Margaret A. Cliff a & Gary J. Pickering b a Agriculture and Agri-Food Canada, Pacific Agri-Food Research

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1 This article was downloaded by: [The University of British Columbia] On: 20 February 2015, At: 14:58 Publisher: Routledge Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Journal of Wine Research Publication details, including instructions for authors and subscription information: Determination of odour detection thresholds for acetic acid and ethyl acetate in ice wine Margaret A. Cliff a & Gary J. Pickering b a Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Box 5000 Highway 97, Summerland, BC, VOH 1ZO, Canada b Department of Biological Sciences, Brock University, St. Catharines, Ontario, L2S 3A1, Canada Published online: 23 Jan To cite this article: Margaret A. Cliff & Gary J. Pickering (2006) Determination of odour detection thresholds for acetic acid and ethyl acetate in ice wine, Journal of Wine Research, 17:1, 45-52, DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

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3 Journal of Wine Research, 2006, Vol. 17, No. 1, pp SHORT COMMUNICATION Determination of Odour Detection Thresholds for Acetic Acid and Ethyl Acetate in Ice Wine MARGARET A. CLIFF and GARY J. PICKERING Original manuscript received, 26 February 2004 Revised manuscript received, 21 January 2006 ABSTRACT Collectively acetic acid and ethyl acetate are responsible for volatile acidity (VA) in wine. The detection limit or threshold for these compounds is well documented in table wine but not for ice wine. Knowledge of the ice wine thresholds is important for understanding perception limits and setting legal standards, particularly for a product with high intrinsic concentrations. Thresholds were determined for each compound using seventeen subjects and an ascending series of paired comparison tests, consisting of five concentrations. The detection threshold, the concentration at which there is 75% correct detection, was determined using least-squares linear regression. The correlation coefficients for the linear regressions for acetic acid and ethyl acetate were r ¼ (p, 0.001) and r ¼ (p, 0.001), respectively. The thresholds for acetic acid and ethyl acetate were and g/l, respectively. The threshold for acetic acid was approximately three times that found in table wines, but was relatively unchanged for ethyl acetate. This work supports the need for the legal limit for VA to be higher in ice wine, but also suggests that the legal requirements for the two compounds should be specified independently, not together. Introduction In general, the presence of acetic acid and ethyl acetate above perceptible levels in wine is considered undesirable, and high concentrations are indicative of microbial spoilage. Conversely, at low levels and in some wine styles they may add complexity and enhance fruitiness ( Jackson, 2002). Collectively acetic acid and ethyl acetate are known as volatile acidity (VA) (although ethyl acetate is not an acid). With standard analysis, these compounds are distilled together and are used to establish legal limits. In Canada the legal limits are1.3 g/l for table wine, and 2.1 g/l for ice wines. In Ontario, VQA approval utilizes the following limits: ice wine and totally botrytis affected, 2.1 g/l: special select late harvest and botrytis affected, 1.8 g/l; late harvest and select late harvest, 1.5 g/l; all Margaret A. Cliff, Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Box 5000 Highway 97, Summerland, BC, VOH 1ZO, Canada ( Gary J. Pickering, Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S 3A1, Canada. ISSN print/issn online/06/ DOI: / # 2006 Institute of Masters of Wine

