyl phenylacetate 74.7±10.1 59.7±16.9 26.4±3.3 8.0±0.6 12.7±0.4 10.9±1.0 6.7±0.6 8.2±0.2 7.5±0.7 Branched-Chain Esters isoamyl acetate 328±17 407±33 455±37 266±10 323±33 286±27 383±24 414±31 395±30 isobutyl acetate 80.4±2.1 a 96.1±2.5 b 68.3±1.9 c 81.9±7.7 78.6±3.4 94.4±3.6 93.7±4.2 99.2±10.5 90.9±2.9 ethyl isobutyrate 282±3 326±63 249±6 291±30 314±5 337±2 233±19 249±13 239±2 ethyl 2-methylbutanoate 25.9±0.3 a 26.5±1.4 b 19.8±0.9 c 19.0±1.4 22.1±1.9 2226.8±0.1 13.5±1.4 11.2±0.1 13.8±0.5 Alcohols propanola 32.3±4.8 50.8±3.7 52.7±2.4 19.5±1.7 a 23.9±0.9 b 19.0±1.9 a 19.4±1.4 19.9±0.9 18.6±1.0 isobutyl alcohola 74.0±3.0 120±1 107±6 60.6±5.4 55.8±1.0 64.8±6.3 69.3±3.8 71.3±1.2 74.9±0.7 isoamyl alcohola 269±8 361±7 352±45 225±8 198±3 221±4 230±15 226±6 234±3

1-hexanol 1319±32 a 1374±30 a 1662±101 b 1373±51 1171±43 1389±186 2289±261 a 1755±236 ab 1519±87 b

2-ethyl-1-hexanol 5.35±0.22 ab 4.25±0.28 a 6.38±0.85 b 6.7±2.3 3.6±0.1 4.9±0.1 4.58±0.26 a 2.85±0.36 b 2.10±0.28 b 1-octanol 253±8 a 229±8 a 424±18 b 83.9±1.6 77.1±10.1 76.4±3.4 167±14 a 118±8 b 146±8 a

72

(Table 3.4 continued) 1-octen-3-ol 4.85±0.28 4.58±0.04 3.73±0.25 3.6±0.4 2.9±0.2 3.8±0.5 3.98±0.35 a 2.85±0.35 b 3.32±0.28 ab benzyl alcohol 577±14 a 543±21 a 730±25 b 498±33 555±29 528±13 524±25 472±29 506±32 2-phenylethyl alcohol 47515±963 a 39359±731 b 49866±1066 c 39194±2445 35207±1346 37208±1224 39674±1414 a 34457±1424 b 34152±1720 b Terpenoids linalool nd nd 7.10±0.35 6.0±0.6 5.7±0.4 5.3±0.2 7.07±0.06 6.92±0.53 7.45±0.23 a-terpineol 27.6±4.1 30.0±2.1 26.9±2.5 17.0±0.8 17.5±1.1 17.1±0.2 17.8±1.1 16.1±1.3 16.3±1.7 β-citronellol 3.25±0.14 1.93±1.87 3.38±0.11 4.1±0.9 2.7±0.1 0.0±0.0 3.82±0.37 5.12±0.57 5.45±0.14 nerol 18.33±1.73 a 19.88±0.81 a 6.08±0.75 b 4.1±0.5 3.0±0.4 5.1±0.8 5.07±0.24 ab 5.58±0.36 a 4.65±0.44 b geraniol 7.00±1.03 a 6.75±0.60 a 3.15±0.57 b 4.5±0.1 1.9±0.2 6.3±0.2 4.95±0.28 a 4.55±0.40 a 2.23±0.08 b

C13-Norisoprenoids β-damascenone 2.17±0.08 a 2.45±0.00 b 1.92±0.18 a 0.9±0.0 a 0.9±0.0 a 1.4±0.1 b 1.73±0.12 a 1.62±0.10 a 1.18±0.08 b β-ionone 0.40±0.00 0.32±0.03 nd 0.30±0.00 a 0.25±0.00 b 0.37±0.03 c 0.25±0.00 0.25±0.00 0.20±0.00 vitispiraneb 132±2 124±31 119±14 48.8±1.0 a 56.2±2.5 b 58.5±2.4 b 45.5±1.8 41.5±1.4 45.8±3.2

TDNb 35.3±2.1 31.2±6.3 34.6±2.5 85.6±56.3 57.0±47.5 65.2±40.0 20.5±0.0 a 20.2±0.1 b 20.3±0.1 c β-damascenone* 7.88±0.23 a 9.22±0.10 b 4.75±0.26 c 17.6±0.7 18.9±1.2 18.3±0.3 16.2±1.4 a 21.9±2.1 b 10.2±0.7 c vitispiraneb* 603±34 a 401±186 ab 317±42 b 815±260 958±86 856±168 634±6 ab 734±62 a 554±86 b

TDNb* 250±32 244±86 227±22 537±114 628±85 465±123 480±9 a 479±47 a 302±21 b Aldehydes acetaldehydea 2.26±0.15 3.79±0.04 4.13±0.08 2.05±0.07 a 1.30±0.06 b 1.61±0.03 c 2.91±0.18 a 2.34±0.05 b 1.64±0.04 c Acids hexanoic acid 868±27 a 673±47 b 1039±40 c 1009±80 905±58 876±31 711±52 a 681±23 a 927±55 b octanoic acid 888±26 a 682±71 b 1027±18 c 1430±155 a 1228±21 ab 1121±74 b 788±36 a 645±10 b 1004±15 c decanoic acid 77.7±5.2 a 60.6±0.2 b 52.4±5.2 ab 77.5±4.0 82.9±10.5 68.7±2.3 71.4±7.5 56.0±4.9 85.1±3.2 Volatile phenolic compounds guaiacol 18.8±0.6 16.9±7.6 14.1±3.2 18.4±5.1 15.7±6.4 17.3±4.8 16.6±10.3 11.1±4.3 6.78±5.98

2-methylphenol 1.94±0.09 a 0.67±0.68 b 1.30±0.47 ab 1.59±0.00 1.68±0.08 1.69±0.49 1.33±0.48 1.39±0.26 1.04±0.91 3-ethylphenol 1.02±0.00 0.63±0.45 0.39±0.10 0.30±0.06 0.29±0.08 0.20±0.05 0.30±0.39 0.35±0.16 0.04±0.03

4-ethylphenol 20.8±0.4 34.0±11.4 36.7±5.5 21.8±2.8 21.7±0.9 11.2±9.3 7.07±5.87 4.86±0.97 1.99±3.41

73

(Table 3.4 continued) 4-ethylguaiacol 10.7±0.7 a 12.8±2.5 a 23.6±4.5 b 2.62±0.47 7.41±2.04 3.00±2.62 3.12±1.87 1.14±0.50 1.81±1.67 Sulfur Containing Compounds methyl thioacetate 8.42±0.11 a 6.36±0.06 b 7.01±0.05 c 13.5±1.0 16.5±5.9 12.2±1.3 6.03±0.07 a 7.22±1.73 a 10.1±0.9 b ethyl thioacetate 0.76±0.05 a 0.62±0.07 a 0.32±0.00 b 1.45±0.12 a 1.42±0.24 a 0.92±0.07 b 0.50±0.00 0.56±0.09 0.33±0.05 dimethyl sulfide 151±4 a 110±1 b 134±7 c 264±5 a 395±27 374±14 b 116±3 137±14 122±5 dimethyl disulfide 0.12±0.01 a 0.11±0.01 a 0.28±0.00 b 0.12±0.01 0.13±0.01 0.13±0.01 1.56±0.15 ab 1.68±0.24 a 2.98±0.36 b dimethyl trisulfide 0.13±0.00 a 0.14±0.01 a 0.22±0.01 b 0.15±0.00 0.18±0.04 0.16±0.00 nd nd 2.23±0.69

Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (Tukey HSD at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis.

74

Table 3.5 Composition of volatile compounds in Pinot noir wines from Winery D with cluster thinning treatments from 2013 to 2015 (ug/L)

Year 2013 2014 2015

Cluster Thinning Treatment 1 cls 1.5 cls No Thin 1 cls 1.5 cls No Thin 1 cls No Thin

Straight-Chain Esters ethyl acetatea 416±6 b 245±8 c 526±10 a 168±9 b 163±1 a 182±4 b 139±0 a 170±3 b ethyl butanoate 155±11 133±7 150±6 261±14 228±3 230±6 127±1 a 175±9 b ethyl hexanoate 177±21 177±24 172±4 110±10 a 171±30 b 147±39 ab 138±1 a 255±14 b ethyl octanoate 117±12 102±4 113±2 398±33 368±16 369±24 142±3 a 188±8 b ethyl decanoate 6.8±0.8 6.6±0.4 7.9±0.2 65.1±4.5 b 66.3±3.5 a 76.8±0.9 b 20.1±0.5 a 23.9±0.9 b hexyl acetate 6.18±0.68 b 3.67±0.49 a 3.90±0.15 a 3.00±0.25 2.00±0.28 2.20±0.21 5.73±0.32 6.73±0.95 octyl acetate 1.23±0.03 a 5.53±0.77 b 0.60±0.00 a 0.65±0.28 1.33±0.04 2.03±0.18 nd 2.20±0.33 octyl butyrate 1.60±0.07 1.15±0.05 0.93±0.04 15.12±1.40 b 14.28±0.18 a 19.25±0.85 b 4.52±0.28 a 11.22±0.75 b diethyl succinate 9074±656 b 2831±74 a 3560±67 a 6102±855 5874±585 5751±612 6282±501 7150±442

2-phenylethyl acetate 70.23±6.16 b 12.95±0.28 a 19.55±0.78 a 11.58±1.55 12.02±1.07 13.43±0.45 19.47±1.41 a 15.68±0.92 b ethyl phenylacetate 30.4±2.5 b 7.4±0.1 a 10.3±0.2 a 7.4±1.0 8.1±0.8 7.9±0.2 9.3±0.8 a 7.1±0.4 b Branched-Chain Esters isoamyl acetate 1072±49 b 855±56 c 1443±61 a 482±90 523±113 456±83 313±12 223±4 isobutyl acetate 301.1±26.1 b 153.9±11.1 a 204.2±1.9 a 53.9±2.8 54.0±4.5 54.8±3.8 77.4±1.5 89.5±7.5 ethyl isobutyrate 443±30 358±63 387±1 304±8 354±33 478±181 208±10 213±28 ethyl 2-methylbutanoate 27.5±0.7 23.6±1.5 26.2±0.4 19.1±1.8 18.2±0.7 21.2±0.3 12.4±0.4 a 15.3±0.30845 b

Alcohols propanola 32.2±1.0 b 29.9±4.0 b 51.2±1.9 a 4.25±0.24 3.70±0.30 34.9±14.4 28.6±1.0 28.9±1.9 isobutyl alcohola 68.7±3.9 69.4±2.1 b 113±4 a 93.9±2.8 88.1±2.9 95.1±1.6 47.8±1.6 45.0±1.1 isoamyl alcohola 243±11 b 249±7 b 403±16 a 383±19 357±28 401±9 142±7 125±5 1-hexanol 1272±21 b 1554±72 ab 1604±203 a 1874±177 b 1550±102 a 1689±17 ab 1528±69 a 2017±81 b

2-ethyl-1-hexanol 2.75±0.18 b 2.63±0.25 b 5.18±0.18 a 5.38±0.43 2.60±0.42 7.03±0.32 2.75±0.18 a 8.78±0.26 b

1-octanol 371±10 443±42 464±48 260±23 249±17 227±34 206±6 249±6 75

(Table 3.5 continued) 1-octen-3-ol 2.73±0.08 5.65±0.64 5.53±0.67 3.75±0.13 3.37±0.19 3.55±0.14 2.93±0.12 a 7.48±0.34 b

benzyl alcohol 1600±24 a 1317±160 b 1556±105 ab 477±30 b 444±25 a 425±19 b 833±19 a 1613±115 b 2-phenylethyl alcohol 43240±589 45835±5254 47877±4979 31361±2210 a 32913±1058 ab 34582±2385 b 24871±203 a 31663±2472 b

Terpenoids

linalool 15.08±1.59 a 9.30±1.48 b nd nd nd nd 7.88±0.20 a 16.65±0.74 b

a-terpineol 55.6±0.8 a 43.7±5.4 b 51.9±2.1 ab 21.8±1.0 20.7±1.2 20.7±1.6 18.8±0.2 a 16.8±1.0 b β-citronellol 3.05±0.00 b 10.47±1.11 ab 12.33±0.81 a nd 1.35±0.13 1.23±0.25 3.30±0.21 a 5.65±0.26 b

nerol 17.00±0.64 a 5.60±0.54 b 6.63±0.23 b 39.57±2.53 30.20±1.90 25.13±1.03 4.80±0.33 a 11.20±0.97 b geraniol 4.65±0.53 b 2.10±0.07 a 2.33±0.04 a 22.17±1.59 17.18±0.23 14.78±1.94 6.87±0.94 a 14.83±2.17 b

C13-Norisoprenoids β-damascenone 2.33±0.16 2.00±0.26 2.38±0.18 3.88±0.10 b 3.47±0.20 a 3.30±0.44 b 1.20±0.00 a 1.41±0.09 b β-ionone nd nd nd 0.73±0.03 0.65±0.05 0.58±0.04 0.40±0.00 0.42±0.03

vitispiraneb 328±28 a 148±15 b 284±30 a 107±2 96.1±5.4 90.8±9.6 46.9±2.7 47.0±2.3

TDNb 34.6±0.6 ab 30.5±2.7 b 37.0±1.2 a 34.5±0.6 33.6±1.6 33.1±2.3 20.3±0.1 a 14.5±3.3 b β-damascenone* 8.05±2.12 a 10.2±0.2 a 7.47±0.60 b 14.9±0.7 13.4±2.1 21.8±2.3 10.6±0.7 a 14.9±1.0 b

vitispiraneb* 1191±459 1119±154 1100±241 496±34 412±108 952±75 532±14 539±14

TDNb* 393±155 358±60 355±27 287±52 a 266±80 a 497±102 b 338±11 a 377±8 b

Aldehydes

acetaldehydea 11.0±0.7 b 8.99±0.45 c 19.6±0.5 a 4.47±0.29 4.12±0.10 6.21±0.30 2.48±0.43 a 5.54±0.48 b

Acids

hexanoic acid 704±101 b 809±98 ab 967±50 a 2115±56 b 1899±52 a 1876±29 b 769±41 a 1626±179 b

octanoic acid 392±22 489±33 642±21 2015±40 b 1962±42 a 1931±64 b 784±58 a 1256±12 b

decanoic acid 7.2±1.8 15.2±0.6 12.9±2.3 160.6±6.8 b 164.9±7.7 a 171.7±4.0 b 41.5±5.2 50.3±4.1

Volatile phenolic compounds

guaiacol 34.0±4.9 a 17.2±2.0 b 29.3±4.2 a 27.8±2.7 29.1±1.2 30.1±17.8 17.9±7.4 10.1±2.0

2-methylphenol 4.16±2.27 2.15±0.51 2.61±2.24 2.19±0.19 1.05±1.30 0.71±0.68 1.69±0.29 0.81±0.93 3-ethylphenol 0.08±0.07 0.48±0.28 0.27±0.02 0.44±0.46 1.37±1.38 0.21±0.01 0.94±1.10 0.74±0.39

76

(Table 3.5 continued) 4-ethylphenol 13.3±2.7 b 22.8±1.4 a 26.8±1.9 a 0.64±0.19 0.73±0.05 0.85±0.83 7.55±12.53 9.43±0.91

4-ethylguaiacol 13.0±3.1 14.1±2.5 16.9±1.9 0.09±0.16 ab 1.35±1.19 a 0.18±0.31 b 11.0±4.0 5.42±0.58 Sulfur Containing Compounds

methyl thioacetate 1.42±0.02 0.44±0.77 1.42±0.02 12.1±0.7 9.99±0.67 11.1±0.5 4.92±0.21 a 3.53±0.14 b

ethyl thioacetate nd nd nd 1.42±0.10 1.14±0.25 1.07±0.17 nd nd dimethyl sulfide 140±3 a 127±6 b 140±3 a 127±7 b 122±5 a 132±13 b 300±6 a 269±11 b

dimethyl disulfide 0.21±0.01 a 0.26±0.02 b 0.21±0.01 a 0.21±0.01 b 0.35±0.00 a 0.20±0.02 ab 0.17±0.01 0.16±0.00

dimethyl trisulfide 0.13±0.01 a 0.16±0.01 b 0.13±0.01 a 0.23±0.02 0.48±0.02 0.20±0.01 0.16±0.00 0.17±0.02

Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (Tukey HSD and student t-test at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis.

