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 Pinot noir Wines Produced from Different Crop Levels
Abstract approved:
______
Michael C. Qian
The production of high-quality wine is an important target for wineries worldwide, and low crop level (yield) has been one vineyard 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 taste intensity by raising the content of quality-important compounds like esters 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 harvest, 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 vintage year. For wine phenolic compounds, the influences of cluster thinning on major phenolic compounds were observed in six wineries but various with the vintages 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 winery 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 Viticulture 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 monoterpene alcohols in grapes...... 5
Figure 1.4 Chemical structures of carotenoid-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, alcohol 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 aroma compound production, indicating known metabolic linkages ...... 19
Figure 1.13 AATase enzyme biosynthetic pathway for ester 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 vineyards 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 Vitis 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 terroir (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 monoterpenes, 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 oak-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. Terpenes
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 terpene compounds to wine aroma, especially Muscat grapes and wines (Barbera, 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, linalool, geraniol, 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. Merlot (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 carotenoids 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), Riesling 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 rose oil (Demole, 1970). These compounds are described as honey-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, Cabernet Sauvignon 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 white wine matrices (Sacks, Gates, Ferry, Lavin, Kurtz, & Acree, 10
2012). TDN concentrations of 1.3±0.8 µg/L in many varietals, including Pinot noir, Pinot Gris and Sauvignon blanc 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, Chardonnay, 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 veraison resulted in increased carotenoid levels, while exposure after veraison increased norisoprenoid levels in Syrah (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 Concord grape (Vitis labrusca) (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 Tannat 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 winemaking 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, Fern ndez,
Peña, Escudero, & Cacho, 1995; Iyer, Sacks, & Padilla Zakour, 2010) and many of them are thought to be responsible for “green” aromas in wines (Kotseridis & Baumes, 2000;
Schwab, Davidovich Rikanati, & 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 varietal 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, Davidovich Rikanati, & 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 maceration, 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 sulfur 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, Fiez Vandal, 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 acids in wine 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 thiols (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, garlic, onion 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 red wine 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 wine color 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 pressing 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 Grenache and Tempranillo 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 canopy 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 Nebbiolo, Cabernet franc, 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 Carignan 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.
44
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
46
(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.
47
Chapter 3 VOLATILE COMPOSITION OF PINOT NOIR WINES PRODUCED FROM DIFFERENT CLUSTER THINNING LEVELS
Jingwen Li, Patricia A. Skinkis, and Michael C. Qian,
48
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
49
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 (Mendez Costabel, 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.
50
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