*3.3. PROP Bitterness*

#### 3.3.1. PROP Taster Groups

Out of 126 participants who completed the consumer test, there were 15 non-tasters, 81 medium tasters, and 30 supertasters. The interaction between taster groups and samples was not significant for all taste evaluations of each Reb A, D, and M solutions. There was also no significant difference when examining the main effect of taster groups on perceived intensity scores of sweet and bitter tastes of the three sweeteners combined over time (in-mouth, immediate, and lingering) (Figure 2).

**Figure 2.** The influence of 6-n-Propylthiouracil (PROP) taster groups on the perceived intensities (± SEM), with sweeteners combined (Reb A, D, and M).

Due to the disproportional ratio of people in each taster groups, agglomerative hierarchical clustering (AHC) was used to group people based on their dissimilarity on the PROP rating (data not shown). Three groups were generated with 55, 44, and 30 people, corresponding to low, medium, and high-sensitive clusters, respectively. However, no significant difference in the main effect of clusters was found.

#### 3.3.2. Relationships with Perceived Intensities of Reb A, D, and M

Pearson correlation tests were conducted to determine the association between PROP bitterness and perceived intensities of sweet and bitter tastes of Reb A, D, and M over time (in-mouth, immediate, lingering). No significant relationships existed between PROP bitterness and the rated intensities of the three steviol glycosides (*p* > 0.05).

#### **4. Discussion**

The present study investigated the sweetness and bitterness of Reb A, D, and M compared to sucrose at a high sucrose equivalent level (14% *w*/*v*) using consumers. To compare with 14% (*w*/*v*) sucrose solution, the solution concentration of the three steviol glycosides was determined by two small scale consumer tests as a preliminary test (see 2.4.1 for details). Briefly, the sweetness of 0.1% (*w*/*v*) Reb M was proved to be not significantly different from a 14% sucrose solution, and the same concentration was used for Reb A and D to compare the sensory characteristics of the three steviol glycosides at the same concentration. Prakash et al. [16] estimated that Reb M was about 200–350 times sweeter than sucrose, and the sweet potency at 10% SE was calculated to be 159. In the present study, the sweet potency of Reb M at 14% SE was calculated as 140. This is in line with the model from Prakash et al. [16], sweet potency of high-intensity sweeteners tended to decrease as the sucrose sweetness equivalent level increased [45].

The three steviol glycosides showed significant differences in sweetness and bitterness at the same concentration (0.1% *w*/*v*). The in-mouth sweetness of Reb D and M at 0.1% were not statistically different from sucrose at 14%, while 0.1% Reb A was less sweet than sucrose. Reb A was significantly less sweet than Reb M as well (*p* < 0.01) but was not significantly different from the in-mouth sweetness of Reb D (*p* = 0.24) with a tendency of being less sweet. Reb D and M were not significantly different in in-mouth sweetness (*p* = 0.06), but there was also a clear tendency of Reb D to be less sweet than Reb M. These results were consistent with the previous studies investigating the sweetness of Reb A, D, and/or M at different concentrations showing that Reb M was the sweetest sweetener and Reb A was the least sweet sweetener among Reb A, D, and M at the same concentration [15,16,46].

The sweetness temporal profile of Reb M was studied by Prakash et al. [16], who compared the sweetness appearance time and extinction time to examine the change in perception over 3 min. The sweetness of Reb M elicited later and persisted longer than sucrose at 10% SE in water. The descriptive panel rated the lingering sweetness of Reb M higher than that of sucrose as well [16]. In the present study, Reb M had a similar in-mouth sweetness to sucrose, but the lingering sweetness was significantly higher than that of sucrose, which corresponded with Prakash's finding. Reb A was also found to have a longer extinction time than sucrose [13] and exhibited persistent flavor duration in the mouth [8]. When at a similar sweetness level (i.e., at 8% SE), the lingering sweetness of Reb A and Reb M were not different [16]. Even though, in this study, there was no significant difference in lingering sweetness between sucrose and Reb A (*p* = 0.12), the lingering sweetness of Reb A became higher than sucrose after being rated less sweet in-mouth, which suggested that if the sweetness of Reb A was at the same level as sucrose, the lingering sweetness might be significantly higher than sucrose. Reb D, like Reb M, also had a similar in-mouth sweetness to sucrose, but had marginally higher lingering sweetness than sucrose (*p* = 0.05). When comparing Reb D to Reb M, the lingering sweetness of Reb D was marginally less than that of Reb M (*p* = 0.05) similar to the in-mouth sweetness of Reb D that was almost significantly less than Reb M (*p* = 0.06). Although the lingering sweetness of Reb M seemed to be stronger than Reb D, it may due to its higher initial sweetness than Reb D.

