Challenging the “Non-Volatile” Taste Assumption: Neural Effects of Retro Nasal Occlusion on Stevia Perception

Challenging the “Non-Volatile” Taste Assumption: Neural Effects of Retro Nasal Occlusion on Stevia Perception | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL European Journal of Neuroscience This is a preprint and has not been peer reviewed. Data may be preliminary. 8 December 2025 V1 Latest version Share on Challenging the “Non-Volatile” Taste Assumption: Neural Effects of Retro Nasal Occlusion on Stevia Perception Authors : Hee-kyoung Ko 0000-0002-8294-9548 , Jingang Shi , Thomas Eidenberger , Weiyao Shi , and Ciara Mccabe 0000-0001-8704-3473 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176519393.35971307/v1 309 views 179 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Against conventional wisdom we have recently shown that sucrose a “non-volatile” compound can reach the retro nasal olfactory region and effect perception, when aerosolized. This suggests other “non-volatile” sweet tastes might also be processed via retro nasal pathways challenging the “non-Volatile” taste assumption that pure tastes are only processed via oral pathways. Stevia is a sweet taste commonly used to replace sugar in food products. Knowing if stevia also activate retro nasal pathways could help us understand how to modulate sweet tastes to improve low calorie food acceptability. We examined the neural activity to stevia with a nose clip on (blocking retro nasal pathways) and nose clip off, in robust sample of healthy adults (N=34, mean 25 yrs.). Neural activity to stevia was reduced with the nose clip on in the olfactory cortex, hypothalamus, the subgenual and pregenual anterior cingulate, and the nucleus accumbens. Stevia pleasantness was tracked by the posterior insula, bitterness by the amygdala and mouth fullness by the olfactory cortex but these were either not apparent with the nose clip on or much weaker. In conclusion, our findings are the first to demonstrate that blocking retro nasal pathways significantly reduces neural responses to stevia taste supporting the proposal that retro nasal pathways play a role in the perception of “non-volatile” tastes like stevia, and that stevia sweetened products could be made more palatable via retro nasal pathways. Challenging the “Non-Volatile” Taste Assumption: Neural Effects of Retro Nasal Occlusion on Stevia Perception Running title: Stevia retro nasal effects Hee-kyoung Ko 1 , Jingang Shi 2 , Thomas Eidenberger 3 , Weiyao Shi 2 , Ciara McCabe 1 * 1 School of Psychology and Clinical Language Sciences, University of Reading, Reading, UK 2 EPC Natural Products Co., Ltd. Building 1, 35 Jinghai 3rd Road, BDA, Beijing, China,101111 3 University of Applied Sciences Upper Austria, Roseggerstrasse 15, 4600, Wels, Austria * Corresponding author: C. McCabe, e-mail: [email protected] Keywords: Neuroimaging, stevia, non-nutritive sweeteners , brain, fMRI, retro nasal, taste, smell Abstract Against conventional wisdom we have recently shown that sucrose a “non-volatile” compound can reach the retro nasal olfactory region and effect perception, when aerosolized. This suggests other “non-volatile” sweet tastes might also be processed via retro nasal pathways challenging the “non-Volatile” taste assumption that pure tastes are only processed via oral pathways. Stevia is a sweet taste commonly used to replace sugar in food products. Knowing if stevia also activate retro nasal pathways could help us understand how to modulate sweet tastes to improve low calorie food acceptability. We examined the neural activity to stevia with a nose clip on (blocking retro nasal pathways) and nose clip off, in robust sample of healthy adults (N=34, mean 25 yrs.). Neural activity to stevia was reduced with the nose clip on in the olfactory cortex, hypothalamus, the subgenual and pregenual anterior cingulate, and the nucleus accumbens . Stevia pleasantness was tracked by the posterior insula, bitterness by the amygdala and mouth fullness by the olfactory cortex but these were either not apparent with the nose clip on or much weaker. In conclusion, our findings are the first to demonstrate that blocking retro nasal pathways significantly reduces neural responses to stevia taste supporting the proposal that retro nasal pathways play a role in the perception of “non-volatile” tastes like stevia, and that stevia sweetened products could be made more palatable via retro nasal pathways. Significance statement Neural activity to stevia decreases with retro-nasal occlusion via nose clip. Pleasantness (insula), bitterness (amygdala), and mouth fullness (olfactory cortex) weaken or disappear under occlusion. In conclusion, blocking retro-nasal pathways reduces stevia taste responses, supporting their role in “non-volatile” taste perception and enhancing stevia product palatability. Introduction We are living in an obesity pandemic [1] and food