77

Table 3.6 Composition of volatile compounds in Pinot noir wines from Winery E with cluster thinning treatments from 2013 to 2015 (ug/L)

Year 2013 2014 2015

Cluster Thinning Treatment 1 cls 1.5 cls 2 cls 1 cls 2 cls No Thin 1 cls No Thin

Straight-Chain Esters ethyl acetatea 216±17 a 150±3 b 260±75 a 273±5 a 236±8 b 250±9 b 196±3 a 123±2 b ethyl butanoate 142±13 156±23 157±18 128±1 107±2 113±3 174±5 a 94±5 b ethyl hexanoate 206±23 200±7 186±22 141±1 a 158±7 b 125±6 c 215±6 a 153±12 b ethyl octanoate 121±8 107±3 117±18 97±8 a 118±3 b 78±3 c 169±4 a 96±4 b ethyl decanoate 10.0±1.1 9.9±0.8 8.3±1.2 5.1±0.5 a 10.6±0.4 a 1.6±0.3 b 36.5±1.1 a 22.9±1.4 b hexyl acetate 2.58±0.18 2.68±0.21 3.60±0.07 2.65±0.85 2.03±0.39 1.60±0.35 4.37±0.28 nd octyl acetate 1.73±0.04 1.45±0.14 1.18±0.25 nd 1.00±0.14 0.85±0.09 nd nd octyl butyrate 1.15±0.00 1.18±0.18 1.23±0.04 1.30±0.21 a 2.93±0.38 b 1.05±0.13 a 10.78±0.29 10.80±0.46 diethyl succinate 3008±326 3222±76 4249±494 5407±54 7397±457 5398±341 8474±1045 a 6027±416 b

2-phenylethyl acetate 9.45±1.19 10.78±0.18 20.00±3.32 14.20±0.35 17.00±1.03 11.37±0.62 18.03±0.46 14.75±1.30 ethyl phenylacetate 7.9±0.9 8.5±0.2 12.0±1.7 8.8±0.2 9.9±0.7 7.5±0.4 8.3±1.1 9.5±0.7

Branched-Chain Esters isoamyl acetate 548±122 ab 474±13 a 676±60 b 482±90 523±113 456±83 313±12 a 223±4 b isobutyl acetate 71.1±8.6 a 82.3±9.7 a 141.6±15.3 b 93.2±1.7 83.8±2.8 71.4±7.0 144.4±5.3 a 85.7±4.8 b ethyl isobutyrate 279±26 302±16 339±30 277±6 258±15 258±9 323±1 a 265±15 b ethyl 2-methylbutanoate 28.8±3.0 31.6±4.1 33.7±5.0 17.5±0.2 17.4±0.6 16.6±0.8 16.4±0.5 17.1±1.3

Alcohols propanola 51.9±7.4 35.4±1.4 34.8±0.4 40.5±5.3 31.2±2.9 41.9±2.8 27.6±1.1 a 18.1±0.8 b isobutyl alcohola 78.9±9.5 53.9±0.5 61.1±1.9 85.6±9.4 95.1±12.6 99.7±5.7 63.5±1.9 a 55.0±1.7 b isoamyl alcohola 278±26 276±6 265±8 266±1 308±8 291±25 128±7 140±5

1-hexanol 1917±152 2065±161 1570±204 907±117 1000±35 1048±83 2162±54 2208±142

2-ethyl-1-hexanol 3.28±0.25 3.50±0.49 2.30±0.10 2.45±0.28 2.62±0.03 3.03±0.26 2.45±0.23 2.85±0.14 78

(Table 3.6 continued) 1-octanol 649±73 a 600±80 ab 470±58 b 257±9 286±25 234±18 244±5 224±1

1-octen-3-ol 4.32±0.23 4.73±0.67 3.25±0.35 2.53±0.28 2.65±0.28 2.27±0.28 7.82±0.60 7.73±0.33 benzyl alcohol 1587±127 1601±147 1510±171 643±33 a 743±46 b 628±22 a 1453±50 a 956±22 b 2-phenylethyl alcohol 50511±3409 50793±4922 47672±4585 36431±1887 a 38870±1968 a 30618±592 b 34605±510 36775±1569

Terpenoids linalool 9.65±0.35 10.73±0.11 9.20±0.64 7.90±0.21 9.90±0.35 6.38±0.95 19.37±0.59 a 17.50±0.85 b a-terpineol 68.3±6.0 67.7±1.5 58.8±5.9 20.1±2.6 ab 21.6±0.5 a 17.6±0.4 b 18.9±1.2 a 16.5±0.4 b β-citronellol 11.73±0.53 13.10±0.57 3.00±0.49 nd 3.73±0.39 nd 5.97±0.28 a 3.38±0.46 b nerol 4.33±0.25 a 4.80±0.28 ab 7.23±0.08 b 8.20±0.57 9.77±1.33 6.98±0.68 3.58±0.39 3.83±0.67 geraniol 1.98±0.25 a nd 4.48±1.87 b 3.13±0.04 a 1.05±0.21 b nd 14.80±0.56 a 20.92±1.41 b

C13-Norisoprenoids β-damascenone 1.63±0.14 1.75±0.10 1.78±0.04 1.75±0.00 a 1.88±0.03 a 1.30±0.10 b 3.22±0.06 3.57±0.29 β-ionone nd 0.23±0.04 0.27±0.03 nd 0.25±0.00 0.37±0.03 0.65±0.00 0.45±0.00 vitispiraneb 220±15 209±37 172±55 60.4±17.3 72.3±1.6 61.9±4.2 49.4±4.0 43.9±1.6

TDNb 30.7±0.9 30.8±1.7 30.3±2.5 27.3±1.4 26.1±0.5 25.7±1.3 13.4±2.9 13.5±0.7 β-damascenone* 10.6±0.5 a 6.65±0.78 b 10.8±0.3 a 6.92±2.84 9.00±2.70 7.62±0.34 12.4±0.4 a 10.1±0.2 b vitispiraneb* 1331±127 1016±104 1131±428 437±32 523±146 462±128 422±38 a 332±16 b

TDNb* 520±36 a 367±65 ab 242±89 b 228±30 264±68 261±79 338±44 a 110±84 b

Aldehydes acetaldehydea 9.84±0.56 a 5.09±0.74 b 7.18±0.08 ab 6.99±0.29 a 7.55±0.12 a 4.79±0.33 b 4.02±0.02 a 7.74±0.48 b

Acids hexanoic acid 1072±133 1099±46 1017±27 701±35 ab 674±16 a 758±32 b 1282±108 1141±67 octanoic acid 577±59 569±13 564±12 429±8 ab 476±4 a 405±43 b 1096±88 a 927±29 b decanoic acid 23.9±1.0 17.4±2.2 17.8±3.5 10.2±1.3 a 21.9±1.7 b 5.9±0.5 c 70.3±7.3 60.1±0.6 Volatile phenolic compounds guaiacol 15.5±0.1 20.3±5.9 17.8±5.7 21.7±4.8 22.7±1.3 32.5±17.4 16.5±5.4 14.1±0.6

2-methylphenol 0.77±0.82 2.03±0.74 1.42±1.18 2.05±0.38 1.75±1.32 2.41±1.17 1.21±0.92 0.75±0.68

79

(Table 3.6 continued) 3-ethylphenol 0.81±0.15 a 0.19±0.11 ab 1.31±0.42 b 0.16±0.07 0.22±0.02 0.30±0.35 0.76±0.94 0.72±0.19

4-ethylphenol 10.0±3.3 10.4±10.0 11.9±0.6 3.79±3.06 6.70±1.53 8.15±3.44 9.39±7.91 a 5.70±1.1 b

4-ethylguaiacol 14.0±9.1 6.60±1.43 4.82±0.77 6.79±2.04 7.98±0.69 8.15±4.40 9.19±3.95 30.5±15.3 Sulfur Containing Compounds methyl thioacetate nd nd nd 2.24±0.10 a 2.13±0.13 a 2.89±0.03 b 3.46±1.10 2.72±0.30 ethyl thioacetate nd nd nd nd nd nd nd nd dimethyl sulfide 160±4 a 127±6 b 162±29 a 100±2 a 131±5 b 150±5 c 333±35 a 235±11 b dimethyl disulfide 0.23±0.01 ab 0.26±0.02 a 0.23±0.01 b 0.14±0.01 a 0.15±0.01 ab 0.17±0.01 b 0.25±0.02 0.44±0.03 dimethyl trisulfide 0.15±0.02 ab 0.16±0.01 a 0.19±0.00 b 0.14±0.01 0.14±0.00 0.15±0.00 0.17±0.00 0.34±0.02

Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (Tukey HSD and student t-test at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis.

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Table 3.7 Composition of volatile compounds in Pinot noir wines from Winery F with cluster thinning treatments from 2013 to 2015 (ug/L)

Year 2013 2014 2015 Cluster Thinning Treatment 0.81 T 1.22 T 0.81 T 1.22 T 0.81 T 1.22 T Straight-Chain Esters ethyl acetatea 155±2 a 142±8 b 170±5 169±6 88.3±0.4 a 77.8±0.9 b ethyl butanoate 259±11 216±23 225±6 233±12 199±28 219±13 ethyl hexanoate 356±8 a 195±21 b 205±17 a 281±27 b 362±27 373±26 ethyl octanoate 278±26 a 135±10 b 156±6 a 189±15 b 227±19 242±29 ethyl decanoate 33.2±0.8 21.7±3.2 31.4±0.7 a 25.6±3.2 b 43.1±15.0 37.4±3.3 hexyl acetate 2.30±0.28 a 7.25±0.60 b 1.80±0.66 2.53±0.32 3.98±0.23 3.30±0.49 octyl acetate nd 0.35±0.49 0.93±0.03 0.85±0.09 1.87±0.37 3.40±0.57 octyl butyrate 11.15±1.48 5.30±0.52 9.45±0.73 a 6.62±0.33 b 7.50±0.79 9.15±0.90 diethyl succinate 3773±174 a 3469±88 b 2192±249 2119±213 7724±790 7084±552 2-phenylethyl acetate 14.75±0.49 14.23±0.04 8.45±1.21 8.42±0.70 14.47±1.55 16.03±1.45 ethyl phenylacetate 12.1±0.7 11.8±0.1 5.2±0.5 5.4±0.4 11.3±0.5 11.2±0.2 Branched-Chain Esters isoamyl acetate 568±70 561±21 459±51 432±18 182±6 193±3 isobutyl acetate 57.9±0.6 a 47.9±2.4 b 43.7±1.1 49.1±7.3 52.6±4.1 51.1±3.5 ethyl isobutyrate 241±20 a 194±11 b 223±6 217±13 230±15 204±15 ethyl 2-methylbutanoate 34.7±2.3 a 20.9±1.9 b 24.9±1.8 24.7±1.6 nd 18.5±1.5 Alcohols propanola 62.9±2.8 a 64.8±9.2 b 94.3±0.9 93.6±3.7 29.7±0.7 a 25.2±1.0 b isobutyl alcohola 111±1 115±15 75.9±1.4 74.0±0.9 45.8±0.6 a 50.2±0.5 b isoamyl alcohola 503±67 a 618±54 b 384±5 a 360±13 b 177±6 184±3 1-hexanol 1547±33 1567±26 1167±31 1179±154 1905±140 1899±203 2-ethyl-1-hexanol 1.83±0.04 a 2.37±0.03 b 3.80±0.23 3.65±0.21 3.52±0.21 a 5.63±0.38 b 81

(Table 3.7 continued) 1-octanol 292±0 a 259±7 b 322±3 339±50 127±18 112±5 1-octen-3-ol 3.53±0.25 3.20±0.26 3.12±0.25 3.75±0.33 7.43±1.07 6.35±0.69 benzyl alcohol 426±25 a 447±35 b 624±18 616±85 651±87 619±39 2-phenylethyl alcohol 37760±1750 43766±818 34330±1085 31287±3992 39247±5176 41160±3082 Terpenoids linalool 6.68±0.23 8.08±0.25 9.32±0.36 a 6.92±0.28 b nd 19.98±0.63 a-terpineol 22.7±1.3 23.9±1.2 22.3±1.6 21.7±1.1 16.7±0.7 a 18.3±0.4 b β-citronellol 3.23±0.38 3.05±0.05 3.72±0.35 4.37±1.50 3.37±0.88 3.20±0.40 nerol 6.45±0.49 a 9.40±1.27 b 7.12±0.64 a 4.02±0.49 b 14.83±1.44 15.40±0.14 geraniol 7.98±0.04 6.48±0.60 7.38±0.63 a 4.05±0.14 b 4.83±0.51 a 30.43±2.08 b

C13-Norisoprenoids β-damascenone 1.18±0.21 1.32±0.54 1.35±0.09 a 1.12±0.12 b 3.84±0.48 3.97±0.48 β-ionone nd 0.22±0.03 0.35±0.00 nd 0.33±0.05 0.40±0.00 vitispiraneb 52.5±12.7 a 47.9±2.9 b 53.6±2.6 a 45.4±1.9 b 51.9±6.8 a 36.3±1.1 b TDNb 28.6±2.0 28.4±0.7 30.9±0.6 30.8±1.1 12.9±1.3 a 17.5±0.5 b β-damascenone* 8.70±0.85 a 6.08±0.26 b 20.8±1.5 22.4±1.8 12.8±0.3 a 14.0±0.3 b vitispiraneb* 368±38 a 293±54 b 555±30 498±40 281±51 262±17 TDNb* 202±28 a 178±24 b 505±51 498±59 307±191 239±43 Aldehydes acetaldehydea 3.34±0.46 a 6.58±0.81 b 2.85±0.14 2.93±0.09 1.90±0.11 a 1.70±0.03 b Acids hexanoic acid 1735±67 1762±54 1496±52 1474±127 2092±100 a 2281±45 b octanoic acid 1338±63 1351±51 1197±25 1232±128 1640±148 a 1954±124 b decanoic acid 71.7±2.8 77.7±7.2 79.2±6.0 84.5±8.6 55.3±12.6 66.5±7.9 Volatile phenolic compounds guaiacol 16.0±1.4 29.6±10.0 52.9±7.9 33.6±11.5 17.2±10.8 14.5±2.3 2-methylphenol 1.27±0.05 a 1.62±0.53 b 0.88±0.00 1.66±0.71 1.19±0.48 1.00±0.20 3-ethylphenol 0.42±0.31 0.39±0.39 0.80±0.39 0.65±0.51 0.28±0.21 a 0.63±0.02 b

82

(Table 3.7 continued) 4-ethylphenol 8.21±3.31 a 0.12±0.05 b 7.03±3.57 6.63±5.11 0.28±0.23 0.60±0.17 4-ethylguaiacol 1.80±0.21 a 1.72±0.25 b nd 2.04±0.20 0.80±0.79 0.03±0.05 Sulfur Containing Compounds methyl thioacetate 8.32±0.12 a 5.97±0.33 b 5.54±1.30 4.68±0.01 8.73±3.63 4.93±0.17 ethyl thioacetate 0.43±0.04 nd 0.47±0.47 nd nd nd dimethyl sulfide 174±2 a 92.5±1.6 b 206±10 158±55 429±34 384±15 dimethyl disulfide 0.18±0.03 a 0.14±0.00 b 0.21±0.10 0.25±0.08 0.70±0.27 0.39±0.02 dimethyl trisulfide 0.19±0.02 a 0.15±0.01 b 0.30±0.12 0.21±0.01 0.35±0.01 0.25±0.04 Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (student t-test at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis.

