Physiology & Behavior 87 (2006) 304 313

Bitter taste markers explain variability in vegetable sweetness, bitterness, and intake
M.E. Dinehart a, J.E. Hayes a,b, L.M. Bartoshuk c, S.L. Lanier a, V.B. Duffy a,b,*
b a Department of Health Promotion and Allied Health Sciences, University of Connecticut, USA Department of Nutritional Sciences, College of Agriculture and Natural Resources, University of Connecticut, Storrs, CT 06269 USA c Department of Surgery, Yale University, New Haven, CT 06510 USA

Received 2 August 2005; received in revised form 8 September 2005; accepted 19 October 2005

Abstract Intake of vegetables falls short of recommendations to lower risk of chronic diseases. Most research addresses bitterness as a sensory deterrent to consuming vegetables. We examined bitter and sweet sensations from vegetables as mediators of vegetable preference and intake as well as how these tastes vary with markers of genetic variation in taste (3.2 mM 6-n -propylthiouracil bitterness) and taste pathology (1.0 mM quinine bitterness, chorda tympani nerve relative to whole mouth). Seventy-one females and 39 males (18 60 years) reported prototypical tastes from and preference for Brussels sprouts, kale and asparagus as well as servings of vegetables consumed, excluding salad and potatoes. Intensity and hedonic ratings were made with the general Labeled Magnitude Scale. Data were analyzed with multiple linear regression and structural equation modeling. Vegetable sweetness and bitterness were independent predictors of more or less preference for sampled vegetables and vegetable intake, respectively. Those who taste PROP as most bitter also tasted the vegetables as most bitter and least sweet. The spatial pattern of quinine bitterness, suggestive of insult to chorda tympani taste fibers, was associated with less bitterness and sweetness from vegetables. Via structural equation modeling, PROP best explained variability in vegetable preference and intake via vegetable bitterness whereas the quinine marker explained variability in vegetable preference and intake via vegetable bitterness and sweetness. In summary, bitterness and sweetness of sampled vegetables varied by taste genetic and taste function markers, which explained differences in preference for vegetables tasted in the laboratory as well as overall vegetable intake outside the laboratory. D 2005 Elsevier Inc. All rights reserved.
Keywords: Vegetables; Taste; Genetics; Bitter; Sweet; Food preference

1. Introduction Mounting evidence exists on the protective role of vegetable intake to decrease risk for chronic disease. Accordingly, The 2005 Dietary Guidelines for Americans advise individuals to consume between 5 and 13 servings of fruits and vegetables daily, based on energy needs, an increase from the 2000 guideline recommendation of 5 to 9 servings [1]. Individuals are encouraged to eat a variety of vegetables including dark green, orange, starchy and other vegetables [1]. National surveillance in the US shows that individual consumption falls below established recommendations [2,3]. Average vegetable
* Corresponding author. Department of Health Promotion and Allied Health Sciences, 358 Mansfield Road, Unit 2101 Storrs, CT 06269-2101, United States. Tel.: +1 860 486 1997; fax: +1 860 486 5375. E-mail address: valerie.duffy@uconn.edu (V.B. Duffy). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.10.018

intake for those aged 2 years and older is approximately 3 servings each day, the majority of which is comprised of starchy vegetables, specifically white potatoes [4]. Vegetable sensations (e.g., taste, texture, appearance) and/or preference are cited among other factors (e.g., cost, availability, preparation time) as important determinants of whether vegetables are ultimately consumed [5 7]. Bitterness has been reported as a sensory deterrent for vegetable preference [8] and consumption [9]. Phenols, flavonoids, isoflavones, terpenes, and glucosinolates are some of the compounds responsible for vegetable bitterness, pungency, and/or astringency (see rev. [9]). For example, the glucosinolates neoglucobrassicin and sinigrin have been explicitly identified as the determinants of cauliflower bitterness [10]. In contrast, little is known about specific compounds that may elicit sweetness in traditionally bitter vegetables. Mono- or disaccharides released by starch hydrolysis during processing may impart sweetness. In

