Determining the capacity of plastic polymers to remove alkyl-methoxypyrazines from wine Andreea Botezatu a,b and Gary Pickering a,b,c Cool Climate Oenology & Viticulture Institute, Environmental Sustainability Research Centre, and Department of Biological Sciences BACKGROUND MATERIALS AND METHODS RESULTS Alkyl-methoxypyrazines (MPs) are an important and potent class of fruit- and insect- derived odorants associated with juice and wine quality (Figure 1 a,b,c) . Figure 1 a 3-isobutyl-2-MP (IBMP) b 3-isopropyl-2-MP (IPMP) c 3-secbutyl-2-MP (SBMP) At high concentrations, they can detract from accepted sensory norms (= off-flavor) (1). In grapes, elevated concentrations of MPs can also be indicative of suboptimal fruit ripeness and can translate into low wine quality in the subsequent wines (2). Humans are vey sensitive to MPs: sensory detection thresholds for MPs in wine range from 0.32 - 16 ng/L (3, 4). Another source of these compounds in juice and wine is from the activity of some Coccinellidae (‘ladybug’) species. Harmonia axyridis Pallas (Coleoptera: Coccinellidae - aka Multicolored Asian lady beetle; MALB- Figure 2) and Coccinella septempunctata (Coleoptera: Coccinellidae – aka 7-Spot, C7) in some years migrate to vineyards in autumn, and become incorporated in with the harvested fruit. When this occurs, they release haemolymph as part of a reflex response. The haemolymph contains high concentrations of MPs, particularly IPMP, which adversely affect the juice and wine, leaving them with undesirable green and vegetal characters (“ladybug taint” (5)). Climate change is exacerbating the problem in Ontario Both MALB and C7 are non-native ladybugs introduced to the USA as biological control agents. Climate change has contributed to a northerly progression in the distribution of these insects, and warmer winters in Ontario over the last few years are associated with much higher survival rates of MALB, with subsequent potential for proliferation the following year (6). MPs from these ladybugs have severely affected some grape/wine vintages in Ontario and parts of the USA, and ladybug taint is also suspected in other wine-making regions, including Germany and Burgundy. Traditional treatments such as fining (7), yeast selection (8), light and temperature (9) or juice settling (10) are not effective or have limited effectiveness. Ideally, a treatment would have high specificity for MPs; not removing desirable aroma and flavour compounds from the juice/wine. Previous work from our lab (11, 12) has shown that some commercially-available corks, when soaked in wine, can reduce MP concentrations by up to 75%. This effect has been attributed to the adsorptive affinity of the plastic polymers found in synthetic corks. Further investigation into the capacity of plastic polymers to lower MPs concentrations in wine was undertaken, and a selection of 14 polymers was tested for their capacity to remove MPs from wine as well as their impact on the organoleptic characteristics of the wine. Of the 14 polymers tested, three were selected for more in-depth investigation (a biodegradable polymer, a silicone based polymer and cellulose fiber) Candidate polymers: * Esterified cellulose (polymer A) * Biodegradable polymer (polymer B) * Silicone polymer (polymer C) All polymers were applied in sheet form, with the exception of the cellulose which was applied as fiber. The wine used for testing was a commercial Merlot. For the surface area trial the contact time was 6 hrs and the surface areas used were 50 sq. cm/L, 200 sq. cm/L and 600 sq. cm/L. For cellulose 2, 4, and 12 grams/L were trialed. Both the treatment and the control wines were spiked with 20 ng/L of each MP (IPMP, SBMP, IBMP). For the time function trial the polymer surface was standardized to 300 sq. cm/L wine. Wines were analyzed both sensorally and analytically. Control wine (spiked with MPs) was used for chemical analysis as well as sensory comparison. Wines were analyzed for MP concentrations pre- and post-treatment as well as for other classes of volatiles (data not shown). The analytical method used for MP quantitation was SPME Multidimensional Gas-Chromatography Mass Spectrometry (13) For the sensory analysis, Descriptive Analysis was performed with 8 assessors evaluating the wines in triplicate (after panel training). Statistical analysis of the data was performed using ANOVA (XLSTAT). Figure 3. Reduction of isopropyl- (IPMP) and isobutyl- (IBMP) methoxypyrazines in red wine after selected polymer treatment, as a function of surface area (50 sq. cm/L, 200 sq. cm/L and 600 sq cm/L) RESULTS Results are presented for polymers B and C only (Polymer A – Cellulose – was found to have unsatisfactory MP reduction capabilities). The MP reduction efficacy of both polymers varied with time (Figures 1 and 2) and surface area (Figure 3). The polymers were found to