time-course of astringent sensations

Chemical Senses Vol.16 no.3 pp. 225-238, 1991

Time-course of astringent sensations

Christopher B.Lee and Harry T.Lawless

Department of Food Science, New York State College of Agriculture and LifeSciences, Cornell University, Ithaca, NY 14853, USA

Abstract. Qualitative and quantitative perceptual reactions to astringent materials were examined for threediverse chemical substances (alum, tannic acid and tartaric acid) at several concentrations producing moderateto strong levels of perceived sensation. Group discussions were held to determine language appropriate todescribe the sensations arising from solutions of the three compounds and a composite ballot of six ratingscales (astringency, mouth drying, puckery feeling, mouth roughing, bitterness and sourness) was developed.For both experiments, two concentrations of each compound were rated on the six attributes for five to sixminutes, a discrete-point time — intensity scaling procedure. All ratings showed roughly exponential decaysover time. The intensity ratings for each attribute were found to depend on both the particular astringentsubstance and concentration tested. The results from experiment 2 suggested that the four tactile attributesof drying, puckery feeling, roughing, and overall astringency may not be totally interchangeable and thatthere may be multiple sub-qualities in the sensory reactions grouped as astringency. It is recommended thatfuture structure-activity studies make use of time - intensity procedures with multiple rated attributes, using1 g/1 alum as a reference material, since it is relatively low in perceived bitterness and sourness, but producespronounced drying, roughing, puckery/drawing sensations.

Introduction

Astringency is an important sensory attribute of foods and beverages that containastringent tannins including coffee, tea, beer, wine, apples, ciders, many berry cropsand nuts (Haslam and Lilley, 1988). Other astringent materials include salts ofmultivalent cations (Al, Cr, Zn, Pb, Ca, B), mineral acids, and dehydrating agentssuch as alcohol and dimethyl ketone (Haslam and Lilley, 1988). In some historicalclassifications astringency has been considered a primary or basic taste category,i.e. equal in importance to the classical four taste qualities of sweet, sour, salty, andbitter (Bartoshuk, 1978). Others (Bate-Smith, 1954) have classified astringency as achemically induced tactile sensation, a position which is supported by the tendency ofastringent sensations to grow in intensity with repeated stimulation (Guinard et al.,1986), rather than showing the adaptation that is characteristic of taste receptors.

Several major gaps exist in the literature concerning astringency. From a perceptualpoint of view, there is little or no consensus about whether astringency is a singlesensation or a general category made up of multiple sub-qualities. The ASTM Committeeon Sensory Evaluation of Materials and Products defined astringency as 'the complexof sensations due to shrinking, drawing or puckering of the epithelium as a result ofexposure to substances such as alums or tannins' (American Society for Testing andMaterials, 1989a). Previous research has not attempted to break down astringency intomultiple sub-qualities during psychophysical evaluation, allowing subjects to simplyrate astringency itself, e.g. Guinard et al. (1986), or some aspect such as the dry nessof the mouth, e.g. Lyman and Green (1990). Another difficulty in the literature is thecommon practice of studying astringency in complex food media or beverages suchas wine (Arnold et al, 1980; Noble et al., 1984; Guinard et al., 1986). While thisis understandable from a practical point of view, it may confuse the picture insofar

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as other chemical components in the food may interact with the astringent substancesor directly alter salivary flow and the degree of oral lubrication (Lyman and Green,1990). In addition, other sensory properties of the food could interact with astringentperceptions at higher neural levels causing effects similar to taste mixture suppression(Lawless, 1979; Noble etal., 1984). Furthermore, alcohol and surfactants in the foodsor beverages may increase the solubility of the hydrophobic phenol-protein complexes(Clifford, 1986), decreasing astringent sensations that may depend upon precipitationof salivary proteins.