83

Table 3.8 Thresholds and odors of some important aromatic volatile compounds in wines.

Compounds Odor Thresholds Straight-Chain Esters ethyl acetate Fruity, sweet 12264[3] ethyl butanoate Sour fruit, strawberry, fruity 20[1] ethyl hexanoate Green apple, fruity, strawberry, anise 14[1] ethyl octanoate Pineapple, pear, floral 5[1] ethyl decanoate Fruity, fatty, pleasant 200[7] hexyl acetate Pleasant fruity, pear 1500[5] octyl acetate Orange floral, jasmine, pear 50000[7] octyl butyrate Fruity 8200[1] diethyl succinate Wine-like, vinous, light fruity 200000[7] 2-phenylethyl acetate Pleasant, floral 250[3] ethyl phenylacetate Sweetish solvent 250[6] Branched-Chain Esters isoamyl acetate Banana 30[3] isobutyl acetate Solvent 1605[4] ethyl isobutyrate Fruity 15[1] ethyl 2-methylbutanoate Fruity 18[1] Alcohols Propanol Fresh, alcohol 50000[2] isobutyl alcohol Fusel, alcohol 40000[3] isoamyl alcohol Cheese 30000[3] 1-hexanol Green, grass 8000[2] 2-ethyl-1-hexanol Mushroom, sweet fruity, citrus, fatty 270[8] 1-octanol Intense citrus, roses 120[3] 1-octen-3-ol mushroom 10[3] benzyl alcohol Citrusy, sweet 200000[4] 2-phenylethyl alcohol pleasant floral 14000[4] Terpenoids linalool Fruity, citric 25[1]

(Table 3.8 continued) a-terpineol Pleasant, sweet, anise 250[1] β-citronellol Green lemon 100[1] nerol rose 290[1] geraniol citrus, floral 30[1]

C13-Norisoprenoids β-damascenone Bark, canned peach, baked apple, dry plum 0.05[3] β-ionone Violet 0.09[1] Aldehydes acetaldehyde Pungent, bruised apple, , nutty 500[3] Acids hexanoic acid Cheese, rancid, fatty 420[1] octanoic acid Rancid, harsh, cheese, fatty acid 500[1] decanoic acid Fatty, unpleasant 1000[1] Volatile phenolic compounds guaiacol Phenolic, spicy 9.50[3] 2-methylphenol Woody, phenolic 31[6] 3-ethylphenol Musty 250[6] 4-ethylphenol Medicine, horse 440[6] 4-ethylguaiacol Caramellic 33[3] [1] Ferreira, Lopez, and Cacho (2000), the matrix was a 11% water/ethanol solution containing 7 g/L glycerol and 5 g/L tartaric acid, with the pH adjusted to 3.4 with 1 M NaOH; [2] Li, Tao, Wang, and Zhang (2008), [5] (Etiévant, 1991) and [7] CortsDiguez, RodriguezSolana, Domnguez, and Daz (2015), the matrix was a 12% ethanol/water solution; [3] Guth (1997) and [6] Jiang and Zhang (2010), the matrix was a 10% water/ethanol solution; [4] Gomez- Miguez, Cacho, Ferreira, Vicario, and Heredia (2007), the matrix was a 10% water/ethanol solution containing 5 g/L of tartaric acid at pH 3.2; [8] Pino and Queris (2011), matrix information is not available.

85

Table 3.9 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery A.

Year 2013 2014 2015 Compound 1 cls 1.5 cls No Thin 1 cls 1.5 cls No Thin 1 cls 1.5 cls No Thin Straight-Chain Esters ethyl acetate 21.49 24.33 22.09 24.49 27.93 23.23 13.26 12.26 0.27 ethyl butanoate 7.06 6.12 8.21 8.63 12.89 11.36 6.39 7.89 8.51 ethyl hexanoate 13.57 9.85 14.14 10.47 22.06 17.09 9.86 13.25 13.89 ethyl octanoate 28.95 22.05 31.25 27.17 54.84 44.10 28.07 30.52 34.40 ethyl decanoate 0.09 0.09 0.11 0.10 0.20 0.14 0.10 0.20 0.21 hexyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 octyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 octyl butyrate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 diethyl succinate 0.01 0.01 0.01 0.02 0.03 0.04 0.03 0.03 0.03 2-phenylethyl acetate 0.01 0.02 0.01 0.01 0.01 0.02 0.02 0.02 0.01 ethyl phenylacetate 0.05 0.04 0.04 0.06 0.06 0.07 0.04 0.06 0.06 Branched-Chain Esters isoamyl acetate 15.15 15.53 14.50 17.65 24.05 19.91 8.16 9.37 9.47 isobutyl acetate 0.03 0.03 0.03 0.03 0.05 0.04 0.05 0.05 0.04 ethyl isobutyrate 23.26 19.44 21.95 29.95 26.59 28.64 14.25 34.55 24.18 ethyl 2-methylbutanoate 2.47 1.54 2.06 2.04 2.21 2.35 2.27 2.29 1.82 subtotal 112.13 99.02 114.40 120.62 170.93 146.98 82.49 110.49 92.89 Alcohols propanol 0.79 0.73 0.69 1.12 0.82 0.95 0.34 0.33 0.34 isobutyl alcohol 1.65 1.97 1.45 2.22 2.49 2.47 1.45 1.61 1.62 isoamyl alcohol 12.47 12.04 5.68 12.58 14.96 14.87 7.39 7.34 7.25 1-hexanol 0.22 0.22 0.26 0.11 0.13 0.14 0.21 0.23 0.22 2-ethyl-1-hexanol 0.02 0.02 0.02 0.02 0.07 0.01 0.04 0.01 0.01 1-octanol 2.31 2.16 2.80 1.53 1.81 1.80 1.71 0.00 1.69 1-octen-3-ol 0.33 0.36 0.49 0.23 0.38 0.36 0.32 0.36 0.43 86

(Table 3.9 continued) benzyl alcohol 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2-phenylethyl alcohol 3.86 3.52 3.75 3.09 2.82 3.10 1.78 2.97 2.81 subtotal 21.64 21.02 15.14 20.90 23.47 23.71 13.24 12.86 14.38 Terpenoids linalool 0.31 0.35 0.33 0.37 0.33 0.33 0.31 0.29 0.29 a-terpineol 0.10 0.10 0.10 0.08 0.08 0.08 0.08 0.09 0.10 β-citronellol 0.06 0.05 0.06 0.03 0.03 0.04 0.03 0.00 0.03 nerol 0.03 0.04 0.02 0.02 0.02 0.06 0.08 0.07 0.02 geraniol 0.08 0.08 0.08 0.16 0.25 0.20 0.24 0.09 0.17 subtotal 0.59 0.62 0.59 0.67 0.71 0.70 0.75 0.54 0.61

C13-Norisoprenoids β-damascenone 45.67 61.67 47.67 61.33 80.00 72.67 24.00 50.00 53.00 β-ionone 0.00 4.44 0.00 2.22 2.78 2.96 4.44 3.61 4.63 subtotal 45.67 66.11 47.67 63.56 82.78 75.63 28.44 53.61 57.63 Aldehydes acetaldehyde 0.02 0.02 0.01 0.02 0.01 0.04 0.04 0.04 0.01 Acids hexanoic acid 1.77 1.81 1.98 1.88 2.77 1.93 1.93 2.22 2.14 octanoic acid 0.08 0.08 0.02 0.08 0.13 0.09 0.08 0.14 0.16 decanoic acid 0.91 0.87 0.95 0.96 1.36 1.08 0.76 0.85 1.10 subtotal 2.76 2.75 2.95 2.93 4.27 3.10 2.77 3.21 3.40 Volatile phenolic compounds guaiacol 1.81 2.80 3.42 1.57 1.33 1.99 2.92 1.20 1.76 2-methylphenol 0.04 0.10 0.09 0.08 0.03 0.02 0.05 0.03 0.05 3-ethylphenol 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.01 4-ethylphenol 0.01 0.03 0.01 0.01 0.03 0.03 0.00 0.00 0.00 4-ethylguaiacol 0.10 0.20 0.67 0.08 0.06 0.38 0.01 0.01 0.02 subtotal 1.86 2.93 3.52 1.66 1.40 2.04 2.98 1.24 1.82 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound

87

Table 3.10 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery B.

Year 2013 2014 2015 Compound 1 cls No Thin 1 cls No Thin 1 cls No Thin Straight-Chain Esters ethyl acetate 21.38 20.93 22.39 22.18 12.86 12.15 ethyl butanoate 13.72 15.20 10.26 10.99 8.76 8.76 ethyl hexanoate 19.66 21.67 16.91 16.45 13.79 12.44 ethyl octanoate 51.71 57.02 39.72 44.48 36.12 37.10 ethyl decanoate 0.16 0.18 0.16 0.16 0.18 0.19 hexyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 octyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 octyl butyrate 0.00 0.00 0.00 0.00 0.00 0.00 diethyl succinate 0.01 0.03 0.01 0.01 0.01 0.01 2-phenylethyl acetate 0.01 0.02 0.01 0.01 0.01 0.02 ethyl phenylacetate 0.03 0.10 0.04 0.04 0.05 0.05 Branched-Chain Esters isoamyl acetate 18.80 13.24 26.60 26.43 10.83 11.14 isobutyl acetate 0.06 0.06 0.09 0.08 0.09 0.09 ethyl isobutyrate 39.21 41.21 36.68 29.59 31.07 35.45 ethyl 2-methylbutanoate 2.41 2.86 1.97 1.65 1.35 1.56 subtotal 167.17 172.51 154.83 152.10 115.13 118.95 Alcohols propanol 1.11 1.02 0.83 0.84 0.27 0.29 isobutyl alcohol 3.92 3.93 5.28 5.11 2.71 2.97 isoamyl alcohol 13.54 16.99 16.04 16.19 7.33 7.99 1-hexanol 0.13 0.16 0.13 0.13 0.19 0.19 2-ethyl-1-hexanol 0.02 0.02 0.02 0.02 0.07 0.01 1-octanol 2.30 2.57 1.97 2.00 1.56 1.41 1-octen-3-ol 0.45 0.52 0.33 0.32 0.40 0.48

88

(Table 3.10 continued) benzyl alcohol 0.00 0.00 0.00 0.00 0.00 0.00 2-phenylethyl alcohol 2.54 2.59 2.46 2.51 1.85 1.88 subtotal 24.01 27.81 27.05 27.12 14.38 15.23 Terpenoids linalool 0.33 0.00 0.50 0.55 0.39 0.39 a-terpineol 0.13 0.15 0.09 0.09 0.06 0.07 β-citronellol 0.02 0.00 0.03 0.03 0.03 0.02 nerol 0.03 0.04 0.02 0.02 0.02 0.06 geraniol 0.10 0.35 0.15 0.10 0.29 0.12 subtotal 0.62 0.55 0.79 0.79 0.79 0.66

C13-Norisoprenoids β-damascenone 48.00 39.50 29.33 26.67 32.00 31.33 β-ionone 3.89 18.33 3.52 3.52 4.17 4.26 subtotal 51.89 57.83 32.85 30.19 36.17 35.59 Aldehydes acetaldehyde 0.02 0.02 0.01 0.02 0.01 0.04 Acids hexanoic acid 3.39 3.20 2.13 2.14 1.99 2.04 octanoic acid 0.15 0.15 0.15 0.15 0.14 0.15 decanoic acid 1.37 1.36 0.95 0.89 0.98 1.00 subtotal 4.90 4.71 3.22 3.18 3.10 3.19 Volatile phenolic compounds guaiacol 1.58 1.37 2.08 2.00 1.58 1.53 2-methylphenol 0.08 0.07 0.08 0.05 0.04 0.04 3-ethylphenol 0.00 0.00 0.00 0.00 0.00 0.00 4-ethylphenol 0.03 0.04 0.02 0.05 0.02 0.02 4-ethylguaiacol 0.14 0.17 0.26 0.26 0.40 0.43 subtotal 1.70 1.49 2.18 2.10 1.64 1.60 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound

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Table 3.11 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery C.

