# Can a certain concentration of rotundone be undesirable in Duras red wine? A study to estimate a consumer rejection threshold for the pepper aroma compound

O. Geffroy, J. Descôtes, E. Serrano, M. Li Calzi, L. Dagan, R. Schneider

► To cite this version:

O. Geffroy, J. Descôtes, E. Serrano, M. Li Calzi, L. Dagan, et al. Can a certain concentration of rotundone be undesirable in Duras red wine? A study to estimate a consumer rejection threshold for the pepper aroma compound. Australian Journal of Grape and Wine Research, 2018, 24 (1), pp.88-95. 10.1111/ajgw.12299. hal-05095436

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# Can a certain concentration of rotundone be undesirable in Duras red wine? A study to estimate a consumer rejection threshold for the pepper aroma compound

O. GEFFROY¹,² ID, J. DESCÔTES¹, E. SERRANO¹, M. LI CALZI², L. DAGAN³ and R. SCHNEIDER³,⁴

¹ Institut Français de la Vigne et du Vin Pôle Sud-Ouest, V'innopôle, 81 310 Lisle Sur Tarn, France; ² École d'Ingénieurs de Purpan, 31 076 Toulouse Cedex 3, France; ³ Nyséos, 34 080 Montpellier, France; ⁴ Institut Français de la Vigne et du Vin Pôle Rhône Méditerranée, Domaine de Pech Rouge, 11 430 Gruissan, France

Corresponding author: Mr Olivier Geffroy, email olivier.geffroy@purpan.fr

## Abstract

**Background and Aims:** Rotundone is responsible for peppery aroma in wine. This note is not appreciated by all consumers, which indicates that rotundone might be considered as a taint above a certain concentration. The current study aimed at determining a consumer rejection threshold for rotundone in Duras red wine.

**Methods and Results:** Consumers (n = 62) received pairs of samples consisting of a base wine and the base wine spiked with an ascending concentration of rotundone and were asked to indicate which sample they preferred. Anosmic respondents detected through triangle tests represented 31% of the panellists. For these panellists, the wine spiked at the lowest concentration was significantly rejected suggesting that rotundone might induce a trigeminal sensation and/or is involved in molecular mechanisms of flavour reduction. For remaining panellists, we were not able to determine any consumer rejection threshold. We identified three clusters of consumer profiles. The first cluster preferred a moderate concentration of rotundone and rejected a high concentration. The second cluster, mainly composed of young consumers, preferred the Control. The last group appreciated peppery wines especially at high concentration.

**Conclusions:** The response of consumers to rotundone is complex, and peppery notes are perceived positively or in a neutral manner by most consumers with the exception of young panellists.

**Significance of the Study:** Our results may assist grape growers producing Duras and other cultivars, where rotundone makes a sensory contribution, to adapt their products to consumer profiles. They also open new fields of investigation into mechanisms involved in specific anosmia to rotundone.

**Keywords:** consumer preference, consumer rejection threshold (CRT), Duras red wine, peppery flavour, rotundone

## Introduction

Rotundone is a sesquiterpene originally isolated from the tubers of the grassy weed nutgrass (*Cyperus rotundus*) by Kapadia et al. (1967). More recently, rotundone has been found in various plants and plant products (Wood et al. 2008), such as white and black peppers (*Piper nigrum*), marjoram (*Origanum majorana*), oregano (*Origanum vulgare*), geranium (*Pelargonium alchemilloides*), rosemary (*Rosmarinus officinalis*), saltbush (*Atriplex cinereal*), basil (*Ocimum basilicum*), thyme (*Thymus vulgaris*) and grape and wine (*Vitis vinifera* L. cv. Shiraz). Up to now, it has been identified in a wide range of grape cultivars including Pinot Noir, Durif, Graciano and Riesling (Herderich et al. 2012), Duras (Geffroy et al. 2014), Gamay (Geffroy et al. 2016a), Malbec and Abouriou (Cullere et al. 2016) and Vespolina, Schioppettino and Grüner Veltliner (Caputi et al. 2011). Recent research showed that two key polymorphisms in a newly discovered allele of the *V. vinifera* TPS24 gene were responsible for the production of α-guaiene (Drew et al. 2016). As rotundone is produced although simple aerial or enzymatic oxidation of α-guaiene (Huang et al. 2014, Takase et al. 2016), these polymorphisms are likely to explain the differences in rotundone concentration observed between cultivars. The compound also plays a significant role in the flavour of oak-aged spirits with rotundone increasing with ageing time

(Genthner 2014). During the same study, rotundone was also found in un-aged tequila, which suggests that the compound may also be present in the agave plant.