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cruciferous vegetables, sweetness has been shown to decline in proportion to an increase in bitter glucosinolates [11]. Although humans are born with an innate dislike for bitter and preference for sweet (see recent review [12]), the ability to taste bitterness and sweetness varies. One source of this variation is geneticthe most studied being the ability to taste bitterness from phenylthiocarbamide (PTC) [13 15] and 6-n propylthiouracil (PROP) [14,16,17]. Concentrated PROP varies in perceived intensity from relatively weak to intensely bitter [18]. The ability to taste PTC [19] or PROP [20,21] results from the presence of a functional TAS2R38 receptor, whereas greater intensity of bitterness from these compounds results from increased number of fungiform papillae density [22] and, possibly, other unidentified factors [23]. Those who taste PROP as more bitter also taste more sweetness from a range of sweeteners [24] probably due to the increased numbers of fungiform papillae and other unidentified factors. Polymorphisms in other bitter taste receptor genes likely contribute to variation in the bitterness of other compounds [25]. Although the receptors for quinine and PROP appear to be independent based on molecular [26] and psychophysical [27 29] evidence, the bitterness is often correlated [24], in part, due to the density of fungiform papillae. Variation in taste and oral sensation also results from exposure to environmental insults including pathogens and trauma [30,31]. Infection (e.g., otitis media, upper respiratory infection) or trauma may damage the chorda tympani nerve [32] and reduce taste on the anterior tongue. Damage to the chorda tympani has been shown most often using quinine [31], but the reductions may be shown by other bitters as well. Counter intuitively, damage to (or anesthesia of) the chorda tympani nerve can increase taste sensations of other qualities (e.g., sweet) [33,34] as well as oral somatosensations [35] due to release of inhibition of cranial nerves V, IX and X. We examined PROP and quinine as markers for variability in tastes from traditionally bitter vegetables, preference for these vegetables as well as overall intake of vegetables. Lower acceptance of cruciferous vegetables [36], spinach [37], bitter citrus and tart fruits [38], green tea, soy products [39], and lower vegetable intake [40] is reported by those who taste PROP as more bitter or express a TAS2R38 taster genotype [20]. Heightened bitterness from these foods and beverages may discourage consumption in those who taste PROP as bitter [41]. Fischer et al. were the first to characterize differences in food preference by examining variability in PROP and quinine tasting: individuals with lower thresholds for PROP and quinine (tasters) displayed more food dislikes and aversions than those with higher thresholds (nontasters) [16]. We have used both bitter markers to explain variability in alcohol [22] and sweet [42] preference and intake and presently, variability in positive (sweet) and negative (bitter) tastes from vegetables as mediators of vegetable preference and intake. Our previous study showed that individuals who tasted PROP as least bitter also tasted alcoholic beverages as sweet with little bitterness [43], possibly due to a lack of suppression of sweetness by bitterness [44,45]. Here, we report similar sweet bitter interactions from traditionally bitter vegetables: those who

taste PROP as least bitter tasted these vegetables as mildly sweet and reported little bitterness. As shown with alcohol tastes [43], vegetable sweetness and bitterness predicted vegetable intake, supporting the conclusion that ingestive behaviors result from the presence of pleasant and the absence of negative oral sensations. 2. Methods 2.1. Subjects Individuals were recruited through posters and word of mouth from Yale University and the University of Connecticut. The posters asked individuals to participate in a study looking at why people eat what they do. No specific emphasis was placed on the examination of vegetable intake per se. Healthy males and females, without severe dietary restrictions (e.g., food allergies or intolerance), participated in one or two sessions in a taste laboratory. Table 1 shows the demographic and body mass indices (BMI calculated from reported height and measured weight) of the participants. Forty-seven of the individuals were considered overweight or obese according to the BMI. Data were collected at both institutions, with Institutional Review Board approval at each. Subjects gave informed written consent and were paid for their time. 2.2. Data collection 2.2.1. Collection of hedonic and intensity data 2.2.1.1. Scaling intensity. Individuals used the general Labeled Magnitude Scale (gLMS) [46,47], a semantically labeled line scale, to rate the perceived intensities and/or level of liking/disliking of experimental stimuli. The gLMS generalizes the Labeled Magnitude Scale [48,49] by changing the top of the scale from strongest imaginable oral sensation to strongest imaginable sensation of any kind. The LMS was developed by spacing the labels so that the scale would have ratio properties [48]. The LMS [48,49] and gLMS [47] produce results equivalent to magnitude matching [50]. For intensity
Table 1 Descriptive characteristics of sample All (n = 110) Age (18 60) BMIa,b BMI category Normal (17 to <25) Overweight (25 to <30) Obesity (! 30) Ancestry Asian African American Caucasian Hispanic Native American Other
a b a