The bitterness of Reb A stood out among the samples when consumers first tasted the sample and the bitterness continued to be significantly different from others even after one minute. Many other researchers have reported the bitterness of Reb A [13,15]. On the other hand, Reb D and M did not show much in-mouth bitterness and had a similar intensity to sucrose. Even though Reb D exhibited a significantly higher immediate bitterness than sucrose, it was still considered low. A trained panel did not detect any significant bitter taste of Reb M when comparing to sucrose at 10% SE [16]. Hellfritsch et al. [15] and Ko et al. [47] indicated that Reb D elicited a lot less bitterness than Reb A. Our results confirmed that naïve consumers like trained assessors did not detect much bitterness from Reb D and M.

Based on the total counts of CATA and the CA, Reb A was associated with some negative perception terms, such as *bitter*, *chemical*, and *artificial*. The *bitter* and *chemical* attributes were significantly more selected for Reb A than Reb D, Reb M, and sucrose. The *bitter* attribute was in agreement with the bitterness intensity rating. The bitterness and chemical sensation of Reb A was reported by Fujimaru et al. [48] as well. Significantly less *sweet* was checked for Reb A than the other sweeteners, suggesting that the bitterness and chemical sensation might overshadow the sweetness of it. Reb D and M appeared to have good taste profiles because they were close to positive terms and sucrose. However, even though Reb D and M had low citations for *bitter* and higher citations for *pleasant* than Reb A, many participants still checked *artificial* significantly more frequent than sucrose. Waldrop and Ross [49] reported that consumers did not like stevia because of its association with *artificial* flavor. Thus, the *artificial* attribute may cause negative consumer perception of Reb D and M even though they are natural sweeteners without the aversive bitter aftertaste. Interestingly, the *artificial* attribute was also selected for sucrose by 38 participants. It is not common to drink pure sugar water in daily life, so the participants may not be familiar with the taste of sucrose solutions, and thus might select *artificial* for sucrose solution.

*Licorice*, *pungent*, *spicy*, *tangy*, and *tingling* were rarely selected by the participants and were not significant to discriminate the samples. Thus, these five terms may not be appropriate terms for consumers to describe the three steviol glycosides, even though *licorice* has been commonly used to describe the aftertaste of Reb A by researchers [10,13,50] and media [18]. The *licorice* taste of Reb A did not exhibit at low SE levels, but was elicited at higher SE levels [13], and this was further proved by Reyes et al. [50] that Reb A at 0.1% had more notable *licorice* taste than at 0.012%. In this study, we did not find the correlation between *licorice* and Reb A at 0.1%, since only 8 people out of 126 selected it, which suggested that *licorice* may not be an appropriate term for consumers to describe the aftertaste of Reb A or to discriminate Reb A, D, and M.

The CATA analysis also found that *vanilla* and *honey* were associated with sucrose. A consumer survey showed that honey was the most popular sugar alternative, which was natural, and natural sweeteners were perceived better than artificial sweeteners in general [3]. In this study, those who checked *honey* for steviol glycosides might imply that the sample gave them a sense of natural. As for *vanilla*, Lavin and Lawless [51] showed that an added vanilla flavor enhanced the perception of sweetness in milk, and Wang et al. [52] also indicated a taste-aroma interaction between perceived sweetness and vanilla flavor in skim milk. Vanilla was congruent with sweetness, so participants might choose the term even though the attribute was not presented in the solution.

A secondary objective of this study was to investigate the influence of consumers' PROP taster status on the sweetness and bitterness of Reb A, D, and M. We found that there were no significant differences in the perceived sweetness and bitterness of Reb A, D, and M (in-mouth, immediate, and lingering) among PROP taster groups. Risso et al. [40] reported that there was no correlation between PROP bitterness and stevioside bitterness. Humans have about 25 bitter taste receptors from the taste 2 receptors (hTAS2Rs) gene family [53]. Each receptor responds to different compounds but may have overlapped molecular range [54]. The sensitivity to the bitterness of PROP/PTC is mainly associated with TAS2R38 bitter taste receptor [55,56]. TAS2R4 and TAS2R14 responded to the bitterness of stevioside and Reb A in vitro, while TAS2R38 did not react [15]. Meyerhof et al. [54] sorted receptors

into 4 groups, and both TAR2S4 and TAS2R14 were not in the same group as TAS2R38. The different responses in bitter taste receptors might explain why no relationship was found between PROP taster status and perceived bitterness intensity of Reb A, D, and M.