Year 2013 2014 2015 Compound 0.71 T 1.01 T 1.31 T 0.71 T 1.01 T 1.31 T 0.71 T 1.01 T 1.31 T Straight-Chain Esters ethyl acetate 13.50 21.97 23.48 14.48 15.54 14.93 15.22 17.57 14.69 ethyl butanoate 9.06 9.11 7.32 9.14 9.14 8.82 5.92 5.62 7.13 ethyl hexanoate 13.62 10.98 10.47 16.46 15.18 15.34 13.04 8.65 12.92 ethyl octanoate 40.77 40.27 28.16 41.91 47.44 46.14 31.51 24.34 36.85 ethyl decanoate 0.19 0.15 0.11 0.15 0.24 0.24 0.14 0.12 0.17 hexyl acetate 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.00 octyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 octyl butyrate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 diethyl succinate 0.02 0.01 0.01 0.04 0.05 0.04 0.01 0.01 0.01 2-phenylethyl acetate 0.01 0.02 0.01 0.01 0.01 0.02 0.02 0.02 0.01 ethyl phenylacetate 0.30 0.24 0.11 0.03 0.05 0.04 0.03 0.03 0.03 Branched-Chain Esters isoamyl acetate 10.93 13.55 15.18 8.87 10.76 9.54 12.77 13.79 13.16 isobutyl acetate 0.05 0.06 0.04 0.05 0.05 0.06 0.06 0.06 0.06 ethyl isobutyrate 18.83 23.96 16.59 19.39 20.94 22.47 15.51 16.59 15.93 ethyl 2-methylbutanoate 1.44 1.47 1.10 1.05 1.23 1.27 0.75 0.62 0.77 subtotal 108.73 121.80 102.58 111.59 120.63 118.92 94.97 87.41 101.73 Alcohols propanol 0.65 1.02 1.05 0.39 0.48 0.38 0.39 0.40 0.37 isobutyl alcohol 1.85 2.99 2.68 1.52 1.39 1.62 1.73 1.78 1.87 isoamyl alcohol 8.97 12.03 11.72 7.48 6.60 7.37 7.66 7.52 7.79 1-hexanol 0.16 0.17 0.21 0.17 0.15 0.17 0.29 0.22 0.19 2-ethyl-1-hexanol 0.02 0.02 0.02 0.02 0.07 0.01 0.04 0.01 0.01 1-octanol 2.11 1.91 3.53 0.70 0.64 0.64 1.39 0.98 1.21 1-octen-3-ol 0.49 0.46 0.37 0.36 0.29 0.38 0.40 0.29 0.33

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(Table 3.11 continued) benzyl alcohol 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2-phenylethyl alcohol 3.39 2.81 3.56 2.80 2.51 2.66 2.83 2.46 2.44 subtotal 17.63 21.40 23.16 13.44 12.14 13.23 14.74 13.66 14.22 Terpenoids linalool 0.00 0.00 0.28 0.24 0.23 0.21 0.28 0.28 0.30 a-terpineol 0.11 0.12 0.11 0.07 0.07 0.07 0.07 0.06 0.07 β-citronellol 0.03 0.02 0.03 0.04 0.03 0.00 0.04 0.05 0.05 nerol 0.03 0.04 0.02 0.02 0.02 0.06 0.08 0.07 0.02 geraniol 0.23 0.23 0.11 0.15 0.06 0.21 0.17 0.15 0.07 subtotal 0.41 0.41 0.55 0.52 0.41 0.55 0.64 0.61 0.51

C13-Norisoprenoids β-damascenone 43.33 49.00 38.33 18.50 17.67 27.00 34.67 32.33 23.67 β-ionone 4.44 3.52 0.00 3.33 2.78 4.07 2.78 2.78 2.22 subtotal 47.78 52.52 38.33 21.83 20.44 31.07 37.44 35.11 25.89 Aldehydes acetaldehyde 0.02 0.02 0.01 0.02 0.01 0.04 0.04 0.04 0.01 Acids hexanoic acid 2.12 1.62 2.45 3.40 2.92 2.67 1.88 1.53 2.39 octanoic acid 0.16 0.12 0.10 0.15 0.17 0.14 0.14 0.11 0.17 decanoic acid 0.87 0.67 1.04 1.01 0.91 0.88 0.71 0.68 0.93 subtotal 3.14 2.42 3.59 4.57 3.99 3.68 2.73 2.33 3.49 Volatile phenolic compounds guaiacol 1.98 1.78 1.49 1.94 1.65 1.82 1.75 1.17 0.71 2-methylphenol 0.06 0.02 0.04 0.05 0.05 0.05 0.04 0.04 0.03 3-ethylphenol 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4-ethylphenol 0.05 0.08 0.08 0.05 0.05 0.03 0.02 0.01 0.00 4-ethylguaiacol 0.33 0.39 0.72 0.08 0.22 0.09 0.09 0.03 0.05 subtotal 2.10 1.88 1.61 2.04 1.76 1.90 1.81 1.23 0.75 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound.

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Table 3.12 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery D.

Year 2013 2014 2015 Compound 1 cls 1.5 cls No Thin 1 cls 1.5 cls No Thin 1 cls No Thin Straight-Chain Esters ethyl acetate 55.46 32.63 70.07 25.35 33.61 25.26 18.55 22.69 ethyl butanoate 7.77 6.63 7.48 7.06 7.44 9.08 6.37 8.77 ethyl hexanoate 12.63 12.61 12.28 7.92 10.99 8.91 9.82 18.21 ethyl octanoate 23.48 20.35 22.57 15.69 24.04 19.80 28.40 37.57 ethyl decanoate 0.03 0.03 0.04 0.01 0.05 0.02 0.10 0.12 hexyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 octyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 octyl butyrate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 diethyl succinate 0.05 0.01 0.02 0.03 0.04 0.04 0.03 0.04 2-phenylethyl acetate 0.01 0.02 0.01 0.01 0.01 0.02 0.02 0.02 ethyl phenylacetate 0.12 0.03 0.04 0.04 0.05 0.05 0.04 0.03 Branched-Chain Esters isoamyl acetate 35.74 28.49 48.10 14.94 18.81 13.80 7.97 8.12 isobutyl acetate 0.19 0.10 0.13 0.04 0.06 0.05 0.05 0.06 ethyl isobutyrate 29.51 21.47 25.78 20.29 23.64 24.96 13.88 14.23 ethyl 2-methylbutanoate 1.53 1.31 1.45 1.08 1.23 1.40 0.69 0.85 subtotal 166.53 123.68 187.99 92.45 119.99 103.39 85.92 110.71 Alcohols propanol 0.64 0.60 1.02 0.85 0.89 0.76 0.57 0.58 isobutyl alcohol 1.72 1.74 2.83 2.37 2.71 2.54 1.19 1.12 isoamyl alcohol 8.11 8.31 13.44 10.44 10.91 11.08 4.74 4.16 1-hexanol 0.16 0.19 0.20 0.11 0.14 0.12 0.19 0.25 2-ethyl-1-hexanol 0.02 0.02 0.02 0.02 0.07 0.01 0.04 0.01 1-octanol 3.09 3.69 3.87 2.31 2.82 2.53 1.72 2.07 92

(Table 3.12 continued) 1-octen-3-ol 0.27 0.57 0.55 0.28 0.33 0.40 0.29 0.75 benzyl alcohol 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.01 2-phenylethyl alcohol 3.09 3.27 3.42 2.98 3.05 3.34 1.78 2.26 subtotal 17.11 18.39 25.35 19.36 20.91 20.78 10.53 11.22 Terpenoids linalool 0.60 0.37 0.00 0.34 0.45 0.32 0.32 0.67 a-terpineol 0.22 0.17 0.21 0.09 0.12 0.09 0.08 0.07 β-citronellol 0.03 0.10 0.12 0.03 0.03 0.03 0.03 0.06 nerol 0.03 0.04 0.02 0.02 0.02 0.06 0.08 0.07 geraniol 0.16 0.07 0.08 0.10 0.12 0.10 0.23 0.49 subtotal 1.04 0.77 0.43 0.58 0.74 0.60 0.73 1.35

C13-Norisoprenoids β-damascenone 46.67 40.00 47.67 32.33 46.33 34.67 24.00 28.11 β-ionone 0.00 0.00 0.00 2.22 3.15 2.78 4.44 4.63 subtotal 46.67 40.00 47.67 34.56 49.48 37.44 28.44 32.74 Aldehydes acetaldehyde 0.02 0.02 0.01 0.02 0.01 0.04 0.04 0.04 Acids hexanoic acid 0.93 1.16 1.53 0.87 1.11 0.92 1.87 2.99 octanoic acid 0.01 0.03 0.03 0.01 0.03 0.01 0.08 0.10 decanoic acid 0.70 0.81 0.97 0.53 0.68 0.55 0.77 1.63 subtotal 1.65 2.00 2.52 1.40 1.83 1.48 2.72 4.72 Volatile phenolic compounds guaiacol 3.58 1.81 3.09 2.40 3.45 3.47 1.89 1.06 2-methylphenol 0.13 0.07 0.08 0.03 0.02 0.06 0.05 0.03 3-ethylphenol 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4-ethylphenol 0.05 0.08 0.03 0.05 0.02 0.01 0.02 0.02 4-ethylguaiacol 0.39 0.43 0.51 0.41 0.53 0.20 0.33 0.16 subtotal 3.76 1.96 3.20 2.48 3.49 3.55 1.96 1.11 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound.

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Table 3.13 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery E.

Year 2013 2014 2015 Compound 1 cls 1.5 cls 2 cls 1 cls 2 cls No Thin 1 cls No Thin Straight-Chain Esters ethyl acetate 28.80 20.06 28.97 36.45 31.49 33.29 26.10 16.46 ethyl butanoate 7.11 7.79 7.85 6.38 5.37 5.63 8.69 4.71 ethyl hexanoate 14.69 14.28 13.29 10.06 11.28 8.91 15.38 10.90 ethyl octanoate 24.26 21.41 23.31 19.43 23.65 15.53 33.86 19.29 ethyl decanoate 0.05 0.05 0.04 0.03 0.05 0.01 0.18 0.11 hexyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 octyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 octyl butyrate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 diethyl succinate 0.02 0.02 0.02 0.03 0.04 0.03 0.04 0.03 2-phenylethyl acetate 0.01 0.02 0.01 0.01 0.01 0.02 0.02 0.02 ethyl phenylacetate 0.03 0.03 0.05 0.04 0.04 0.03 0.03 0.04 Branched-Chain Esters isoamyl acetate 15.94 15.79 22.52 14.37 19.59 13.68 10.44 7.42 isobutyl acetate 0.04 0.05 0.09 0.06 0.05 0.04 0.09 0.05 ethyl isobutyrate 18.57 20.14 22.57 18.43 17.17 17.17 21.54 17.66 ethyl 2-methylbutanoate 1.60 1.76 1.87 0.97 0.96 0.92 0.91 0.95 subtotal 111.13 101.39 120.59 106.26 109.72 95.27 117.29 77.64 Alcohols propanol 1.04 0.71 0.70 0.81 0.62 0.84 0.55 0.36 isobutyl alcohol 1.97 1.35 1.53 2.14 2.38 2.49 1.59 1.37 isoamyl alcohol 9.25 9.19 8.82 8.86 10.26 9.70 4.27 4.66 1-hexanol 0.24 0.26 0.20 0.11 0.13 0.13 0.27 0.28 2-ethyl-1-hexanol 0.02 0.02 0.02 0.02 0.07 0.01 0.04 0.01 1-octanol 5.41 5.00 3.92 2.14 2.38 1.95 2.03 1.87 1-octen-3-ol 0.43 0.47 0.33 0.25 0.27 0.23 0.78 0.77 94

(Table 3.13 continued) benzyl alcohol 0.01 0.01 0.01 0.00 0.00 0.00 0.01 0.00 2-phenylethyl alcohol 3.61 3.63 3.41 2.60 2.78 2.19 2.47 2.63 subtotal 21.97 20.63 18.91 16.94 18.88 17.54 12.01 11.95 Terpenoids linalool 0.39 0.43 0.37 0.32 0.40 0.26 0.77 0.70 a-terpineol 0.27 0.27 0.24 0.08 0.09 0.07 0.08 0.07 β-citronellol 0.12 0.13 0.03 0.00 0.04 0.00 0.06 0.03 nerol 0.03 0.04 0.02 0.02 0.02 0.06 0.08 0.07 geraniol 0.07 0.00 0.15 0.10 0.04 0.00 0.49 0.70 subtotal 0.87 0.88 0.80 0.52 0.58 0.38 1.48 1.56

C13-Norisoprenoids β-damascenone 32.67 35.00 35.50 35.00 37.67 26.00 64.33 71.33 β-ionone 0.00 2.50 2.96 0.00 2.78 4.07 7.22 5.00 subtotal 32.67 37.50 38.46 35.00 40.44 30.07 71.56 76.33 Aldehydes acetaldehyde 0.02 0.02 0.01 0.02 0.01 0.04 0.04 0.04 Acids hexanoic acid 1.37 1.35 1.34 1.02 1.13 0.96 2.61 2.21 octanoic acid 0.05 0.03 0.04 0.02 0.04 0.01 0.14 0.12 decanoic acid 1.07 1.10 1.02 0.70 0.67 0.76 1.28 1.14 subtotal 2.49 2.49 2.40 1.74 1.85 1.73 4.03 3.47 Volatile phenolic compounds guaiacol 1.63 2.14 1.88 2.29 2.39 3.42 1.73 1.48 2-methylphenol 0.02 0.07 0.05 0.07 0.06 0.08 0.04 0.02 3-ethylphenol 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 4-ethylphenol 0.02 0.02 0.03 0.01 0.02 0.02 0.02 0.13 4-ethylguaiacol 0.43 0.20 0.15 0.21 0.24 0.25 0.28 0.92 subtotal 1.69 2.23 1.95 2.36 2.46 3.52 1.80 1.64 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound.

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Table 3.14 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery F.

Year 2013 2014 2015 Compound 0.81 T 1.22 T 0.81 T 1.22 T 0.81 T 1.22 T Straight-Chain Esters ethyl acetate 20.70 18.91 22.61 22.54 11.78 10.37 ethyl butanoate 12.97 10.81 11.24 11.63 9.93 10.94 ethyl hexanoate 25.46 13.90 14.65 20.06 25.87 26.63 ethyl octanoate 55.60 27.05 31.23 37.71 45.41 48.40 ethyl decanoate 0.17 0.11 0.16 0.13 0.22 0.19 hexyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 octyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 octyl butyrate 0.00 0.00 0.00 0.00 0.00 0.00 diethyl succinate 0.02 0.02 0.01 0.01 0.04 0.04 2-phenylethyl acetate 0.01 0.02 0.01 0.01 0.01 0.02 ethyl phenylacetate 0.05 0.05 0.02 0.02 0.05 0.04 Branched-Chain Esters isoamyl acetate 18.93 18.69 15.31 14.41 6.08 6.43 isobutyl acetate 0.04 0.03 0.03 0.03 0.03 0.03 ethyl isobutyrate 16.07 12.96 14.85 14.47 15.34 13.59 ethyl 2-methylbutanoate 1.93 1.16 1.38 1.37 0.00 1.03 subtotal 151.95 103.70 111.50 122.39 114.76 117.71 Alcohols propanol 1.26 1.30 1.89 1.87 0.59 0.50 isobutyl alcohol 2.76 2.88 1.90 1.85 1.15 1.25 isoamyl alcohol 16.77 20.61 12.81 12.01 5.89 6.12 1-hexanol 0.19 0.20 0.15 0.15 0.24 0.24 2-ethyl-1-hexanol 0.02 0.02 0.02 0.02 0.07 0.01 1-octanol 2.43 2.16 2.68 2.83 1.06 0.94 1-octen-3-ol 0.35 0.32 0.31 0.38 0.74 0.64

96

(Table 3.14 continued) benzyl alcohol 0.00 0.00 0.00 0.00 0.00 0.00 2-phenylethyl alcohol 2.70 3.13 2.45 2.23 2.80 2.94 subtotal 26.48 30.61 22.21 21.34 12.54 12.64 Terpenoids linalool 0.27 0.32 0.37 0.28 0.00 0.80 a-terpineol 0.09 0.10 0.09 0.09 0.07 0.07 β-citronellol 0.03 0.03 0.04 0.04 0.03 0.03 nerol 0.03 0.04 0.02 0.02 0.02 0.06 geraniol 0.27 0.22 0.25 0.14 0.16 1.01 subtotal 0.69 0.71 0.77 0.57 0.28 1.98

C13-Norisoprenoids β-damascenone 26.00 32.50 27.00 22.33 76.88 79.33 β-ionone 0.00 2.41 3.89 0.00 3.72 4.44 subtotal 26.00 34.91 30.89 22.33 80.60 83.78 Aldehydes acetaldehyde 0.02 0.02 0.01 0.02 0.01 0.04 Acids hexanoic acid 3.18 3.22 2.85 2.93 3.91 4.65 octanoic acid 0.14 0.16 0.16 0.17 0.11 0.13 decanoic acid 1.73 1.76 1.50 1.47 2.09 2.28 subtotal 5.06 5.14 4.50 4.58 6.11 7.07 Volatile phenolic compounds guaiacol 1.69 3.11 5.57 3.54 1.81 1.53 2-methylphenol 0.04 0.05 0.03 0.05 0.04 0.03 3-ethylphenol 0.00 0.00 0.00 0.00 0.00 0.00 4-ethylphenol 0.02 0.00 0.02 0.02 0.00 0.00 4-ethylguaiacol 0.05 0.05 0.00 0.06 0.02 0.00 subtotal 1.75 3.17 5.62 3.61 1.85 1.56 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound.

