Preharvest Methyl Jasmonate and Postharvest UVC Treatments: Increasing Stilbenes in Wine
In varieties of Vitis vinifera, a number of different stilbenes are present in several parts of the grapevine as constitutive compounds of the lignified organs (roots, canes, seeds, and stems) and as induced substances (in leaves and berries) acting as phytoalexins in the mechanisms of grape resistance against pathogens.
This chapter describes the strategies and recent advances regarding ways to increase the stilbene concentration in grapes through the use of a combination of elicitors. Special attention is paid to the treatment combining MEJA (Methyljasmonate)+UVC (Ultraviolet C light), which results in grapes enriched in stilbenes. The effectiveness of treatments is subject to many vinicultural factors, as is the transfer of stilbene compounds into the wine. Maximum skin contact with the must and minimum amounts of fining agent is recommended. However, the production of stilbene-enriched wines is a complex process which is difficult to standardize.
KeywordsBioactive Phytoalexins Trans-resveratrol ε-viniferin Induction Dm Stress Elicitors Biosynthesis Functional
Time required for reaching maximum concentration of resveratrol
Tyrosine ammonia lyase
Ultraviolet C light
Stilbene are natural non-flavonoid phenolic compounds that are synthesized by a wide range of plant families: Pinaceae, Moraceae, Liliaceae, Myrtaceae, Fagaceae, Gnetaceae, Cyperaceae, Dipterocarpaceae, Leguminoseae, and Vitaceae. Although polyphenols display enormous chemical diversity, stilbenes seem to constitute a rather restricted group of molecules, the skeleton of which is based on resveratrol (3,4′,5-trihydroxystilbene), with a structure consisting of two aromatic rings substituted by hydroxyl groups linked by an ethyl bridge, especially in Vitaceae and Fabaceae, and also based on pinosylvin (3,5-dihydroxystilbene) in Pinaceae .
In Vitis a number of different hydroxystilbenes are present in several parts of the grapevine as constitutive compounds of the lignified organs (roots, canes, seeds, and stems), and as induced substances (in leaves and berries) acting as phytoalexins in the mechanisms of grape resistance against pathogens.
As phytoalexins, stilbenoids are induced by infections and mechanical stress, such as that caused by UV damage or insects. Their production affords protection against many Vitis pathogens. Stilbenoids are, therefore, of great interest due to their activity defending vines from many devastating diseases and pests. In fact, stilbenoids have recently been reported to be effective against Plasmopara viticola [2, 3], and one of their potential uses is therefore as a naturally occurring pesticide for nonresistant species, such as Vitis vinifera L., the most widely cultivated grape species in winemaking.
Furthermore, stilbenes have been reported to possess health promoting compounds with cardioprotective, neuroprotective, and anticancer properties [4, 5 ]. Trans-resveratrol seems to be one of the most promising compounds due to its bioactivity. Other stilbenes, such as piceatannol and viniferins, are usually found in grapes and wine at a lower concentration than resveratrol. Although their bioactivity has received less attention as a consequence, some of their health-promoting properties are currently being investigated . To sum up, stilbenes are of great interest thanks to their health-promoting properties.
Stilbenoid in grape and wine
Grape (mg Kg−1 fw)
Wine (mg L−1)
A prospective study involving 40.685 subjects estimated the intake of stilbenes among the Spanish population. The authors established that main source of stilbenes in diet was wine (98.4%), followed by grapes and grape juices (1.6%). The main stilbenes ingested were trans-piceid (53.6%), followed by trans-resveratrol (20.9%), cis-piceid (19.3%), and cis-resveratrol (6.2%) .
Studies conducted on 120 grape germplasm cultivars of Vitis for 2 years showed that berries of interspecific rootstock cultivars had very high levels of extractable resveratrol (<210 μg g−1 fw−1). The various genotypes of V. riparia tested usually contained high levels of resveratrol, whereas most genotypes of V. vinifera and their hybrids with V. labrusca usually contained relatively low levels (<2 μg g−1 fw−1) . Moreover, red varieties contain higher stilbene concentrations than white ones . Additionally, ripe grapes lose their ability to respond to elicitors.
Seasonal meteorological conditions, especially temperature, rainfall, and relative humidity during the last month before harvest, all affect stilbene synthesis because they are all related to fungal-disease pressure [22, 25]. Very few studies have been found with regards to soil. They are unequivocally linked with climate conditions.
