April 2018 Issue of Wines & Vines

Managing Green Flavors in the Winery

Sorting, clarification and heat treatments are among the most successful options

by Gary J. Pickering

Green, vegetal flavors in wine are typically associated with fruit that has not achieved the desired level of ripeness. Often such wines suffer from other quality issues linked to grape immaturity, including high acidity, poor color and lack of varietal aroma. Thus, green flavors in wine are almost universally rejected by consumers. The exceptions are when these notes can be considered (at low levels) part of the expected varietal character, such as in cool-climate Cabernet Sauvignon, Carménère and Sauvignon Blanc.
     The main compounds eliciting green/vegetal flavors in wine are alkl-methoxypyrazines (also known as MPs, and often simply referred to as pyrazines), C6 green leaf volatiles (particularly hexanols and hexanals) and some thiols. MPs are the most important. In nature, they are produced by microbes, plants and insects.
     With the exception of some rare cork-taint issues from microbial contamination and even rarer cases of deliberate adulteration of wine, grapes and beetles from the Coccinellidae family are the most common sources of MPs in juice and wine. A fuller understanding of the impact of MPs and where they come from during winegrowing is important to prevent greenness and remediate affected juice and wine.

The four MPs most commonly associated with greenness in wine are 3-isobutyl-2-MP (IBMP), 3-isopropyl-2-MP (IPMP), 3-secbutyl-2-MP (SBMP) and 2,5 dimethyl-3-MP (DMMP).7 All are capable of eliciting herbaceous or vegetative aroma and flavor, although subtle differences in aroma quality have been noted; for example, IBMP is often described as more bell pepper-like than the others. While DMMP and IBMP are often the most prevalent in wine, the relative contribution of each MP depends on its odor threshold, the grape variety, vinification practices and presence or absence of lady beetles during harvest. (See graph on this page)
     One thing shared by all MPs is their very low human detection threshold in wine, which for smell ranges from 320 pg/L for IPMP to 31 ng/L for DMMP.3,18 As a means of reference, 1 picogram (pg) is one-trillionth of a gram, or the average weight of the DNA in one cell of a hummingbird.11 Of interest to how we perceive greenness in wine, there is significant variation in sensitivity between individuals; for example, tasters report a 400-fold difference in IPMP thresholds for some white wines. (See "Sensitivity to Methoxypyrazines Varies Between Tasters" on page 71.) Also, greater familiarity with ladybug taint may be associated with higher sensitivity to IPMP.20
     What might smell or taste too green to you may be barely detectable to your colleague. This disparity has important implications for vintners assessing levels of greenness in wine and making decisions on treatment and market acceptability; it is important to know your own sensitivity and, if needed, use additional assessors to complement your perceptions.

Sources of methoxypyrazines
Grapes: MPs are secondary metabolites that accumulate early during berry development and likely serve to discourage frugivores (fruit eaters) from eating unripe grapes. They typically reach maximum levels a few days before véraison, after which their concentration decreases, particularly during early stages of ripening.25 Their distribution within grapes varies greatly, however, with implications for how vinification decisions impact the final MP load in wines.
     In Cabernet Sauvignon, for example, stems contain approximately 53% of the MPs, skins and seeds 46% and the pulp less than 1%.24 Climate, terroir and viticultural practices all have been implicated in affecting grape MP concentration, however there is considerable debate in literature about the impact of specific factors during the growing season (refer to D. Sidhu et al. [2015] for a review of these considerations). There is good agreement that the lowest MP levels are found in grapes exposed to techniques applied pre-véraison that promote cluster exposure (such as leaf removal) and in fruit that has achieved full ripeness.
     Lady beetles: Recently, it has become clear that elevated MP levels in juice and wine can come from infestation of grapes by members of the Coccinellidae (lady beetle) family. If either Harmonia axyridis (multicolored Asian lady beetle) or Coccinella septempunctata (seven-spotted ladybird) are incorporated with the fruit into the processing stream, MPs contained in their haemolymph (the insect equivalent of blood) can produce greenness in the final wine, a fault known as ladybug taint.6
     Harmonia axyridis in particular is becoming widely recognized as a significant problem for this reason. It is an invasive pest in many of the world's established and developing wine regions, where it tends to outcompete local lady beetle species and reach large populations rapidly. Typically, these beetles migrate from non-grape crops such as soy in the fall and take temporary residence in vineyards within grape clusters (see photos of seven-spotted ladybird and multicolored Asian lady beetle above).
     Due to the high potency of MPs, as few as one beetle per vine can be sufficient to affect the final wine, with typical sensory descriptors of peanut and green pepper. Wines affected by ladybug taint have much higher levels of IPMP than found naturally in mature V. vinifera grapes. In fact, the ratio of IPMP to IBMP can be used as a chemical indicator that lady beetles are the source of the undesired greenness.7
     Adulteration: A much less common source of MPs is their deliberate introduction as flavorants during winemaking. Such practice is not permitted, yet it was documented in Sauvignon Blanc by the South African media in 2003 and 2004.9,10,17 Adding MPs directly or in the form of green pepper extract, as suggested in these articles, would presumably increase the aromatic intensity and cool-climate nature of the wines, potentially conferring a competitive advantage in markets where this style of Sauvignon Blanc is desired. Hopefully tighter control by regulatory bodies and enhanced analytical techniques have led to cessation of the practice.