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Chapter 4 GENERAL CONCLUSION

In this research, the impacts of cluster thinning practices on non-volatile phenolic and volatile compositions in Pinot noir wines were investigated. The non-volatile phenolic compounds were analyzed with UV-spectrometry and HPLC techniques. The results showed that four anthocyanins (Dp, Pt, Pn, and Mv) could be detected in Pinot noir wines while Pt and Dp could only be detected in some of the wine samples. Mv possessed the highest concentration among four detected anthocyanins for all wines. Flava-3-ols (catechin and epicatechin) and hydroxycinnamic acid were also determined. The impacts of cluster thinning treatments on major phenolic compounds were varied with vintage years and wineries. However, the results showed that cluster thinning increased TMA or TP in wines from Winery A, B, C, D and E in some vintages. But no significant difference of TP and TMA was observed with cluster thinning practices for most wineries for three vintages. In addition, cluster thinning had limited influence on phenolic compounds in wines from Winery F.

For volatile compositions in Pinot noir wines, results showed that no obvious relationship between wine volatile compositions and cluster thinning treatments. Certain volatile compounds were mainly influenced by cluster thinning treatments in different ways, depending on wineries and vintage years. Low thinning (1.5 cls or non-thinned treatments) resulted in significantly higher free-from β-damascenone for Winery A, while 1 cls treatment for Winery E to resulted in significantly higher free-from or total β-damascenone. Cluster thinning introduced significantly lower 2-phenylethyl alcohol in wines from Winery A, C, D and F, although the influences were varied with vintages. Moreover, it was indicated that wines from Winery B were insensitive to cluster thinning practices. Based on OAVs of aroma compounds, C13-norisoprenoids, esters and alcohols contributed a lot to Pinot noir wine aroma. There was no consistent trend figured from quality-important compounds. Contrary to the common belief in the wine industry, our results point out that cluster thinning does not guarantee higher Pinot noir wine aroma quality. Cluster thinning only served to reduce yields of Pinot noir grapes, rather than increase wine quality. However, narrowly defined yield targets are a metric of quality used by industry to estimate quality, and industry will continue 98 to reduce vine yields in pursuit of quality until research is shared with them and allow them to make more informed vineyard management decisions in their vineyards and under certain seasonal conditions.

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APPENDIX

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Table A2.1The information of wine companies and vineyards and cluster thinning treatments in the fields with yields (Kg/meter) and percent thinned in 2013 to 2015 vintage years.

Yield Year Treatment Percent Thinned Winery (cluster/shoot or ton/hectare) Kg/meter 0.5 cls 0.57 62.9% G 2013 1 cls 1.05 37.9% Half Crop 0.54 49.4% 2014 1 cls 0.72 25.8% H 1 cls 0.57 28.7% 2015 No Thin 0.80 0.0% 0.5 cls 0.67 66.1% 2013 1 cls 1.32 33.8% 1.5 cls 1.85 18.6% I 0.5 cls 1.03 74.0% 2015 1 cls 1.49 53.5% 1.5 cls 2.12 30.6% 1 cls 1.09 44.5% J 2014 2 cls 0.77 22.5% 1 cls 1.31 39.0% 2014 1.5 cls 1.82 23.1% No Thin 2.79 0.0% K 1 cls 1.72 43.1% 2015 1.5 cls 2.56 26.8% No Thin 4.25 0.0% 1 cls 2.36 42.3% 1.5 cls 2.67 28.9% L 2015 2 cls 3.68 9.2% estate1 - - 1: estate means unknown information. Cls: clusters per shoot. No Thin: no clusters were removed

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Table A2.2 Total phenols and total monomeric anthocyanins in Pinot noir wine with different cluster thinning treatments from 2013 to 2015 vintage

Winery Year Treatments Total Phenolsa Total Monomeric Anthocyaninsb 0.5 cls 4682±352 11.3±1.1 G 2013 1 cls 4866±92 13.4±0.0 Half Crop 8451±101 32.0±2.7 a 2014 1 cls 8669±372 19.3±2.0 b H 1 cls 7461±560 28.2±0.1 2015 No Thin 7623±393 33.6±3.7 0.5 cls 5314±165 4.20±1.18 2013 1 cls 5305±88 4.20±1.13 1.5 cls 5414±40 6.85±0.07 I 0.5 cls 7445±648 30.5±0.9 a 2015 1 cls 7863±415 33.0±3.0 a 1.5 cls 7414±430 22.5±3.0 b 1 cls 5556±539 a 21.5±2.0 a J 2014 2 cls 4710±192 b 30.9±4.2 b 1 cls 5498±104 20.9±0.6 2014 1.5 cls 5056±398 20.6±1.2 No Thin 5121±109 24.2±0.1 K 1 cls 4810±441 36.3±1.5 2015 1.5 cls 5134±656 28.3±3.1 No Thin 5012±220 28.4±3.3 1 cls 6539±456 ab 17.6±2.1 1.5 cls 5888±415 a 18.3±2.3 L 2015 2 cls 6775±525 ab 13.9±2.7 estate 7722±582 b 18.8±5.0 Mean ± SD presented (n=2). Different letters indicating differences in means (Tukey HSD and student t-test at α=0.05). estate: unknown; aas peak area; bas mg/kg malvidin-3-monoglucoside equivalent.

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Table A2.3 Phenolic composition in Pinot noir wine with different cluster thinning treatments from 2013 to 2015 vintage. Hydroxycinnamic Flavan-3-olsa Anthocyaninsb acid estersa Delphinidin-3- Petunidin-3- Peonidin-3- Malvidin-3- Winery Year Treatments Caffeoyltartaric acid Catechin Epicatechin monoglucoside monoglucoside monoglucoside monoglucoside 0.5 cls 1215±74 518±38 518±63 2.3±0.0 2.6±0.1 3.1±0.0 8.8±0.4 G 2013 1 cls 1422±8 630±40 486±1 2.2±0.0 2.4±0.0 3.0±0.4 9.6±1.5 Half Crop 3424±54 623±2 452±28 2.6±0.0 a 3.3±0.1 a 3.3±0.2 a 17.4±0.2 a 2014 1 cls 3292±4 611±4 470±17 2.2±0.1 b 2.7±0.0 b 2.6±0.2 b 11.0±0.3 b H 1 cls 2209±19 823±3 a 369±0 3.6±0.1 4.1±0.0 4.6±0.7 26.3±1.6 2015 No Thin 2506±28 939±16 b 348±11 3.8±0.1 5.2±1.1 5.5±1.3 29.2±2.3 0.5 cls 514±2 1044±20 a 565±19 nd 1.9±0.1 2.2±0.1 5.4±0.1 ab 2013 1 cls 560±46 980±5 b 557±16 nd 1.8±0.0 2.1±0.0 5.1±0.1 b 1.5 cls 545±41 1061±8 a 516±2 nd 1.9±0.0 2.2±0.0 5.7±0.0 a I 0.5 cls 1062±44 a 1142±1 a 697±38 2.7±0.1 a 3.6±0.5 3.9±0.0 31.6±0.1 a 2015 1 cls 1650±27 b 1073±1 b 629±4 2.4±0.0 b 3.3±0.2 4.4±0.3 26.1±0.4 b 1.5 cls 1423±7 c 1093±5 c 603±2 2.7±0.1 a 4.0±0.2 4.3±0.0 32.7±0.7 a 1 cls 1437±55 a 936±22 a 490±9 2.7±0.1 a 3.8±0.2 3.8±0.2 25.2±1.2 J 2014 2 cls 1316±57 b 746±69 b 487±25 2.6±0.1 b 3.7±0.1 3.9±0.1 26.8±1.1 1 cls 2764±19 ab 1303±18 a 533±21 2.2±0.0 3.6±0.0 3.2±0.0 a 31.4±0.5 a 2014 1.5 cls 2833±33 a 1167±4 b 554±5 2.1±0.1 3.6±0.0 2.9±0.1 b 26.2±0.2 b No Thin 2724±19 b 1192±15 b 520±3 2.0±0.0 3.4±0.2 3.2±0.0 a 28.2±0.2 c K 1 cls 2458±28 995±11 a 415±5 a 2.6±0.1 5.0±0.3 a 4.5±0.0 52.0±1.9 a 2015 1.5 cls 2875±125 1068±11 b 373±2 b 2.5±0.1 5.1±0.3 a 4.9±0.4 46.8±2.1 b No Thin 2560±7 912±2 c 334±3 c 2.3±0.1 4.1±0.2 b 4.6±0.2 38.8±0.9 c 1 cls 1725±6 a 1264±13 b 419±3 2.3±0.3 2.7±0.2 3.4±0.6 12.8±2.4 L 2015 1.5 cls 1636±1b 624±0 a 380±1 2.1±0.1 2.5±0.1 3.6±0.6 12.6±1.1 125

(Table A2.3 continued) 2 cls 2056±31 c 1163±4 b 419±4 2.0±0.0 2.6±1.5 3.2±0.3 10.1±1.6 estate 1796±26 a 929±7 c 499±11 2.1±0.1 2.8±0.7 2.7±0.4 9.4±2.0 Mean ± SD presented (n=2). nd: not detected. Different letters indicating differences in means (Tukey HSD and student t-test at α=0.05). estate: unknown; aas peak area; bas mg/kg malvidin-3-monoglucoside equivalent.

126

Table A3.1 Composition of volatile compounds in Pinot noir wines from Winery G with cluster thinning treatments in 2013 (ug/L)

Year 2013 Cluster Thinning Treatment 0.5 cls 1 cls Straight-Chain Esters ethyl acetatea 107±3 106±5 ethyl butanoate 208±5 a 226±3 b ethyl hexanoate 212±8 228±9 ethyl octanoate 197±16 217±8 ethyl decanoate 28.8±1.6 29.8±0.8 hexyl acetate 1.33±0.48 0.90±0.10 octyl acetate 0.55±0.00 0.55±0.07 octyl butyrate 7.10±0.28 7.70±0.49 diethyl succinate 2688±172 2792±153 2-phenylethyl acetate 4.20±0.35 4.08±0.19 ethyl phenylacetate 12.1±0.8 14.7±0.8 Branched-Chain Esters isoamyl acetate 336±34 398±40 isobutyl acetate 46.2±4.2 46.5±5.2 ethyl isobutyrate 461±39 437±19 ethyl 2-methylbutanoate 41.5±1.9 43.9±1.1 Alcohols propanola 41.8±2.7 42.5±1.0 isobutyl alcohola 88.5±9.2 96.2±6.6 isoamyl alcohola 328±90 419±46 1-hexanol 1331±117 1140±74 2-ethyl-1-hexanol 4.08±0.45 4.57±0.13 1-octanol 337±2 301±16 127

(Table A3.1 continued) 1-octen-3-ol 4.98±0.58 4.05±0.39 benzyl alcohol 708±31 704±15 2-phenylethyl alcohol 38706±1533 39028±1336 Terpenoids linalool 8.75±0.78 10.70±2.55 a-terpineol 25.4±0.9 23.4±0.7 β-citronellol 2.75±0.14 2.25±0.00 nerol 8.80±0.87 a 12.95±0.48 b geraniol 1.58±0.04 2.37±0.25

C13-Norisoprenoids β-damascenone 4.03±0.03 a 4.83±0.34 b β-ionone 0.30±0.00 0.25±0.00 vitispiraneb 114±12 101±7 TDNb 35.3±1.6 a 30.9±0.9 b β-damascenone* 14.9±2.5 9.40±3.04 vitispiraneb* 686±42 485±118 TDNb* 246±3 226±13 Aldehydes acetaldehydea 8.44±0.43 10.0±0.6 Acids hexanoic acid 790±72 a 1006±17 b octanoic acid 897±99 1132±120 decanoic acid 41.3±5.6 a 61.6±8.8 b Volatile phenolic compounds guaiacol 19.2±2.1 20.6±5.8 2-methylphenol 2.32±0.17 a 1.27±0.20 b 3-ethylphenol 0.28±0.04 0.37±0.07 4-ethylphenol 2.33±0.42 a 5.77±0.73 b

128

(Table A3.1 continued) 4-ethylguaiacol 1.79±0.11 a 3.16±0.69 b Sulfur Containing Compounds methyl thioacetate 5.72±0.40 5.97±0.16 ethyl thioacetate nd nd dimethyl sulfide 136±7 132±1 dimethyl disulfide 0.18±0.00 0.18±0.01 dimethyl trisulfide 0.16±0.01 0.16±0.02 Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (student t-test at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis

129

Table A3.2 Composition of volatile compounds in Pinot noir wines from Winery H with cluster thinning treatments from 2014 to 2015 (ug/L)