Cultural practices may also affect the resveratrol concentration in grapes. No general recommendations can be given since each assay has been performed under specific conditions. For example, leaf removal at veraison increased the concentration of piceid in grapes from the Barbera cultivar but resulted in decreased resveratrol in the Croatina and Malvasia cultivars under cool meteorological conditions; leaf removal had no effect on the stilbene content of grapes in warmer and drier weather conditions according to a 4-year trial carried out in Piacenza viticultural area . Indeed, other factors are considered that may interfere in the results, which should be discussed taking all these factors as a whole.
Moreover, winemaking techniques are also key to obtaining stilbene-enriched wines, as is discussed below.
This chapter describes the strategies and recent advances regarding ways to increase the stilbene concentration in grapes through the use of a combination of elicitors. Special attention is paid to the treatment combining MEJA+ UVC. Moreover, ways to transfer stilbenes into wine to achieve the production of added-value wines (stilbene-enriched wines) are also reviewed and discussed.
2 Elicitors to Increase Stilbene Concentration in Grapes
Stilbenes are known to act as phytoalexins, plant defensive substances of low molecular weight synthesized de novo in response to stress. The process by which the grapevine is stimulated to produce secondary metabolites is called “elicitation,” indicating an external stressful stimulus applied to the plant. Therefore, the stilbene concentration in grapes can be significantly increased by both biotic and abiotic stresses. The fungi that attack grapevines, such as Botrytis cinerea (grey mould), Plasmopara viticola (downy mildew), or Erysiphe necator (powdery mildew) are considered biotic stresses. Meanwhile, abiotic stresses can be classified as chemical and physical elicitors. Below is a review of how abiotic stress affects the stilbene content in grapes.
2.1 Chemical Elicitors
BTH has been shown to increase grey mould resistance in grapes by increasing the levels of phenolic compounds . Preharvest treatment of Syrah grapevines with BTH (0.3 mM) significantly increased the resveratrol content in grapes by up to three times with regard to the control ones .
CHIT (β-1,4-d-glucosamine) is a polysaccharide obtained from the deacetylation of chitin and is a natural structural compound within the cell wall of several fungi and crustaceous shells. CHIT is described as having antimicrobial properties as well as being able to elicit plant defenses . CHIT has been reported to induce stilbenes in cell cultures  and grapevine leaves . However, when used as a preharvest treatment, controversial results have been described [33, 37].
MEJA is the most active derivative of jasmonic acid. Both are endogenous plant regulators that act as signaling molecules upon biotic stress and are involved in plant defense mechanisms triggering the synthesis of secondary compounds .
Some studies have shown that the application of MEJA to grape bunches may exert a profound effect on the phenolic content of both the grapes and wine, particularly anthocyanins and stilbenes [39, 40].
In the case of stilbenes, MEJA treatment improved both the resveratrol and ε-viniferin content of Barbera berries in an accumulative manner until ripeness . In contrast, more recent results have described how resveratrol was induced a few days after treatment but subsequently decreased throughout ripening until harvest . In fact, the final level of stilbenes after MEJA treatment depends on other factors, such as variety, climate, and even viticultural conditions , which makes the treatment difficult to apply from a technological point of view.
Additionally, apart from the stimulation of stilbene compounds, other studies suggest that MEJA is also able to enhance wine quality, increasing the content of both anthocyanins, and therefore chromatic parameters and aroma compounds [43, 44].
2.2 Physical Elicitors
Many physical treatments have also been studied as elicitors in grapevines, the following being particularly significant: ultrasonication (US), ozone (OZ), anoxic treatments (AT), and UVC light .
US treatment has been proposed as a tool to produce a resveratrol-enriched grape juice . In all the grape varieties tested, a significantly higher elicited amount of resveratrol was found in grape juice manufactured from fruit treated with US for 5 min followed by 6 h of incubation. The accumulation of resveratrol is transcriptionally controlled by the enzyme stilbene synthase and ultrasonication treatment was found to elicit the activity of stilbene synthase, demonstrating the underlying mechanism behind increases in resveratrol. However, in comparison with grape skin or wine, the amount of resveratrol in grape juice was much lower. Indeed, stilbenes are more soluble in alcoholic solutions such as wine than in aqueous media .
