Vol. 12, No. 1 January - February, 1997

Bruce Zoecklein

Department of Food Science and Technology

VPI & SU

Blacksburg, VA 24061-0418

I. Secondary Metabolites 1

II. Viticulture Practices Influencing Flavor and Aroma Compounds 2

Mesoclimate 2

Soil Conditions 3

Light and Shade 3

Crop Load 3

Asynchronous berry, cluster or vine development 4

III. Current Research 4

IV. On Site Monitoring and Production Short Course 5

Grape-derived secondary metabolites are the principal sources of wine aroma, flavor, color and taste (Hardie et al., 1996). Most grape aroma/flavor compounds are present in the grape either as free volatiles, which may contribute directly to odor, or as non-volatile bound sugar conjugates (Fig.1). The free volatiles are a diverse group composed of potent aroma/flavor compounds such as monoterpenes, norisoprenoids, volatile phenols and methoxypyrazines, which collectively are responsible for varietal aroma/flavor. For example, monoterpenes contribute floral and fruity aromas while methoxypyrazines and some isoprenoids contribute herbaceous and earthy aroma 'notes'.

The bound sugar conjugates or glycoconjugates are nonvolatile and, for the most part, represent aroma/flavor precursors. They can undergo acid or enzyme hydrolysis, releasing free volatiles and potentially enhancing aroma (Williams et al., 1982). Glycosidically bound grape conjugates include a wide range of compounds which represent, in part, the potential aroma/flavor of a grape variety.

The glycosidation process occurs with the help of glycosyltransferase enzymes. This enzyme system catalyzes the transfer of carbohydrates to bind with free volatiles creating the bound or glycosidic form (Fig.1). The possible regulation (either genetically or biochemically) of the process which binds free volatile, odors aroma/flavor metabolites to the non-odor glycosidic form is an important area of plant research.

There is a tremendous interest in the possible manipulation of grape aroma/flavor. One approach may be to control the activity of glycosyltransferase enzymes which cause the binding of free volatiles to the sugar moiety. If we are able to increase the ratio of free to bound components we likely would observe an increase in aroma/flavor intensity. Another possible approach to manipulating grape aroma/flavor is to increase the quantity of glycosides, thus increasing the pool of potential aroma/flavor compounds. Indeed, it has been demonstrated that there is a positive correlation between the concentration of bound secondary metabolites and ultimate wine quality.

II. Viticulture Practices Influencing Flavor and Aroma Compounds

The production of wine grapes with desirable concentrations of secondary metabolites at harvest is the result of the convergence of two broad influences (Hardie et al., 1996). The first is the reproductive process that, through seed development, determines potential berry size. Because the skin contains a high concentrations of most secondary metabolites, the berry surface to volume ratio is important in determining the concentration of these metabolites. The second influence on the production of secondary metabolites comes from environmental factors and viticultural management that condition the genetic potential and influence berry growth. Viticulture conditions and practices that influence secondary metabolites include: macro and mesoclimate (Jackson and Lombard, 1993), regulation of water supply (Hardie and Martin, 1990), regulation of leaf area to crop ratio (Iland et al.,1993), and canopy management practices that increase light exposure of leaves and fruit (Smart and Robinson, 1991). Table 1, adapted from Jackson and Lombard (1993) outlines important environmental and viticultural practices influencing aroma/flavor compounds.

Mesoclimate: Among the viticultural options affecting the production of aroma/flavor components either directly or indirectly, mesoclimate (site climate) is considered one of the most important. Becker(1977) and others have shown that white wines produced from cool climates are fresher in aroma and have lower alcohol than their warm-climate counterparts. Warm regions tend to produce wines which have less varietal aroma and are harsher on the palate, due primarily to the increase in grape phenols. The belief that warm climates produce wines with less and/or different aroma/flavor is well accepted but has been difficult to quantify. Ewart (1987) compared cool and warm climate vineyard sites in south Australia and found that in the former, the terpene content (responsible for the floral aroma of White Riesling, Gewurztraminer, and Muscates) increased more slowly, but was in greater concentration at the end of the season. On the other hand, the concentration of methoxypryrazines, responsible in part, for the herbaceousness of some wines, may be undesirably high in fruit from cool climates. This is particularly true with certain cool climate varieties grown in dense canopies with excessive leaf and cluster shading.