Year 2014 2015 Cluster Thinning Treatment Half Crop 1 cls 1 cls No Thin Straight-Chain Esters ethyl acetatea 167±3 a 291±19 b 198±2 a 100±3 b ethyl butanoate 240±12 a 207±10 b 206±19 206±4 ethyl hexanoate 234±23 a 176±9 b 191±24 197±13 ethyl octanoate 251±3 151±11 235±11 241±1 ethyl decanoate 33.4±0.7 19.9±1.1 40.9±2.5 46.4±4.4 hexyl acetate 1.60±0.14 2.10±0.07 3.63±0.46 3.18±1.45 octyl acetate 2.23±0.18 a 2.0±0.22 b 1.03±0.04 nd octyl butyrate 8.53±0.04 5.20±0.52 8.05±0.71 8.58±0.25 diethyl succinate 5926±529 4585±39 4735±254 5125±296 2-phenylethyl acetate 14.03±1.10 a 21.93±0.18 b 37.68±3.50 23.33±1.17 ethyl phenylacetate 10.3±0.8 10.6±1.6 17.4±1.3 16.7±1.0 Branched-Chain Esters isoamyl acetate 649±4 712±61 505±51 a 324±40 b isobutyl acetate 77.6±1.7 a 85.4±5.8 b 118.9±10.4 a 63.5±5.6 b ethyl isobutyrate 448±34 a 289±15 b 350±36 303±29 ethyl 2-methylbutanoate 40.8±0.6 a 27.8±2.4 b 25.8±3.4 27.7±0.6 Alcohols propanola 48.8±2.6 56.1±1.6 25.1±2.2 23.4±1.0 isobutyl alcohola 121±16 92.4±2.4 42.1±5.0 50.8±2.2 isoamyl alcohola 439±28 444±7 121±15 202±1 1-hexanol 1042±23 1034±101 1290±14 1366±75 2-ethyl-1-hexanol 10.28±0.46 a 9.37±0.28 b 3.05±0.28 2.65±0.21 130

(Table A3.2 continued) 1-octanol 274±40 a 250±21 b 280±3 271±18 1-octen-3-ol 3.05±0.28 a 3.17±0.41 b 37.03±0.25 35.32±1.90 benzyl alcohol 479±19 a 443±49 b 315±26 349±19 2-phenylethyl alcohol 38483±535 42129±3374 24890±493 26738±1537 Terpenoids linalool 10.75±0.35 7.38±0.60 7.65±0.21 8.95±0.00 a-terpineol 30.9±2.0 a 31.9±2.1 b 19.4±0.6 18.2±2.4 β-citronellol 2.13±0.32 a 2.00±0.14 b 0.95±0.14 nd nerol 9.82±0.99 12.50±0.14 19.12±2.68 a 24.65±1.62 b geraniol 4.35±4.31 5.48±0.18 7.35±0.78 8.47±0.68

C13-Norisoprenoids β-damascenone 1.58±0.11 a 2.20±0.13 b 2.58±0.04 2.67±0.28 β-ionone nd 0.23±0.04 nd 0.20±0.00 vitispiraneb 94.2±23.7 a 95.7±8.3 b 82.6±12.1 73.5±18.0 TDNb 35.4±4.1 a 31.6±0.8 b 23.5±3.2 20.7±0.8 β-damascenone* 5.62±0.28 a 6.60±0.76 b 25.1±1.8 22.8±0.6 vitispiraneb* 573±63 a 556±87 b 843±97 676±38 TDNb* 393±236 a 360±30 b 741±87 588±43 Aldehydes acetaldehydea 3.43±0.29 a 2.82±0.22 b 1.51±0.11 1.24±0.00 Acids hexanoic acid 964±43 a 1225±50 b 1170±68 1235±29 octanoic acid 969±35 a 1128±37 b 1057±107 1223±33 decanoic acid 72.7±7.3 a 73.0±3.5 b 86.2±2.7 94.3±6.1 Volatile phenolic compounds guaiacol 21.6±5.0 28.6±11.2 12.6±3.1 15.8±3.5 2-methylphenol 3.45±0.75 2.97±0.33 1.75±0.60 0.69±0.46 3-ethylphenol 1.69±1.03 a 4.27±1.00 b 1.03±0.37 a 2.22±0.63 b

131

(Table A3.2 continued) 4-ethylphenol 1.08±0.08 0.92±0.04 0.89±0.19 a 2.32±0.58 b 4-ethylguaiacol 1.23±0.08 1.06±0.19 0.78±0.31 a 2.76±1.02 b Sulfur Containing Compounds methyl thioacetate 8.23±0.32 13.3±0.5 5.80±0.69 6.70±0.16 ethyl thioacetate nd 0.55±0.03 nd nd dimethyl sulfide 121±7 a 130±2 b 120±4 113±7 dimethyl disulfide 0.26±0.04 0.25±0.01 3.85±0.80 a 7.05±0.40 b dimethyl trisulfide 0.20±0.05 0.16±0.01 1.70±0.61 3.44±2.35 Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (student t-test at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis

132

Table A3.3 Composition of volatile compounds in Pinot noir wines from Winery I with cluster thinning treatments from 2013 and 2015 (ug/L)

Year 2013 2015 Cluster Thinning Treatment 0.5 cls 1.5 cls 1 cls 0.5 cls 1.5 cls 1 cls Straight-Chain Esters ethyl acetatea 151±6 a 94.0±1.5 b 180±13 c 94.9±1.2 a 93.2±1.4 a 120±1 b ethyl butanoate 184±8 a 189±2 a 149±4 b 175±1 177±0 166±8 ethyl hexanoate 177±7 a 172±17 a 133±4 b 205±6 214±17 176±10 ethyl octanoate 137±8 163±1 101±4 171±15 195±13 178±20 ethyl decanoate 13.7±0.2 a 12.4±1.5 a 8.7±0.4 b 49.1±3.4 48.6±5.0 45.4±3.0 hexyl acetate 1.55±0.78 2.48±1.31 1.63±0.04 0.18±0.25 nd 0.20±0.28 octyl acetate nd 1.33±0.04 0.68±0.04 0.73±0.18 nd 0.90±0.00 octyl butyrate 3.92±0.18 5.93±3.50 2.23±0.11 7.60±1.34 8.68±0.08 8.53±0.70 diethyl succinate 3640±268 4377±790 1626±230 2251±318 a 2826±409 a 5745±188 b 2-phenylethyl acetate 21.03±1.48 24.18±5.27 10.32±1.52 7.75±1.56 9.78±1.59 12.95±0.21 ethyl phenylacetate 22.2±1.4 24.5±4.3 9.9±1.4 6.6±0.9 8.6±1.1 9.8±0.3 Branched-Chain Esters isoamyl acetate 437±49 a 243±24 b 481±39 a 209±25 201±30 239±34 isobutyl acetate 47.0±4.2 58.8±2.4 41.5±3.2 46.2±4.8 51.2±2.8 57.0±1.3 ethyl isobutyrate 380±19 337±13 335±23 332±20 338±7 422±24 ethyl 2-methylbutanoate 41.9±0.9 a 36.3±2.7 b 37.8±0.8 ab 21.4±2.6 22.1±0.3 29.9±1.3 Alcohols propanola 87.5±1.3 a 46.4±1.1 b 50.7±1.3 b 44.4±1.7 a 39.9±0.5 b 42.6±0.4 ab isobutyl alcohola 65.7±2.1 a 47.8±0.7 b 67.0±4.8 a 46.9±1.1 47.9±1.0 47.4±0.3 isoamyl alcohola 339±23 a 246±4 b 386±31 a 176±7 179±2 180±2 1-hexanol 1210±47 a 1364±14 b 1399±81 b 1513±75 a 1563±58 a 1221±132 b 2-ethyl-1-hexanol 3.05±0.00 ab 2.65±0.39 a 4.70±0.57 b 2.72±0.26 2.38±0.25 2.45±0.09 133

(Table A3.3 continued) 1-octanol 354±9 a 392±10 a 293±23 b 197±12 a 192±7 a 155±15 b 1-octen-3-ol 3.47±0.21 3.03±0.25 2.38±0.32 4.00±0.23 a 3.63±0.10 ab 3.28±0.25 b benzyl alcohol 670±20 a 712±19 a 807±12 b 386±5 367±13 386±22 2-phenylethyl alcohol 43671±1121 a 44411±704 a 50180±1381 b 19021±559 20228±369 19382±629 Terpenoids linalool 9.93±1.03 nd 11.62±1.28 7.48±0.25 7.30±0.09 7.52±0.33 a-terpineol 25.7±0.8 22.4±1.3 27.6±0.9 13.2±1.7 13.3±1.1 14.7±0.5 β-citronellol 2.25±0.21 3.00±0.28 3.63±0.53 4.57±0.40 a 3.87±0.28 ab 3.68±0.18 b nerol 15.53±1.59 15.53±0.78 7.35±1.08 3.88±0.35 a 4.08±0.23 a 6.63±0.24 b geraniol 46.95±1.70 6.05±0.18 3.43±0.60 2.53±0.25 2.97±0.19 3.30±0.42

C13-Norisoprenoids β-damascenone 2.28±0.03 2.00±0.13 2.33±0.20 0.92±0.08 a 0.92±0.03 a 1.33±0.10 b β-ionone 0.65±0.00 a 0.48±0.03 b 0.73±0.08 a 0.20±0.00 0.20±0.00 0.25±0.00 vitispiraneb 115±6 95.2±25.8 86.2±3.4 46.5±4.0 50.0±2.7 56.8±5.6 TDNb 29.1±0.5 a 26.6±1.3 b 29.4±0.2 a 20.4±0.1 20.5±0.1 22.4±3.5 β-damascenone* 8.02±1.15 7.85±1.56 7.03±1.98 16.1±0.9 a 7.68±0.06 b 8.93±0.53 b vitispiraneb* 536±46 407±94 493±130 930±184 a 682±25 ab 622±48 b TDNb* 185±27 154±11 201±80 546±56 a 279±32 b 202±33 b Aldehydes acetaldehydea 4.21±0.13 a 2.62±0.05 b 4.00±0.17 a 2.65±0.03 a 3.32±0.10 b 1.75±0.06c Acids hexanoic acid 795±30 a 856±64 a 1076±25 b 1044±17 a 1008±18 a 894±45 b octanoic acid 512±35 a 500±57 a 713±44 b 887±60 a 881±26 a 779±19 b decanoic acid 19.7±1.5 16.3±0.2 18.9±2.9 95.8±8.3 a 91.1±6.5 ab 77.8±2.6 b Volatile phenolic compounds guaiacol 20.4±2.5 a 41.4±7.8 b 10.7±3.5 a 11.5±2.7 14.8±4.5 20.9±13.0 2-methylphenol 1.79±1.44 1.95±1.52 2.11±0.55 1.09±0.66 0.49±0.85 0.83±0.45 3-ethylphenol 0.23±0.06 0.41±0.20 0.31±0.21 0.55±0.45 0.04±0.04 0.33±0.29

134

(Table A3.3 continued) 4-ethylphenol 14.1±0.4 9.07±7.48 5.53±4.27 2.42±3.63 9.05±15.63 20.4±31.0 4-ethylguaiacol 11.2±0.8 ab 15.0±1.3 a 8.27±3.28 b 0.37±0.27 0.03±0.06 0.46±0.80 Sulfur Containing Compounds methyl thioacetate 9.50±0.04 a 5.38±0.51 b 1.60±0.18 c 4.48±0.22 5.72±0.39 5.23±0.04 ethyl thioacetate 0.43±0.02 nd nd nd nd nd dimethyl sulfide 161±6 a 116±4 b 142±4 c 186±10 a 194±3 ab 232±27 b dimethyl disulfide 0.41±0.03 a 0.20±0.02 b 0.22±0.01 b 0.30±0.03 ab 0.28±0.01 a 0.53±0.04 b dimethyl trisulfide 0.25±0.05 a 0.15±0.00 b 0.14±0.03 b 0.17±0.01 0.17±0.01 0.19±0.02 Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (Tukey HSD at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis

135

Table A3.4 Composition of volatile compounds in Pinot noir wines from Winery J with cluster thinning treatments in 2013 (ug/L)

Year 2013 Cluster Thinning Treatment 1 cls 2 cls Straight-Chain Esters ethyl acetatea 153±36 208±102 ethyl butanoate 232±8 244±20 ethyl hexanoate 353±25 349±31 ethyl octanoate 400±50 383±28 ethyl decanoate 81.6±16.0 a 65.7±3.7 b hexyl acetate 2.73±0.44 2.24±1.03 octyl acetate 1.33±0.62 0.97±0.67 octyl butyrate 20.7±5.6 a 14.7±1.0 b diethyl succinate 6086±822 5988±667 2-phenylethyl acetate 14.4±1.8 a 11.8±1.2 b ethyl phenylacetate 8.30±0.98 7.71±0.90 Branched-Chain Esters isoamyl acetate 412±209 564±266 isobutyl acetate 55.1±5.8 53.9±3.4 ethyl isobutyrate 187±12 181±10 ethyl 2-methylbutanoate 20.9±0.7 a 18.6±1.3 b Alcohols 497732±111573 654647±355173 propanola 28.9±16.0 29.9±46.9 isobutyl alcohola 81.6±19.6 115±58 isoamyl alcohola 349±88 475±251 1-hexanol 1590±197 1712±219 2-ethyl-1-hexanol 4.54±2.12 4.29±1.40 1-octanol 236±23 254±19 136

(Table A3.4 continued) 1-octen-3-ol 3.02±0.46 a 3.56±0.26 b benzyl alcohol 431±16 461±31 2-phenylethyl alcohol 35786±2007 a 32137±1768 b Terpenoids linalool 1.83±2.84 1.55±2.70 a-terpineol 19.8±1.6 21.2±1.1 β-citronellol 1.68±1.06 0.93±0.73 nerol 24.5±7.8 a 34.9±5.5 b geraniol 8.65±5.73 a 19.7±2.9 b

C13-Norisoprenoids β-damascenone 3.24±0.28 a 3.68±0.27 b β-ionone 0.60±0.08 a 0.69±0.06 b vitispiraneb 84.0±9.6 a 102±7 b TDNb 32.2±1.8 34.1±1.2 β-damascenone* 18.9±3.6 a 14.2±1.6 b vitispiraneb* 797±190 a 454±85 b TDNb* 473±71 a 276±61 b Aldehydes acetaldehydea 4.97±1.12 5.25±2.28 Acids hexanoic acid 1907±41 2007±128 octanoic acid 2012±99 1988±46 decanoic acid 190±21 a 163±7 b Volatile phenolic compounds guaiacol 23.5±13.5 28.5±2.0 2-methylphenol 1.50±1.00 1.62±1.04 3-ethylphenol 0.36±0.19 0.90±1.05 4-ethylphenol 3.78±3.31 a 0.69±0.14 b

137

(Table A3.4 continued) 4-ethylguaiacol 1.26±1.32 0.72±1.02 Sulfur Containing Compounds methyl thioacetate 1.51±0.27 1.25±0.15 ethyl thioacetate 0.61±0.14 0.59±0.11 dimethyl sulfide 1.80±0.15 a 1.61±0.08 b dimethyl disulfide 0.07±0.01 0.09±0.03 dimethyl trisulfide 0.10±0.03 0.14±0.05 Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (student t-test at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis

138

Table A3.5 Composition of volatile compounds in Pinot noir wines from Winery K with cluster thinning treatments from 2014 to 2015 (ug/L)