Females (n = 71) 37 T 13 25 T 6 39 22 10 5 3 56 5 1 1

Males (n = 39) 30 T 12 26 T 6 21 11 7 4 2 29 2 0 2

34 T 13 26 T 6 60 33 17 9 5 85 7 1 3

Mean T SD. BMI (body mass index) is weight (kg)/height squared (m2)).

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ratings, the 100-point scale ranges from Fno sensation_ at the bottom (0) to Fthe strongest imaginable sensation of any kind_ at the top (100) and has adjectives spaced in a logarithmic appearance; Fbarely detectable_ (1.4), Fweak_ (6), Fmoderate_ (17), Fstrong_ (35), and Fvery strong_ (53). For orientation to the scale, subjects rated the intensities of 16 oral and non-oral sensations. For hedonic ratings, the subjects were instructed to anchor the top of the scale either Fstrongest imaginable liking_ (+ 100) or the Fstrongest imaginable disliking_ (100) with zero being neutral [46]. 2.2.1.2. Food sampling. Subjects tasted vegetables typically thought of as bitter and/or having flavors that are often disliked (asparagus, Brussels sprouts, kale) as well as foods with predominantly sweet taste (Hersheys\ milk chocolate, Marshmallow Fluffi). To control consistency in the vegetable stimuli, the vegetables were store-brand (Shaws Supermarket, Inc, www.shaws.com), purchased frozen in bulk from a single location, and stored frozen in their respective packages. Before testing, they were thawed enough to portion into sample-sizes (1 asparagus spear, 1 Brussels sprout, 4 g kale), individually stored in plastic bags with zip-lock closures, and refrigerated less than 24 h before serving. For testing and to minimize variability, vegetables were allowed to come to room temperature without heating. Individuals were instructed on use of the gLMS to rate level of liking/disliking and perceived sweetness, sourness, saltiness, and bitterness of vegetables and sweet foods based on present experiences, not previous beliefs or values (e.g., I have always hated this food, I like this food because it is good for me). Subjects were free to swallow or expectorate the samples and rinsed with deionized (>15 MV) water after each. Mean bitterness and sweetness from sampled vegetables as well as mean sweetness from sampled sweet foods were used for the analyses. Salty and sour ratings were also collected to minimize attribute dumping (i.e., subjects putting sensory ratings in inappropriate categories) but were not used in any models to predict intake. 2.2.1.3. Vegetable intake. From a validated food frequency questionnaire (Block version 98.1) [51], a registered dietitian asked subjects to approximate how many servings of vegetables they consume per day or per week, excluding salad and potatoes. The question served as a rapid method to assess vegetable intake, was the initial food-related question on the food frequency questionnaire [51] and is similar to that used in nutrition surveillance (e.g., [52]). Because the question was asked in the context of a larger battery of foods, the participants were unaware of our particular interest in vegetable intake. The responses were based on nine frequency categories ranging from Fless than once per week_ to F4+ per day_. To give each response a comparable value, the categorical variable was expressed as frequency per year (e.g., 3 per day = 1095 servings). 2.2.2. Quinine ratio This measure came from modification of the spatial taste test [53], which measures intensities of 1.0 M NaCl, 1.0 M