producers are encouraged to make foods low in calories. Retro nasal pathways might be one mechanism by which the experience of low/no-calorie sweet tastes could be enhanced via aroma modulation. This would help producers create foods low in sugars that are more palatable for consumers . However, there is very little data on the contribution of retro nasal pathways to sweeteners in the human brain. Recent studies using high speed cameras show that “non-volatile” tastes such as sucrose can be transferred to the nasal cavity in the form of aerosol particles [2]. This suggests that retro nasal sensation and not just oral sensation is involved in the consumption of “pure” tastes. P revious studies show that disabling retro nasal sensation through reversed nasal airflow or using a nose clip can significantly impair participants’ ability to identify sucrose [3-5], and its perceived sweetness intensity [6-8] and mouth fullness [9, 10] suggesting an important olfactory component even with “non-volatile” tastes [7]. Consistent with this, our recent study found that blocking retro nasal pathways effects not only reduced the subjective experience of mouth fullness but also neural activity [11]. Specifically, we found reduced neural activity in the primary taste, olfactory, attention and reward regions of interest (ROI), and in the rolandic operculum, lingual gyrus and precuneus in the whole brain analyses. Further olfactory and prefrontal cortex ROIs tracked subjective mouth fullness, but this was not apparent with the nose clip on . Hence we concluded that blocking retro nasal sensation could play a role in “pure” sweet taste perception [11]. Stevia is a natural sweetener derived from the stevia plant native to South America that has been used as a sweetener for hundreds of years [12] and is considered a “non-volatile” substance [13]. Stevia, as high-purity stevia leaf extract, is being used globally to reduce energy and added sugar content in foods and beverages [12] [14]. It’s main sweetening compounds are steviol glycosides such as stevioside and rebaudioside A and has trace amounts of rebaudioside D and M [15-17]. Stevia has shown advantageous effects on appetite and energy intake [17-19] and doesn’t negatively affect appetite [20]. The neural response to stevia has only been examined in a few studies. A previous study by Stamataki et al. (2022) examined the effects of caloric (glucose and maltodrextin) and non caloric (stevia) sweet tastes on the human brain focusing on effects over time (up to 30 min post ingestion). They confirmed that consumption of the caloric and non-caloric sweet drinks activated similar brain regions such as the prefrontal cortex, striatum, insula and cingulate cortex [21]. They also found that stevia resulted in decreased activity over time in regions such as the motor, frontal areas, and insula, similar to previous studies with glucose [22]. This finding could relate to stevia’s prolonged sweet lingering effects [17]. Stevia also has a slow onset of sweetness, bitterness at higher concentrations, and has less mouth fullness compared to sucrose [17, 23] . We recently also examined the neural response to stevia in a study on flavour modifiers and found that stevia activated similar brain regions to those of Stamataki et al., study and to the taste of sucrose [24]. However, if retro nasal pathways might also be involved in stevia processing, as they are with sucrose [11], is currently unknown. If stevia does activate retro nasal pathways, we suggest stevia sweetened foods could be made more palatable with the addition of modifiers. These could potentially reduce stevia bitterness and enhance mouth fullness. Therefore, in this study the aim was to examine the neural response to the taste of stevia in the human brain and how it might be affected by retro nasal blockade via a nose clip. As sweet tastes are known to activate the insula (primary taste cortex), postcentral gyrus (somatosensory region), the hypothalamus and the orbitofrontal cortex (OFC) (possibly secondary taste cortex) we identified these areas as regions of interest [25-27]. We also aimed to examine the amygdala as it responds to taste and oral texture [28] and projects to olfactory regions, responding to both pleasant and unpleasant tastes [29-31] and the nucleus accumbens, implicated in the rewarding aspects of sweet tastes [32] . Given our interest in retro nasal pathways we also aimed to examine the olfactory region, piriform cortex and the pregenual cingulate (pgACC) highlighted in a study examining neural differences to ortho nasal vs retro nasal odour delivery [33]. Finally we aimed to examine the subgenual anterior cingulate cortex (sgACC) as a region of interest as it has been shown involved in involuntarily attention to odours [34]. We also aimed to collect the subjective experiences of the taste of stevia in the scanner (pleasantness, bitterness and mouth