4 46 MARGARET A. CLIFF AND GARY J. PICKERING other wines, 1.3 g/l. Whereas, in the United States the legal limits are 1.4 g/l for red wines and 1.2 g/l for all other wines (Amerine and Ough, 1980). Under aerobic conditions, wine not only oxidizes, but supports the growth of microorganisms, such as Acetobacter pasteurianus, Acetobacter aceti and Gluconobacter oxydans (Fleet, 1993). These organisms utilize ethanol and produce acetaldehyde and acetic acid. The acetic acid concentration of table wine is also influenced by yeast species and strain, juice sugar concentration, ph, nitrogen content, and fermentation temperature (Bisson, 2003). The higher levels of acetic acid in ice wine compared to table wine (Nurgel et al., 2004), are believed to be produced as part of the process of maintaining redox balance by the yeast in response to osmotic stress imposed by high sugar levels (Pigeau et al., 2002; Pitkin et al., 2002). Once acetic acid is formed it can recombine or esterify with ethanol to produce ethyl acetate, a compound with a solvent or nail polish remover odor. The concentrations of acetic acid and ethyl acetate occurring in wine are interdependent, with the relationship between the compounds at equilibrium expressed using the equilibrium constant (K). At equilibrium, if one concentration is known the other can be calculated, if one knows the ethanol concentration, wine density and extract values. When discussing taste and odor thresholds, it is important to identify the type of threshold evaluated: absolute, recognition, difference or terminal. An absolute or detection threshold is the concentration range below which the odor or taste of a substance is not detectable and above which individuals with a normal sense of smell or taste can readily detect the presence of the substance (ASTM, 1991). For populations, the detection threshold is defined as the concentration at which 50% of the group can detect the odorant or tastant. While individual thresholds may have a high variability, group thresholds are much more reliable (Brown et al., 1978; Punter, 1983). The detection threshold is not to be confused with the recognition threshold, the concentration at which the stimuli takes on the characteristic taste or smell. Odor recognition thresholds are particularly difficult to obtain because of the many variables which influence recognition such as: articulateness, sensitivity, familiarity, adaptation, interest, motivation, memory and nasal integrity, to name a few. In contrast, the difference threshold is the change in concentration that is perceptible. The terminal threshold is the concentration at which there is no further increase in response, with increasing stimulus concentration (Brown et al., 1978). Interestingly, this level is rarely achieved in food products for odor because the saturation level is obscured by other sensations such as pain or irritation, which can have an inhibitory effect on odor intensity (Cain, 1976; Cain and Murphy, 1980). It is believed that many wine defects would fall into this category, for odorants have irritant qualities (Doty et al., 1978), particularly at high concentrations. The most common methods for threshold determination are: the ascending method of limits (ASTM E679, 1979), the method of limits (average) (McBurney and Collings, 1977), the semi-ascending paired difference (Lundahl et al., 1986), the staircase method (Cornsweet, 1962), the up-down transformed response (Wetherill and Levitt, 1965) and CHARM analysis (Acree et al., 1984). For a complete overview of these methodologies, readers are referred to Lawless and Heymann (1999). Currently, the forced-choice methods for threshold determination are preferred because they are criteria-free; that is, the experimenter knows if the judge is guessing or faking. The fraction of correct responses due to chance alone in the pair comparison and triangle tests are 1 in 2 (0.5) or 1 in 3 (0.33), respectively. In forced-choice methods, the detection threshold is the concentration, above chance, at which 50% of the population can detect the odorant. Therefore, for the paired comparison

5 ETHYL ACETATE AND ACETIC ACID THRESHOLDS 47 and triangle test, the detection threshold is at 75% and 83.3% correct response, respectively. Corison et al. (1979) reported the thresholds for acetic acid in white and red table wine to be 1130 ppm (1.1 g/l) and 740 ppm (0.74 g/l), respectively, and the thresholds for ethyl acetate to be 170 ppm (0.17 g/l) and 160 ppm (0.16 g/l). Other publications (Amerine and Roessler, 1980) suggest that the threshold of acetic acid in table wine is 0.7 g/l and that the threshold for ethyl acetate is 0.07 g/l. While thresholds are available for table wine they are not for ice wine; therefore the objective of this research was to determine the detection threshold for acetic acid and ethyl acetate in ice wine. Materials and Methods Sample Preparation and Composition Ice wine was produced from both vidal and riesling grapes from the 1999 Ontario vintage and vinified in the pilot winery at the Cool Climate Oenology and Viticulture Institute (CCOVI), Brock University, in accordance with VQA-Ontario regulations governing ice wine production. After fermentation and stabilization, individual wines were assessed by a panel of experts (n ¼ 12), and those that showed acceptable typicity for ice wine were selected, blended, and bottled. This vidal riesling blended ice wine was used as the base wine for the study. Table 1 gives the basic composition of the ice wine. The ph and titratable acidity were assessed using the methods of Amerine and Ough (1974). Reducing sugar was measured using the Luff-Schoorl method (Anon, 1990). Ethanol, acetic acid, ethyl acetate and iso-amyl alcohol were analyzed using a gas chromatography (GC) (Agilent, 6890 CA, US) equipped with a Carbowax (30 X 0.23 mm X 0.25 mm) column. 0.5 ml wine was injected into the injection port heated to 250 8C. The carrier gas was helium with a column head pressure of 20 psig. The oven temperature was programmed to start at 60 8C, increase to 125 8C at68c/min, and then increase to 225 8C at258c/min and hold for 1 min. The detector temperature was 250 8C, and 1-butyl alcohol was used as an internal standard. Standards were purchased from Sigma (Sigma Chemical Co., Oakville, Canada), and measurements of all samples and standards were performed in triplicate. Specific gravity (SG) was measured using a calibrated 25 ml pycnometer equipped with thermometer (KIMAX Chemistry, NY) and immersed in a 20 8C water bath. Physical viscosity was determined using a 50 ml Cannon Fenske Capillary Viscometer (Cannon Instrument Co, State College, PA) immersed in a 20 8 C water bath. All viscometer flux times were determined in triplicate (stop-watch, Heuer). The intrinsic levels (Table 1) of acetic acid (0.816 g/l) and ethyl acetate (0.145 g/l) were both below perceptible levels as determined by a small panel of experts. Table 1. Composition of base ice wine used for threshold tests PH Titratable acidity (g/l) Reducing sugar (g/l) Ethanol (%, v/v) Acetic acid (mg/l) Ethyl acetate (mg/l) Iso-amyl alcohol (mg/l) Specific gravity Viscosity (cp) Mean a sd Note: a Values shown are the means of duplicate or triplicate assessments.