Remediating green musts and wine
Grape sorting: There are several interventions in the vineyard that can help reduce potential MP load the vintner might face when grapes arrive at the winery, and the reader (refer to D. Sidhu et al. [2015] and Botezatu and Pickering [2018]).
In the case of harvested grapes that contain large numbers of lady beetles, shaker tables have proven particularly effective at separating them from the fruit, thus preventing extraction of MPs from the beetles during grape processing. These tables are typically custom-built and consist of a vibrating metal mesh conveyor, along which clusters move. The shaking motion forces beetles through the mesh into a collection and disposal area below. Several wineries also report that harvested grape clusters can be immersed in water, where beetles float to the surface and can be removed. Sorting is also important to remove leaves, which contain MPs and other compounds that can contribute to greenness.
     Crush, destem and juice treatment: Destemming is critical, as the presence of stems during vinification significantly increases wine MP concentration.12 Given that about one-half of MPs are located in the grape skin, minimizing skin contact pre-fermentation is very important to reduce the potential for green wines. Thus, crushing should be avoided where possible. In the case of red grapes, this may even require serious consideration of making a white or blush wine rather than red, should the concern about potential greenness be sufficiently high.
     Fortunately, one of the first options in the vinification process for white wine turns out to be one of the most effective: settling and clarifying the juice prior to yeast inoculation. Thoroughly settling and racking the juice before fermentation can reduce MP content by more than half, although it should be noted that this particular juice had a high initial level of solids at 1,280 NTU (see "Juice Settling Can Significantly Reduce Greenness in Wines," left).
     Thermo-vinification has been shown to be effective at reducing MP content in must. It involves heating red grapes and must for a short period to between 140° and 176° F (typically to aid in phenolic and color extraction), and is used widely in France and Germany. A reduction in IBMP content of up to 67% has been reported using thermo-vinification,23 with more modest decreases found for other MPs.14
     A newer heat treatment, flash-détente, involves heating the grapes for less than one minute to around 185° F and then cooling them in a vacuum. A reduction in the herbaceousness of the resulting wine is widely claimed anecdotally; whether this is due to a loss of MPs, other green compounds and/or a change in other volatile constituents remains to be determined in the peer-reviewed literature.

Low temperature pre-fermentation maceration (cold soaking) to extract color and aromatic compounds is not recommended with high-MP grapes. While yet to be widely investigated, there is no evidence that commercial yeast strains can degrade MPs during fermentation. In fact, red wine fermented with the Lalvin BM45 cerevisiae yeast showed increased levels of IPMP,21 suggesting it should be avoided in musts with high MP levels.
     Yeast strains may also impact perceived greenness in wine indirectly, particularly through suppression and enhancement effects. For example, Cabernet Sauvignon wine fermented with Lalvin D2 produced wines that were less green in aroma and flavor than those fermented with three other commercial Saccharomyces cerevisiae strains (see "Green Characters in Red Wine are Affected by Yeast Strain" on this page), demonstrating the capacity for vintners to mask green characters to some degree by selecting yeast that produce high levels of fruit characters.
     As most MPs are extracted from grapes during the first 24 hours of fermentation (and largely in the absence of alcohol), the duration of fermentation and maceration may not play a significant role in the MP content of the final wine.28 Whether this is true for must that contains lady beetles has not yet been determined. Adding stems back during fermentation to help fix red wine color should be avoided,12 given that about one-half of the total MP concentration in grapes is found in the stems.
     There is no evidence that malolactic fermentation affects MP levels.28 If fermenting or aging in oak, it is important that the wood is well-seasoned to avoid extraction of green aromatic compounds or other constituents (such as harsh tannins) that may accentuate greenness. Well-seasoned, medium-toast oak chips are, in fact, effective as a masking agent for a range of herbaceous and green notes characteristic of high-MP wine.19