Rotundone was reported in grape leaves and stems (Capone et al. 2012) and more recently in Shiraz flower caps at pre-veraison (Zhang et al. 2016). The compound does not appear to be translocated through the phloem tissues, and the hypothesis of an in situ production is more likely (Geffroy et al. 2016c, Zhang et al. 2016). Viticultural and environmental factors have a substantial impact on rotundone concentration in grape berries and in finished wines. Cool and wet vintages promote the production of red wines with a higher rotundone concentration (Caputi et al. 2011, Zhang et al. 2015b). Within a single vineyard, large spatial variability in rotundone associated with variation in the land underlying the vineyard and vine water status were reported (Scarlett et al. 2014, Geffroy et al. 2015, Zhang et al. 2015a). Patterns of this spatial variation are temporally stable from year to year (Bramley et al. 2017).

Clonal differences in rotundone concentration were identified among certified clones of Duras (Geffroy et al. 2015), Shiraz (Siebert and Solomon 2010) and Grüner Veltliner (Caputi et al. 2011). The compound might be involved in the vine's natural defence mechanisms as a positive correlation was identified between rotundone and severity
of powdery mildew on bunches (Geffroy et al. 2015). Rotundone concentration increases with the ripening of the fruit (Caputi et al. 2011, Geffroy et al. 2014, Logan 2015). It is enhanced by irrigation and lowered by leaf removal in the bunch zone (Geffroy et al. 2014). The spraying of 1-naphthaleneacetic acid, a plant growth regulator (Davies et al. 2015), and the implementation of a viticultural system combining irrigation and on-vine grape drying with cutting of the fruit bearing cane (Geffroy et al. 2016c) were successfully tested to enhance rotundone in vineyard.

Rotundone is mainly located in the grape exocarp (Siebert and Solomon 2011, Caputi et al. 2011), and most of it is extracted from the berries between day 2 and 5 of fermentation (Siebert and Solomon 2011). Winemaking techniques and fermentation variables have an impact on rotundone in finished wines (Geffroy et al. 2017). Compared with a traditional maceration wine, wines made from a rosé vinification and from thermovinification treatments, which involved a preferment removal of skins, resulted in a low rotundone concentration. None of the studied treatments including the use of macerating enzymes and the increase in temperature or time of maceration resulted in enhanced rotundone concentration.

The compound is potent and has an aroma detection threshold of 8 ng/L in water and 16 ng/L in red wine (Wood et al. 2008). It has been described as one of the 16 most important aroma compounds in wine (Ferreira 2012). Specific anosmia has been reported for rotundone (Wood et al. 2008), and during the first sensory trials, 20-25% of panellists could not detect this compound at high concentration in water. A significant correlation (r² = 0.66) was identified between peppery aroma scores and rotundone concentration in 21 Gamay wines (Geffroy et al. 2016a). During the same study on consumer preference, the cluster of consumers who preferred peppery wines frequently rated a wine with an amylic note as the least appreciated and vice versa. This cluster was mainly composed of managers who are willing to pay more for a bottle of wine. When assessing the liking responses of Chinese consumers for red wines, the cluster of consumers preferring peppery wines represented 20% of the whole population, while for 49% of the panellists, the black pepper attribute was negatively related to liking (Williamson et al. 2012). These results suggest that rotundone might be considered as a taint by certain consumers or that rotundone might be undesirable above a certain concentration.