Research on sensory reactions to astringent substances has faced difficultmethodological problems. One problem concerns the build-up of sensations over time(Hinreiner et al., 1955; Lyman and Green, 1990), which makes paired testing methods(usually very sensitive) somewhat problematic. One solution to this problem has beena reversed paired comparison technique in which the second substance is judged relativeto the first in counterbalanced order, and then statistical methods are used to 'factorout' order effects as covariates. This allows adjusted marginal means to reflect pureestimates of astringency, a technique first proposed by Scheffe' (1952). A secondapproach to this problem has been to focus specifically on the relative intensity ofastringency during sequential ingestions (Guinard et al., 1986; Lyman and Green, 1990).This has the advantage of resembling the pattern of repeated stimulation in the normalsipping and drinking of a beverage. Finally, recent studies have used the techniquesof time — intensity scaling to give a fuller picture of astringent reactions, either throughcontinuous tracking techniques that generate an analog signal in proportion to sensationintensity (Guinard et al., 1986) or through repeated intensity estimates at discrete pointsin time (Lyman and Green, 1990). To date, there is relatively little informationconcerning the time course of single sipped stimuli in model systems such as aqueoussolutions, using current time-intensity methods, and little comparison of differentcompounds.

Another difficulty in studying astringency is that many untrained observers confuseastringency and bitterness. Lea and Arnold (1978) went so far as to classify bitternessand mouth drying as 'twin sensations' because nearly all phenolic astringents are alsobitter, and untrained panelists sometimes confuse the two qualities. Furthermore, Clifford(1986) has suggested that small flavolans (condensed phenols) such as caffeoylquinicacid may bind to both bitter receptors and salivary proteins in the oral cavity. Thusthe sensory intensity and character of such compounds may depend on their relativeaffinity for the two interactions. It is difficult, however, to provide psychophysicalevidence relevant to such ideas in the face of confusion between bitterness andastringency among observers. In addition to being bitter, many astringent materials(particularly the organic acids) also have a sour side-taste associated with them.According to McAuliffe and Meiselman (1974) and O'Mahony et al. (1979) there isa common linguistic confusion between sour and bitter. Therefore, the data on sensoryastringency derived from the opinions of naive subjects (i.e. those who have not receiveddefinitions and reference samples for sour and bitter) should be viewed with caution.

The general goals of this study were to examine the qualitative and quantitativeperceptual responses to selected astringent materials. One goal was to providedocumentation on the time-course of astringent materials having diverse chemicalstructures, at several concentration levels, using current time-intensity methods. That

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Perception of astringency

is, we wished to provide psychophysical characterization of reactions to astringentcompounds with respect to perceived intensity as a function of compound, concentra-tion, and time since exposure to oral tissues. Since there is substantial interest inastringency in the applied flavor analysis, a second goal was to examine a potentialstandard methodology by which astringency could be studied in future structure—activitystudies or in sensory evaluation of foods. Along these lines, an additional objectivewas to determine the presence and time course of side tastes produced by astringentmaterials, particularly sourness and bitterness. Potential reference standards orbenchmarks that are low in side-tastes are needed for use as panel training standards(Civille and Lawless, 1986; Rainey, 1986) and as references in structure —activitybioassays.

Another question of interest was whether astringency can be broken down into multiplesubqualities. This issue has received surprisingly little attention. Astringency is widelyrecognized to encompass both drying and puckering sensations (Bate-Smith, 1954;Josslyn and Goldstein, 1964; Guinard etal., 1986), although Lyman and Green (1990)chose to have subjects attend only to drying sensations. In the work described below,a qualitative exploration of terms was conducted to explore the specific attributes thatmight be relevant to astringent substances, consisting of non-directive small groupdiscussions. Such discussion groups are similar to the 'Focus groups' used in marketingresearch, and are widely used in applied sensory evaluation in the terminologydevelopment phase of descriptive analysis (Civille and Lawless, 1986; Stone and Sidel,1985; Johnsen et al., 1987). One variation of time - intensity scaling is the rating ofmultiple sensory attributes at discrete points in time, rather than the continuous trackingof a single attribute. This procedure provides for efficient characterization of stimulithat are both qualitatively complex and highly fatiguing and was given the name 'intensityvariation descriptive methodology' by Gordin (1987). We applied this methodologyto astringent materials through the rating of perceived qualities of sensation determinedfrom the qualitative phase of the study, specifically, mouth drying, drawing/puckerysensations, mouth roughing, and overall astringency, over time.