have limited ability to reduce other unwanted volatile compounds (up to 25% reduction of 4 EP and 4EG after 24 hrs contact, data not shown). Polymers were found to have minimal impact on the sensory profiles of wines (no significant differences were found between treatments for any of the sensory descriptors used; Figures 4, 5). Decreases in “green type” aroma characters are noted for treated wines, as anticipated. Figure 2. Harmonia axyridis (MALB) beetle Figure 4. Intensity Ratings for AROMA Descriptors (no significant differences were found) FUTURE WORK Treated wines will be further tested to assess the potential impact of the polymers on classes of non-target volatile wine constituents, including esters, ketones and higher alcohols. Polymers will also be assessed as part of a dual-system that makes use of odorant-binding protein technology. Selected polymer(s) will be tested this vintage on a commercial scale at the Niagara College Winery. Wines treated with the selected polymer(s) will be analyzed both chemically and sensorally. PURPOSE Your caption can go here. The purpose of this work was to (i) test the capacity of selected polymers to remove MPs from wine under various working conditions, (ii) assess their specificity for MPs, and (iii) establish their impact on the sensory quality of the wine. The research questions were: How does the adsorptive capacity of the selected polymers vary as a function of contact time with the wine and surface area used for treatment? To what extent do the polymers affect desirable volatile constituents of treated wines? Do the polymers reduce other unwanted taint-related compounds (such as 4EP, 4EG, ethyl acetate) 4. Can they be used as a juice/wine treatment without deleterious effects to sensory quality? References: 1)-Allen, Malcolm S., Michael J. Lacey, and Stephen Boyd. "Determination of methoxypyrazines in red wines by stable isotope dilution gas chromatography-mass spectrometry." Journal of agricultural and food chemistry 42.8 (1994): 1734-1738. 2)-Roujou de Boubée, Dominique, Cornelis Van Leeuwen, and Denis Dubourdieu. "Organoleptic impact of 2-methoxy-3-isobutylpyrazine on red Bordeaux and Loire wines. Effect of environmental conditions on concentrations in grapes during ripening." Journal of Agricultural and Food Chemistry 48.10 (2000): 4830-4834. 3)-Pickering, G. J., et al. "Determination of Ortho‐and Retronasal Detection Thresholds for 2‐Isopropyl‐3‐Methoxypyrazine in Wine." Journal of food science 72.7 (2007): S468-S472.4)-Sala, C., et al. "Factors affecting the presence of 3-alkyl-2-methoxypyrazines in grapes and wines." A review 7 (2004): 169-176. 5)- Pickering, Gary, et al. "Influence of Harmonia axyridis on the sensory properties of white and red wine." American journal of enology and viticulture 55.2 (2004): 153-159. 6)-Brown, P. M. J., et al. "Harmonia axyridis in Europe: spread and distribution of a non-native coccinellid." BioControl 53.1 (2008): 5-21. 7)-Pickering, Gary, et al. "The evaluation of remedial treatments for wine affected by Harmonia axyridis." International journal of food science & technology 41.1 (2006): 77-86. 8)- Pickering, Gary J., et al. "Yeast strain affects 3‐isopropyl‐2‐methoxypyrazine concentration and sensory profile in Cabernet Sauvignon wine." Australian Journal of Grape and Wine Research 14.3 (2008): 230-237. 9)-Pickering, G. J., A. Blake, and Y. Kotseridis. "Czech J. Food Sci." Czech J. Food Sci 27 (2009): S62-S65. 10)- Kotseridis, Y. S., et al. "Quantitative analysis of 3-alkyl-2-methoxypyrazines in juice and wine using stable isotope labeled internal standard assay." Journal of Chromatography A 1190.1 (2008): 294-301. 11)- Blake, Amy, et al. "Effect of closure and packaging type on 3-alkyl-2-methoxypyrazines and other impact odorants of Riesling and Cabernet Franc wines." Journal of agricultural and food chemistry 57.11 (2009): 4680-4690. 12)- Pickering, Gary J., et al. "Remediation of wine with elevated concentrations of 3-alkyl-2-methoxypyrazines using cork and synthetic closures." International journal of food, agriculture and environment 8.2 (2010): 97-101. 13)-Botezatu, Andreea I., et al. "Occurrence and contribution of alkyl methoxypyrazines in wine tainted by Harmonia axyridis and Coccinella septempunctata." Journal of the Science of Food and Agriculture (2012). Figure 1. Reduction of isopropyl- (IPMP) and isobutyl- (IBMP) methoxypyrazines in red wine after selected polymer treatment (8 hrs contact). Surface area was standardized (300 sq.cm/L) Figure 2. Reduction of isopropyl- (IPMP) and isobutyl- (IBMP) methoxypyrazines in red wine after selected polymer treatment (24 hrs contact). Surface area was standardized (300 sq.cm/L) This research was supported by : The Wine Council of Ontario; Grape Growers of Ontario; Ontario Grape and Wine Research Inc.; Ontario Research Fund- Research Excellence, Round 5.