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Effect of viticultural and winemaking practices on the phenolic composition of grapes and wines Part II
Key words: phenolic maturity, phenol composition, viticultural and winemaking practice
Hierdie is 'n populêre samevatting van `n volledige literatuurstudie oor die onderwerp deur Anita Oberholster. Die volledige studie "The influence of viticultural and winemaking practices on the phenolic composition of grapes, wine and the resulting mouth-feel implications" is beskikbaar by:
SAWIS Bedryfsbiblioteek, Posbus 238, Paarl,7629 Tel: 021-8075756 Faks: 021-8722354 library@sawis.co.za
Phenolic compositional changes in grapes during ripening
The concentration, nature and structure of tannins vary with grape maturity but also with the type of technology used during winemaking. The variables traditionally used for determining grape ripening, sugars and acidity, are known as industrial maturity, while tartaric acid and phenolic compounds are associated with technological maturity. Technological maturity is determined according to the optimum phenolic composition for obtaining a specific wine style.
The hydroxycinnamic tartaric acids decrease in concentration, while the hydroxycinnamates increase over the ripening period on a per berry basis (Fernández de Simón et.al., 1992). They are broken down if the grapes become overripe. The tannin concentration in grapes increases accordingly, although it is already fairly high at colour change. This pattern is valid for all grape varieties and most vineyard conditions, but the accumulation of anthocyanins and the maximum value obtained vary widely according to the environment and climate.
In seed extracts, the tannin concentration generally decreases after colour change, as the grapes ripen. In certain cases, the decrease occurs at an earlier stage, before colour change, and the concentration then remains relatively constant throughout the ripening period. Others have found an increase in the degree of polymerisation of the procyanidins and an average increase in concentration of the monomers after veraison (Kennedy et.al., 2000a). The tannins had a relatively low degree of polymerisation at colour change that increased during ripening, with the quantity of dimers and trimers decreasing by 90%, while Katalinic (Katalinic and Males, 1997) found that seed maturation was characterised by a relative enrichment in dimeric compared to monomeric flavanol forms. Galloyation decreased from 31 to 22% from before to after veraison and the extractable polyphenol amount decreased by 64% (Kennedy et.al., 2000b).
Seeds of Shiraz berries underwent distinct visual changes (see Picture 1) during development from 6.9 to 20 øBrix. Initially seeds were plump, bright green and pliable, but from veraison they changed, shrinking and becoming hard and brown. Hardening of the seed coat with a change in colour makes the polyphenols less extractable. After 20 øBrix they changed very little. Ripe grape seed tannins are described as soft, round, velvety and silky, while those from unripe seeds are described as hard, green, harsh and aggressive.
The more complex skin tannin varies little in its degree of polymerisation during ripening. A high concentration of anthocyanins in the skin is mostly accompanied with a high concentration of skin tannins. A decline in skin-to-berry ratio during fruit ripening suggests that the skins become thinner towards maturity. The tannin concentration in the stalks is very high at colour change, and little variation occurs during ripening.
Skins contain all anthocyanins and variable portions of the flavanols, and all of the small amount of flavonol glycosides. The seeds contain the rest of the flavanol monomers and a large portion of the berry's oligomeric and polymeric procyanidins. In general, providing the grapes get ripe, cooler conditions give more tannin as well as more anthocyanins. A large amount of contradictory information has been obtained and there is a need to determine whether this is introduced by differences in vineyard and climatic conditions, the varieties investigated or inaccurate experimental procedures.