6 48 MARGARET A. CLIFF AND GARY J. PICKERING To determine the appropriate concentration range for the threshold assessments, acetic acid and ethyl acetate were added to the base using values obtained from the literature for still wine. Concentrations were gradually increased in the base ice wine until an odour was detected by a small group of laboratory staff (N ¼ 4). This concentration was then utilized as the upper-end of the concentration range to obtain six concentrations, of which the lower five were used for the threshold determinations. The five concentrations of acetic acid (glacial acetic acid, Fisher) were 1.0, 1.5, 2.0, 2.5 and 3.0 g/l and the five concentrations of ethyl acetate (99.9% pure, Fisher) were 0.000, 0.015, 0.030, and g/l. All solutions were quantitatively prepared in 500 ml volumetric flasks using the base ice wine. These concentrations represented final or total concentrations of 1.816, 2.316, 2.816, and g/l of acetic acid and 0.145, 0.160, 0.175, and g/l of ethyl acetate. All solutions were prepared approximately 2 hrs prior to sensory analysis. Samples (30 ml) were poured into 210 ml egg-shaped ISO glasses and covered with small plastic petri-dishes one-half hour prior to evaluation. All samples were paired with the base Ice wine. Pairs were arranged in an ascending series, low to high concentration, and the order of presentation within each pair was randomized. All evaluations were conducted at room temperature (22 o C) between the hours of 10 : 30 and 11 : 30. Subjects Eighteen subjects participated in the study. All were students or staff in the Oenology and Viticulture program at Brock University ranging in age between 22- to 50-yearsold. Evaluations were conducted in individual tasting booths. Data from one subject was excluded because they showed no discrimination. Sensory Methodology Prior to the threshold determinations, subjects were familiarized with the odor of acetic acid and ethyl acetate, commonly referred to as vinegar and nail polish remover. All subjects had extensive experience with wine quality evaluation and easily recognized the odorants. Subjects proceeded to the booths and were asked to evaluate five pairs and to select the sample in each pair with the named odorant either vinegar or nail polish remover. This was a forced choice assessment; subjects were required to make a selection regardless of their degree of certainty. Samples were assessed orthonasally (sniffed) only. Half the judges on the panel evaluated the acetic acid series first; whereas, the other half evaluated the ethyl acetate series first. After a short break (5 min), subjects evaluated the other series. All judges then took a 30 min break before performing a duplicate evaluation. Data Analysis Data were tabulated as the percent correct for each concentration and plotted against the concentration (total). Least squares linear regressions were calculated and correlation coefficients determined using MS Excel. Results and Discussion Linear regressions for acetic acid and ethyl acetate are shown in Figure 1 and Figure 2, respectively. For acetic acid, the regression equation was Y ¼ 15.88X þ with a