fullness) so that we could correlate them with neural activity and examine the contribution of retro nasal pathways to these processes. Materials and Methods Thirty-four healthy, right-handed adults (10 male and 24 female) were recruited for the fMRI study. All participants were between 18 and 45 years old and had a current body mass index (BMI, weight in kg/height in m 2 ) or waist-to-height ratio (WTH) in the healthy range. Participants were excluded if they had any current/previous psychiatric history using the Structured Clinical Interview for DSM-IV Axis I Disorder Schedule (SCID), or if they took psychoactive medication or had an eating disorder (measured with Eating Attitude Test (EAT) > 20), food allergies, diabetes, smoked, or had any contraindications to fMRI scanning. We also recorded the frequency, liking and craving for sugary and sweetened foods [35]. The questions in this scale consisted of “How frequently do you eat sugary foods?” with answers of either; a few times per month; 1-2 times per week; 3-4 times per week; or more than 5 times per week and “How frequently do you eat/drink foods with sweeteners?”, with answers of either; Never; Rarely; Sometimes; Often; Usually or Always. The Craving and Liking for sugary foods were scored as 1 for low craving and 10 for high craving on a Likert scale. All procedures contributing to this work comply with the ethical standards of the Helsinki Declaration of 1975, as revised in 2013 and ethical approval was obtained from the University of Reading Ethics committee, ethics ref: 2023-130-CM all participants provided written informed consent. Pre-test 1 (Triangle test or Taste perception test) The 34 participants were entered into the study if they could distinguish 2% sucrose from a control. This standard taste perception test was as follows: The participants were randomly allocated to the following sequences of two samples A (distilled water) and B (20 g sucrose/litre [2 % Sucrose]): ABB, AAB, ABA, BBA, BAA and BAB. For the individual performance, each participant received all six sequences in random order. In a sequence, the participants took the whole 10 mL of each sample into their mouth, swirled and coated the solution around their mouth for 3 seconds and then spit it into a spittoon. On each trial after tasting all three, they indicated which was different from the other two. Participants who yielded correct identification of at least 5 out of the 6 trials on a second attempt, were recruited to the study. Pre-test 2 (Candy smell test) We used the candy smell test to check participants retro nasal olfactory performance [36]. This test examines participants ability to identify the flavour of a candy (500 mg) placed on the middle of the tongue from 6 possible choices (6-alternative, forced-choice procedure) strawberry, banana, orange, coffee, cherry, or pineapple. Participants can suck the candy or chew it if necessary. The participants wrote down one of the choices. If they could not identify the candy they could skip to the next trial. Each participant performed 5 trials with nose clip on and 5 trials with nose clip off. Between trials participants rinsed their mouths with water. There was no feedback to the participants about whether their responses were correct or incorrect. We expected 80-100% correct (4 or 5 corrects / 5 trials) for nose clip off condition and less than 40 - 50% correct (1 or 2 corrects / 5 trials) for nose clip on condition in line with previous studies [36]. Pre-test (Smell test ortho nasal) To check participants ortho nasal olfactory performance and to exclude anosmia we used the coffee smell test [37]. This is reported to have excellent validity with sensitivity of 93% and specificity of 96% in comparison to a 12 item Sniffin Sticks test kit [38]. We prepared a 100 ml cup with grounded coffee beans and one empty cup. In a trial, the participants were asked to close their eyes and sniff from a cup that was presented to them blind (either coffee or empty) they had to report the smell by marking on 0 – 10 scales the smell intensity. 0 indicated no smell at all with 10 indicating a very strong smell. Each participant performed 5 trials with nose clip on and 5 trials with nose clip off. Stimuli for the scan For the fMRI scan, stevia was the basic stimulus. The stevia sample consisted of > 95 % steviol glycosides, thereof > 97 % Rebaudioside A provided by EPC Natural Products Co., Ltd. The concentration of stevia was 0.036% Stevia was diluted and delivered in distilled water. A tasteless solution (containing the main ionic components of saliva, 25 mM KCl + 2.5 mM NaHCO 3 ) was used as a control rinse condition used at the end of each trial. Nose clips Soft plastic foam nose clips were used to block retro nasal smell (size approx. 