Determination of phenolic maturity
Glories (Glories, 1984) uses the extractability assay as a indicator of optimal ripeness. A sudden decrease in this assay will indicate breakdown of the cellular structure of the skin and the beginning of over ripeness. The extractability assay is measured as the difference in colour extracted from skins at wine pH and pH 1. Low pH breaks down the cell structure, releasing the phenolics and is thus an indication of maximum extraction. Grape skin tasting is another general method used in the vineyard where flavours are mostly assessed. This method should be optimised to define clear descriptors for tannin development. Methods to assess the 280 nm versus 520 nm absorbing components, including spectrophotometric measurements and HPLC, are important. Enologists agree that this ratio has an important influence on colour stability and aging potential. Generally a ratio of 4:1 (tannin:anthocyanin) is seen as favourable although there is no published scientific data to support this. Following the 420 nm absorbance changes during ripening might proof important. If the skins of grapes start to deteriorate during over ripeness, an increase in the 420nm absorbance with a simultaneous decline in anthocyanin concentration (520 nm absorbance) could indicate possible oxygen induced polymerisation. The flavonol concentration (maximum absorbance at 365 nm), especially the quercetin derivatives, an indication of co-pigmentation potential, may proof important for colour extraction and stabilisation. Seeds also need to be investigated, as unripe seeds can contribute undesirable mouth-feel attributes to the wine and optimal ripeness for seed and skin tannins may not occur simultaneously. Some seeds stay green even in overripe fruit, while others mature weeks before optimal skin ripeness. Mouth-feel descriptors describing the optimal ripeness of seeds should be developed with a standardised extraction method.
Effect of the environment and vine management on the phenolic composition of grapes and wine quality
Designing the wine in the vineyard by understanding both the quantitative and qualitative changes occurring in grape phenols as a result of vineyard management practices is the new focus. Variables such as climate (macro-, meso- and micro-), particularly light exposure to the fruit, fruit maturity (including berry softness), berry size, fruit-to-leaf ratio, and moisture stress, affect the grape phenol composition.
The effect of the climate and environment
The general effect of climate is known, mild to cool and wet winters followed by warm springs, then warm to hot summers with little precipitation provide adequate growth potential and increase the likelihood of higher wine quality. Therefore, there is an optimum seasonal climate regime that contributes greatly to the overall quality of a given vintage, with the most important developmental stages being d‚bourrement (budburst), floraison (flowering), veraison (colour change and maturation ascent), and harvest (grape maturity). Rainfall during physiologically important periods (flowering and maturation) tends to decrease crop production, while the interactions between the local climate, soil, and site location (termed the 'terroir' by the French) play a varied role in the composition of grapes and yield of the grapevines.
Grape ripening is affected by a number of environmental factors and of these, light and nitrogen are of particular importance. High rates of nitrogen supply delay the accumulation of phenolic compounds, particularly the flavonols and anthocyanins, in the grapes. The exact nature of the vine's response to nitrogen is not clear as the effect is partly restored by increased light intensity (Keller et.al., 1999).
Irrigation and canopy management
Viticultural practices such as irrigation and canopy management are designed to control vine vigour and yield, improve fruit ripening and improve colour development. There have been numerous studies on the effects of water stress on berry growth and ripening; see reviews by Williams (Williams and Matthews, 1990) and Smart (Smart, 1985). Water deficit during the period after flowering resulted in the greatest reduction in berry weight for Shiraz compared with that of well-watered vines especially in years with high temperature summation (Ginestar et.al., 1998). This resulted in an increase in the concentration of anthocyanins and total soluble phenolics. In contrast, water deficit after veraison has only a minor effect on berry weight at maturity and berries are insensitive to water deficit during the month before harvest (McCarthy, 1999).
The extraction of anthocyanins during fermentation was greater from pre-veraison irrigation cut-off fruit, however, the loss of anthocyanins at the end of fermentation was also greater, thereby cancelling out differences in the concentration of anthocyanins attributable to the irrigation treatment (Sipiora and Gutierrez, 1998). The compositional differences from the previous studies were not investigated during winemaking or maturation.
Partial root zone drying (PRD) is the irrigation method that is showing a lot of promise (Dry et.al., 2000). In practice this means applying water to one side of the vine for 10 to 15 days and then changing to the other side. Experiments have also shown that the dry roots are maintained in a healthy condition by water supplied to them from the wet roots. It has been a consistent feature that there was no significant reduction in yields or berry size due to PRD treatment even though the irrigation amount was halved. The reduction in canopy density that results from PRD appears to be a likely cause of higher berry anthocyanins and phenolics than controls. When bunch exposure of the controls was increased by a combination of basal leaf removal and training system (the vines of both treatments were converted to the Smart Dyson trellis), there were no significant differences in fruit composition parameters. The potentially-useful technique of partial root zone drying, needs to be applied with great caution and understanding so as to prevent shock and stress to the vine in the last two to three weeks of ripening.