OZ treatment is also known to stimulate the synthesis of resveratrol in grapes under storage conditions. However, although OZ treatment (3.88 g h−1 for 5 h) increased the resveratrol concentration in grapes as much as UVC light did, the treatment decreased the quality of the grapes enormously .
AT of grapes placed in a vacuum chamber with nitrogen gas enhanced the resveratrol content . After testing different times, 6 and 15 h were recommended to both improve resveratrol concentration and grape quality.
UVC light with a wavelength range between 200 and 280 nm is a germicidal, nonionizing radiation that has been used extensively to sterilize fresh fruit and vegetables . Moreover, UVC light is also very popular in the field of enhancing the production of resveratrol in grape berries and its derivatives, including grape juice and wine. In fact, UVC treatment is the most efficient elicitor at increasing the stilbene content in grapes, in particular trans-resveratrol.
The use of UVC light as an elicitor in grapevines was first described by Langcake and Pryce (1977) . The authors observed an increase in trans-resveratrol concentration in leaves after irradiation (in vitro). Thereafter, many studies were developed in vineyards to determine the mechanisms and possible applications of this tool [51, 52]. However, the majority of the above studies were performed on leaves due to their availability during the vegetative cycle of grapevines.
In 2000, UVC light was applied for the first time in the postharvest treatment of table grapes . Later, the treatment was optimized to maximize the resveratrol concentration in grapes . The optimized treatment (1040w. 40 cm and 1 min) has been widely applied to different Vitis subspecies, varieties, areas of production, conditions, years, etc.
Regarding postharvest UVC treatment, two parameters should be taken into account. First, the maximum resveratrol concentration achieved, and second, the time required to reach this maximum concentration after the postharvest UVC treatment (called Dm). Obviously, the activation of plant defense mechanisms requires few days. Dm is a key factor to sure the quality of the grapes. If Dm is too long, grapes will lose quality, making them unsuitable for producing wine. Therefore, varieties achieving a high resveratrol concentration in a short time period guarantee quality stilbene-enriched grapes .
The induction capacity of grapes from three varieties of Vitis vinifera sylvestris (VS9, VS15, and VS16), seven of Vitis vinifera sativa (Merlot, Syrah, Graciano, Tempranillo, Palomino fi Graciano, Tempran, and Tintilla de Rota), and two hybrid direct-producer vines (Regent and Orion) after postharvest UVC treatment has been described for two harvests. Four compounds were identified in UVC treated grapes: piceatannol, trans-resveratrol, ε-viniferin, and δ-viniferin . Varieties belonging to the sylvestris group and the Merlot variety presented high stilbene production. Syrah, Vitis vinifera sylvestris V15, Pinot noir, and Graciano stood out for their capacity to induce piceatannol, while Vitis sylvestris V9 and Syrah stood out for presenting the highest ε-viniferin concentrations after UVC treatment. Therefore, it could be established that the effectiveness of the UVC treatment depends on both variety and year, but not on the subspecies. As mentioned above, effectiveness is determined not just for the maximum concentration but also for Dm. Moreover, the authors concluded that from a technological point of view it is vitally important to consider the variability between years, since the number of days to reach the maximum concentration might also vary.
Terroir (climate and soil) is also considered a key factor for the effectiveness of postharvest UVC treatments . Indeed, terroir factor was stronger than variety factor regarding stilbene induction capacity upon UVC treatment. In that study, the induction capacity of stilbenes was studied on four red grape varieties cultivated in four Andalusian terroirs. In agreement with previous data, the Syrah variety obtained the highest stilbene concentration, especially in Cabra (Córdoba) terroir (up to 33 mg kg−1 f.w.) with limestone soil, a high average temperature and low average relatively humidity.
Therefore, the use of postharvest UVC light to increase grape stilbene content is quite difficult to standardize due to the numerous factors involved. However, certain conclusions can be reached: (i) each variety seems to be influenced to a different degree by the climate and harvest; (ii) varieties with high induction capacity and consequently a short Dm are highly recommended; (iii) Syrah is the particular variety proposed for projects aimed at producing wine enriched in stilbenes.
More recently, UV-C light application as a preharvest treatment tool to induce stilbenoid production was tested in open field experiments for table grapes . UV-C light application preharvest day, output power, exposure time, and storage conditions were optimized.