Fining and other additives
Micro-oxygenation involves the injection of small, controlled quantities of oxygen into wine, mainly to moderate the tannin content. Anecdotally, it has also been reported to reduce greenness, although there is little evidence in the peer-reviewed literature supporting this. However, the observation is widespread enough in the industry to suggest it is real. What (if any) aromatic compounds are being altered to explain this purported effect remains to be determined. It is possible that it is largely psychological (which makes it no less important). By altering some of the harsh tannins associated with fruit that has not achieved optimal ripeness, the greenness that often accompanies such wine is also perceived to be less. With the exception of oak chips and some enological tannins, traditional additives and fining agents have not proved effective at reducing greenness from MPs in either red or white wines.19
     Two materials not traditionally used in winemaking hold considerable promise for remediating wine with elevated MPs. Treatment of both red must and wine with silicone resulted in a large reduction in several MPs and, importantly, without significantly changing desirable aromatic compounds.4,8,26 Similar results have been reported in red wine with elevated concentrations of IPMP, IBMP and SBMP when treated with a biodegradable polylactic acid-based polymer.8 (See "Reduction in Methoxypyrazine Content in Wine After Treatment with Silicone and Polylactic Acid Polymers" on page 72.)
     How these polymers can best be integrated into the winemaking process remains to be determined, but there are several promising avenues. For example, polylactic acid can be manufactured in a variety of forms with different physical properties, allowing for flexibility in how it might be used. Potentially, it could be integrated into existing filtration systems, manufactured as solid tank inserts or added as pellets directly to the juice/wine and later removed when the target reduction in MP content has been attained.8
     A prototype fining system using a protein (mMUP Odorant Binding Protein) with very high specificity for MPs has recently been developed.13 The protein, when introduced into grape juice, binds to MPs with the MP-protein complex then efficiently removed with bentonite fining. Optimizing the system for use on a commercial scale and in a wine matrix is under development at the Cool Climate Oenology and Viticulture Institute at Brock University.

Finishing, closing and storing wine
Depending on the extent of greenness, blending can be a useful tool, particularly if it is used in conjunction with other treatments to reduce the concentration of MPs to below their detection threshold. If a vintner is only using sensory analysis to arrive at the optimum blend, it is important that several palates guide the decision-making, given the wide range of sensitivities to MPs outlined above.
     Bottling both white and red wine in Tetrapak cartons has been shown to reduce MP content by 26% to 45% after 18 months of storage compared to glass bottles.1 However, the concurrent scalping of desirable flavor compounds from wines finished in multilayer aseptic cartons such as Tetrapak also needs to be considered,27 as does the relatively rapid loss of free sulfur dioxide.1
     Some research has indicated that exposure to light after bottling can reduce IBMP by up to 57%, particularly with storage in clear bottles.16 Other studies have found no consistent effect from light exposure, bottle hue (clear, green and amber) or storage temperature on MP levels.2
     Some synthetic closures have shown marked capacity to reduce MP content in soaking trials,22 and significant but smaller effects have been observed in bottled wines closed with synthetic closures compared with screwcaps and natural cork.1 Simply aging bottled wine can reduce IBMP and IPMP by up to 30% after 18 months,2,18 possibly through binding reactions with tannins.29 However, this does not guarantee a corresponding decrease in greenness,18 as other volatile chemicals also are changing during the aging process, influencing masking effects and other sensory interactions.
     The winery team has the ability to influence consumer perception of wines with elevated greenness through marketing and advertising, including label information. Figure 7 shows an interesting approach one winery has taken with its back label on a wine with high MP levels.

Eliminating greenness
Methoxypyrazines are a potent class of grape- and insect-derived odorants that contribute greenness and herbaceousness to wine. Viticultural interventions that can reduce levels in wine are those associated with increased cluster exposure pre-véraison, full grape ripeness and elimination of Coccinellidae beetles.
     Grape-sorting and destemming can further reduce the potential for elevated methoxypyrazine concentration in wine. In grapes and juice with high MP loads, minimizing skin contact where possible and juice clarification prior to fermentation are very advantageous. Heat treatment of grapes and must, including thermo-vinification, can further reduce greenness, and oak can partially mask it in wine.
     Several polymers and a high-specificity protein are under development and show significant promise for remediating MPs in the future. This may be particularly important, given the impact that more volatile weather conditions have on grape ripeness, and a warming climate on the spread and prevalence of insects associated with ladybug taint.