From the studies above, it is clear that it is not possible to investigate the influence of irrigation treatments on the phenolic compositional changes of grapes in isolation. The resulting change concerning berry weight and canopy density, influencing the level of sun exposure is one of the most important contributions to phenol maturation as discussed below.
In South Africa it often happens that vines are excessively vigorous, resulting in too dense canopies, which in turn has a negative effect on the quantity and quality of grapes produced. Archer and Strauss (Archer and Strauss, 1989) found that an increase in shading significantly decreased the skin colour of Cabernet Sauvignon, which is in agreement with the findings of wine tasters that the wine quality was reduced in proportion to the degree of shading. The difference between the effects of leaf shading and cluster shading on grape composition was investigated by Morrison and Noble (Morrison and Noble, 1990). They found that shaded bunches caused a reduction in the phenol and anthocyanin concentrations, while shading of the leaves caused a delay in berry growth and sugar accumulation. In warm regions, grapes hanging in direct sunlight become up to ten degrees Celsius warmer than the ambient temperature. Bergqvist found that the anthocyanin concentration in grapes increased linearly as sunlight exposure increased, up to exposure exceeding 100 mmol m-2 sec-1 (Bergqvist et.al., 2001). It has been noted that colour improves with greater light exposure, but due to the inherent dangers of heat, grapes from warm to hot areas cannot be exposed to the same degree as in the cooler regions. Vineyards should therefore be judged on an individual basis, with the designed canopy management system complementing the specific climatic conditions.
Cluster sun exposure appears to be the primary factor determining quercetin-3-glucoside (flavonol) levels in grapes and wine (Haselgrove et.al., 2000), with high concentrations in wines from warmer climates. In wines made from Pinot noir clusters from three different sun-exposure levels: shaded, moderately exposed and highly exposed; the concentration of quercetin glycosides increased respectively three and eight times with increase in sun exposure, while the level of quercetin aglycone also increased. The magnitude of the flavonol response to sun exposure seemed large enough to affect wine composition and quality. Wines from highly and moderately exposed cluster positions had higher total anthocyanin levels than those from shaded clusters, while wines from highly exposed clusters had 40% greater polymeric anthocyanin levels than those from shaded treatments (Price et.al., 1995). It appears that the anthocyanin metabolism responds to changes in both light and temperature conditions. Studies by Pirie suggest that the optimum temperature for the enzymes involved in the anthocyanin biosynthetic pathway is between 17 and 26§C (Pirie and Mullins, 1977). As a guide to the degree of openness, scientific studies and opinion would suggest that a desirable canopy for vines grown in hot climatic conditions is one where bunches are moderately exposed.
The caftaric acid, catechin, and epicatechin concentrations in wine were inversely related to cluster sun exposure. The low levels of caftaric acid in wines from sun-exposed clusters appeared to be related to hydrolysis of the tartaric ester, with wines from highly sun-exposed clusters having 50% more caffeic acid than moderate and 130% more than shaded clusters (Price, 1994). It is clear from the examples mentioned that sun exposure has a significant effect on the phenolic composition of grapes.
Crop thinning has become an increasingly common practice in Australian vineyards. However, there is little scientific basis to what a vine can carry - usually long experience of the vineyard and an understanding of the vine's capacity is the best guide to an appropriate cropping level. As a rough rule of thumb, approximately eight to 10 leaves per shoot (average length 1 to 1.2 metre) are required to ripen two bunches per shoot in a warm to hot climate, 12 to 15 leaves per shoot in a cooler climate and more than 15 leaves in a cool climate (Archer, 2002). If it is necessary to remove crop to allow the vine's canopy to cope with and ripen the remaining crop, then those bunches should be cut off prior to veraison. When done at this stage, the vine continues to push its photosynthates into the leaves and growth of leaf and shoot is encouraged. If the decision is left too late, until after veraison, then the vine continues to develop its shoots in the similar way to when all fruit was present. Shoot thinning is necessary for young vines that tend to over crop, and should be addressed early in the growing season while the shoot are only 5-10 cm long, and too much energy in growing those shoots and potential berries has not been expended (Davidson, 2002). Controlling the leaf area/crop weight will improve berry colouration and accelerate ripening (Kliewer and Dokoozlian, 2000).