UV-C light preharvest treatment was applied at different days before grape ripeness to establish the optimum application day to reach the maximum trans-resveratrol concentration. Grapes were illuminated with an UV-C light dose of 1866 J m−2 (1040 W, 1 min) on 7, 5, 3, and 1 days prior to the optimal harvest day. Maximum trans-resveratrol concentration was achieved after 24 h regardless of application day. An increase between 22- and 46-fold as compared with the control concentration was achieved. trans-Resveratrol concentration reached a maximal concentration in grape (19 mg kg−1 f.w. skin), 24 h after illumination (10.000 J m−2), and subsequently declined. Preharvest UV-C light treatment might reduce the required time in 2 days to reach maximum trans-resveratrol concentration in comparison with postharvest UV-C light treatment. In 2002, a Dm value for Red Globe equal to five when postharvest UV-C light was established .
trans-Resveratrol was affected not only by the dose but also by how the dose was applied in terms of output power and exposure time. When postharvest storage was studied, trans-resveratrol increased for 3 days, after which time a reduction was observed. The mechanism for hormesis proposed by Luckey  suggested that low doses of UV-C could inflict repairable damage to DNA and this slight trauma would activate repair mechanisms for radiation-induced DNA damage. This suggests that sublethal radiation may stimulate vital processes inside the cells and create a positive change in the homoeostasis of the plant physiology. A dose over approximately 10,000 J m−2 approximately seemed to reduce trans-resveratrol concentration compare with lower dose . Similar trends were found for ε-viniferin, whose concentration at days 3 and 4 reached similar levels to those of trans-resveratrol. UV-C light increased its concentration between 5- and 31-fold depending on the treatment and sampling day. Maximum concentrations of 28,42 and 26,75 mg kg−1 f.w. skin were found on postharvest day 4 in grapes treated with 520 W for 10 min and 1040 W for 5 min, respectively.
Moreover, daily periodic preharvest UV-C light treatment showed a cumulative effect on grape stilbenoids, reaching a trans-resveratrol concentration around (120 mg kg−1 f.w. skin) . The results for grape trans-resveratrol after daily periodic preharvest UV-C light treatment compared with the results after a single treatment demonstrated that a higher concentration was reached with each periodic treatment under comparable conditions. Periodic preharvest UV-C light treatment maintained a high grape stilbenoid concentration over time, similar to that achieved by postharvest UV-C light treatment, but its maximal effects can be observed sooner, 24 h after each daily treatment. The variability of this UVC preharvest treatment regarding grape variety and season has not been studied yet, but it is expected to be highly variable.
2.3 Combination of Elicitors: Preharvest Treatment with MEJA Plus Postharvest UVC Treatment
All the above stresses or their combinations can be used to target increases in health-promoting stilbenes. A synergistic effect on phytoalexin production has been described between MEJA and ethephon , CHIT and UVC [33, 60], MEJA and UVC [33, 61], BTH and UVC , and MEJA and cyclodextrins [62, 63], among others .
Treatments with MEJA and UVC have been studied in Vitis vinifera L. cv. suspension cultures of Cabernet Sauvignon cells . Both treatments improved both the production of stilbene within the cells and the accumulation of trans-resveratrol in the culture medium. UV-C irradiation for 20 min or MEJA at 100 μM was efficient in promoting stilbene accumulation. The combined treatment of UV-C and MEJA highly induced the production of total intracellular stilbene at the maximum of 2005.05 ± 63.03 μg g−1 dw and showed a synergistic effect on the accumulation of extracellular trans-resveratrol at 3.96 ± 0.2 mg l−1.
3 Stilbene-Enriched Wines
Numerous epidemiological studies have shown that long-term moderate consumption of wine is linked to a lower level of cardiovascular illnesses. A study conducted by Renaud and De Lorgeril  revealed that the incidence of coronary heart disease in France is about 40% lower than in the rest of Europe; this was termed the “French paradox,” which appeared to be related to regular consumption of red wine. Numerous beneficial qualities with positive effects on health have been attributed to wine, particularly red, including antioxidant, anticarcinogenic, and antispasmodic properties; enhancement or activation of bile secretion; and antibacterial and antihistaminic agents . The finding that red wine presents more health-promoting activity than beer or spirits has led to research focusing its attention on phenolic compounds; within this group, stilbenes (in particular, trans-resveratrol) seem to show high bioactivity. Therefore, a great deal of effort has been devoted to increasing the stilbene content of wines.