Experiment 1

Method

Subjects. Eleven healthy adult subjects (three male, eight female) between the ages of21 and 35 were paid to participate in the experiment.

Stimuli. The test stimuli consisted of two concentrations each of alum (aluminumammonium sulfate, Sigma), tannic acid (Fisher), and tartaric acid (Fisher) dissolvedin deionized water. These compounds, representing a large tannin polymer, a smallorganic acid and a complex salt, were chosen for their chemical diversity, for theirpresence in the literature as examples of astringent materials (Guinard et al., 1986;Lyman and Green, 1990), and on the basis of discussions with workers in wine researchwho suggested them as potential reference standards or prototypes suitable for the trainingof taste panels for astringency evaluation. The samples were adjusted via benchtopestimates to provide approximately equal overall sensory impact, giving the followingconcentrations: alum, 0.50 and 1.00 g/1, tannic acid, 0.75 and 3.00 g/1, and tartaric

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acid, 0.50 and 1.00 g/1. Samples were 20 ml in volume, maintained at 36°C, and servedin 2 ounce glasses.

Procedure, qualitative term generation. Group interviews were used to explore termsthat might be relevant to astringent substances. Small, non-directive group discussionswere held to develop the terminology to be used for the time-intensity profiling ofthe three compounds (Civille and Lawless, 1986; Stone and Sidel, 1985; Johnsen etal., 1987). Three groups of four to five paid volunteers generated a list of responsesafter tasting each of the compounds, namely alum (1.81 g/1), tannic acid (1.70 g/1)and tartaric acid (0.45 g/1). Subjects first wrote down their impressions independently.A discussion was subsequently held during which time terms were recorded on a flipchart, and potential redundancies discussed. Two additional groups were also run beforeexperiment 2 using six and seven paid volunteers with no special background in foodsor sensory analysis. Since many of the subjects from the first three groups were FoodScience students and staff, these additional groups were run to guard against the possibiltythat the choice of terms may have been influenced by the group's familiarity with theconcept of astringency and its occurrence in foods.

Two experimenters examined the list of terms common to all three substances andindependently constructed composite lists based on frequency of response, logicalnon-redundancy, elimination of vague hedonic terms (fresh, clean) and elimination ofterms referring to specific ingestibles (aspirin, walnuts). The most frequendy mentionedterms over the 15 separate discussions (three samples by five groups) were drying (14),puckering (11), sour/tart (11), astringent (9), bitter (9), and rough (5) (frequencies inparentheses). Discrepancies between the two lists were resolved through discussion toyield the following list of terms to be used in the subsequent quantitative scaling phase:astringency, mouth drying, puckery feeling, mouth roughing, bitterness and sourness.While the discussion procedure entails a certain subjectivity in interpretation, somereliability was insured through the use of multiple groups and two interpreters (somewhatanalogous to inter-rater reliability methods in psychological testing).

Procedure, quantitative ratings. Ratings were made on 9-point category scales by circlingan integer (1 through 9) on the ballot for each attribute at each time interval. Eachscale was labeled with the phrases 'not ' (1) and 'very ' (9). In order to makethe task manageable for the subjects, all the scales were placed on the same page fora given time interval, and the same order of appearance on the page was maintainedin order to reduce confusion. Rating scales for each time interval were placed on separatepages, and subjects turned the page after completing the scales to minimize their abilityto refer to previous ratings. A technician with a stopwatch controlled the timing ofthe ratings via warning signals and verbal commands. A warm-up session was conductedto familiarize participants with the scales and procedures, to provide verbal definitionsof attributes, and to answer any questions about the definitions of the terms. This sessionalso served to orient the subjects to the approximate level of sensations they wouldexperience. Since one general question of interest was how subjects would use theattribute scales for these compounds, no additional training or physical references weregiven, beyond simple verbal explanations of the meanings of the attributes.