This demonstrates the importance of pruning and other factors that control vine vigour. Wine quality parameters, such as wine colour density, total anthocyanins, and phenolics are negatively correlated with bunch weight and berry weight (Clingeleffer et.al., 2000).
Influence of wine-making practices on the phenolic composition of wine
Many questions remain regarding the optimal winemaking practices to employ for the achievement of a specific wine style. What is the appropriate tannin level for the type and style of wine being produced, and how does this relate to ageing? Questions concerning primary fermentation such as, how do varying amounts of whole berries and/or stems affect the wine? How is skin contact best achieved, and for what schedule and duration? What are the desirable fermentation temperature, maceration period and degree of aeration during and following skin contact? Many questions also remain regarding yeast and malolactic fermentation. Although most of these variables have been investigated, it is difficult to propose any specific course of action as grape variety, composition and intended wine style have unknown consequences. Studies so far have lacked long term investigations with replicates from different 'terroirs' and varieties.
Fermentation technology
During vinification, colourless phenolics increase during alcoholic fermentation, reaching maximum values at pressing, and remain stable during malolactic fermentation and subsequent storage. Anthocyanins and colour density, on the other hand, increase during the early stages of alcoholic fermentation, reaching maximum values 2-3 days (3% to 6% ethanol) after the start of fermentation, and decrease during storage. Others measured maximum colour extraction within four days of skin fermentation with optimum maturity fruit, but only at day six with later maturity fruit, while the non-coloured phenolics increased with increased skin fermentation time (Auw et.al., 1996). Vigorous crushing favours the extraction of the astringent and bitter tannins.
The interest in partial whole cluster or destemmed berries resides from the perceived benefits of lowering the extraction of non-coloured phenols. Catechin, gallic acid, caftaric acid and both total and polymeric phenol content were higher in whole cluster fermentations, compared with those made without stem contact (Kovac et.al., 1992). Polymeric pigments were also higher in whole cluster fermentations, suggesting anthocyanins were combining with stem phenolic compounds (Watson et.al., 1995). Kovac found that whole cluster wines with pomace contact of 7 days were more acceptable sensorially than wines made with extended maceration. Carbonic maceration causes a reduction in the amount of phenols extracted from the grape skins, with the total phenol content reaching a level only one half as high as the normally fermented control. It creates a wine for immediate consumption, but incapable of ageing for any length of time. On the other hand cold maceration ('cold-soak') may be useful to increase anthocyanin to tannin ratio for lightly coloured varieties by increasing the time of extraction from the skins without the simultaneous increase in extraction from the seeds. Investigations of the use of different yeasts for primary fermentation have indicated no significant effect on the phenol composition of the wine.
The maceration temperature greatly affects the transfer of polyphenols from skins to must, with a linear increase in colour extraction by increasing the temperature from 15°C to 33°C (Lee et al., 1977). A fermentation temperature around 30°C has been determined to be optimal for the extraction of anthocyanins and the promotion of stable polymers for Pinot noir wines (Gao et.al., 1997), while a fermentation temperature of between 28°C and 32°C was also found optimal for the production of high quality Pinotage wines. Contrary, G¢mez-Plaza determined that low-temperature maceration (<20°C) produced wines with higher anthocyanin and hydroxycinnamic acid derivative concentrations, which could indicate better aging potential but the wines were unfortunately not analysed after bottling (Gomez et.al., 2000).
During fermentation, the cap formed is mixed with the juice on regular intervals to promote contact between the juice and skins and thus enhance extraction. Different cap management techniques were compared to determine any differences on the extraction of phenolics. It was found that mechanical punch down and pump over treatments significantly enhanced the extraction of all phenolic compounds and their polymerisation in comparison to the traditional manual punch down treatments. The total polyphenol concentration and wine quality was also higher for the punching-down and rotor treatments, in comparison with the pumping-over treatments. The pump over regime gave all varieties of wines significantly higher quercetin levels. Traditional pumping over tended to cause juice to infiltrate through fissures in the cap, leaving much of the pomace untouched. Therefore many wineries now use sprinklers or splash plates.