Some studies suggested that PROP bitterness sensitivity influenced other oral sensations, such as sweetness [36,37,57–61]. Drewnowski et al. [36] found a weak and marginal significant difference in sweetness perception of sucrose and saccharin between PROP tasters and non-tasters, and the difference was more significant at lower concentrations. Allen et al. [59] reported that the sweetness of acesulfame potassium was positively associated with PROP bitterness. A large sample size study (*n* > 1500) found a weak association between sweetness and PROP bitterness, suggesting that a bigger size sample is required to detect weak association with PROP [60]. A recent study confirmed that PROP bitterness was positively correlated with sweetness of sucrose [61]. However, some of the previous studies also indicated that there was no relationship between PROP sensitivity and sweet taste responsiveness [62–64]. Here, we found no significant differences in perceived sweetness intensity among PROP groups and further, no correlation between PROP bitterness and sweetness of the steviol glycosides. In this study, the test stimuli were singles (i.e., each sweetener solution), but the aftertaste of the three steviol glycosides, especially the sweet-bitter Reb A at a high concentration, might cause difficulties for participants to evaluate intensities of sweet and bitter tastes. Horne et al. [38] reported that sweet-bitter stimuli might be more difficult to evaluate than single taste stimuli due to taste–taste interactions. Expansive, linear, and compressive phases of psychophysical functions could be used to predict how taste stimuli would behave when mixed at low, medium, and high intensity/concentration [65]. For example, perceptual enhancement and suppression has been extensively reported at low and high intensity/concentration mixtures, corresponding to the expansive phase and compressive phase of the psychophysical function, respectively [65]. Ly and Drewnowski [63] showed a reduced difference in bitterness between PROP taster groups was found when the caffeine solution was sweetened, even though PROP tasters rated caffeine solution without sweetener as more bitter than non-tasters [63]. The perceptual suppression as a result of sweet-bitter interaction at a high intensity/concentration may explain no differences in perceived sweetness intensity among PROP groups and no correlations between PROP bitterness and perceived sweetness of Reb A, D, and M.

One possible limitation of the study was that consumers did not swallow the solution, which limited the number of taste buds utilized for the evaluation. Taste buds are distributed not only in the mouth but also in the throat [66]. Consumers were asked to expectorate the sample to reduce fatigue, however, the swallowing sensation could be different and might impact the perceived intensities. No hedonic question was asked in this study because it might be difficult for naïve consumers to rate the likings of pure sweetener solutions when the solutions were not regularly consumed in daily life. However, no association could be drawn between the negative CATA attributes and the likings of the sweeteners. Another limitation was the disproportional size of PROP groups, which only had 15 non-tasters. The data from non-tasters might be less variable if more non-tasters were recruited.

#### **5. Conclusions**

The present study investigated the sensory profile of Reb A, D, and M at 14% SE using a consumer panel, and the influence of PROP taster groups on the perceived sweet and bitter tastes of the three glycosides. Reb D and M had sensory profiles that were closer to sucrose, compared to Reb A, but were still associated with negative sensation, such as *artificial*, which may cause negative perception toward Reb D and M. The lingering sweetness of Reb D and M was also a concern. The sensory characteristics of Reb A, D, and M in this study can be used as a reference for the food industries working with steviol glycosides in high-sugar applications, such as frozen desserts. Furthermore, there were no significant differences among non-tasters, medium tasters, and supertasters on the perceived sweetness and bitterness of Reb A, D, and M as well as no significant correlations between PROP bitterness and perceived sweet and bitter tastes, suggesting that supertasters who experience more intense taste

sensations may not report aversive sensations from stevia. Further studies on the consumer acceptance of Reb A, D, and M are needed to determine if these characteristics would affect the likings of these sweeteners. Since the sweeteners may perform differently in a food matrix than in aqueous solution, more research using steviol glycosides in final food products are needed to determine the sensory profile and acceptance of them.

**Author Contributions:** R.T. contributed to study design, data collection, data analysis, and manuscript writing. S.C. contributed to study design, data collection, data analysis, and critical revision of the manuscript. S.C. supervised the project. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the United States Department of Agriculture Specialty Crop Research Initiative (grant number 2017-5181-26828).

**Acknowledgments:** The authors thank Ingredion Incorporated for providing Reb A, D, and M.

**Conflicts of Interest:** The authors declare no conflict of interest.