6.8 x 4 cm/ 2.7 x 1.6 inches in length and width) and sourced from Frienda Ltd., China). The pleasantness, pain and comfort of the nose clips was piloted before the study, on 8 subjects. All participants rated the nose clip on the nose between 4 and -4 for pleasure, pain and comfort, once at baseline and again after wearing the nose-clip for 5 minutes (the length of time they would be wearing the nose clip in each condition in the scanner). To examine the effects of the nose clip on subjective ratings we used a repeated measures ANOVA with ratings (3 levels, pleasantness, pain, and comfort) as one within subject factor and time (2 levels, time1 and time2) as a second within subject factor. We found no main effect of ratings (F=0.165 (2,14) p=0.85) or time (F=0.1 (1,7) p=0.75) and no ratings * time interaction (F=2 (2,14) p=0.17) (Table 1). Table 1 here Table 1: Nose clip test Pleas Pain Comfort Pleas Pain Comfort -0.16 (1.09) -0.76 (1.90) -0.26 (1.51) -0.21 (1.41) -0.42 (1.81) -0.78 (1.69) Study design The fMRI scans took place at the Centre of Integrative Neuroscience and Neurodynamics (CINN) at the University of Reading. If scheduled for a morning scan participants fasted overnight, if having an afternoon scan participants fasted for 3 h (no food, only water) before the scan. 10 participants had a morning scan, and 24 participants had an afternoon scan. 60-90 minutes before scanning all the participants were given a standardized meal similar to previous studies (a banana, a cup of orange juice, 2 crackers, ~261 total calories) with the instruction to “eat until feeling comfortably full, without overeating” similar to our previous study [39, 40]. We asked participants to rate their hunger and mood, before the scan, on a visual analogue scale from 0 being not at all to10 indicating the most ever felt. Subjects were screened for potential pregnancy and metal in their body before being placed in the fMRI scanner. Taste delivery Tastes were delivered to the subject via separate long (~3 m) thin Teflon tubes with a mouthpiece (~1 cm in diameter) at one end, which was held by the subject comfortably between the centre of the lips. At the other end of the tubes were connected to separate reservoirs via syringes and one-way Syringe Activated Dual Check Valves (Model 14044–5, World Precision Instruments, Inc) which allowed any stimulus to be delivered manually by the researcher at exactly the right time indicated by the programme [41] thus avoiding the delays and technical issues experienced when using computerised syringe drivers. fMRI Task At the beginning of a trial, a white cross at the centre of the screen appeared for 2 s indicating the start. Then, stevia was delivered in a 0.5 mL aliquot to the subject’s mouth, the green cross was presented at the same time on the visual display for 5 s. The instruction given to the subject was to move the tongue once as soon as a stimulus was delivered in order to distribute the solution round the mouth to activate receptors, and then to keep still until a red cross was shown, when the subject could swallow. Swallowing was 2 s, then the subject was asked to rate the ‘pleasantness’ (+2 to –2) hedonic value, asked to rate the ‘bitterness’ (0 to +4), and asked to rate the ‘mouth fullness’ (richness) sensory aspect (0 to +4) of the taste in their mouth on a visual analogue scale by moving a bar to the appropriate point using a button box, similar to previous taste/fmri studies [25]. Each rating period was 5 s long. After the last rating on each trial 0.5 mL tasteless control solution was administered in the same way as the stevia stimulus and a green cross was again presented at the same time on the visual display for 5 s. The control was used as the comparison condition to allow somatosensory effects produced by liquid in the mouth, and the single tongue movement made to distribute the liquid throughout the mouth, to be subtracted in the fMRI data analysis [31, 42]. The tasteless control condition was not subjectively rated. A grey cross was presented for a duration between 0.8 s and 2 s (jittered) to indicate the end of the trial. Then the screen was black for 2 s before a new trial started. Each trial lasted ~30 sec. Using a block design there were 7 trials of stevia taste with nose clip on followed by a second block of 7 trials of stevia taste with nose clip off, block order was counterbalanced across participants. Between blocks the scanner was also stopped for ~5 to 10 min, to allow the participant to either place the nose clip on or take it off, depending on the order of blocks. During the break participants were told they could let go of the taste tubes and just relax and they could close their eyes. Stopping the scanner between nose clip addition or removal also allowed for movement to be minimised as the second block began with a new localiser scan. The whole task took ~30 minutes, including stopping and starting the scanner. fMRI data acquisition Blood oxygenation level dependent (BOLD) functional MRI images were acquired using a three-Tesla Siemens scanner (Siemens