Harmonia axyridis and Wine Quality Source: (www.ncpest.com/ladybeetlefrom_usda.htm) G. J. Pickering 1,2,*, J.Y. Lin 2, R. Riesen 4, A. Reynolds 1,2,3, G. Soleas 5, K. Ker 1, I. Brindle 3 1 Cool Climate Oenology and Viticulture Institute, 2 Department of Biological Sciences,3 Department of Chemistry, Brock University, 4 Lake Erie Enology Research Center, Youngstown State University, Ohio, USA; 5 Quality Control Division, Liquor Control Board of Ontario, Ontario * gary.pickering@brocku.ca Background Harmonia axyridis (Coleoptera: Coccinellidae) (HA) introduced into the US from Japan, the Republic of Korea, and the former USSR during the late 1970s and early 1980s as a biocontrol tool for aphids and other insect pests Now widespread throughout much of these regions and has also been recorded in some western areas of the United States and Canada Acknowledgements Financial support: Materials and Manufacturing Ontario Other support: NSERC; Ker Crop Management Services; Brock sensory panel; Gail Higenell, Dr. Canan Nurgel, Palmina DeMiglio, CCOVI; Steve Cater, LCBO; American Society of Enology and Viticulture Background Over the last decade intermittent reports from winemakers in the eastern wine regions of North America of an atypical aroma and flavor, reminiscent of crushed lady beetles, in some wines Typically, this has coincided with observation of high numbers of HA beetles in vineyards and on the fruit at harvest Background HA have a reflex bleeding response of haemolymph when stressed Methoxypyrazines (low odor threshold – green, vegetal, bell pepper aroma) have been identified in HA Plausible that HA capable of influencing wine quality via transfer of haemolymph onto grapes, or directly into juice Study 1: Impact of HA on the Sensory Properties of White and Red Wine 1 Research objectives/questions 1. Quantify the impact of HA on wine quality Sensory and chemical 2. How does this impact change with time? Bottle ageing Experimental Design White and red wines fermented in the presence of different levels of Harmonia axyridis Std microvinification procedures (from concentrate) Treatments: No beetles (16L x4, & 10L) 1 beetle/L (16L x3) 10 beetle/L (16L x3) Chemical analysis - methoxypyrazines (Soleas et al., 2003); other measurements – Iland (1988) Sensory - descriptive analysis (Lawless & Heymann, 1988) BITTER ACID SWEET SULFUR DIOXIDE HUMUS melon 7 6 5 4 3 2 1 0 citrus floral 0 Beetles/L peanut** bell pepper*** asparagus* ASPARAGUS*** humus BELL PEPPER** PEANUT*** FLORAL sulfur dioxide CITRUS MELON Mean intensity scores for white wine aroma and FLAVOR attributes for control wine BITTER ACID SWEET SULFUR DIOXIDE HUMUS melon 8 7 6 5 4 3 2 1 0 citrus floral 0 Beetles/L 1 Beetle/L peanut** bell pepper*** asparagus* ASPARAGUS*** humus BELL PEPPER** sulfur dioxide PEANUT*** FLORAL CITRUS MELON Mean intensity scores for white wine aroma and FLAVOR attributes for control wine and I level of HA addition to juice BITTER ACID SWEET SULFUR DIOXIDE HUMUS melon 8 7 6 5 4 3 2 1 0 citrus 0 Beetles/L 1 Beetle/L floral 10 Beetles/L peanut** bell pepper*** asparagus* ASPARAGUS*** humus BELL PEPPER** sulfur dioxide PEANUT*** FLORAL CITRUS MELON Mean intensity scores for white wine aroma and FLAVOR attributes for control wine and 2 levels of HA addition to juice BITTER* ACID* SWEET* SULFUR DIOXIDE EARTHY/HERBACEOUS*** red berry* 6 5 4 3 2 1 0 plum** cherry* 0 Beetles/L peanut* asparagus/bell pepper** cheesy