sucrose, 3.2 mM citric acid and 1.0 mM quinine from chorda tympani (CTN) and glossopharyngeal nerves as well as whole mouth. Using a cotton-tipped applicator, the experimenter painted tastants individually onto areas innervated by these nerves, thus stimulating both sides of the tongue tip (CTN) and the posterior tongue (circumvallate papillae). After all localized testing for a single tastant, subjects filled their mouths sequentially with each tastant, swished, expectorated and then swallowed the residual for whole mouth intensity. Subjects rinsed with deionized (> 15 MV) water before each stimulus and made intensity ratings on the gLMS. A quinine ratio was calculated from the mean of the bitterness on the right and left tongue tip (CTN) divided by the whole mouth bitterness, as reported previously [54]. 2.2.3. PROP scaling At the end of the session subjects rated the intensity of replicate sets of PROP solutions (.5 log steps, from 0.032 to 3.2 mM) using a protocol including intensity ratings of sodium chloride (NaCl, .5 log steps from 0.01 to 1 M) and tones (1000 Hz, 50 to 98 dB) as sensory standards as reported previously [55]. Mean intensity of the two 3.2 mM PROP stimuli was used as a continuous variable throughout the analyses for comparisons across individuals. 2.3. Statistical analyses Using linear regression in Statistica version 6.0 (StatSoft, Tulsa, OK) and a strategy similar to that reported previously [43], taste markers (PROP, quinine) were used to explain variability from vegetable tastes (bitterness, sweetness), which in turn, were used to explain variability in vegetable preference and vegetable intake. We also tested the ability of taste markers to predict vegetable intake directly. Variables were appropriately transformed and outliners (univariate, multivariate) were removed by the standardized residual (! 2.5) and the Mahalanobis distance criteria (critical chisquare table with p < 0.001, with degrees of freedom equaling the number of independent variables) [56]. Significance criterion was p 0.05 and mean T SD are presented. Independent contributions of sex and age were accounted for in all models, particularly since the mean age of females (37 T 13 years) exceeded that of males (30 T 12 years, t = 2.5, p = 0.01). BMI was also added into the models but was not a significant predictor because of its covariance with age: older participants showed a greater BMI, whether male or female (sr = 0.22, p < 0.05). Pearsons r is reported for bivariate relationships; in multiple linear regression analyses, the semipartial correlations (sr) of significant contributors to the multiple R are presented. A Structural Equation Model (SEM) was constructed and tested using AMOS 5.0 (Smallwaters, Inc., Chicago, IL). A confirmatory approach was used to describe relationships among bitter taste markers, vegetable taste, preference and intake. SEM is a superior approach to a series of multiple linear regression models as it tests the relationships between all variables simultaneously [57]. For the present study, this

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statistical approach has two advantages. First, it eliminates the need for a correction for multiple comparisons. Second, by testing all relationships at once, it controls for spurious (3rd variable) relationships between measured variables. Default options (Maximum Likelihood Estimation) were used and missing data was handled using the estimate means and intercepts option. Measures of global fit v 2, Tucker Lewis Index (TLI) and the Root Mean Square Error of Approximation (RMSEA) were chosen a priori. A nonsignificant v 2 is desirable, and the closer RMSEA is to zero and TLI is to one, the better the model fit [58]. Nonsignificant paths ( p > 0.10) were trimmed from a model described with a series of multiple regression analyses. The fit of the respecified model was tested before being provisionally accepted [57]. 3. Results 3.1. The sample: taste markers and vegetable intake 3.1.1. Taste markers Individuals varied in their ratings of PROP bitterness (Fig. 1) with 23 being nontasters (3.2 mM PROP 22 on the gLMS), 66 medium tasters (3.2 mM PROP 23 49), and 21 supertasters (3.2 mM PROP ! 50). In multiple regression analysis, PROP bitterness neither associated with age ( p = 0.47) nor varied by sex ( p = 0.65). The observed distribution of BMI categories [normal (17 to 25) vs. overweight and obese (! 25)] across PROP taster groups was not significantly different from expected (v 2(3) = 3.739, p = 0.15). There were no significant age effects for quinine from the CTN, whole mouth or quinine ratio. Females exceeded males for the quinine ratio (t = 2.75, p < 0.01), but neither differed significantly for quinine from the CTN (t = 0.94, p = 0.35) or
80

Table 2 The table shows individual taste qualities from sampled vegetables reported on the gLMS [46,47], where 1.4=barely detectable, 6=weak, 17=moderate, 35=strong, and 53=very strong Mean Asparagus Bitterness Sweetness Sourness Saltiness Brussels sprouts Bitterness Sweetness Sourness Saltiness Kale Bitterness Sweetness Sourness Saltiness 10.3 2.3 2.6 2.5 12.7 3.9 4.8 2.7 14.3 1.2 3.9 2.7 Standard deviation 11.0 4.8 6.6 6.6 15.4 6.9 10.5 7.0 14.8 4.7 10.3 7.3 Range 0 60 0 26 0 50 0 49 0 71 0 50 0 70 0 47 0 64 0 40 0 70 0 49