AG, Erlangen, Germany) with a 32-channel head coil. During the task, around 1500 volumes were obtained for each participant, using a multiband sequence with GRAPPA and an acceleration factor of 6. Other sequence parameters included a repetition time (TR) of 700 ms, an echo time (TE) of 30 ms, and a flip angle (FA) of 90°. The field of view (FOV) covered the whole brain with a voxel resolution of 2.4 x 2.4 x 2.4 mm 3 . Moreover, structural T1-weighted images were acquired utilizing a magnetization prepared rapid acquisition gradient echo sequence (TR = 2020 ms, TE = 3.02 ms, FA = 9°) with a FOV covering the whole brain and a voxel resolution of 1 x 1 x 1 mm 3 . fMRI data analysis The imaging data were analysed using SPM12 (Wellcome Centre for Human Neuroimaging, University College London). Pre-processing of the data used SPM12 realignment, coregister, segment, normalization to the MNI coordinate system (Montreal Neurological Institute; Collins et al., 1994), and spatial smoothing with a 6 mm full width at half maximum isotropic Gaussian kernel. The time series at each voxel was low pass filtered with a haemodynamic response kernel. Time series non-sphericity at each voxel was estimated and corrected for, with a high-pass filter with cut-off period of 128 s. In the single-event design, a general linear model was then applied to the time course of activation in which stimulus onsets were modelled as single impulse response functions and then convolved with the canonical hemodynamic response function. Linear contrasts were defined to test specific effects. Time derivatives were included in the basis functions set. Following smoothness estimation, linear contrasts of parameter estimates were defined to test the specific effects of each condition with each individual dataset. Voxel values for each contrast resulted in a statistical parametric map of the corresponding t statistic (transformed into the unit normal distribution (SPM z)). Movement parameters were added as additional regressors. At the second level, we report the main effects of stevia with nose clip off vs the corresponding control tasteless conditions with nose clip off (supplemental data), and stevia with nose clip on vs stevia with nose clip off, thresholded at p<0.05 corrected (familywise-error (FWE) and p values cluster corrected at both p<0.05 False Discovery Rate (FDR) and p<0.05 FWE. We also added gender, hunger level and scan time as covariates of no interest. We then examined regions of interest (ROI) spheres (10 mm) for the anterior insula (primary taste cortex, [-32, 16, 2]), posterior insula [-38, -2, -12], and postcentral gyrus [60, -16, 24] using WFU pickatlas, and the hypothalamus using aal atlas, and identified from meta-analyses on sweet tastes in humans [26, 27]. We examined the olfactory regions; the piriform cortex, olfactory cortex and the orbitofrontal cortex using aal atlas anatomical masks in WFU pickatlas. Given our interest in retro nasal effects (Small et al., 2005) and attention to odors [34] we also created a sphere (10 mm) in the pgACC [3, 42, -9] (Small et al., 2005) and examined anatomical masks of the mOFC (Small et al., 2005) and sgACC, (BA25) [34] using aal atlas in WFU pickatlas . Finally, as we are interested in the retro nasal contribution to the rewarding/aversive effects of stevia we also examined the nucleus accumbens [32] and amygdala [29, 30] using IBASPM71 atlas and the aal atlas anatomical masks, respectively, in WFU pickatlas. For the ROI analyses, data were extracted using the SPM ROI analysis Matlab code and SPM’s spm_get_data command and analysed with paired-sample t tests, in excel and SPSS, and then corrected for multiple comparisons across the 18 ROIs, i.e. p=0.05/18=0.003. We also examined correlations between the extracted ROI data and the subjective ratings of pleasantness, bitterness, and mouth fullness. Results Demographic data for fMRI study 34 participants took part with a mean age of 25.71 yrs. See Table 2 for demographics. Table 2 here Age, years 25.71 (8.25) Gender, female/male: n 24/10 Body mass index 22.00 (2.68) EAT 3.09 (3.20) Craving for sugary foods 5.11 (1.99) Liking for sugary foods 5.85 (1.98) Freq eating sugary foods 3.44 (2.09) Freq eating/drinking foods with sweeteners 3.97 (2.11) Pre-test results of sensitivity to 2% sweet taste Twenty-one participants passed the pre-test with 6 out of 6 trials correct the first time. Ten participants passed the pre-test with 5 out of 6 trials correct the first time and three participants got 6 of the 6 trials correct on their second attempt, so were also included in the study. Pre-test Candy Smell Test As expected we found that with the nose clip off participants could identify the flavours in the candy smell test with average accuracy of 84% (± 14) and when the nose clip was added this accuracy dropped to 31% (± 20) in line with previous studies [36]. Pre-test (Smell test ortho nasal) As