Year 2014 2015 Cluster Thinning Treatment 1 cls 1.5 cls No Thin 1 cls 1.5 cls No Thin Straight-Chain Esters ethyl acetatea 133±3 134±5 149±7 50.8±2.2 a 55.8±1.3 b 52.4±1.1 ab ethyl butanoate 260.7±24.3 261±24 222±4 321±20 a 274±8 b 270±9 b ethyl hexanoate 375.3±35.0 375±35 298±15 566±14 544±52 537±40 ethyl octanoate 319.9±19.2 320±19 281±10 465±11 437±4 440±24 ethyl decanoate 62.6±9.3 62.6±9.3 56.4±0.7 145.9±0.1 125.8±0.9 113.8±6.7 hexyl acetate 4.6±0.5 4.58±0.53 2.60±0.07 4.78±1.45 4.45±0.85 4.33±0.32 octyl acetate nd nd nd nd nd 0.65±0.07 octyl butyrate 15.5±1.8 15.45±1.84 16.10±1.13 9.78±0.46 10.02±0.64 9.78±0.10 diethyl succinate 1505.1±21.8 1505±22 1632±157 3557±93 2709±181 2696±74 2-phenylethyl acetate 10.3±0.1 10.30±0.07 11.45±0.99 15.00±0.64 15.63±0.55 14.78±1.60 ethyl phenylacetate 4.3±0.2 4.3±0.2 5.4±0.7 7.1±0.5 6.8±0.5 7.0±1.0 Branched-Chain Esters isoamyl acetate 557±40 704±58 694±62 289±13 a 238±7 b 264±26 ab isobutyl acetate 41.2±2.1 41.2±2.1 42.5±5.7 36.7±4.2 35.1±3.2 33.7±1.5 ethyl isobutyrate 91.7±3.1 92±3 85±4 67±2 76±1 63±6 ethyl 2-methylbutanoate 10.7±0.3 14.2±1.2 12.0±0.4 10.4±0.6 10.5±0.2 12.2±1.1 Alcohols propanola 78.0±1.6 73.8±1.6 55.3±20.7 51.2±1.8 44.2±0.2 39.7±1.2 isobutyl alcohola 70.3±2.4 64.2±1.3 72.2±5.2 34.4±2.2 36.1±0.6 35.2±1.1 isoamyl alcohola 377±22 348±2 383±48 185±7 184±4 181±4 1-hexanol 996.0±46.5 996±46 1188±48 3051±352 3047±123 2934±197 2-ethyl-1-hexanol 2.1±0.3 2.07±0.31 1.47±0.13 2.40±0.00 2.05±0.28 2.25±0.33 1-octanol 141.5±3.4 142±3 145±5 106±5 118±3 103±4 139

(Table A3.5 continued) 1-octen-3-ol 1.4±0.1 1.40±0.07 1.23±0.18 nd nd nd benzyl alcohol 641.5±6.3 642±6 714±10 1494±38 1421±43 1389±71 2-phenylethyl alcohol 31053.9±239.6 31054±240 35742±1029 38933±1524 40171±1147 38890±1580 Terpenoids linalool 7.4±0.0 7.43±0.04 10.15±0.73 20.72±0.88 19.58±0.25 18.97±0.64 a-terpineol 35.7±0.4 35.7±0.4 17.7±0.8 12.3±1.1 14.7±1.2 13.3±0.8 β-citronellol 6.9±0.1 6.85±0.14 5.65±0.33 12.27±0.83 11.28±0.55 10.32±1.03 nerol 9.4±0.5 9.43±0.53 10.13±1.24 11.95±0.35 13.17±0.86 11.72±0.46 geraniol 2.5±0.3 2.53±0.32 2.30±0.49 5.92±0.32 a 5.68±0.33 a 4.62±0.34 b

C13-Norisoprenoids β-damascenone 2.9±0.1 2.85±0.14 2.60±0.07 4.70±0.44 5.33±0.44 5.15±0.20 β-ionone 0.25±0.00 b 0.25±0.00 b 0.45±0.05 a 0.43±0.03 0.47±0.06 0.45±0.00 vitispiraneb 40.3±0.6 36.5±5.3 32.7±7.7 13.6±7.8 18.9±1.0 20.1±3.7 TDNb 21.6±1.9 25.2±1.9 21.6±1.6 9.15±1.73 7.77±0.53 9.58±1.70 β-damascenone* 21.5±3.5 23.8±1.1 22.1±0.4 14.8±0.2 a 17.1±0.7 b 16.4±0.9 ab vitispiraneb* 834±196 806±207 915±25 428±96 365±77 293±19 TDNb* 331±21 368±14 381±73 142±64 148±80 148±78 Aldehydes acetaldehydea 4.23±0.22 4.82±0.26 3.55±1.18 3.06±0.42 2.39±0.06 2.95±0.06 Acids hexanoic acid 1732±22 1732±22 1709±55 3745±40 3682±78 3622±231 octanoic acid 1638±66 1638±66 1772±33 3841±58 3851±129 3799±202 decanoic acid 174.7±14.7 174.7±14.7 184.5±3.9 321.5±4.3 a 372.2±17.0 b 301.5±12.8 a Volatile phenolic compounds guaiacol 11.1±0.9 12.3±8.9 18.8±2.8 10.0±2.6 15.3±10.0 12.6±3.3 2-methylphenol 1.79±0.25 ab 1.35±0.32 b 2.07±0.29 a 1.46±0.62 1.68±0.28 1.27±0.12 3-ethylphenol 0.25±0.18 0.19±0.22 0.25±0.28 0.64±0.27 0.52±0.37 0.54±0.57 4-ethylphenol 0.43±0.56 0.97±0.82 0.54±0.26 1.66±1.02 0.61±0.37 0.57±0.50

140

(Table A3.5 continued) 4-ethylguaiacol 0.53±0.17 0.47±0.25 0.25±0.25 0.60±0.53 0.34±0.58 0.09±0.16 Sulfur Containing Compounds methyl thioacetate 0.2±12.9 13.3±0.5 14.0±3.5 11.4±0.5 9.99±0.69 9.64±0.91 ethyl thioacetate 0.79±0.19 0.92±0.04 0.91±0.17 0.69±0.09 nd 0.71±0.03 dimethyl sulfide 162±13 160±2 191±27 266±7 a 238±6 b 256±6 a dimethyl disulfide 0.16±0.79 0.13±0.02 0.19±0.05 0.30±0.02 0.28±0.03 0.19±0.00 dimethyl trisulfide 0.19±0.00 0.16±0.02 0.21±0.04 0.18±0.00 0.21±0.01 0.16±0.03 Mean ± SD presented (n=3). nd: not detected. Different letters indicating differences in means (Tukey HSD at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis.

141

Table A3.6 Composition of volatile compounds in Pinot noir wines from Winery L with cluster thinning treatments in 2015 (ug/L)

Year 2015 Cluster Thinning Treatment 1 cls 1.5 cls 2 cls estate Straight-Chain Esters ethyl acetatea 78.8±9.0 a 81.5±0.5 a 58.8±1.1 c 98.2±1.5 b ethyl butanoate 180±3 ab 194±10 a 163±9 a 231±11 c ethyl hexanoate 234±15 ab 238±16 a 145±21 b 323±25 a ethyl octanoate 188±5 a 162±5 a 92±6 b 177±8 a ethyl decanoate 48.9±4.7 b 36.3±4.8 a 25.5±2.0 ac 41.7±1.6 ab hexyl acetate 1.63±0.74 ab 0.58±0.81 a nd 4.63±0.25 ab octyl acetate nd nd 0.88±0.04 nd octyl butyrate 9.32±0.70 10.20±0.07 10.95±1.44 9.82±0.33 diethyl succinate 6096±554 4359±535 4885±13 4733±253 2-phenylethyl acetate 28.75±2.33 ac 23.20±2.40 a 17.53±0.25 ab 11.78±0.49 b ethyl phenylacetate 31.1±4.4 b 22.6±2.0 ab 25.8±0.7 ab 15.2±0.6 a Branched-Chain Esters isoamyl acetate 329±30 319±11 199±21 286±29 isobutyl acetate 77.6±4.1 ad 73.1±2.9 a 44.4±3.3 c 125.8±4.5 b ethyl isobutyrate 414±20 a 409±60 a 339±26 a 770±18 b ethyl 2-methylbutanoate 45.3±2.4 ad 48.8±1.6 a 36.2±1.2 c 62.7±1.8 b Alcohols propanola 16.4±2.1 16.8±1.0 15.6±0.8 18.6±0.8 isobutyl alcohola 69.8±8.4 a 68.8±0.7 a 75.7±2.4 a 112±1 b isoamyl alcohola 247±36 ab 255±1 ab 288±4 b 238±9 a 1-hexanol 3285±467 3027±410 2831±205 2774±97 2-ethyl-1-hexanol 1.70±0.00 2.58±0.25 2.15±0.35 1.85±0.14 1-octanol 121±5 218±0 97±13 162±2 142

(Table A3.6 continued) 1-octen-3-ol 5.48±0.66 ab 4.33±0.21 a 3.95±0.44 ac 6.48±0.31 b benzyl alcohol 1274±43 1228±35 1231±91 1153±40 2-phenylethyl alcohol 74109±2772 a 71905±2828 a 75943±3583 a 39190±927 b Terpenoids linalool 19.28±1.10 16.55±0.58 nd 17.70±0.28 a-terpineol 17.0±1.4 13.5±1.7 17.1±1.5 17.1±0.9 β-citronellol 5.88±0.32 5.18±0.63 2.37±0.33 2.25±0.07 nerol 24.45±1.74 19.28±1.36 31.18±0.39 24.45±0.21 geraniol 28.93±3.57 ab 21.48±3.16 a 45.85±4.60 c 34.85±1.25 b

C13-Norisoprenoids β-damascenone 5.87±0.32 5.73±0.47 4.93±0.25 5.38±0.31 β-ionone 0.62±0.03 ab 0.52±0.03 a 0.63±0.04 ab 0.65±0.05 b vitispiraneb 82.8±7.0 ab 57.9±8.7 a 101±24 b 110±5 b TDNb 25.6±22.4 11.8±1.0 13.6±1.3 15.6±0.6 β-damascenone* 14.6±0.8 a 16.8±0.9 b 13.7±0.5 a 11.7±0.3 c vitispiraneb* 373±10 a 384±26 a 388±11 a 280±63 b TDNb* 146±3 ab 133±3 a 158±14 b 140±9 ab Aldehydes acetaldehydea 3.90±0.38 4.32±0.36 2.66±0.11 4.92±0.11 Acids hexanoic acid 1460±76 1458±178 1632±113 1684±64 octanoic acid 1255±42 ab 1107±80 a 1071±67 a 1294±48 b decanoic acid 91.5±7.0 b 75.6±9.1 ab 65.8±5.4 a 82.1±7.5 ab Volatile phenolic compounds guaiacol 12.2±5.3 13.7±5.1 11.2±2.2 11.1±2.2 2-methylphenol 0.91±0.01 b 1.76±0.09 a 1.69±0.21 a 1.51±0.18 a 3-ethylphenol 0.53±0.29 0.56±0.13 0.59±0.36 0.50±0.35 4-ethylphenol 62.8±25.5 47.4±5.3 71.9±4.8 58.1±3.8

143

(Table A3.6 continued) 4-ethylguaiacol 35.2±11.7 23.8±7.8 36.4±2.5 33.4±1.3 Sulfur Containing Compounds methyl thioacetate 16.3±0.1 21.1±3.6 20.3±1.2 19.9±1.5 ethyl thioacetate 1.45±0.45 1.49±0.31 0.86±0.05 0.89±0.02 dimethyl sulfide 225±13 ab 233±4 a 207±3 b 211±10 b dimethyl disulfide 0.79±0.52 0.33±0.00 0.42±0.02 0.41±0.01 dimethyl trisulfide 0.25±0.02 0.24±0.04 0.34±0.03 0.32±0.00 Mean ± SD presented (n=3). nd: not detected. estate: unknown. Different letters indicating differences in means (Tukey HSD at α=0.05); a: concentration expressed as mg/L; b: vitispirane and TDN estimated on the basis of β-damascenone; *: after hydrolysis

144

Table A3.7 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery G.

Year 2013 Compound 0.5 cls 1 cls Straight-Chain Esters ethyl acetate 14.32 14.12 ethyl butanoate 10.42 11.30 ethyl hexanoate 15.17 16.30 ethyl octanoate 39.48 43.39 ethyl decanoate 0.14 0.15 hexyl acetate 0.00 0.00 octyl acetate 0.00 0.00 octyl butyrate 0.00 0.00 diethyl succinate 0.01 0.01 2-phenylethyl acetate 0.01 0.02 ethyl phenylacetate 0.05 0.06 Branched-Chain Esters isoamyl acetate 11.19 13.27 isobutyl acetate 0.03 0.03 ethyl isobutyrate 30.71 29.12 ethyl 2-methylbutanoate 2.31 2.44 subtotal 123.84 130.20 Alcohols propanol 0.84 0.85 isobutyl alcohol 2.21 2.41 isoamyl alcohol 10.92 13.97 1-hexanol 0.17 0.14 2-ethyl-1-hexanol 0.02 0.02 1-octanol 2.81 2.51 1-octen-3-ol 0.50 0.41 benzyl alcohol 0.00 0.00 2-phenylethyl alcohol 2.76 2.79 subtotal 20.22 23.09 Terpenoids 145

(Table A3.7 continued) linalool 0.35 0.43 a-terpineol 0.10 0.09 β-citronellol 0.03 0.00 nerol 0.03 0.04 geraniol 0.05 0.08 subtotal 0.56 0.65

C13-Norisoprenoids β-damascenone 80.67 96.67 β-ionone 3.33 0.00 subtotal 84.00 96.67 Aldehydes acetaldehyde 0.02 0.02 Acids hexanoic acid 2.13 2.70 octanoic acid 0.08 0.12 decanoic acid 0.79 1.01 subtotal 3.01 3.83 Volatile phenolic compounds guaiacol 2.02 2.16 2-methylphenol 0.07 0.04 3-ethylphenol 0.00 0.00 4-ethylphenol 0.01 0.01 4-ethylguaiacol 0.05 0.10 subtotal 2.10 2.22 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound.

146

Table A3.8 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery H.