Strategies for increasing stilbenes levels in grapes have been already described in the current chapter. Regarding factors which affect the stilbene concentration in wine, oenological practices such as skin contact maceration, yeast strain, fining agent, filtration, and ageing seem to be important (Fig. 1). In general, all the processes that maximize the extraction of phenols from skin are recommended . Moreover, the use of yeasts which have the gene of overexpressed STS have been suggested . However, this method is not allowed in Europe. On the other hand, the use of fining agents such as bentonite, casein, albumin, or PVPP reduced the resveratrol content in wines enormously [67, 69]. In fact, although PVPP is one of the most-widely used fining agents, it lacks selectivity and therefore it has a limited applicability. A new polymer (P-NIOA) has shown a similar removal to PVPP, but with a lower affinity to resveratrol . Likewise, a filtration step may reduce resveratrol content by up to 58% , while ageing hardly affected resveratrol content in red wines aged in oak barrels . To sum up, all processes involved in the final stage of wine production, but ageing, reduce importantly the content of stilbenes in wine (Fig. 5).
A study performed on stilbene-enriched grapes concluded that the concentration of trans-resveratrol decreased progressively during winemaking, especially during AF, probably due to the interaction with yeast and/or other organic compounds in the fermentation media .
In another more recent study, stilbene-enriched grapes obtained through a combination of MEJA+UVC treatments were used to make red wine following traditional methods to obtain a stilbene-enriched wine . The results showed that wines whose grapes were treated first with methyl jasmonate (before harvest) and secondly with UVC light (after harvest) presented a twofold higher stilbene concentration than the control. However, the concentration in bottled wine was not very high (up to 2.32 mg l−1 of total stilbenes). The stilbenes were lost during the winemaking process, not only during alcoholic fermentation, as reported by previously , but also during the following steps. At pressing, at racking, and at cold stabilization wastes are generated (pomaces, lees and tartrates respectively) (Fig. 5). Above wastes are stilbene-enriched by products (up to 25 gr Kg−1 fw waste). They were even proposed as a valuable source for manufacturing nutraceutical products . It is also remarkable that in the study by Guerrero et al. , the treated wines, with higher stilbene content, showed better chromatic properties and obtained the highest scores at tasting.
Stilbene-enriched wines are claimed to be a rich source of bioactives. They provide consumers with added value since their intake of stilbenes is significantly increased while the consumption of ethanol remains the same.
Many strategies have been tested to increase the stilbene content of grapes. Among them, the combination of preharvest MEJA treatment with postharvest UVC treatment on grapes is suggested to be the most powerful tool. Grapes treated in this way present a significantly higher concentration of trans-resveratrol, piceatannol, and ε-viniferin. The most difficult task is transferring those compounds into the wine. Stilbenes, as phenolic compounds do, interact with solids in the media (yeast, tartrates, and lees), precipitating and reducing their concentration in wine. In fact, winemaking by-products have been suggested as a valuable source of stilbenes for the manufacture of nutraceutical products.
To sum up, the production of stilbene-enriched wines is a complex process that is difficult to standardize. Many factors should be taken into account. Terroir and variety are key factors influencing both the constitutive stilbene concentration and induction capacity. The Syrah variety can be highlighted as a good candidate for undergoing induction experiments.
Regarding winemaking, some recommendations can be given. First, it is important to maximize skin and must contact during alcoholic fermentation. Secondly, post-fermentative maceration should be avoided as far as possible. Finally, the number of operations after fermentation (raked, filtrations, etc.) should be kept to a minimum.
Taking all the above into consideration, it is possible to produce stilbene-enriched wines, although it is difficult to make accurate predictions regarding their stilbene concentration due to the large number of factors involved.
The authors thank INIA and FEDER for their financial support of the projects “Stilbenes as a sustainable tool to replace SO2 in winemaking” (RTA2015-00005-C02-01) and “Research and Technological Innovations in Viticulture” (AVA.AVA201601.3). Susana Cruz and Maria I. Fernandez thanks FEDER program (2014–2020) for supporting her contract.
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