expected, all participants could identify the cup that had coffee in compared to no coffee and rated the coffee smell as above average intensity and rated the coffee smell (6.70 ±1.78) higher than the intensity of the empty cup smell (1.17 ±1.90), (t(22) = 12.04, p higher with the nose clip off than with the nose clip on (0.26 ±0.59), (t(22) = 16.7, p <0.001). fMRI scan day Subjective hunger and Mood Participants had relatively high mood and low hunger levels before the scan (Table 3). Table 3 here Appetite How hungry do you feel right now? How full do you feel right now? 4.35 ± 2.30 4.05 ± 2.11 Mood Alertness 6.08 ± 2.40 Disgust 0.91 ± 1.23 Drowsiness 3.05 ± 2.66 Anxiety 1.79 ± 1.55 Happiness 6.11 ± 1.93 Nausea 0.70 ± 0.97 Sadness 0.55 ± 1.05 Withdrawn 1.08 ± 1.76 Faint 1.08 ± 1.84 Rate between 0 and 10, where 0 = Not at all, 10 = Most ever felt Subjective ratings of stevia during the scan with nose clip on and off To examine the effects of the nose clip on subjective ratings, we used a repeated measures ANOVA with ratings made during the scans as a within factor (3 levels, pleasantness, bitterness, mouth fullness) and condition (2 levels, nose clip on, nose clip off) as a second within subject factor. We found a main effect of ratings (F=17.2 (1.5, 50) p<0.001), but no main effect of condition (F=0.9 (1,33) p=0.34), or ratings*condition interaction (F=0.594 (1.69,56) p=0.53) (Figure 1). Figure 1 here Figure 1 . Pleasantness, Bitterness, and Mouth Fullness ratings for stevia with nose clip on (NC1) and nose clip off (NC0). Whole brain analysis Main effects of taste stimuli The stevia taste vs the control taste activated parts of the brain such as the primary taste cortex (insula), primary somatosensory cortex (postcentral gyrus), the precentral gyrus, caudate and putamen, as expected and similar to previous studies on sweet tastes [27] ( Table S1). There were no significant increased activations for the control vs stevia contrast. ROI analysis Stevia: Nose clip off vs nose clip on: We found activity in the olfactory cortex, sgACC, and pgACC, hypothalamus and right NAcc reduced with the retro nasal pathways occluded with a nose clip, controlled for multiple comparisons (Table 4). There were no regions that had greater neural activity for the opposite contrast of nose clip on vs off. Table 4. Effect of nose clip on stevia in ROIs ROI t value p value cohens D Olfactory 4.16 0.0001* 0.71 Piriform 1.63 0.06 0.28 sgACC 3.43 0.0008* 0.59 mOFC 1.76 0.04 0.30 pgACC 3.03 0.002* 0.52 Hypothalamus 3.34 0.001* 0.57 Left Right t value p value cohens D t value p value cohens D Postcentral gyrus 2.29 0.01 0.39 1.64 0.05 0.28 Anterior Insula 2.81 0.004 0.48 2.05 0.02 0.35 Posterior Insula 0.56 0.29 0.10 1.47 0.08 0.25 NAcc 2.82 0.004 0.52 2.91 0.003* 0.51 OFC 2.3 0.014 0.39 0.15 0.44 0.03 Amygdala 1.89 0.03 0.32 2.35 0.01 0.40 Survives correction for multiple comparisons, (0.05/18 ROIs, p = 0.003) Figure 2 A. Olfactory ROI. B. Contrast estimates extracted from ROI using marsbar for stevia nose clip off and nose clip on (error bars, SEM). Figure 3 A. sgACC (BA 25) ROI. B . Contrast estimates extracted from ROI using marsbar for stevia nose clip off and nose clip on (error bars, SEM). Figure 4 A. pgACC ROI. B . Contrast estimates extracted from ROI using marsbar for stevia nose clip off and nose clip on (error bars, SEM). Parametric modulation Pleasantness We found a negative correlation between stevia pleasantness and ROI activity in the anterior insula (left: rho = -0.41, p = 0.01; right: rho = -0.42, p = 0.01, two-tailed) and the left posterior insula ( rho = -0.56, p = 0.0006, two-tailed) for nose clip off. The posterior insula survived correction for multiple comparisons. We also found a negative correlation between pleasantness for stevia and activation in ROI right anterior insula ( rho = -0.35, p = 0.04, two-tailed) for nose clip on, but this did not survive correction for multiple comparisons (p=0.05/18=0.0028). Figure 5 A . Left posterior insula ROI. B . Correlations between stevia pleasantness ratings and contrast estimates extracted from ROI using marsbar. Bitterness For bitterness we found a weak positive correlation between stevia and ROI activity in the right amygdala ( rho = 0.29, p=0.05, one-tailed) for nose clip off, but this did not survive correction for multiple comparisons. There were no relationships between brain activity and subjective bitterness with the nose clip on. Mouth fullness We found a positive correlation between stevia mouth fullness and activity in the olfactory ROI ( rho = 0.36, p = 0.04, two-tailed) and the piriform cortex ( rho = 0.35, p = 0.04, two-tailed) for nose clip off, but not for nose clip on. We also found a positive correlation between mouth fullness for stevia and activation in ROI right amygdala ( rho = 0.34, p = 0.04, two-tailed) for nose clip on. None of these correlations survived correction for multiple