Year 2014 2015 2014 Compound Half Crop 1 cls 1 cls No Thin Straight-Chain Esters ethyl acetate 22.29 38.76 26.35 13.40 ethyl butanoate 12.00 10.36 10.28 10.28 ethyl hexanoate 16.70 12.56 13.61 14.10 ethyl octanoate 50.13 30.15 47.00 48.14 ethyl decanoate 0.17 0.10 0.20 0.23 hexyl acetate 0.00 0.00 0.00 0.00 octyl acetate 0.00 0.00 0.00 0.00 octyl butyrate 0.00 0.00 0.00 0.00 diethyl succinate 0.03 0.02 0.02 0.03 2-phenylethyl acetate 0.01 0.02 0.01 0.01 ethyl phenylacetate 0.04 0.04 0.07 0.07 Branched-Chain Esters isoamyl acetate 21.65 23.74 16.83 10.81 isobutyl acetate 0.05 0.05 0.07 0.04 ethyl isobutyrate 29.89 19.28 23.31 20.23 ethyl 2-methylbutanoate 2.27 1.54 1.44 1.54 subtotal 155.23 136.64 139.20 118.87 Alcohols propanol 0.98 1.12 0.50 0.47 isobutyl alcohol 3.02 2.31 1.05 1.27 isoamyl alcohol 14.64 14.79 4.03 6.75 1-hexanol 0.13 0.13 0.16 0.17 2-ethyl-1-hexanol 0.02 0.02 0.02 0.02 1-octanol 2.28 2.08 2.34 2.26 1-octen-3-ol 0.31 0.32 3.70 3.53 147

(Table A3.8 continued) benzyl alcohol 0.00 0.00 0.00 0.00 2-phenylethyl alcohol 2.75 3.01 1.78 1.91 subtotal 24.12 23.78 13.58 16.38 Terpenoids linalool 0.43 0.30 0.31 0.00 a-terpineol 0.12 0.13 0.08 0.07 β-citronellol 0.02 0.02 0.01 0.00 nerol 0.03 0.04 0.02 0.02 geraniol 0.15 0.18 0.25 0.28 subtotal 0.75 0.67 0.66 0.38

C13-Norisoprenoids β-damascenone 31.50 44.00 51.50 53.33 β-ionone 0.00 2.50 0.00 2.22 subtotal 31.50 46.50 51.50 55.56 Aldehydes acetaldehyde 0.02 0.02 0.01 0.02 Acids hexanoic acid 2.31 2.68 2.52 2.91 octanoic acid 0.15 0.15 0.17 0.19 decanoic acid 0.96 1.22 1.17 1.24 subtotal 3.42 4.06 3.86 4.34 Volatile phenolic compounds guaiacol 2.27 3.01 1.32 1.66 2-methylphenol 0.11 0.10 0.06 0.02 3-ethylphenol 0.01 0.02 0.00 0.01 4-ethylphenol 0.00 0.00 0.00 0.01 4-ethylguaiacol 0.04 0.03 0.02 0.08 subtotal 2.40 3.12 1.38 1.70 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound

148

Table A3.9 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery I.

Year 2013 2015 Compound 0.5 cls 1 cls 1.5 cls 0.5 cls 1 cls 1.5 cls Straight-Chain Esters ethyl acetate 20.08 23.97 12.53 12.66 16.04 12.43 ethyl butanoate 9.22 7.45 9.46 8.73 8.31 8.85 ethyl hexanoate 12.64 9.47 12.28 14.61 12.58 15.25 ethyl octanoate 27.37 20.12 32.51 34.16 35.64 38.90 ethyl decanoate 0.07 0.04 0.06 0.25 0.23 0.24 hexyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 octyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 octyl butyrate 0.00 0.00 0.00 0.00 0.00 0.00 diethyl succinate 0.02 0.01 0.02 0.01 0.03 0.01 2-phenylethyl acetate 0.01 0.02 0.01 0.01 0.01 0.02 ethyl phenylacetate 0.09 0.04 0.10 0.03 0.04 0.03 Branched-Chain Esters isoamyl acetate 14.57 16.04 8.09 6.97 7.98 6.72 isobutyl acetate 0.03 0.03 0.04 0.03 0.04 0.03 ethyl isobutyrate 25.30 22.30 22.45 22.13 28.11 22.57 ethyl 2-methylbutanoate 2.33 2.10 2.02 1.19 1.66 1.23 subtotal 111.73 101.59 99.55 100.77 110.66 106.28 Alcohols propanol 1.75 1.01 0.93 0.89 0.85 0.80 isobutyl alcohol 1.64 1.68 1.19 1.17 1.19 1.20 isoamyl alcohol 11.31 12.86 8.21 5.88 5.99 5.97 1-hexanol 0.15 0.17 0.17 0.19 0.15 0.20 2-ethyl-1-hexanol 0.02 0.02 0.02 0.02 0.07 0.01 1-octanol 2.95 2.44 3.27 1.64 1.29 1.60 1-octen-3-ol 0.35 0.24 0.30 0.40 0.33 0.36 149

(Table A3.9 continued) benzyl alcohol 0.00 0.00 0.00 0.00 0.00 0.00 2-phenylethyl alcohol 3.12 3.58 3.17 1.36 1.38 1.44 subtotal 21.29 22.00 17.26 11.55 11.25 11.58 Terpenoids linalool 0.40 0.46 0.00 0.30 0.30 0.29 a-terpineol 0.10 0.11 0.09 0.05 0.06 0.05 β-citronellol 0.02 0.04 0.03 0.05 0.04 0.04 nerol 0.03 0.04 0.02 0.02 0.02 0.06 geraniol 1.57 0.11 0.20 0.08 0.11 0.10 subtotal 2.12 0.77 0.34 0.51 0.53 0.54

C13-Norisoprenoids β-damascenone 45.67 46.67 40.00 18.33 26.67 18.33 β-ionone 7.22 8.15 5.37 2.22 2.78 2.22 subtotal 52.89 54.81 45.37 20.56 29.44 20.56 Aldehydes acetaldehyde 0.02 0.02 0.01 0.02 0.01 0.04 Acids hexanoic acid 1.22 1.70 1.19 2.11 1.86 2.10 octanoic acid 0.04 0.04 0.03 0.19 0.16 0.18 decanoic acid 0.79 1.08 0.86 1.04 0.89 1.01 subtotal 2.05 2.81 2.08 3.35 2.91 3.29 Volatile phenolic compounds guaiacol 2.14 1.13 4.36 1.21 2.20 1.55 2-methylphenol 0.06 0.07 0.06 0.04 0.03 0.02 3-ethylphenol 0.00 0.00 0.00 0.00 0.00 0.00 4-ethylphenol 0.03 0.01 0.02 0.01 0.05 0.02 4-ethylguaiacol 0.34 0.25 0.46 0.01 0.01 0.00 subtotal 2.23 1.21 4.44 1.25 2.27 1.59 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound.

150

Table A3.10 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery J.

Year 2013 Compound 1 cls 2 cls Straight-Chain Esters 0.00 0.00 ethyl acetate 23.43 22.13 ethyl butanoate 11.58 12.21 ethyl hexanoate 25.25 24.94 ethyl octanoate 80.06 76.56 ethyl decanoate 0.43 0.33 hexyl acetate 0.00 0.00 octyl acetate 0.00 0.00 octyl butyrate 0.00 0.00 diethyl succinate 0.03 0.03 2-phenylethyl acetate 0.01 0.01 ethyl phenylacetate 0.03 0.03 Branched-Chain Esters 0.00 0.00 isoamyl acetate 17.31 15.14 isobutyl acetate 0.03 0.03 ethyl isobutyrate 12.44 12.06 ethyl 2-methylbutanoate 1.16 1.03 subtotal 171.77 164.51 Alcohols 0.00 0.00 propanol 0.68 0.08 isobutyl alcohol 2.35 2.27 isoamyl alcohol 13.48 12.33 1-hexanol 0.21 0.21 2-ethyl-1-hexanol 0.02 0.01 1-octanol 1.96 2.12 1-octen-3-ol 0.32 0.36 151

(Table A3.10 continued) benzyl alcohol 0.00 0.00 2-phenylethyl alcohol 2.56 2.30 subtotal 21.58041253 19.68728717 Terpenoids 0.00 0.00 linalool 0.00 0.00 a-terpineol 0.08 0.08 β-citronellol 0.02 0.01 nerol 0.08 0.12 geraniol 0.35 0.66 subtotal 0.52 0.87

C13-Norisoprenoids 0.00 0.00 β-damascenone 64.83 73.50 β-ionone 6.34 7.69 subtotal 71.18 81.19 Aldehydes 0.00 0.00 acetaldehyde 0.01 0.01 Acids 0.00 0.00 hexanoic acid 4.79 4.73 octanoic acid 0.38 0.33 decanoic acid 1.91 2.01 subtotal 7.08 7.07 Volatile phenolic compounds 0.00 0.00 guaiacol 2.47 3.00 2-methylphenol 0.05 0.05 3-ethylphenol 0.00 0.00 4-ethylphenol 0.01 0.00 4-ethylguaiacol 0.04 0.02 subtotal 2.52 3.05 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound.

152

Table A3.11 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery K.

Year 2014 2015 Compound 1 cls 1.5 cls No Thin 1 cls 1.5 cls No Thin Straight-Chain Esters ethyl acetate 17.76 17.83 19.88 6.77 7.43 6.98 ethyl butanoate 13.04 13.04 11.08 16.05 13.70 13.48 ethyl hexanoate 26.81 26.81 21.29 40.46 38.83 38.34 ethyl octanoate 63.97 63.97 56.30 92.92 87.39 88.09 ethyl decanoate 0.31 0.31 0.28 0.73 0.63 0.57 hexyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 octyl acetate 0.00 0.00 0.00 0.00 0.00 0.00 octyl butyrate 0.00 0.00 0.00 0.00 0.00 0.00 diethyl succinate 0.01 0.01 0.01 0.02 0.01 0.01 2-phenylethyl acetate 0.01 0.02 0.01 0.01 0.01 0.02 ethyl phenylacetate 0.02 0.02 0.02 0.03 0.03 0.03 Branched-Chain Esters isoamyl acetate 18.56 23.47 23.13 9.62 7.93 8.80 isobutyl acetate 0.03 0.03 0.03 0.02 0.02 0.02 ethyl isobutyrate 6.12 6.12 5.66 4.47 5.08 4.22 ethyl 2-methylbutanoate 0.59 0.79 0.67 0.58 0.59 0.68 subtotal 147.23 152.40 138.37 171.68 161.67 161.23 Alcohols propanol 1.56 1.48 1.11 1.02 0.88 0.79 isobutyl alcohol 1.76 1.61 1.80 0.86 0.90 0.88 isoamyl alcohol 12.58 11.59 12.77 6.18 6.14 6.03 1-hexanol 0.12 0.12 0.15 0.38 0.38 0.37 2-ethyl-1-hexanol 0.02 0.02 0.02 0.02 0.07 0.01 1-octanol 1.18 1.18 1.20 0.88 0.98 0.86 1-octen-3-ol 0.14 0.14 0.12 0.00 0.00 0.00 153

(Table A3.11 continued) benzyl alcohol 0.00 0.00 0.00 0.01 0.01 0.01 2-phenylethyl alcohol 2.22 2.22 2.55 2.78 2.87 2.78 subtotal 19.57 18.36 19.73 12.14 12.23 11.72 Terpenoids linalool 0.30 0.30 0.41 0.83 0.78 0.76 a-terpineol 0.14 0.14 0.07 0.05 0.06 0.05 β-citronellol 0.07 0.07 0.06 0.12 0.11 0.10 nerol 0.03 0.04 0.02 0.02 0.02 0.06 geraniol 0.08 0.08 0.08 0.20 0.19 0.15 subtotal 0.62 0.64 0.63 1.22 1.16 1.13

C13-Norisoprenoids β-damascenone 57.00 57.00 52.00 94.00 106.67 103.00 β-ionone 2.78 2.78 5.00 4.81 5.19 5.00 subtotal 59.78 59.78 57.00 98.81 111.85 108.00 Aldehydes acetaldehyde 0.02 0.02 0.01 0.02 0.01 0.04 Acids hexanoic acid 3.90 3.90 4.22 9.15 9.17 9.04 octanoic acid 0.35 0.35 0.37 0.64 0.74 0.60 decanoic acid 1.73 1.73 1.71 3.74 3.68 3.62 subtotal 5.98 5.98 6.30 13.53 13.60 13.27 Volatile phenolic compounds guaiacol 1.17 1.29 1.98 1.05 1.61 1.33 2-methylphenol 0.06 0.04 0.07 0.05 0.05 0.04 3-ethylphenol 0.00 0.00 0.00 0.00 0.00 0.00 4-ethylphenol 0.00 0.00 0.00 0.00 0.00 0.00 4-ethylguaiacol 0.02 0.01 0.01 0.02 0.01 0.00 subtotal 1.23 1.34 2.04 1.11 1.67 1.37 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound.

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Table A3.12 Odor Active Values (OAVs)a of Pinot noir wine volatiles produced from different treatments in Winery L.

Year 2015 Compound 1 cls 1.5 cls 2 cls estate Straight-Chain Esters ethyl acetate 10.50 10.87 7.84 13.09 ethyl butanoate 9.01 9.68 7.91 11.53 ethyl hexanoate 16.74 17.01 11.23 23.08 ethyl octanoate 37.65 32.41 18.44 35.32 ethyl decanoate 0.24 0.18 0.13 0.21 hexyl acetate 0.00 0.00 0.00 0.00 octyl acetate 0.00 0.00 0.00 0.00 octyl butyrate 0.00 0.00 0.00 0.00 diethyl succinate 0.03 0.02 0.02 0.02 2-phenylethyl acetate 0.01 0.02 0.01 0.01 ethyl phenylacetate 0.12 0.09 0.10 0.06 Branched-Chain Esters isoamyl acetate 10.98 10.64 6.64 9.53 isobutyl acetate 0.05 0.05 0.03 0.08 ethyl isobutyrate 27.58 27.30 21.84 51.35 ethyl 2-methylbutanoate 2.52 2.71 2.01 3.48 subtotal 115.45 110.98 76.21 147.77 Alcohols propanol 0.33 0.34 0.31 0.37 isobutyl alcohol 1.75 1.72 1.89 2.80 isoamyl alcohol 8.25 8.51 9.61 7.92 1-hexanol 0.41 0.38 0.41 0.35 2-ethyl-1-hexanol 0.02 0.02 0.02 0.02 1-octanol 1.01 1.82 0.84 1.35 1-octen-3-ol 0.55 0.43 0.41 0.65 155

(Table A3.12 continued) benzyl alcohol 0.01 0.01 0.01 0.01 2-phenylethyl alcohol 5.29 5.14 5.42 2.80 subtotal 17.60 18.35 18.92 16.27 Terpenoids linalool 0.77 0.66 0.00 0.71 a-terpineol 0.07 0.05 0.07 0.07 β-citronellol 0.06 0.05 0.02 0.02 nerol 0.03 0.04 0.02 0.02 geraniol 0.96 0.72 1.53 1.16 subtotal 1.89 1.53 1.64 1.98

C13-Norisoprenoids β-damascenone 117.33 114.67 98.50 107.67 β-ionone 6.85 5.74 6.94 7.22 subtotal 124.19 120.41 105.44 114.89 Aldehydes acetaldehyde 0.02 0.02 0.01 0.02 Acids hexanoic acid 2.99 2.64 2.54 3.08 octanoic acid 0.18 0.15 0.13 0.16 decanoic acid 1.46 1.46 1.68 1.68 subtotal 4.63 4.25 4.34 4.93 Volatile phenolic compounds guaiacol 1.29 1.45 1.18 1.17 2-methylphenol 0.03 0.06 0.05 0.05 3-ethylphenol 0.00 0.00 0.00 0.00 4-ethylphenol 0.14 0.11 0.16 0.13 4-ethylguaiacol 1.07 0.72 1.10 1.01 subtotal 1.46 1.61 1.40 1.36 a: Odor activity value calculated by dividing concentration by odor threshold value of the compound.

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