The consumer rejection threshold (CRT) approach has been used to determine the concentration of a compound that gives a significantly lower preference. Based on a novel application of the paired preference test, the method has been applied to answer several off-flavour questions in wine, such as 2,4,6-trichloroanisole (Prescott et al. 2005), phenolic substances (Yoo et al. 2011), 1,1,6-trimethyl-dehydronaphthalene (Ross et al. 2014), ethyl phenyl-acetate and phenyl acetic acid (Campo et al. 2012) and 1,8-cineole (Saliba et al. 2009). According to Francis and Williamson (2015), the approach provides, more generally, a valuable measure as to when consumers will respond negatively to a flavour.

The aim of the current study was to determine a CRT for rotundone in a Duras red wine according to the methodology established by Prescott et al. (2005). Duras is one the most planted red grape cultivars in the Protected Designation of Origin Gaillac in the south-west of France. As this grape cultivar is known to be a regular rotundone producer with a final concentration in wine, in most cases, over its detection threshold (Geffroy et al. 2014, 2015, 2016c, 2017), the base wine

spiked with several concentration values of rotundone was made following a viticultural and oenological process conceived to minimise rotundone and therefore having a negligible concentration of this compound in the finished wine. A questionnaire was used to assess whether there are any differences in preference for rotundone across the variables of demographic and level of knowledge. Anosmic respondents were also identified using a triangle test.

# Material and methods

## Panellists

The panel was composed of 49 wine consumers and 13 wine professionals (i.e. winemakers, winegrowers and estate managers). Wine consumers, all of whom were self-declared regular consumers of red wine (at least once per month), were recruited in the Toulouse area (south-west of France) through an advertisement published in the regional daily newspaper. Wine professionals from the Gaillac Protected Designation of Origin were recruited via the local association of winegrowers and winemakers. Remuneration for participating in the study consisted of a bottle of wine. The only information about the samples provided to the participants was that they were evaluating some red wines. They were asked to provide some demographic information and to respond to several questions regarding their purchase behaviour and their knowledge of peppery notes at the end of the tasting session. The questionnaire also contained ample space for free comments. The panel included 28 women and 34 men, and the age of panellists was between 18 and 24 (23%), 25 and 34 (26%), 35 and 44 (16%), 45 and 54 (19%) and over 55 (16%) years. Most of the panellists were managers (39%) and employees (29%) according to French National Institute for Statistics and Economics categories, and 89% reported consuming wine at least once per week. Subjects reported consuming wine for a mean of 17.9 years (standard deviation = 13.9) and described their level of knowledge as 'experts' (21%), 'knowledgeable wine drinkers' (31%) and 'interested in wine' (48%). The label 'expert' was only used for wine professional panellists. Average prices spent on a bottle were €3-5 (16%), €5-10 (73%) and >€10 (11%). The questionnaire revealed that 79% of the panellists had previously heard of peppery notes in wine and that 69% of them appreciated this kind of flavour.

## Base wine and other materials

To have a negligible level of rotundone, the base wine used for this study was made in our experimental cellar (Lisle sur Tarn, France) following a special process. Duras grapes (100 kg) from the 2015 vintage – a hot and dry vintage not favourable to the expression of intense peppery notes in wines – were sourced from a non-irrigated vineyard experiencing a moderate to severe water deficit. A two-face leaf removal was performed at mid-veraison, and harvest took place early in the maturation period exactly 38 days after the mid-veraison date. The grapes were destemmed and crushed, and sulfur dioxide (40 mg/L) was added using a 10% bisulfite liquid solution (Solution 10, Laffort, Bordeaux, France). The must was inoculated with 200 mg/L rehydrated active dried *Saccharomyces cerevisiae* yeast (Lalvin Rhône 2056, Danstar Ferment, Zug, 91 Switzerland), and 300 mg/L of diammonium phosphate was added. Grapes were fermented on skins for 8 days at 25°C controlled temperature, and to minimise the extraction of skin compounds (i.e. rotundone), a single punch down per day was performed during the first 4 days of fermentation. The wine was then racked and inoculated with lactic acid bacteria
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Proportion of non-anosmic panellists preferring the Control sample (n = 43). No preference (—) and the Control is significantly preferred at the 5% threshold using paired comparison tests (---) are indicated.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Proportion of anosmic panellists preferring the Control sample (n = 19). No preference (—) and the Control is significantly preferred at the 5% threshold using paired comparison tests (---) are indicated.</figcaption></figure>