No differences were observed in the anthocyanin content of wines made with different sulphur dioxide levels added at crushing (0, 50, and 100mg/L) however, the wine with the 100mg/L addition had the lowest polymeric pigment and caftaric acid content (Watson et.al., 1995).
The use of processing enzymes
Enzymes are used during juice and wine processing to increase juice yield, facilitate colour extraction and stability, as well as clarification. Commercial enzymes are typically crude fungal preparations, containing impurities such as proteins, mucilage and extraneous enzymes, such as b-glucosidase (Martino et.al., 1994). The latter can cleave the sugar from anthocyanins, leaving the unstable aglycone, which spontaneously transform into a colourless form. The free hydroxycinnamic acid concentration can also be greatly increased from enzymatic treatments during vinification as a consequence of esterase activity on hydroxycinnamic esters and on p-coumarate anthocyanins. Some studies have found a decrease in the anthocyanin content in wines treated with processing enzymes as a result of anthocyanin destruction (Pardo et.al., 1999), with no research showing any long-term enhancement in colour. It looks as if enzyme treatments may increase the initial release of pigments from the skin cells during fermentation, but that the colour extracted is not stable, therefore the loss of colour by the end of fermentation would have more than compensated for the initial increase. Enzyme manufacturers are trying to identify and eliminate these side activities by producing more substrate specific enzymes.
Extended maceration
Different skin contact times were investigated for the possible benefit of enhanced extraction of anthocyanins and skin tannins to promote the stabilisation of colour. Assume 5 to 6 days of skin contact until the end of fermentation. Although the polyphenol extraction rates differed between the skin contact treatments, the concentrations in the final wines increased slightly with an increase in skin contact time. Wines made with extended skin contact of 4, 5 and 10 days, still exhibited increased colour characteristics after one year of ageing. The extraction of 280 nm absorbing phenols as well as cinnamic acids was found to reach an optimum at 36 days of skin contact while 520 nm absorbing pigments increased very steeply from days 1 to 4 and then decreased gradually (Yokotsuka et.al., 2000). According to sensory evaluations astringency and bitterness of the wines increased steeply with increasing pomace contact time for the period from 0 to 8 days, and gradually from 8 to 32 days. The colour intensity and amount of body also increased sharply with the pomace contact time from 0 to 8 or 16 days. The conclusion was that red wines made with pomace contact for 4 to 16 days were judged to have higher complexity, acceptable bitterness and astringency and better appearance than wines made with pomace contact for less than 4 or more than 16 days. This is in agreement with another study that determined that pre- and post-fermentation maceration for 15 days (approximately 10 days of extended skin contact) resulted in a wine with lower anthocyanin concentration than the control with 7 days skin contact, suggesting degradation or precipitation with extended skin contact. Scudamore determined that although the colour densities of extended pomace treatment wines were higher initially than traditional fermented wines, after 14 months of ageing they were similar (Scudamore et.al., 1990).
It appears to be generally true that factors controlling the solubility and retention of pigments in young wines are more important than contacting methods in determining wine colour. That is one explanation for why a wide range of alternative contacting and extraction practices continues to be used, with no single method being significantly better than any other in terms of colour retention.
Natural addition of grape tannin
Several microvinification experiments were carried out with different red and white grapes to elucidate the effect of adding supplementary quantities of seeds (two-fold) during fermentation on the phenolic composition of wines. The presence of a higher quantity of seeds in contact with the must during fermentation resulted in wines with a higher phenolic content especially catechins and proanthocyanidins (Revilla et.al., 1998). According to Revilla the addition of seeds increased the stabilisation of wine colour, as indicated by a slight increase in colour intensity and free anthocyanin content after fermentation (Kovac et.al., 1995). Another process used to increase tannin extraction, is the addition of supplementary pomace to the fermentation tank, by racking away a certain volume of must some hours after the beginning of fermentation, and adding crushed grapes to fill the tank. The term doble pasta makes reference to this technological approach, and may be translated into English as "double pomace". These types of wines are deeply coloured and rich in dry extract, containing an appreciable amount of phenolic compounds. Unfortunately, there is no published data available about this winemaking process.