Gary Pickering is a professor and research scientist at the Cool Climate Oenology and Viticulture Institute, the Department of Biological Sciences and the Environmental Sustainability Research Centre at Brock University in St. Catharines, Ontario. He is also an adjunct professor at the National Wine and Grape Industry Centre at Charles Sturt University in Wagga Wagga, Australia.

Many students and colleagues have contributed to this research over the past several years, including: Dr. David Adams, Dr. Ai-Lin Beh, Amy Blake, Dr. Andreea Botezatu, Ryan Brewster, Dr. Ian Brindle, Neil Carter, X Chen, Dr Christoph Hoffmann, Dr. Thomas Hudlicky, Eric Hulmes, Dr. Debbie Inglis, Anna Karthik, Dr. Belinda Kemp, Dr. Kevin Ker, Dr. Susanne Kögel, Dr. George Kotseridis, James Lin, Hannah Pickering, Dr. Andy Reynolds, Dr. Roland Riesen, Gavin Robertson, Dr. Mark Sears, Dr. George Soleas, Mason Spink, Terrance van Rooyen, Lynda van Zuiden, Tony Wang, Shufen Xu, Fei Yang, and Dr. Paul Zelisko. Funding support from the following organizations is gratefully acknowledged: Ontario Research Fund, Ontario Grape & Wine Research Inc., Natural Sciences and Engineering Research Council of Canada, and the Faculty of Mathematics and Science Brock University.