(Lalvin 31, Danstar Ferment). When the concentration in malic acid, determined using an enzymatic kit (Thermo Fisher Scientific, Waltham, MA, USA), was less than 0.2 g/L, the wine was racked and 50 mg/L of sulfur dioxide was added. The wine was stored in the cellar for 3 months at room temperature and then was tartrate stabilised (1 month at 0°C). Prior to bottling, the wine was filtered through a cartridge filter (Pall France, Saint Germain-en-Laye, France) equipped with 5 and 1 µm filtration cartridges (Prédel, Saint-Loubès, France). Analysis of the bottled wine revealed the following: alcohol concentration 12.1% (Alcoquick L200 Infralyser, Unisensor, Karlsruhe, Germany), pH 3.66 (Titromatic pH meter, Hachlange, Düsseldorf, Germany), TA 4.23 g/L of tartaric acid measured according to the Organisation Internationale de la Vigne et du Vin (OIV) method (Organisation Internationale de la Vigne et du Vin 2009), 0.1 g/L of glucose + fructose and volatile acidity 0.36 g/L acetic acid (enzymatic kits provided by Megazyme, Wicklow, Ireland). The wine had a low concentration of phenolic substances, as anthocyanin and Total Phenolic Index quantified according to Ribéreau-Gayon and Stonestreet (1965) and Ribéreau-Gayon (1970) were 309 and 36 mg/L, respectively. The concentration of free and total sulfur dioxide, determined according to the iodometric method (Organisation Internationale de la Vigne et du Vin 2009), was 22 and 48 mg/L, respectively.

Tasting trials suggested no peppery notes in the base wine, which was described as ‘fruity’. The rotundone concentration in the base wine was 9 ng/L determined by the method of Mattivi et al. (2011). To our knowledge, this concentration is the lowest ever detected for a Duras red wine made using the traditional maceration technique (i.e. without preferment removal of skins) from grapes collected at least 37 days after mid-veraison. In fact, for this cultivar, a concentration ranging from 18 (Geffroy et al. 2014) to 341 ng/L – an unexpected high concentration found in wine made from powdery mildew infected grapes (Geffroy et al. 2015) – has been previously reported. Such variation combined with a pilot study using a small group of tasters allowed us to determine the amount of rotundone to add to the base wine. Food grade rotundone (purity > 99%) provided by Firmenich (Geneva, Switzerland) was used to spike the wine at the rate of 25, 50, 100, 200 or 400 ng/L. The spiked wines and an unspiked Control wine

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Dendrogram of agglomerative hierarchical clustering showing the existence of three groups of non-anosmic consumers: clusters 1 (—), 2 (—) and 3 (—). Numbers on the x-axis refer to identification numbers from 1 to 62 provided to each panellist at the beginning of the study.</figcaption></figure>
were prepared 24 h before the tasting. A 200 ng/L rotundone solution was also prepared for triangle tests with the aim to identify anosmic respondents among the panellists. Samples were coded with three-digit codes, and a constant volume of 15 mL was poured in clear wine-tasting glasses at 18°C. After the tasting, determination of rotundone confirmed that the concentration of rotundone added was appropriate with a recovery rate of 86–97%. The study was organised in Toulouse, south-west of France, in a professional tasting room with booths.

### Procedures

The procedure for CRT determination was described by Prescott et al. (2005) and is based on replicate series of five paired comparison tests, one for each rotundone concentration. Each pair consisted of a sample of the base wine alone and a sample of the base wine with added rotundone. Presentation order was randomised, and each pair was presented in order of ascending concentration. Panellists were asked to proceed with an olfactory assessment, to place the whole sample in their mouth, to move it around for a few seconds and to expectorate it. Then they had to indicate on a sheet which coded sample of the pair they preferred. Panellists received a new pair of samples every 5 min.