7 ETHYL ACETATE AND ACETIC ACID THRESHOLDS 49 Figure 1. Linear regression for determination of acetic acid thresholds in ice wine, using the paired comparison method. correlation coefficient of (df ¼ 3, p, 0.05). For ethyl acetate, the regression equation was Y ¼ X with a correlation coefficient of r ¼ (df ¼ 3, p, 0.001). Interestingly, the regression coefficient for ethyl acetate (431.37) was approximately 20 times that for acetic acid (15.88), suggesting that the psychophysical function, at sub-threshold concentrations, rises more quickly than that for acetic acid. However, as Pangborn (1981) points out relative sensitivity to compounds at threshold does not always reflect the relative intensities at supra-threshold concentrations. The detection thresholds for acetic acid and ethyl acetate acid in ice wine were and g/l respectively. While the detection threshold for ethyl acetate is similar to that (160 ppm; 0.16 g/l) obtained by Corison et al. (1979) in table wines, the detection threshold for acetic acid is approximately three times larger in ice wine than table wine Figure 2. Linear regression for determination of ethyl acetate thresholds in ice wine, using the paired comparison method.

8 50 MARGARET A. CLIFF AND GARY J. PICKERING (1130 ppm; g/l) (Corison et al., 1979). The carrier in which the stimuli are dispersed (e.g. water, oil, air, alcohol) is known to be extremely important; for example, the detection threshold for acetic acid in wine is an order of magnitude higher than that reported in 10% ethanol (Corison et al., 1979). Residual sugar is also known to mask perception of volatile acidity (Zoecklein et al., 1995). However, this suppression appears to take place preferentially for acetic acid and not ethyl acetate. The mechanism for this purported selective suppression is unknown, but may be related to the greater chemical reactivity of acids compared to esters (Zoecklein et al., 1995). The viscosity of this ice wine (2.5 cp) is also well above the range normally encountered in table wine (1.2 to 1.7 cp; Lopez et al., 1989; Bayindirli, 1993; Kosmerl et al., 2000). Increasing viscosity has been shown to decrease the perceived intensities of volatile compounds in solutions containing varying concentrations of hydroxyl propyl methylcellulose (Hollowood et al., 2002), and a similar mechanism may be occurring here. The effect may be due to overlapping hydrocolloids decreasing compound mobility and therefore the dynamics of flavour release (Morris, 1981). The threshold value for acetic acid obtained here, is above the concentration range reported in Canadian ice wine ( g/l; Nurgel et al., 2003). This is interesting given anecdotal observations from wine professionals that many ice wines do elicit a low but perceptible intensity of vinegar aroma, associated with the odor of acetic acid. Possible synergies between ethyl acetate and acetic acid would be worth investigating in this regard. Conclusions This research has shown that the odour detection threshold for acetic acid is increased threefold in ice wine, but remains relatively unchanged for ethyl acetate when compared to table wine. This work justifies that the legal limit for VA should indeed be higher in ice wine. However, it also suggests that the limits for the two compounds would be more appropriately specified separately, not together, as is currently the case. Acknowledgments Many thanks to the OEVI students at Brock University who participated in this study and to Gail Higenell for her technical assistance. The National Science and Engineering Research Council of Canada, is gratefully acknowledged for its financial support. Thanks also to the following CCOVI individuals for their input and assistance with the chemical analyses: Dr Canan Nurgel, Amanda Bartel, Lynda van Zuiden, and James Lin. Finally; thank you to Dr Andy Reynolds for providing the ice wine for this trial. References ACREE, T.E., BARNARD, J. and CUNNINGHAM, D.G. (1984) A procedure for the sensory analysis for gas chromatographic effluents, Food Chemistry, 14, AMERINE, M.A. and OUGH, C.S. (1974) Wine and Must Analysis. New York: John Wiley. AMERINE, M.A. and ROESSLER, E.B. (1980) Wines: Their Sensory Evaluation. New York: W. H. Freeman and Company.