After rinsing their mouths with distilled water, subjects swirled 20 ml of sample in

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their mouths for 15 s and then expectorated. Ratings began 30 s following expectora-tion and continued every 30 s thereafter for a total of six min. One substance was testedeach day with the lower concentration being tested first. A 15-min break was takenbetween the low and high concentrations. Subjects could cleanse their palates withdistilled water and crackers during this time.

Results

Data for each rating scale were submitted to separate three-way analyses of variance(with repeated measures) with time, compound and concentration as factors. All listedeffects are significant at P < 0.05 unless stated otherwise. The mean ratings for thesix attributes and six stimuli over time are shown in Figure 1 (l°wer concentrations)and Figure 2 (higher concentrations). The general pattern of results regarding intensityover time may be summarized as follows: all substances and scales showed significant(P < 0.0001) decrements in sensation over time. Despite the attempt to approximate

2 3 4 5TIME (MIN)

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Fig. 1. Mean ratings for overall astringency, mouth drying, puckery feeling, mouth roughing, bitternessand sourness as a function of time, for the lower concentrations of tannic acid, alum and tartaric acid usedin experiment 1.

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Fig. 2. Mean ratings for overall astringency, mouth drying, puckery feeling, mouth roughing, bitternessand sourness as a function of time, for the higher concentrations of tannic acid, alum and tartaric acid usedin experiment 1.

equal overall sensory impact for the high and low concentrations of the three compounds,significant compound effects were observed for all six scales [F (2,20) ranged from4.0 to 18.9, P < 0.05]. This occurred in part because the mean ratings for 3.00 g/1tannic acid, for every attribute except sourness, were much higher than any of the otherstimuli. Differences between low and high concentrations of each compound were alsoobserved for all scales.

There was a pattern of convergence observed when different concentrations (collapsedover compounds) were plotted versus time, and also when different compounds(collapsed over concentrations) were plotted versus time. That is, the mean ratings forthe initially higher intensity sensations tended to drop faster than those for the initiallylower intensity sensations with all the curves converging at later time intervals. Thesensations, which were moderate to strong in perceived intensity, decreased to halftheir peak intensity after two to four minutes. However, even at the termination of the6-min time period, many of the sensations had not yet returned to baseline. Exponentialfunctions of the form R = Roe"b were fit, where R = mean response, RQ = initial

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response, k = the time constant (time to reach 1/e of initial response), and t = time.Time constants were in the range of 4 to 5 min. No systematic differences betweentaste and 'tactile' (i.e. astringency-related) terms were seen in time course.

The general pattern of results regarding qualitative differences among compoundsmay be summarized as follows: as might be expected, the organic acids (tannic acidand tartaric acid) were highest in perceived sourness. Furthermore, tannic acid washighest in perceived bitterness, with both concentrations being rated more bitter thanany of the other samples. The 3.00 g/1 tannic acid also showed the highest ratings forastringency, puckery, roughing and drying. The lower (0.75 g/1) tannic acid stimuluspulled away from the other four stimuli in overall astringency, even though it clusteredwith the high and low levels of alum for the other three tactile attributes (drying, puckeryand roughing).

Experiment 2

Several refinements were made in the procedure for Experiment 2. Intensity levels inexperiment 1 were not entirely comparable in terms of the overall sensory impact andspacing. Therefore concentrations were adjusted for experiment 2. Second, shorter timeintervals between initial scaling points were used in order to provide a more detailedpicture of the initial changes in astringency that might not have been captured by the30-s delay between ratings. A 15-point unlabeled box scale was used rather than a 9-pointinteger scale to provide for finer gradations in judgements by the subjects and helpto eliminate potential number biases. Finally, subjects were trained in sourness andbitterness using reference standards (O'Mahony et al., 1979; Rainey, 1986) in orderto avoid any semantic/linguistic confusion that may have occurred in experiment 1.