At the end of the session and just before to completing the post-experiment questionnaire, anosmic respondents were identified using a triangle test in which one the three coded samples contained a 200 ng/L rotundone solution and the other two samples water alone. The position of the rotundone sample within each test was randomised across each series. Those who were not able to identify the different sample after the olfactory and taste assessment were considered as anosmic respondents.

### Data treatment

The binomial distribution for paired comparison test (Roessler et al. 1978) was used to determine when a wine was significantly rejected. Statistical analyses including principal component analysis (PCA), agglomerative hierarchical clustering and chi-squared test were conducted with XLSTAT software (Addinsoft, Paris, France). The Kolmogorov–Smirnov test was used to assess whether preference for rotundone differed according to gender, knowledge and self-reported liking of peppery notes. For age, socio-economic classification, frequency of consuming, average price spent on bottle and level of knowledge, the Kruskal–Wallis test was used. In the case of statistically significant results at the 5% threshold for the Kruskal–Wallis test, post hoc comparisons were performed using Conover–Iman test.

### Results

Nineteen anosmic respondents that represent 31% of the whole panel were identified. As they are likely to add noise to the data, they were considered separately. A chi-squared test showed that the proportion of women within the non-anosmic (46.5%) and anosmic populations (42.0%) was equivalent (P = 0.748). The same statistical method revealed that the proportion of panellists over 55 was significantly larger (P = 0.028) within the anosmic population.

Figures 1 and 2 show, respectively, the proportion of non-anosmic and of anosmic panellists who preferred the Control wine at each concentration of rotundone tested. Surprisingly, the wine spiked at 25 ng/L was significantly dispreferred by anosmic panellists using the 5% significance criterion (0.79)

for paired preference tests (n = 19). For non-anosmic respondents, no CRT was determined, which suggests the existence of several groups of consumer behaviour among this population. To determine these clusters, preference data were first treated with PCA. The data set consisted of a matrix of order 43 × 5, where the rows represented the 43 non-anosmic panellists and the columns represented the preferred rotundone concentration for each of the five paired comparison tests. Then an agglomerative hierarchical clustering (Figure 3) was performed on the PCA coordinates. The analysis was made using the Euclidian distance and with Ward's method as aggregation criterion. Figure 4 illustrates the proportion of panellists from the three identified clusters who preferred the unspiked wine. The proportion of consumers in

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Proportion of panellists preferring the Control sample: (a) for the cluster 1 of non-anosmic consumers (n = 20); (b) for the cluster 2 of non-anosmic consumers (n = 9); and (c) for the cluster 3 of non-anosmic consumers (n = 14). No preference (—) and the Control is significantly preferred or dispreferred at the 5% threshold using paired comparison tests (—–) are indicated. CRT, consumer rejection threshold.</figcaption></figure>
each cluster by demographic, consumption habit and wine knowledge categories is presented in Table 1.

It appears that consumers from cluster 1 (n = 20) significantly prefer wines with a moderate concentration of rotundone (less than 46 ng/L) and reject wines with a high concentration (more than 309 ng/L). This consumer group is mainly composed of male participants (65%) and panellists over 35 (55%). It presents a substantial proportion of managers

(40%) and a good wine knowledge as 60% of the panellists are experts or knowledgeable wine drinkers. Most of them (65%) declared liking peppery notes.

Those from cluster 2 (n = 9) significantly prefer the unspiked wine except when the rotundone concentration exceeds 275 ng/L, in which case neither the Control wine nor the spiked wine is preferred. This group has more female participants (67%) and is composed of young panellists as 66% of