whole mouth (t = 1.83, p = 0.07). PROP bitterness was correlated with whole mouth quinine (r = 0.40, p < 0.05), but not significantly with CTN quinine (r = 0.15, p = 0.11) or the quinine ratio (r = 0.01, p = 0.99). The quinine ratio thus served as a PROP-independent marker of taste function. 3.1.2. Vegetable intake The sample varied in intake of vegetables ranging from 35 to 1460 servings per year, the latter equivalent to 4 servings per day. Compared to national surveillance of adults from ages 18 to above 65 years in the 2000 Behavioral Risk Factor Surveillance System [52], our sample had slightly lower vegetable intakes in females (geometric mean = 1.04 vs. 1.12 servings) and males (0.71 vs. 0.91). This finding is expected as national surveillance indicates that vegetable intake shows agerelated increases [52] and our sample had no individuals over the age of 65 years. Within our sample and with multiple regression analyses, females (sr = 0.20, p < 0.05) and those with lower BMIs (sr = 0.19, p < 0.05) reported consuming more vegetables. Intakes did not vary significantly with age ( p = 0.95). 3.2. Vegetable tastes

perceived intensity (gLMS)

60

Nontasters (n=23) Medium taster (n=66) Supertasters (n=21)

40

20

0 .01

.1

10

PROP concentration (log mM)

Fig. 1. The bitterness of a PROP concentration series measured on the gLMS with subjects classified by 3.2 mM PROP bitterness into 23 nontasters ( 22 Fbarely detectable_ to Fmoderate_ on the gLMS), 66 medium tasters (between 23 and 50 Fstrong_), and 21 supertasters (! 50 Fvery strong_).

Bitterness averaged as the most intense taste quality and showed the most variability across all three sampled vegetables. The mean bitterness ratings ranged from no sensation to greater than very strong (Table 2). Subjects also reported the vegetables as having sour tastes; there was a strong correlation between average sourness and average bitterness of the sampled vegetables (r = 0.45, p < 0.05). Although subjects frequently confuse sour and bitter taste qualities [59,60], subjects in our study were least likely to report sourness in the absence of bitterness and more likely to report on the bitterness only or both bitterness and sourness (Fischers exact probability test, p = 0.05). Thus, we focused our analyses on bitterness. Sweet and salty tastes were the lowest in intensity. Fifty-four of the subjects

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12.25

Vegetable Sweetness (sqrt)

9 6.25 4 2.25 1 .25 0 0 20 40 60 80 100

reported no sweetness from the sampled vegetables. Across all vegetables, individuals who reported no sweetness from the vegetables were most likely to report bitterness greater than the sample mean (v 2 = 4.983, p < 0.05). 3.3. Explaining variability in vegetable sweetness and bitterness 3.3.1. Using PROP as a marker Those who tasted PROP as most bitter also tasted more bitterness from sampled vegetables (sr = 0.45, p < 0.001), independent of age ( p = 0.47) and sex ( p = 0.65), and more sweetness from sampled sweet foods (Marshmallow Fluffi, chocolate; sr = 0.25, p = 0.01), independent of sex ( p = 0.12), but not age (sr = 0.19, p = 0.05). PROP appeared to show a nearly flat association with sweetness of vegetables in multiple regression analyses (sr = 0.05, p = 0.58) across the entire sample; however, among those who reported some

3.2 mM PROP
Fig. 2. The scatterplot shows a nearly flat association between PROP bitterness and average sweetness of sampled vegetables across all subjects (n =111; dashed regression line). Among those who reported some sweetness (n =56), PROP bitterness was negatively associated with vegetable sweetness (solid regression line).