comparisons. Figure 6 A . Right olfactory ROI. B . Correlations between stevia mouth fullness ratings and contrast estimates extracted from ROI using marsbar. Exploratory whole brain analysis When examining the effects of the stevia nose clip on vs off and nose clip off vs on, there were no effects at the whole brain. Discussion Stevia activated brain regions similar to those activated by other sweet tastes such as sucrose reported in previous studies [11, 21, 24, 26, 27, 40]. We also provide novel first evidence that retro nasal occlusion with a nose clip reduces neural activity to stevia in the olfactory cortex, hypothalamus, sgACC, pgACC, and nucleus accumbens. The reduced activity with the nose clip on in areas related to taste, odour and reward processing (olfactory, hypothalamus, sgACC, pgACC, NAcc) demonstrates a potential biological mechanism for the reduced subjective experience of stevia with retro nasal blockade [13] and provides further support for the notion of retro nasal pathways as contributors to the perceptual processing of “non-volatile” substances [2, 11, 13]. Consistent with our findings p revious work by Small et al. also found greater pgACC activity to retronasal vs orthonasal smell delivery [33]. Reduced olfactory and hypothalamic activity with the nose clip on suggests that when tasting stevia the retro nasal pathways are activating regions involved in smell, appetite and metabolic responses. The olfactory cortex receives projections from retro nasal pathways and including the piriform cortex and orbitofrontal cortex, integrates this retro nasal information with other sensory inputs like taste and texture [43] and was found reduced in activity with retro nasal blockade during sucrose tasting [11]. The hypothalamus has been implicated in appetite and metabolic processes, and studies also report its involvement in taste perception [44] therefore reduced hypothalamus with nose clip on during stevia taste could indicate a reduced ability to fully interpret stevia. Reduced anterior cingulate and nucleus accumbens activity to stevia with the nose clip on, is consistent with our previous study on retro nasal blockade for sucrose taste [11] and indicates retro nasal pathways are activating regions involved in emotion, reward and decision-making. Further, previous studies show that pre frontal cortex regions such as the pgACC are multi-modal regions integrating taste and smell information [45], and show greater activation to tastes when combined with odours than to the sum of the activations by the taste and olfactory components presented separately [25, 46]. This could suggest that the nose clip reduces the integration of taste and olfactory components making it more difficult to perceive stevia. Further, as studies find that odour/preference learning in rats is more effective from retro vs ortho nasal routes [47] our findings could indicate that blocking retro nasal processing of stevia with a nose clip could alter decision making in relation to stevia. Future studies using a fMRI decision-making task could therefore examine if retro nasal blockade with a nose clip would disrupt neural responses during decision-making and hence interrupt subsequent food choice behaviour, further implicating a role for retro nasal pathways. We also found reduced activity in the NAcc, a hub related to feeding, homeostatic and hedonic circuits, that facilitates behaviour via its downstream projections [48] when examining the effects of retro nasal blockade, similar to our previous study on retro nasal blockade during sucrose tasting [11]. The ventral striatum i s at the crossroads of olfactory and reward pathways and receives direct projections from the primary olfactory cortex [49] and the dopaminergic midbrain [50] and is greatly involved in odour-guided eating behaviour [51]. Therefore, reduced activity in the NAcc supports the idea that retro nasal olfactory signals related to stevia are being occluded. We also found that correlations between the subjective ratings (pleasantness, bitterness, mouth fullness) and neural activity (insula, amygdala, olfactory) respectively, was weaker/non-significant with the nose clip on vs off. This further supports our proposal that retro nasal pathways contribute to the neural processing of stevia [2, 13], but at an unconscious level, as we found no significant difference between nose clip on vs off when examining the subjective reports alone. These findings therefore highlight the power of neuroimaging to detect objective biological sensory effects, outside of subjective conscious awareness. Future studies should test if the regions that correlated with subjective report can be used to predict subsequent choice behaviour in relation to stevia and if these predictions are affected by a nose clip. In summary, we have shown with neuroimaging that retro nasal pathways may be playing a role in the neural processing of stevia and