<table><caption>**Table 1.** Proportion of consumers in each cluster of non-anosmic panellists by gender, age, socio-economic classification, frequency of consuming, average price spent on bottle, levels of knowledge in wine and peppery notes and self-declared appreciation of peppery notes.</caption><thead><tr><th rowspan="2">Category</th><th rowspan="2"></th><th colspan="3">Proportion (%)</th></tr><tr><th>Cluster 1<br>n = 20</th><th>Cluster 2<br>n = 9</th><th>Cluster 3<br>n = 14</th></tr></thead><tbody><tr><td rowspan="2">Gender</td><td>Male (n = 23)</td><td>65</td><td>33</td><td>50</td></tr><tr><td>Female (n = 20)</td><td>35</td><td>67</td><td>50</td></tr><tr><td rowspan="5">Age (years)</td><td>18–24 (n = 11)</td><td>30</td><td>44</td><td>7</td></tr><tr><td>25–34 (n = 11)</td><td>15</td><td>22</td><td>43</td></tr><tr><td>35–44 (n = 9)</td><td>30</td><td>22</td><td>7</td></tr><tr><td>45–54 (n = 8)</td><td>15</td><td>12</td><td>29</td></tr><tr><td>&gt;55 (n = 4)</td><td>10</td><td>0</td><td>14</td></tr><tr><td rowspan="7">Socio-economic classification</td><td>Farmers (n = 1)</td><td>5</td><td>0</td><td>0</td></tr><tr><td>Middle-level occupations (n = 1)</td><td>0</td><td>0</td><td>7</td></tr><tr><td>Managers (n = 17)</td><td>40</td><td>11</td><td>50</td></tr><tr><td>Service, sales and support workers (n = 1)</td><td>5</td><td>11</td><td>0</td></tr><tr><td>Employees (n = 15)</td><td>30</td><td>55</td><td>29</td></tr><tr><td>Students and unemployed workers (n = 8)</td><td>20</td><td>23</td><td>14</td></tr><tr><td>Once a day (n = 6)</td><td>15</td><td>11</td><td>14</td></tr><tr><td rowspan="5">Frequency of consuming</td><td>3–4 times a week (n = 5)</td><td>20</td><td>22</td><td>50</td></tr><tr><td>Once a week (n = 19)</td><td>55</td><td>22</td><td>29</td></tr><tr><td>Twice a month (n = 5)</td><td>10</td><td>45</td><td>7</td></tr><tr><td>Once a month (n = 0)</td><td>0</td><td>0</td><td>0</td></tr><tr><td>&lt;3 (n = 0)</td><td>0</td><td>0</td><td>0</td></tr><tr><td rowspan="4">Average price spent on bottle (€)</td><td>3 to 5 (n = 6)</td><td>5</td><td>22</td><td>21</td></tr><tr><td>5 to 10 (n = 29)</td><td>75</td><td>67</td><td>57</td></tr><tr><td>&gt;10 (n = 8)</td><td>20</td><td>11</td><td>22</td></tr><tr><td>Interested in wines (n = 23)</td><td>40</td><td>78</td><td>57</td></tr><tr><td rowspan="2">Level of wine knowledge</td><td>Knowledgeable wine drinkers (n = 13)</td><td>35</td><td>11</td><td>36</td></tr><tr><td>Experts (n = 7)</td><td>25</td><td>11</td><td>7</td></tr><tr><td rowspan="2">Have you heard of peppery notes in wine?</td><td>Yes (n = 36)</td><td>85</td><td>100</td><td>71</td></tr><tr><td>No (n = 7)</td><td>15</td><td>0</td><td>29</td></tr><tr><td rowspan="2">Do you appreciate peppery notes in wine?</td><td>Yes (n = 30)</td><td>65</td><td>44</td><td>93</td></tr><tr><td>No (n = 13)</td><td>35</td><td>66</td><td>7</td></tr></tbody></table>