Bitter sr=0.03 Sweet sr=0.26, p<0.01 Age sr=0.47, p<0.001 Bitter sr=-0.36, p<0.001 Sweet sr=0.27, p<0.01 Age sr=0.25, p<0.01 Multiple R=0.59, p<0.001 Brussels Sprouts Preference (mean=0.227.6 SD) 'Moderately Dislike to Moderately Like' Multiple R=0.54, p<0.001 Asparagus Preference (mean=7.129.1 SD) 'Moderately Dislike to Strongly Like'

Bitter sr=-0.38, p<0.001 Sweet sr=0.23, p=0.01 Age sr=0.06 Bitter sr=-0.22, p=0.01 Sweet sr=0.34, p=0.001 Age sr=0.28, p=0.001

Multiple R=0.48, p<0.001

Kale Preference (mean=-3.126.5 SD) 'Moderately Dislike to Moderately Like'

Multiple R=0.55, p<0.001

Vegetable Preference (mean=1.523.6 SD) 'Moderately Dislike to Moderately Like'

Fig. 3. Multivariate models predicting preference for each vegetable from bitterness, sweetness, and age. Semipartial correlation coefficients (sr) and the multiple regression coefficients (r ) are reported. The dotted line represents a non-significant association.

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vegetable sweetness (n = 56), those who tasted PROP as least bitter reported greater sweetness (sr = 0.28, p = 0.04) (Fig. 2). This effect was independent of sex, although vegetable sweetness increased with age (sr = 0.29, p = 0.03). 3.3.2. Using quinine as a marker Adding the quinine ratio to the multiple regression analyses with PROP, sex and age increased the explained variance in vegetable bitterness by 7%. A lower ratio was associated with less bitterness from the sampled vegetables (sr = 0.23, p < 0.01). For vegetable sweetness, adding the quinine ratio to the multiple regression analyses with vegetable bitterness, sex and age increased the amount of variance explained by 10%. A lower ratio was associated with less sweetness from the sampled vegetables (sr = 0.22, p = 0.02). 3.4. Vegetable liking is driven by sweetness and bitterness Vegetable preference averaged from strongly disliked to strongly liked, with similar variance across all vegetables. Vegetable sweetness was a significant predictor of preference across all vegetables, while bitterness predicted preference for Brussels sprouts and kale, but not asparagus. Across all vegetables, the regression model with age, sex, vegetable bitterness and vegetable sweetness explained 32% of the variance in vegetable preference. Multiple regression models predicting preference for each of the sampled vegetables and overall is summarized in Fig. 3.

3.5. Explaining variability in vegetable intake 3.5.1. Using multiple regression Sixteen percent of the variance in vegetable intake ( p < 0.001) was predicted by vegetable sweetness, bitterness and sex. Those who tasted the vegetables as most sweet (sr = 0.28, p < 0.005), but least bitter (sr = 0.19, p < 0.04) consumed vegetables most frequently. Level of adiposity (BMI) did not increase the prediction of vegetable intake ( p = 0.20). Vegetable preference was a direct predictor of intake (sr = 0.26, p < 0.01), independent of age ( p = 0.55) and sex ( p = 0.16). Those who tasted PROP as most bitter (sr = 0.28, p < 0.005) consumed vegetables least frequently, independent of sex (females > males; sr = 0.21, p = 0.03) and age ( p = 0.55). The quinine ratio failed to be a direct predictor of vegetable intake ( p = 0.77). Fig. 4 shows a summary of the relationships between taste markers and vegetables tastes to predict vegetable preference and intake. 3.5.2. Using structural equation modeling Structural equation modeling allowed testing of the relationships among markers of variation in taste, vegetable tastes, preference and intake suggested by the multiple regression analyses summarized in Fig. 4. Although the initial model (Fig. 4, assessed via SEM) had excellent global fit (v 2 = 8.77, df = 11, p = 0.643; TLI = 1; RMSEA= 0.000, 90% C.I. 0.000 0.082), it contained three non-significant paths ( p > 0.10)relationships between PROP bitterness and vegetable intake, vegetable sweetness and intake, and vegetable bitterness and intake.

PROP

Quinine Ratio
sr=0.45 sr=0.22 sr=0.23

Age

sr=0.28

Vegetable Bitterness
sr=-0.28 sr=-0.20 sr=-0.22

Vegetable Sweetness
sr=0.34 sr=0.34

Vegetable Preference
sr=0.26

Vegetable Intake
Fig. 4. A model summarizing the relationships between bitter markers, bitterness and sweetness of sampled vegetables, vegetable preference and intake. Partial correlation coefficients are reported.