Method

Subjects. Twelve adult subjects (seven male, five female) between the ages of 21 and35 were paid to participate in the experiment. One of the subjects had participated inexperiment 1.

Stimuli. Concentrations were adjusted to attempt to bring the overall sensory impactof the samples into the same range. The following concentrations resulted: alum(aluminum ammonium sulfate), 1.36 and 2.72 g/1, tannic acid, 0.64 and 2.55 g/1, andtartaric acid, 0.53 and 1.05 g/1. Samples were once again 20 ml in volume, maintainedat 36°C, and served in 2-ounce glasses.

Procedure. Ratings were made on a 15-point box scale. Each scale was labeled with'not ' (1) and 'very ' (15). Once again, a warm-up session was conductedto familiarize participants with the scales and procedures and to answer any questionsabout the definitions of terms on the ballot. In addition, subjects were trained to recognizeand differentiate bitterness and sourness via reference standards. Quinine hydrochloride(0.09 g/1) was used as a reference standard for bitterness and citric acid (1.68 g/1) wasused as a reference standard for sourness.

Shorter time intervals between the initial scale points were used to attempt to providea more detailed picture of the initial changes in sensation. After 15 s of swirling 20 mlof sample in their mouths, subjects expectorated and immediately rated the six attributes.

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Ratings continued every 20 s for 1 min, and then every 30 s for 4 additional minutes.Once again only two substances were tested each day and a 15-min break was takenbetween the low and high concentrations, during which time subjects were providedwith distilled water and crackers.

Results

Data for each scale were once again submitted to separate three-way analyses of variance(with repeated measures) with time, compound and concentration as factors. All listedeffects are significant at P < 0.05 unless stated otherwise. The mean ratings for thesix attributes and six stimuli over time are shown in Figures 3 (lower concentrations)and 4 (higher concentrations).

Once again, all substances and scales showed significant (P < 0.0001) decrementsin sensation over time. In addition, significant compound effects were observed forall of the attributes except puckery and sourness. The stimuli used in experiment 2 were

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Big. 3. Mean ratings for overall astringency, mouth drying, puckery feeling, mouth roughing, bitternessand sourness as a function of time, for the lower concentrations of tannic acid, alum and tartaric acid usedin experiment 2.

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better matched for overall sensory impact than those used in experiment 1, with themajor exception being that both concentrations of tannic acid were rated much higheron perceived bitterness than any of the other stimuli [significant compound differencesfor bitterness F (2,22) = 21.38, roughing F (2,22) = 6.71, drying F (2,22) = 5.47,and astringency F (2,22) = 3.99]. Over the four tactile attributes, the higherconcentrations of alum and tannic acid were rated about equivalent at the initial timeintervals, but alum was the more persistent of the two stimuli at the later time intervals.Moreover, the higher concentration of tartaric acid was rated noticeably lower on allof the tactile attributes. Significant concentration differences between low and high levelsof each compound were observed for all attributes except bitterness.

Once again, plots of different concentrations (collapsed over compounds) versus timeand different compounds (collapsed over concentrations) versus time showed a patternof convergence, with initially higher intensity sensations dropping faster in perceivedintensity than initially lower intensity sensations. All sensations decreased to half theirpeak intensity after 2 to 4 min and most of the curves did not reach baseline. The decline

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Fig. 4. Mean ratings for overall astringency, mouth drying, puckery feeling, mouth roughing, bitternessand sourness as a function of time, for the higher concentrations of tannic acid, alum and tartaric acid usedin experiment 2.

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in intensity generally followed an exponential function, with larger correlations beingfound for the exponential fit (as opposed to linear) in 35 out of the 36 curves. In mostof the cases, however, the improvement in fit was minor. Exponential time constantswere in the range of 3 to 4 min, in most cases.