contributing to its perceptual properties. Knowing this could help us potentially improve the palatability of stevia sweetened foods via the addition of aerosols attributes. In conclusion, this study contributes to a broader understanding of how retro nasal pathways contribute to the neural processing of natural, zero-calorie, sweeteners such as stevia. Acknowledgements: We would like to thank Dr Shan Shen and the staff at the Centre for Neuroscience and Neurodynamics (CINN) at the University of Reading for their help with the scanning. Declaration of Interest Statement: The authors declare the following financial interests/personal relationships which may be considered as potential competing interest. Weiyao Shi and Jingang Shi are employees of EPC Natural Products Co., Ltd. who provided the compounds and funded the study. The work was conducted independently at the NRG laboratory of Prof. McCabe at the University of Reading solely for the purpose of scientific understanding. All authors declare that they have no other known competing financial interests or personal relationships that could have appeared to influence the findings reported in this paper. Authors’ Contributions and Agreement: CMcC, TE, JS, WS conceived and designed research. JS, WS and TE prepared and supplied the study samples. 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Contrast estimates extracted from ROI using marsbar for stevia nose clip off and nose clip on (error bars, SEM). Figure 5 A . Left posterior insula ROI. B . Correlations between stevia pleasantness ratings and contrast estimates extracted from ROI using marsbar. Figure 6 A . Right olfactory ROI. B . Correlations between stevia mouth fullness ratings and contrast estimates extracted from ROI using marsbar. Supplementary doc Challenging the “Non-Volatile” Taste Assumption: Neural Effects of Retro Nasal Occlusion on Stevia Perception Main effects of taste stimuli Table S1 Region x(mm) y(mm) z(mm) Z-score voxels p(FWE-corr) p(FDR-corr) Precentral gyrus -35 -28 64 6.481 196 < 0.0001 < 0.0001 Postcentral gyrus -40 -21 52 6.170 Caudate 13 15 4 6.148 152 < 0.0001 < 0.0001 Caudate -8 10 2 5.380 Putamen -18 15 0 5.073 Insula -30 25 7 5.992 78 < 0.0001 < 0.0001 Insula -32 18 2 5.844 Insula 30 27 2 5.899 72 < 0.0001 < 0.0001 Insula 35 18 7 5.128 Supp Motor Area -4 15 45 5.632 26 < 0.0001 =0.000623 Supp Motor Area 4 3 60 5.371 46 < 0.0001 < 0.0001 Threshold: 0.0001 uncorrected Region x(mm) y(mm) z(mm) Z-score voxels p(FWE-corr) p(FDR-corr) Precuneus -8 -69 43 4.902 45 =0.004325 =0.018615 Rolandic Operculum 40 -4 12 4.729 27 =0.02498 =0.067898 unknown (Temporal Sup L) -35 -38 7 4.717 30 =0.018268 =0.056552 Precentral gyrus 32 -6 50 4.424 38 =0.008281 =0.02976 Superior frontal gyrus 23 -11 55 3.968 Table S2 Region x(mm) y(mm) z(mm) Z-score voxels p(FWE-corr) p(FDR-corr) Postcentral gyrus -47 -18 52 5.748 122 < 0.0001 < 0.0001 Precentral gyrus -37 -26 60 5.665 Supp Motor Area -6 13 50 5.502 29 < 0.0001 =0.00430 Supp Motor Area 4 15 48 4.743 unknown (caudate L) 1 10 4 5.250 10 =0.00140 =0.08882 Supp Motor Area -8 -6 62 5.210 9 =0.00178 =0.09070 unknown (caudate L) -16 30 0 5.175 11 =0.00110 =0.08882 Threshold: 0.0001 uncorrected Region x(mm) y(mm) z(mm) Z-score voxels p(FWE-corr) p(FDR-corr) unknown (caudate L) 1 10 4 5.250 104 =0.00014 =0.00077 Caudate 8 15 4 4.466 Frontal Inf Oper -47 10 9 5.139 168 < 0.0001 < 0.0001 Insula -32 25 7 4.848 Insula 30 27 7 4.918 68 =0.00141 =0.0052 unknown (Temporal Mid L) -37 -47 0 4.668 24 =0.04702 =0.0819 unknown (Temporal Sup R) 40 -45 4 4.635 55 =0.00357 =0.0099 Unknown 42 -38 -8 4.508 Unknown 35 -50 0 4.471 Inferior parietal gyrus -30 -50 43 4.526 58 =0.00287 =0.00908 unknown (caudate R) 16 27 0 4.512 25 =0.04276 =0.08061 Superior frontal gyrus -23 -4 52 4.471 29 =0.02956 =0.06036 Superior parietal gyrus -20 -62 43 4.351 29 =0.02956 =0.06036 Information & Authors Information Version history V1 Version 1 08 December 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection European Journal of Neuroscience Keywords brain reward system neuroimaging non-nutritive sweeteners retro nasal taste Authors Affiliations Hee-kyoung Ko 0000-0002-8294-9548 University of Reading View all articles by this author Jingang Shi EPC Natural Products Co Ltd View all articles by this author Thomas Eidenberger University of Applied Sciences Upper Austria View all articles by this author Weiyao Shi EPC Natural Products Co Ltd View all articles by this author Ciara Mccabe 0000-0001-8704-3473 [email protected] University of Reading View all articles by this author Metrics & Citations Metrics Article Usage 309 views 179 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Hee-kyoung Ko, Jingang Shi, Thomas Eidenberger, et al. Challenging the “Non-Volatile” Taste Assumption: Neural Effects of Retro Nasal Occlusion on Stevia Perception. Authorea . 08 December 2025. 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