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e6
.00

Quinine Ratio
.15 .17

e2
.15 .03

e1 Vegetable Bitterness Vegetable Sweetness

Age

-.34 .36

.26

.30

e3 e4
.00

.27

3.2mM PROP Bitterness

Vegetable Preference

.30

e5

.09

Frequency of Vegetable Consumption

Fig. 5. Same as Fig. 4, except that Structural Equation Modeling was used to assess all relationships simultaneously. Numerical values next to arrowed lines represent path coefficients, which may be interpreted like standardized regression weights. Values adjacent to boxed variables represent the variance explained for that variable. Errors (represented by circles) are required computationally, but are not of theoretical interest.

After removing these paths [57], the respecified model (Fig. 5) was provisionally accepted as all paths were significant and global fit remained excellent (v 2 = 12.98, df = 14, p = 0.523; TLI = 1; RMSEA= 0.000, 90% C.I. 0.000 0.085). By comparing the conceptual model (Fig. 4) and the final SEM (Fig. 5) we find the two models are quite similar; collectively these models demonstrate that bitter taste markers and vegetable tastes act on intake via preference. 4. Discussion 4.1. General findings We sought to use markers of variation in taste to explain taste qualities from, preference for and intake of vegetables. Starting with separate multiple regression analyses and confirming the findings with structural equation modeling, we were able to describe how taste influences vegetable intake. Building upon the work of Fischer et al. [16] and previous work from our laboratory, PROP and quinine (tongue tip / whole mouth ratio) were unique contributors to explaining the level of bitterness and sweetness from sampled vegetables that are thought of as bitter and/or disliked. PROP was more strongly related to the bitterness of the vegetables whereas

quinine was more related to both bitterness and sweetness from vegetables. Preference serves as an intermediary step in associations between taste and intake. That is, relationships seen between PROP bitterness and intake (i.e., [42,54]) are mediated through the bitterness of vegetables. The preference for vegetables is influenced by the presence of positive (e.g., sweetness) and negative (e.g., bitterness) sensations. Thus, using both negative and positive taste sensations increased the ability to associate sensation with intake, similar to findings with alcohol intake [43]. 4.2. Vegetable bitterness and vegetable sweetness Bitter and sweet tastes result from the interaction of tastants with G protein-coupled receptors (GPCR) located on taste receptor cells, and these GPCRs may share similar transduction cascades [26]. Adding to this complexity, the ability to taste bitter varies genetically [14 16]. We found that those tasting PROP as most bitter tasted more bitterness from sampled vegetables, consistent with other findings [41] and more sweetness from sampled sweet foods, also consistent with previous work [43]. The positive relationship between PROP and sweet taste was blunted in vegetables. Heightened bitterness appears to suppress vegetable sweetness, consistent

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with prior work [43 45,61]. However, in the present study, a full reversal of PROP effects on sweetness was seen only after removing ratings of zero, which could indicate no perceived sweetness, or, alternatively, could represent the failure of subjects to attend to weak tastes [62,63]. By removing zeros from the analysis, we eliminated this potential source of noise, and in doing so, uncovered a reversal of PROP effects on sweetness, consistent with our findings on PROP effects on sweet taste of scotch [43]. Using quinine as a marker of taste function further enhanced the ability to predict vegetable sweetness and bitterness. Those with lower quinine ratios (low taste function on the tongue tip relative to whole mouth) perceived less sweetness and less bitterness from sampled vegetables. A low ratio may result from diminished bitterness via the CTN and increases in bitterness from cranial nerves IX and X. Damage to (or anesthesia of) the CTN has been shown to reduce taste on the anterior tongue, particularly to quinine bitterness [31] and increase taste sensations from other taste nerves [33,34,64]. Thus, the complexity surrounding vegetable bitterness and sweetness likely results from variability in perception, physiology, genetics, and environmental insults as well as interactions among these factors. 4.3. Preference Collectively, the tastes from vegetables, sweetness and bitterness, contribute more towards explaining preference than does age. Additionally the relationship between age and preference appears direct (versus mediated by vegetables tastes), suggesting it results from non-taste influences (e.g., cohort effect, cognitive factors). Taste influences on vegetable preference have been reported previously. Kaminski et al. [41] found that greater PROP bitterness was associated with greater vegetable bitterness, in turn resulting in lower acceptability for these foods. Building upon these findings, we found that vegetable sweetness and vegetable bitterness were positive and negative predictors of vegetable preference, respectively. Our findings match those of Schonhof et al. [11], who found that consumers prefer vegetables with lower contents of bitter glucosinolates and higher sucrose contents and that flavor profile was closely associated with the sugar and bitter content of vegetables. The question of how variation in perceived bitterness and sweetness contributes to conditioning preference for vegetable flavor is of interest. A predominate bitterness could condition a dislike for a vegetable flavor, thereby negatively influencing intake of that particular vegetable. Conversely, if individuals perceive sweetness in a vegetable, even if mild, it may be enough to condition a preference for the flavor of that vegetable and encourage its consumption. Sweet taste plays an important role in the acquisition of preference, independent of energy (i.e., post-ingestive consequence). In rats, Ackroff and Sclafani [65] found that following conditioning with intragastric fructose, saccharin sweetened conditioned stimuli were more preferred compared to unsweetened conditioned stimuli. The same study also found that training with a