The production of secondary metabolites is influenced by the temperature during stage 3 or the finial period of fruit maturation (Jackson and Lombard, 1993). Therefore, it has been suggested that the best variety for a site is one that matches the length of the growing season so that fruit maturation occurs during the portion of the season that is cool but warm enough to allow the fruit to continue to accumulate aroma/flavor.

Meso-climate has been divided into two general temperature zones, Alpha and Beta (Jackson, 1987). In Alpha zones maturity occurs just before the mean monthly temperature drops to 10 C (Jackson, 1991). Specifically, Alpha zones are those where the mean temperature during stage 3 ripening, for a particular variety, is between 9-15C. In warm climates the length of the growing season is more than adequate to ripen most grape varieties which, therefore, mature in the warm part of the season. In Alpha zones, day temperatures are moderate and night temperatures usually cool, creating desirable conditions for the development of secondary metabolites including aroma/flavor compounds.

On the other hand, in Beta zones the majority of grapes ripen well before temperatures begin to drop. Specifically, Beta zones are those with a mean temperature above 16C at the time of ripening for a particular variety. Thus, days and nights are still warm, and attainment of adequate Brix (either by sugar production or dehydration) is not a problem. In many Beta zones grape varieties with short, moderate and long seasons are grown. One value of this meso-climate classification is that it can allow a positive correlation between soluble solids and wine quality. This relationship is much more direct in Alpha, compared to Beta zones, a feature noted repeatedly in Virginia. Additionally, a more negative correlation exists between yield and quality in Alpha vs Beta zones.

Soil conditions. Soil conditions can influence secondary metabolites either directly or indirectly. Important factors include water holding capacity, degree of moisture stress and timing (Becker and Zimmerman, 1983). Holding water after varaison reduces vegetative growth and is reported to increase the perception of fruity aromas/flavors (McCarthy et al., 1987). In regions which experience frequent summer rains, such as Virginia, the water holding capacity of the soil may be an important factor influencing secondary metabolites.

Light and Shade. Excessive fruit and leave shading can affect the concentration of secondary metabolites directly and as a result of influencing berry size. For example, light exposure increases the production of glycosides and phenolics including anthocyanins, while decreasing the concentration of methoxypyrazines. Vines, the foliage and fruit of which already receive adequate exposure may not benefit and indeed may suffer from increased exposure.

Crop load. We know that a high fruit weight to leaf area ratio or fruit weight to pruning weight ratio can influence secondary metabolites, possibly directly but certainly indirectly. The indirect affects are changes in berry size and the rate of fruit maturation. In an ongoing study conducted by myself and Dr. T.K. Wolf, we have seen a consistent influence of crop level on the rate of fruit maturation and secondary metabolites (Zoecklein, et al., 1997).

Asynchronous berry, cluster or vine development. Asynchronous berry, cluster or vine development can be a major factor influencing secondary metabolites. A crop with asynchronous berries has a mixture of developmental stages, resulting in a proportion of berries with optimal qualities diluted by those that are inferior. The hypothetical effect of an asynchronous mix on the aroma/flavor profile is shown in fig. 2 adapted from Coombe and Iland (1987). In addition to the effects of sunlight exposure, asynchronicity may be enhanced by the varying leaf/fruit ratio of individual shoots and by fruit development on weak shoots, e.g., those that are less than 30 cm (Long, 1987).