9 ETHYL ACETATE AND ACETIC ACID THRESHOLDS 51 ANONYMOUS (1990) Recueil des Methods International d Analyses des Vins et des Mouts. Paris: Office International de La Vigne et du Vin. ASTM STANDARD PRACTICE E679 (1979) Determination of odor and taste thresholds by a forced-choice ascending concentration series method of limits. American Society of Testing and Materials, Philadelphia. BAYINDIRLI, L. (1993) Density and viscosity of grape juice as a function of concentration and temperature, Journal of Food Processing and Preservation, 17, BISSON, L.F. (2003) Stuck and sluggish fermentation, American Journal of Enology and Viticulture, 50, BROWN, D.G.W., CLAPPERTON, J.F., MEILGAARD, M.C. and MOLL, M. (1978) Flavor thresholds of added substances, American Society of Brewing Chemists Journal, 36, CAIN, W.S. (1976) Olfaction and the common chemical sense: some psychophysical contrasts, Sensory Processes, 1, CAIN, W.S. and MURPHY, C.L. (1980) Interaction between chemoreceptive modalities of odor and irritation, Nature, 284, CORISON, C.A., OUGH, C.S., BERG, H.W. and NELSON, K.E. (1979) Must acetic acid and ethyl acetate as mold and rot indicators in grapes, Am. J. Enol. Viti., 30(2), CORNSWEET, T.M. (1962) The staircase method in psychophysics, American Journal of Psychology, 75, DOTY, R.L., BRUGGER, W.E., JURS, P.C., ORNDOROFF, M.A., SYNDER, P.J. and LOWRY, D.L. (1978) Intranasal trigeminal stimulation from odorous volatiles: psychometric responses from anosmic and normal humans, Physiol. Behavior, 20, FLEET, G.H. (1993) Wine Microbiology and Biotechnology. Switzerland: Hardwood Academic Publishers. HOLLOWOOD, T.A, LINFORTH, R.S.T. and TAYLOR, A.J. (2002) The effect of viscosity on the perception of flavour, Chemical Senses, 27, JACKSON, R.S. (2002) Wine Tasting: A Professional Handbook. UK: Academic Press KOSMERL, T., ABRAMOVIC, H. and KLOFUTAR, C. (2000) The rheological properties of Slovenian wines, Journal of Food Engineering, 46, LAWLESS, H.T. and HEYMANN, H. (1998) Measurement of sensory thresholds, in: Sensory Evaluation of Food: Principles and Practices. New York: Chapman & Hall, LOPEZ, A., IBARZ, A., PAGAN, J. and VILAVELLA, M. (1989) Rheology of wine musts during fermentation, Journal of Food Engineering, 10, MCBURNEY, D.H. and COLLINGS, V.B. (1977) Introduction to Sensation and Perception. Englewood Cliffs, NJ: Prentice-Hall. MORRIS, E.R. (1981) Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions, Carbohydrate Polymers, 1, NURGEL, C., PICKERING, G.J. and INGLIS, D. (2004) Sensory and chemical characteristics of Canadian ice wines, Journal of the Science of Food and Agriculture, 84, O MAHONY, M. (1986) Sensory Evaluation of Food: Statistical Methods and Procedures. New York: Marcel Dekker. PANGBORN, R.M. (1981) A critical review of threshold, intensity and descriptive analysis in flavour research, in: Flavor 81. Berlin: Walter de Gruyter. PIGEAU, G., MARTIN, S., PITKIN, C. and INGLIS, D. (2002) The hyperosmotic stress response of wine yeast induced by fermentable sugars during ice wine fermentation and its relation to gene expression and metabolites specific to this wine, in: Proceedings of the International Bacchus to The Future Conference, St Catharines, Ontario: Brock University Press, May 2002, pp PITKIN, C., KONTKANEN, D. and INGLIS, D. (2002) The effects of varying soluble solids concentration in ice wine juice on metabolite production by Saccharomyces cerevisiae K1-V1116 during fermentation, in: Proceedings of the International Bacchus to The Future Conference, St Catharines, Ontario: Brock University Press, May 2002, p 179.

10 52 MARGARET A. CLIFF AND GARY J. PICKERING PUNTER, P.H. (1983) Measurement of human olfactory thresholds for several groups of structurally related compounds, Chemical Senses, 7, SOLMS, J., OSMAN-ISMAIL, F. and BEYELER, M. (1973) The interaction of volatiles with food components, Can. Inst. Food Sci & Tech. J., 6(1), A10 A16. WETHERHILL, G.B. and LEVITT, H. (1965) Sequential estimation of points on a psychometric function, British Journal of Mathematical and Statistical Psychology, 18(1), ZOECKLEIN B.W., FIGELSANG, K.C., GUMP, B.H. and NURY, F.S. (1995) Wine Analysis and Production. New York: Chapman and Hall.

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