Effect of fining treatments
Fining treatments are an important step in enology as they allow clarification and are reported to decrease the astringency of wines. Fining agents such as polyvinylpyrrolidone (PVPP), gelatin, or bentonite have been shown to reduce phenolic levels and presumably alter the colour and sensory characteristics of wine. These studies lack extensive investigation with ageing for the determination of the long-term effects as well as the descriptive sensorial analysis that is needed to determine any alteration in astringency and/or quality. It has been determined that the more polymerised and galloylated tannins are selectively precipitated by gelatin (Maury et.al., 2001), whereas PVPP typically binds and removes smaller molecular weight phenolic compounds (Sims et.al., 1995), while bentonite reduces the protein content of wine. Bentonite also absorbs polyphenol oxidase, phenols, and other positively charged molecules. Clarification with bentonite and gelatin results in lower anthocyanin and hydroxycinnamic acid derivative concentrations, while having little effect on the monomeric flavanols, which are correlated to browning. The most suitable fining agent for young red wine was PVPP resulting in a wine with lower astringency and a little reduction in colour (Gomez et.al., 2000).
The application of micro-oxygenation
Red wine is often made by aerating must during pump-overs, which then stimulates yeast growth as well as the formation of tannin-anthocyanin bonds. The amount of oxygen added during fermentation is normally 2-ml/L/day for the two days during peak fermentation. Near the end of fermentation no oxygen should be added. The main time micro-oxygenation is used on wine and specifically on press wine, is before malolactic fermentation. At this time the tannins are more susceptible to oxidation due to the lack of SO2. It can take between eight and ten days for the added oxygen to be absorbed, depending on the temperature and the phenolic composition of the wine (Vivas and Glories, 1995). The amount of oxygen normally taken up during the racking of wine ranges from 2.2 mg/L for a protected pump over to 7.4 mg/L for a pump over with deliberate splashing. It was found that the amount of oxygen taken up during normal ageing in a barrel is on average 2.5 mg/L/month. The suggested rate of micro-oxygenation post-fermentation is between 0.75 and 3 mg/L/month (Rowe and Kingsbury, 1999). The outcome of micro-oxygenation is difficult to predict, as every wine is a unique system of equilibriums containing a range of phenolic compounds with closely related characteristics. The above mentioned amounts offer only a general indication of what is typical for a range of red wines, and are based on the personal experience of winemakers. Scientific data is not yet available on this subject. Although the practice has been used for years in the old wine countries, it's relatively new and untested in South Africa. Detailed analysis of the promoted compositional changes during micro-oxygenation and the resulting effect on the mouth-feel profile of the wine should be investigated. Popular believe is that through micro-oxygenation a higher concentration of phenols can be retained in wine by the stabilisation of anthocyanins through the stimulation of polymerisation. The controlled aeration of red wine may help evolve and soften tannins, and improve lightly structured wines by providing body.
Conclusion
Correlation of the changing phenolic composition of wine resulting from different viticultural and winemaking practices applied and quality will be possible with continued investigation.
The discussed vineyard management practices are considered as important variables determining the phenolic composition of grapes and wines. The optimal canopy should be developed for a vineyard in a specific 'terroir' by investigating the influence of irrigation and trimming and/or pruning on sun exposure, as well as the resulting effect on bunch weight and bunch/leaf ratio. There is a significant amount of scientific data available on one or more of the variables discussed, without fully taking the accumulative effect of the resulting canopy density changes on the determined phenolic composition of the grapes, into account. There is also a need to extend investigation to the resulting wine, not only for compositional but also sensorial differences, specifically the mouth-feel attributes of the wine. At the moment the incentive is to promote the formation of colour components and flavonols in grapes using viticultural practices.
Different winemaking practices are applied in an effort to obtain as much stable colour as possible before maturation. There is a close correlation between wine quality and colour. Several fermentation conditions such as fermentation temperature, skin contact time, and skins to must ratio (berry size) influence the extraction of anthocyanins and other phenolic compounds. Therefore, the possibility exists that vinification practices may either augment or diminish the potential differences in wine colour and phenolic composition established by different viticultural practices.
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Anita Oberholster, Department of Enology and Viticulture,
University of Stellenbosch, Stellenbosch
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