CHEESY earthy/herbaceous ** ASPARAGUS/BELL PEPPER** sulfur dioxide PEANUT** CHERRY PLUM RED BERRY* Mean intensity scores for red wine aroma and FLAVOR attributes for control wine 2 BITTER* ACID* SWEET* SULFUR DIOXIDE EARTHY/HERBACEOUS*** red berry* 7 6 5 4 3 2 1 0 plum** cherry* 0 Beetles/L 1 Beetle/L peanut* asparagus/bell pepper** cheesy CHEESY earthy/herbaceous ** ASPARAGUS/BELL PEPPER** sulfur dioxide PEANUT** CHERRY PLUM RED BERRY* Mean intensity scores for red wine aroma and FLAVOR attributes for control wine and I level of HA addition to juice BITTER* ACID* SWEET* SULFUR DIOXIDE EARTHY/HERBACEOUS*** red berry* 8 7 6 5 4 3 2 1 0 plum** cherry* 0 Beetles/L 1 Beetle/L 10 Beetles/L peanut* asparagus/bell pepper** cheesy CHEESY earthy/herbaceous ** ASPARAGUS/BELL PEPPER** sulfur dioxide PEANUT** CHERRY PLUM RED BERRY* Mean intensity scores for red wine aroma and FLAVOR attributes for control wine and 2 levels of HA addition to juice Chemical results No or minimal effect of beetles in white or red wines on: – rate of fermentation – pH – titratable acidity – ethanol – residual sugar – free and bound terpenes – hue, hue density, browning, pinking, phenolic conc. (spectro) Small reduction in volatile acidity for both whites & reds with beetles at any level! Concentration of IP and IB methoxypyrazines Wine: Isopropyl MP (ppt) Isobutyl MP (ppt) White, No Beetle White, 1 Beetle/L White, 10 Beetle/L <5 26 157 Red, No Beetle Red, 1 Beetle/L Red, 10 Beetle/L <5 7* 23 (duplicate measurements, GC-MS, LCBO) <5 <5 6* <5 7* 5* But are methoxypyrazines responsible for the HA character? B IT T E R A C ID SW EET SO 2 HUM US m e lo n 8 6 4 2 0 citru s flo ral 1 0 B e e tle s/L 15ppt peanut b e llp e p p e r asp arag u s ASPAR AG U S hum us B ELL PEPPER S O 2 (aro m a) PEAN U T FLO R AL M ELO N C IT R U S Comparison between HA and 2-methoxy-3-isopropylpyrazine in white wine 3 B ITTE R A C ID SW EET S O 2 (flavor) A R TH Y /H E R B A C E O U S red berry 8 6 4 2 0 p lu m c h e rry 1 0 B e e tle s/L 15ppt peanut a s p a ra g u s /b e ll p e p p e r cheesy CHEESY e a rth y /h e rb a c e o u s A S P A R A G U S /B E LL P E P P E R S O 2 (arom a) PEANUT RED BERRY CHERRY P LU M Comparison between HA and 2-methoxy-3-isopropylpyrazine in red wine Okay, but what happens with bottle age? Mean intensity scores for white wine aroma and FLAVOR at bottling and after 8 months (1 HA beetle/L juice treatment) BITTER ACID SWEET SULPHUR DIOXIDE HUMUS melon 8 6 4 2 0 citrus floral At bottling After 8 months peanut bell pepper asparagus ASPARAGUS BELL PEPPER PEANUT FLORAL humus sulphur dioxide MELON CITRUS Mean intensity scores for white wine aroma and FLAVOR at bottling and after 8 months (10 HA beetle/L juice treatment) BIT T ER ACID SWEET SULPHUR DIOXIDE HUMUS melon 8 6 4 2 0 citrus floral At bottling After 8 months peanut bell pepper asparagus ASPARAGUS BELL PEPPER PEANUT FLORAL humus sulphur dioxide MELON CIT RUS Mean intensity scores for red wine aroma and FLAVOR at bottling and after 8 months (1 HA beetle/L juice treatment) BITTER ACID red berry 8 6 SWEET 4 plum cherry At bottling After 8 months peanut SULPHUR DIOXIDE 2 asparagus/bell pepper EARTHY/HERBACEOUS 0 cheesy CHEESY ASPARAGUS/BELL PEPPER PEANUT CHERRY earthy/herbaceous sulphur dioxide RED BERRY PLUM Mean intensity scores for red wine aroma and FLAVOR at bottling and after 8 months (10 HA beetle/L juice treatment) BITTER ACID SWEET SULPHUR DIOXIDE EARTHY/HERBACEOUS