III. Current Research

We have several research projects evaluating the relationship between vineyard management and secondary grape metabolites. Fig. 3 shows the effect of fruit zone leaf removal on Chardonnay glycosides and phenol-free glycoside at harvest. Selective leaf removal significantly influenced the glycoside content expressed as glycoside (M) and on a fresh fruit weight bases. Increased fruit exposure is reported to lower berry weight, although differences in berry weight were not seen in this study. A high skin-to-pulp ratio is believed to be an important feature in premium quality grapes because it provides an adequate concentration of secondary metabolites in the skins. Thus, a high glycoside content on a weight basis for non-shriveled berries may indicate a potential for high quality wines. While phenolic glycosides are important color and structural components of wines, their impact on aroma and flavor is minimal. The goal was to reduce the phenolic glycoside content, leaving a remaining pool of glycosidically bound aroma and flavor precursors such as norisoprenoids and terpenes. Leaf removal generally had no effect on the total phenol, flavonoid phenol and hydroxycinnamic acid content of the fruit (data not shown). At harvest, the phenol-free glycosides per 100 grams was 20% higher in leaf pulled vs. control fruit. The relative increase in leaf-pulled phenol-free glycosides per fresh fruit weight was similar to the increase noted for total glycosides. At harvest, Chardonnay phenol-free glycosides averaged 66.5% of the total glycoside content. There is ample evidence that the direct stimuli of fruit by light can affect the content of secondary metabolites such as colored and non-pigmented phenols.

Several grapevine modification studies that have reported increases in canopy light interception have also reported increased photosynthetic efficiency of the remaining leaves. Increases in secondary metabolites would be expected to accompany increases in photosynthetic activity. Possible increases in photosynthesis were not consistently reflected in increased Brix or sugar per berry from leaf pulled vines, in this study. Leaf removal influenced canopy light interception and fruit temperature. Berries exposed to direct sunlight were 11.5C + 4.8C and 3.1C + 3.0C warmer than the air temperature for exposed and shaded berries, respectively.

Secondary metabolites may also be influenced by changes in the pattern of assimilate movement. The increase in glycoconjugates noted in this study may be the result of reducing both cluster shading and intra-plant competition through removal of non-functional or low efficiency leaves, as suggested by Reynolds and Wardle, (1989). Older basal leaves, which were removed, have photosynthesis rates 1/3 of recently expanded leaves.

Varietal grape aroma and aroma intensity are needed for high quality wines. While this study demonstrated the influence of leaf removal on increasing grape glycosides, these products themselves have no direct and immediate aroma flavor value. However, there is some reclamation of aroma/flavor by hydrolysis. Therefore, the increase in glycoconjugates represents, in part, an increase in the pool of potential aroma components. This research was made possible through a grant from the Virginia Winegrowers Advisory Board.

IV. On Site Monitoring and Production Short Course

A three-day monitoring and production short course is being planned for June 7, 8, and 9, 1997 and will be held in northern Virginia. The objective of this program is to provide an intensive production and on site monitoring based program to improve stylistic wine production and quality. This program is designed to increase the in-house monitoring performed by the industry and to discuss and encourage the implementation of new technologies.

The program will involve demonstrations, discussions, and sensory evaluation. This workshop will be for those who have experience with routine winery activities and are seeking advanced knowledge. A supplemental section will be scheduled at the beginning of this event for those who would like answers to various basic methodologies.

The workshop is for winemakers interested in receiving an integrated package of information covering such topics as: grape and wine aroma, flavor and phenol management; what's 'hot' in fermentation and problems along the way; strategies for finishing bottling and corks; related on-site monitoring (analytical procedures).

This program will be cosponsored by VPI&SU and the Viticulture and Enology Research Center, CSU-Fresno and will be a regional meeting of the American Society of Enology and Viticulture. Instructors will include Bruce Zoecklein, Department of Food Science and Technology, VPI&SU; Barry Gump and Ken Fugelsang, Viticulture and Enology Research Center, California State University, Fresno. Registration material will be sent to each producer.

Table 1: Effect of Environmental and Viticultural Practices on Grape

Secondary Metabolites at Stage 3 Development

Meso Soil Canopy Crop

climate conditions management load

High

aroma/flavor night temp. deficit exposed canopy moderate

5-15C, moisture leaf layers 3 <8kg/kg

mean temp.

9-29C

High

herbaceousness excessive excessive shade

moisture > 3 leaf layers

< 40% cluster exp.

High

Phenols/ night temp. deficit > 60% cluster moderate

anthocyanins 5-15C, moisture exp. <8kg/kg

mean temp.

9-29C