1. Blake, A., Kotseridis, Y., Brindle, I.D., Inglis, D., Sears, M., and G.J. Pickering. 2009 Effect of closure and packaging type on 3-Alkyl-2-methoxypyrazines and Other Impact Odorants of Riesling and Cabernet Franc Wines. J. Agric. Food. Chem. 57, 4680-4690.
2. Blake, A., Kotseridis, Y., Brindle, I.D., Inglis, D., and G.J. Pickering. 2010 Effect of light and temperature on 3-alkyl-2-methoxypyrazine concentration and other impact odourants of Riesling and Cabernet Franc wine during bottle ageing. Food. Chem. 119, 935-944.
3. Botezatu, A. and G.J. Pickering. 2012 Determination of Ortho- and Retronasal Detection Thresholds and Odor Impact of 2,5-Dimethyl-3-Methoxypyrazine in Wine. J. Food. Sci. 77(11), S394-S398.
4. Botezatu, A., and G.J. Pickering. 2015 Application of plastic polymers in remediating wine with elevated alkyl-methoxypyrazine levels. Food. Addit. Contam. 32 (7), 1199-1206.
5. Botezatu A, and G.J. Pickering. 2018 Ladybug (Coccinellidae) taint in wine. IN A.G. Reynolds (ed). Managing Wine Quality, Volume 2: Oenology and Wine Quality (2nd ed). Woodhead Publishing Limited, Sawston, Cambridge, In Press.
6. Botezatu A, Kotseridis Y, Inglis D, and G. Pickering. 2013 Occurrence and contribution of alkylmethoxypyrazines in wine tainted by Harmonia axyridis and Coccinella septempunctata. J. of the Science of Food and Agriculture. 93(4): 803-810.
7. Botezatu, A., Kotseridis, Y., Inglis, D., and G.J. Pickering. 2016a A Survey of Methoxypyrazines in Wine. J. Food. Agri. Environ. 14(1), 24-29.
8. Botezatu, A., Kemp, B.S., and G.J. Pickering. 2016b Chemical and Sensory Evaluation of Silicone and Polylactic Acid-Based Remedial Treatments for Elevated Methoxypyrazine Levels in Wine. Molecules. 21, 1238. doi:10.3390/molecules21091238.
9. Fridjhon, M. 2003 Fragrant sauvignon belies SA conditions. Business Day, Nov. 13.
10. Galpin, V.C. 2006 A comparison of legislation about winemaking additives and processes. February 2006. Retrieved Dec 13th 2017 from homepages.inf.ed.ac.uk/vgalpin1/pubs/Gal-wine06.html
11. Gregory, T.R., Andrews, C.B., McGuire, J.A. and C.C. Witt. 2009 The smallest avian genomes are found in hummingbirds. Proc. R. Soc. B; DOI: 10.1098/rspb.2009.1004. Published August 5.
12. Hashizume, K. and T. Samuta. 1997 Green Odorants of Grape Cluster Stem and Their Ability To Cause a Wine Stemmy Flavor. J. Agric. Food Chem. 45, 1333−1337.
13. Inglis, D., Beh, A-L., Brindle, I. D., Pickering, G., and E.F. Humes. 2014 Method For Reducing Methoxypyrazines in Grapes and Grape Products. United States. US 8,859,026 B2.2013/10/01.
14. Kögel, S., Botezatu, A., Hoffmann, C., and G.J. Pickering. 2013 Methoxypyrazine composition of Coccinellidae-tainted Riesling and Pinot noir wine from Germany. J. Sci. Food. Agric. 95, 509-514.
15. Kotseridis, Y., Spink, M., Brindle, I., Blake, A.J., Sears, M., Soleas, G.J., Inglis, D., and G.J. Pickering. 2008 Quantitative analysis of 3-alkyl-2-methoxypyrazines in juice and wine using Stable Isotope Dilution Assay. J. of Chromatography A. 1190(1/2): 294-301.
16. Maga, J.A. 1990 Sensory and stability properties of added methoxypyrazines to model and authentic wines. In: Flavors and Off Flavors: Proceedings of the 6th International Flavor Conference, Amsterdam. pp. 61-70.
17. Morris, R. 2004 KWV to pull plug on 67,000 litres of dodgy wine. Business Report, 7th December. Retrieved Dec. 13, 2017 from .
18. Pickering, G.J., Yong, L., Reyonolds, A., Soleas, G., Riesen, R., Brindle, I., 2005 The Influence of Harmonia axyridis on Wine Composition and Aging. J. Food. Sci. 70 (2), S128-S135.
19. Pickering, G.J., Lin, J., Reynolds, A., Soleas, G., and R. Riesen. 2006 The evaluation of remedial treatments for wine affected by Harmonia axyridis. IJFST. 41, 77-86.
20. Pickering, G.J., Karthik, A., Inglis, D., Sears, M., and K, Ker. 2007 Determination of Ortho- and Retronasal Detection Thresholds for 2-Isopropyl-3-Methoxypyrazine in Wine. J. Food Science, 7 (7), S468-S472.
21. Pickering, G.J., Spink, M., Kotseridis, Y., Inglis, D., Brindle, I.D., Sears, M., and A-L. Beh. 2008 Yeast strain affects 3-isopropyl-2-methoxypyrazine concentration and sensory profile in Cabernet Sauvignon wine. Australian J.l of Grape & Wine Research. 14: 230-237.
22. Pickering, G.J., Blake, A.J., Soleas, G.J., and D.L. Inglis. 2010 Remediation of wine with elevated concentrations of 3-alkyl-2-methoxypyrazines using cork and synthetic closures. J Food, Agric & Envir. 8:97-101.
23. Roujou de Boubee, D.R. 2004 Research on the vegetal green pepper character in grapes and wines. Rev. des Oenol. 110, 6-10.
24. Roujou de Boubée, D., Cumsille, A., Pons, M., and D. Dubourdieu. 2002 Location of 2- methoxy-3-isobutylpyrazine in Cabernet Sauvignon grape bunches and its extractability during vinification. Am. J. Enol. Vitic. 53(1), 1-5.
25. Ryona, I., Pan, B.S., Intrigliolo D.S., Lakso, A.N., and G.L. Sacks. 2008 Effects of Cluster Light Exposure on 3-Isobutyl-2-methoxypyrazine Accumulation and Degradation Patterns in Red Wine Grapes. Vitis vinifera L. Cv. Cabernet Franc. J. Agric. Food. Chem. 56, 10838-10846.
26. Ryona, I., Reinhardt, J., and G.L. Sacks. 2012 Treatment of grape juice or must with silicone reduces 3-alkyl-2-methoxypyrazine concentrations in resulting wines without altering fermentation volatiles. Food Res Int. 47, 70-79.
27. Sajilata,M., Savitha, K., Singhal, R., and V. Kanetkar. 2007 Scalping of flavor in packaged foods. Comp. Rev. Food Sci. Food Saf. 6, 17-35.
28. Sala, C., Busto, O., Guasch, J., and F. Zamora. 2004 Influence of Vine Training and Sunlight
Exposure on the 3-Alkyl-2-methoxpyrazines Content in Musts and Wines from the Vitis vinifera
Variety Cabernet Sauvignon. J. Agric. Food. Chem. 52, 3492-3497.
29. Sidhu, D., Lund, J., Kotseridis, Y., and C. Saucier. 2015 Methoxypyrazine Analysis and Influence of Viticultural and Enological Procedures on their Levels in Grapes, Musts and Wines. Crit. Rev. Food Sci. Nutr. 55 (4), 485-502.

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