<table><caption>**Table 2.** Comparison of preference within the non-anosmic population for each rotundone concentration, for gender, age, socio-economic classification, frequency of consuming, average price spent on bottle, level of knowledge in wine and self-reported liking of peppery notes.</caption><thead><tr><th rowspan="2">Category</th><th colspan="5">Significance according to rotundone concentration</th></tr><tr><th>34 ng/L</th><th>59 ng/L</th><th>109 ng/L</th><th>209 ng/L</th><th>409 ng/L</th></tr></thead><tbody><tr><td>Gender†</td><td>0.097</td><td>0.999</td><td>1.00</td><td>0.70</td><td>1.00</td></tr><tr><td>Age‡</td><td>0.564</td><td>0.342</td><td>0.047</td><td>0.115</td><td>0.044</td></tr><tr><td>Socio-economic classification‡</td><td>0.121</td><td>0.725</td><td>0.201</td><td>0.427</td><td>0.550</td></tr><tr><td>Frequency of consuming‡</td><td>0.499</td><td>0.101</td><td>0.100</td><td>0.167</td><td>0.166</td></tr><tr><td>Average price spent on bottle‡</td><td>0.795</td><td>0.491</td><td>0.681</td><td>0.537</td><td>0.415</td></tr><tr><td>Level of wine knowledge‡</td><td>0.156</td><td>0.555</td><td>0.395</td><td>0.584</td><td>0.214</td></tr><tr><td>Familiarity with peppery notes in wine†</td><td>0.261</td><td>1.00</td><td>1.00</td><td>1.00</td><td>0.564</td></tr><tr><td>Self-report liking of peppery notes‡</td><td>0.514</td><td>0.988</td><td>0.169</td><td>0.258</td><td>0.377</td></tr></tbody></table>

†The Kolmogorov–Smirnoff (two-tailed) test was performed. ‡The Kruskall–Wallis test was performed.
them are less than 35 years of age. These consumers drink wine more occasionally (no more than once per week for 77% of them) and have a basic wine knowledge (78%). Most of them declared disliking peppery notes in wine (66%).

Consumers from the third cluster (n = 14) significantly prefer peppery wines when the rotundone concentration exceeds 94 ng/L. They are mainly managers (50%), drink wine several times per week (64%) and declare an appreciation of peppery notes in wine (93%).

Non-parametric statistical tests showed that the age of panellists had an impact on the appreciation of wines for two levels of rotundone concentration, 100 and 400 ng/L (Table 2). For the wine spiked at 100 ng/L, consumers' preference differed significantly between the youngest panellists (18-24) who preferred the Control wine and the oldest panellists (>55) who appreciated the spiked wine. For the highest level of rotundone concentration tested in our study, differences were observed between the 25-34 and 35-44 year age categories. The first category preferred the complemented wine while the second one the unspiked wine.

## Discussion

The proportion of anosmic respondents within our population (31%) appears to be higher than that observed in Australia (20-25%). Specific anosmia, which refers to the reduced ability (10-1000 times) of an individual to detect a particular odorant, has been widely documented. For androsterone, one of the most notable and studied examples, differences in perception were found between populations from continental Europe, Asia and Australia (Wysocki and Gilbert 1989). These observations corroborate our hypothesis that the rate of specific anosmia to rotundone might vary between French and Australian populations. Our findings also indicate that older panellists (>55 years) are less likely to detect rotundone. In a large-scale study, it was noticed that a steep decline in detection began more abruptly in the fifth decade for aroma compounds (i.e. androstenone and galaxolide) with previously documented specific anosmia (Wysocki and Gilbert 1989).

The fact that the lowest concentration of rotundone addition (25 ng/L) induced, within the anosmic population, a stimulus leading to the rejection of the spiked sample deserves further comment. Among anosmic panellists who preferred the Control wine for this series (n = 15), 47 and 27% of them indicated, in the space available for free comments, that they found the complemented wine less fruity and more acidic, respectively. It is well known that most odorants stimulate the trigeminal system, and it has been demonstrated that anosmic respondents were capable of detecting trigeminal sensations (Doty et al. 1978). Even if our observations suggest that rotundone might induce a trigeminal stimulus, reasons why no sensation was perceived by anosmic panellists at a higher rotundone concentration remain unclear. It was recently shown that 2,4,6-trichloroanisole (TCA) attenuated olfactory transduction even at extremely low concentration without evoking odorant responses (Takeuchi et al. 2013). Suppression by TCA was correlated with the lipophilicity of TCA and appeared to be mediated by a partitioning of the compound into the lipid bilayer of plasma membranes. Rotundone is known to be a lipophilic compound (Caputi et al. 2011), and such molecular mechanisms of flavour reduction might also be involved. As these mechanisms are concentration-dependent (Takeuchi et al. 2013), we might suppose that rotundone concentration in the spiked wine from the first pair was not sufficient to suppress the olfactory signal transduction,

while for the four following pairs of wines with higher rotundone concentration, the suppression was effective. Molecular mechanisms involved in specific anosmia to rotundone appear to be complex and deserve further studies.