red berry 8 6 4 2 0 plum cherry At bottling After 8 months peanut asparagus/bell pepper cheesy CHEESY ASPARAGUS/BELL PEPPER PEANUT CHERRY earthy/herbaceous sulphur dioxide RED BERRY PLUM 4 Chemical changes after 8 months Isopropyl MP lower in all beetle treatments for both white and red – For 10 beetle/L treatments: 90% lower in white 25% lower in red – For 1 beetle/L treatments: Now ND (analytically) Isobutyl MP ND Little change with other parameters (pH, titratable acidity, ethanol, residual sugar, spectro. measures.) Study 2: Evaluation of Potential Remedial Treatments Bench-tested range potential remedial treats., incl: ™ Fining agents Bentonite: various levels Issinglass Bentonite + Issinglass Activated charcoal: various periods ™ Oak chips - various brands & levels + de-odorized chips ™ Light sources UV: 254nm, 18.4W for different periods Broad spectrum: halogen bulb, 120W for different periods ™ (Irradiation: 90, 110, and 180 Gry ) Settled on: ™ Fining agents Bentonite: 1g/L, 3 days Activated charcoal: 0.2g/L, 3 days ™ Oak chips ‘Normal’: French medium toast chips @ 4g/L for 3 days De-odorized: Per above but chips treated: 40% ethanol overnight, washed & boiling in water for 10 mins. ™ Light UV: only for red wines, 254nm, 18.4W; for 5 min. Broad spectrum: white wines only, halogen bulb, 120W;5min. ™ (Irradiation - 90 and 110 Gry ) Experimental Design These wines treated and analyzed in duplicate as before: Chemical analysis - Methoxypyrazines (Soleas et al., 2003) - Other measurements – Iland (1988) Sensory - Descriptive analysis (Lawless & Heymann, 1988) Results - summary • Chemical: No to minimal change in isopropyl MP conc. for all treatments except ….. Activated charcoal - 34% decrease in white wine Activated charcoal - 11% decrease in red wine • Sensory: White wine: • ‘Normal’ oak chips significantly reduced HA-associated attributes (average 17% for aroma & 52% for flavor [masking]) • Other treatments ‘less successful’ Red wine: • Red wine aroma was improved with the use of 'normal' oak chips, but not with the other treatments used • Decrease in both asparagus and earthy/herbaceous flavours in all treatments except for UV & irradiation • ‘Normal’ oak chips (↓34%) & bentonite (↓33% ) most effective across all HA-associated flavor attributes 5 Study 3 – HA-related wine taint: Identification of critical stages during wine processing & determination of sensory thresholds (2003 vintage) (With support from Wine Council of Ontario and Grape Growers of Ontario) Experimental Plan 1. 690 kg of Riesling sourced from commercial vineyard believed to be free from HA & with a history of low-no occurrences of HA 2. Fruit carefully hand-sorted and any beetles (very few) carefully removed. Following treatments instigated using CCOVI’s teaching & research winery: I. Control (no beetles) HA beetles introduced at different stages: II. Whole fruit – agitation treatment to simulate mechanical harvester (@ 3 beetles/kg fruit) III. Crush/de-stemmer (@ 3 beetles/kg fruit) IV. Crush/de-stemmer (@ 0.3 beetles/kg fruit) V. Whole bunch press (@ 3 beetles/kg fruit) VI. Directly into wine (per 2001 study) (@ 3 beetles/kg fruit) … Analyses Methoxypyrazines (Soleas et al., 2003) (and other wine analytes – Iland, 1988) measured at each stage of treatment & processing → an objective estimate of the influence of HA throughout the winemaking process → better inform prevention measures Sensory (triangle) tests to determine which treatments (if any) different from Control Sensory threshold tests conducted (ascending limits, forced-choice, paired comparison - Lawless and Heymann, 1998) Data then related back to tolerance level for fruit (i.e. # of beetles/weight of grapes req’d to contribute detectable ‘character’) Results - summary Critical Stage of Processing ….. • Isopropyl methoxypyrazine < 5 ppt (LOQ) in all treatments except direct addition of beetles into wine (10 ppt) • In triangle tests, only the direct addition of beetles into wine treatment was different from Control (p=0.001) Ethanol, TA, RS, spectrophotometric, and other quality parameters showed little or no change compared with Control % correct response 100 75 50 25 0 0% y = 0.4135x + 50.76 R2 = 0.852 20% 40% 60% 80% Wine Blend (% 3 beetles/kg grapes / control) 100% Sensory threshold for HA in Riesling wine ©G.J. Pickering Results - summary Sensory Threshold …… • Using the established “50% above chance” criteria (i.e. 75% correct response), threshold is at 58.6% of the 3 beetles/kg grapes blend Translates to 1.758 beetles / kg grapes Which equals 1 758 beetles/ton of grapes 6 Results - summary Sensory Threshold …… Caution Assumptions: beetle behavior at the microprocessing level is the same as at commercial level Extrapolating to other wine styles (particularly reds with skin/beetle maceration) and alternative processing regimes Detection ≠ consumer rejection Overall Summary & Conclusions Fermenting in the presence of H. axyridis had minimal impact on basic wine composition ↑ in IPMP conc. with ↑ # of beetles (greater in white wine) Sensory profile modified: – Beetle-derived aroma and flavor attributes – Decrease in ‘varietal’ attributes – Profile can now be used as tool for measuring consumer acceptance, effectiveness of remedial treatments, etc IPMP strongly implicated in taint, BUT data suggest relationship between conc. & sensory impact not simple – may not be the only culprit (isobutyl and sec-butyl MP have been detected in HA – [Brindle, unpublished data]) – trained nose still the best instrument Bottle aging appears to ↓ MP content, but not MALB-‘taint’ ‘Traditional’ remedial wine treatment results mixed – ↓ in HA characters depends on wine style and aroma vs flavor – No panacea Non-traditional approaches (UV light and irradiation) not effective Current repertoire of treatments not selective enough for MPs Overall, results reinforce the importance of controlling H. axyridis in the vineyard • Sensory threshold for HA-character in Riesling wine equates to ca. 1 750 beetles/ton of grapes Further work Stable-isotope-dilution method to quantify MPs at lower levels than currently possible Other remedial juice/wine options, including ‘designer’ molecules to selectively bind MPs Development & evaluation of exclusion practices in the vineyard and winery – e.g. sprays, deterrents, decoys, shaking sorting trays Determine threshold/tolerance levels in other wine styles, particularly red wines – ? how to estimate HA numbers in a bin of grapes Determine tolerance level of consumers for HA Further information CCOVI, Brock University web site: Pickering lab MALB research: www.brocku.ca/ccovi/res/results/result6.html General MALB questions & answers: www.brocku.ca/ccovi/news/Que_ALB2003.html Pickering et al. (2004). Influence of Harmonia axyridis on the Sensory Properties of White and Red Wine. Am. J. Enol. Vitic. (in press). 7