sweetened stimulus could convert an aversion into a preference in male rats, and transform an indifference to a preference in female rats. In humans, flavor conditioning has been shown to occur independently of post-ingestive consequences [66,67]. Diminishing bitterness of vegetables through horticultural techniques and minimizing flavor changes during storage and processing may promote optimal sweetness in vegetables from naturally occurring sugars. Studies on taste and odor mixtures reveal that the addition of a pleasant stimulus to an unpleasant stimulus increases pleasantness directly as well as indirectly by decreasing unpleasantness of the negative stimuli [68]. Use of seasonings or cooking techniques to blunt bitterness and enhance positive sensations (e.g., sweetness) may also promote conditioning a preference for vegetable flavor. 4.4. Intake Self-reported preference for vegetables has been shown to associate with frequency of consuming these foods [41]. Here we found that taste sensations from and preference for sampled vegetables in a laboratory setting were associated with frequency of consuming vegetables with a question that probed for total intake of vegetables from a validated food frequency questionnaire [51]. Consistent with previous findings, using both PROP and quinine enhanced the ability to explain variability in preference and/or intake [16,42]. In the final Structural Equation Model, the taste markers were not direct predictors of vegetable intake, instead acting indirectly via vegetable bitterness (PROP) or bitterness and sweetness (quinine). Vegetable bitterness and sweetness were independent predictors of intake supporting the need to examine taste qualities that both hinder and promote consumption [43]. The opportunity exists to build on previous research on PROP/PTC tasting and vegetable consumption (e.g., [69,70]) by improving psychophysical measurement, accounting for non-sensory determinants of vegetable intake (e.g., economics, health beliefs), and considering how genetics and the environment interact to influence the total sensory profile from vegetables. 4.5. Summary Most studies examining the relationship between PROP/ PTC and vegetable preference and intake have focused solely on bitterness as a sensory deterrent to acceptance and consumption. Here we show through structural equation modeling that the ability of PROP and quinine to predict intake of vegetables is mediated through vegetable bitterness and sweetness. To the best of our knowledge, this is the first study to examine how bitterness and sweetness are negative and positive predictors of vegetable preference and intake, and how both contribute to explaining dietary behaviors. Limitations of the study should be noted. The sample was relatively homogenous in ancestry and the findings may not generalize to samples that are more diverse. To minimize experimental variability, the vegetables were served in a form that may not be usual to cultural food patterns, including temperature, as well as the lack of seasonings that could

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diminish bitterness (e.g., salt) and/or enhance pleasant sensations (e.g., herbs). The assessment of intake, although similar to that used in nutrition surveillance [52], could have been more quantitative for portion consumed, individual vegetables, and preparation techniques. Nonetheless, the findings of the study increase the knowledge of how genetic and environmental variation in taste mediates taste qualities from, preference for and consumption of vegetables. The study findings support the need to increase attention on the taste and sensory qualities of vegetables to improve the intake of vegetables for the promotion of health and prevention of chronic disease. Acknowledgements The project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2003-35200-12943 and NIH Institutes of Deafness and Communication Disorders grant number DC00283. Author JEH is the Pangborn Sensory Science Scholar for 2006, an award given in memory of Rose Marie Pangborn and generously underwritten by GlaxoSmithKline Consumer Healthcare Division. References
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