When focusing on non-anosmic respondents, we were not able to determine any concentration of rotundone from which peppery notes become undesirable in Duras red wine. This illustrates the complexity of the response to this aroma compound and the existence of several clusters of consumer behaviour. Our data indicate that rotundone, unless it is present at unusually high concentration (>309 ng/L), is globally positively perceived or in a neutral manner by two of the three clusters representing 79% of the non-anosmic population. The second cluster, much smaller and mainly composed of young consumers with less experience in wine, preferred the fruity Control wine. This opposition of appreciation between young (18-24 years) and older (>55 years) consumers is particularly marked for the 100 ng/L series, a common level of rotundone concentration found in Duras wine. In accordance with previous studies conducted on Gamay wines (Geffroy et al. 2016a), these findings suggest that winemakers should promote moderate to significant levels of rotundone in their Duras wine unless the product is specifically targeted to young consumers.

Interestingly, the real appreciation of peppery notes for each cluster, assessed through our study, was in accordance with consumers' self-report liking. This coherence tends to indicate that panellists had a good overall knowledge in wine and were able to analyse their olfactory sensations. Our study was conducted in Toulouse, south-west of France. This place is favourable notably because of its climatic conditions (Geffroy et al. 2014, 2016c) to the expression of peppery notes in wines. The post-experiment questionnaire revealed that most panellists had previously heard of peppery notes in wine. Unfamiliarity is susceptible to impact the appreciation of a given flavour (Saliba et al. 2009), and participants less familiar with peppery notes in wine could have rejected spiked samples. It is not possible to exclude that, in other French cities or countries, we might have been able to calculate a CRT for rotundone for the non-anosmic population.

Our data are, of course, valid for a particular red wine made from the Duras cultivar. It is important to mention that the base wine made especially for the study is likely the Duras wine with the lowest rotundone concentration ever produced. To generalise our results, we should have measured the detection threshold and conducted the CRT experiment in several wines. During the pre-experiment pilot study, the wine spiked at 25 ng/L had slightly spicier and less fruity notes in comparison with that of the Control, which is in accordance with the experience of the authors with this cultivar. Therefore, the hypothesis that our results might be transposable to other grape cultivars for which rotundone makes an important contribution from the same concentration (30 to 35 ng/L) cannot be completely discarded.

If the number of consumers used in our study (62) exceeds the panel size of previous research work on CRT that varied from 35 (Campo et al. 2012) to 58 (Prescott et al. 2005), it is important to mention the relative weakness of our clustering data. According to MacFie (2007), 40 responses per cluster are a minimum to make meaningful interpretations, and we cannot completely exclude the likelihood of a significant result by chance especially for the smallest group (cluster 2). In addition, the clustering may have simply detected artefacts such as those individuals who have a specific sample presentation order or those individuals susceptible to fatigue.
## Conclusion

Our study provides winemakers from the Protected Designation of Origin Gaillac with key parameters to adapt their products to consumer profiles. It also opens new fields of investigation into the mechanisms involved in specific anosmia to rotundone.

## Acknowledgements

This study was carried out with financial support from FranceAgriMer and Midi-Pyrénées region. We are grateful to Mrs Laure Gontier and Mrs Fanny Prezman, Institut Français de la Vigne et du Vin, for the support in the organisation of the study, to Dr Christian Vial, Firmenich, for providing us with the rotundone sample and to Dr Patricia Taillandier, French National Diploma of Enologist (DNO), for giving us the possibility to use their tasting facilities.

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Manuscript received: 11 April 2017

Revised manuscript received: 04 May 2017

Accepted: 16 May 2017