A pattern of qualitative differences was observed among the four tactile attributes(astringency, drying, puckery feeling and roughing). For the attributes of astringencyand puckery, the curve for the 1.36 g/1 alum was clustered with the curves for the 0.64 g/1tannic acid and both levels of tartaric acid (i.e. the four functions of lowest intensityformed a group). However, this function was statistically higher at the initial timeintervals for the attributes of drying and roughing. For the first two points of roughingand the first seven points of drying, low alum was rated statistically higher than eitherlow tannic acid, low tartaric acid, or high tartaric acid (Duncan Multiple Range Tests,P < 0.05). Alum was not statistically higher than the other three stimuli for the attributesof astringency and puckery. In order to compare the slopes of the curves for low alumover the four tactile attributes, z values were calculated for differences among the simplelinear slopes (Guilford, 1956, p. 185). Significant differences were not found betweenthe slopes of astringency and puckery or between the slopes of drying and roughing.Significant differences were, however, found between all other pairs of tactile attributeslopes.

As in experiment 1, tannic acid was rated highest in perceived bitterness with boththe low and high concentrations being rated more bitter than any of the other samples.Both concentrations of tartaric acid and the highest concentration of tannic acid wereonce again rated fairly high in perceived sourness. In addition, the perceived sournessof alum was comparable to the organic acids, especially at the higher level.

Considering both experiments, no systematic pattern of differences in decay rate wasobserved comparing taste to astringency descriptors. There was a tendency for strongerstimuli to decay at a slower rate. For example, the adjusted Qowered) tannic acid haddecay constants in the range of 2.6 to 4.7 across descriptors, as compared to 4.2 to5.3 in the first study.

Discussion

Astringent sensations produced by individual compounds have received little attentionfrom a psychophysical perspective in spite of their importance in many foods andbeverages (Clifford, 1986). This work was designed to help remedy this deficiencyby providing empirical time—course data of astringent materials in aqueous solutions.These data may help to guide future workers in their selections of materials, concentra-tions, and psychophysical methods.

While the pattern for the exponential decline in sensation intensity was similar forthe four tactile attributes of overall astringency, drying, puckery feeling and roughing,the functions were not totally interchangeable. This was seen most clearly for the functionof 1.36 g/1 alum in experiment 2. The decay function for this compound clustered withthose of tartaric acid (both levels) and 0.64 g/1 tannic acid for the attributes of overallastringency and puckery sensations, but had higher initial ratings of drying and roughing.This difference was confirmed by an examination of the simple linear slopes of thedecay functions for 1.36 g/1 alum, which showed a similarity of the decay rate for

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astringency and puckery, and a similarity for drying and roughing, but significantdifferences between these two pairings. While these variations in temporal and intensitypatterns across attributes are admittedly subtle, they do suggest that sub-attributes ofastringency may differ for some compounds and concentration ranges. Future workshould supplement these preliminary results and address the question of multiplesubqualities using a wider range of stimulus molecules. Alternative statistical procedures,such as factor analysis or principal components analysis could be used to examine thepattern of intercorrelation among sensory descriptors.

As noted by other workers (Lea and Arnold, 1978; Clifford, 1986), astringentmaterials will also show major differences in the presence and intensity of side tastes,particularly sourness and bitterness. The results from experiment 1 suggest that alummay be better than tannic acid or tartaric acid as an example of an astringent material,since it was rated relatively high on the four tactile attributes, and at the same time,fairly low in perceived bitterness and perceived sourness. Experiment 2, however,indicated that, at higher concentrations, alum might also have a sour side-taste associatedwith it. Furthermore, several of the subjects from both experiments commented thatalum also exhibited a sweet side-taste. For both experiments, the organic acids wererated high in perceived sourness, while tannic acid was also rated high in perceivedbitterness. A difficulty that arose during the present study involved equating thecompounds in order to provide approximately equal overall sensory impact. Althoughthe stimuli used in experiment 2 were better matched than those used in experiment1, significant compound differences were still observed for four of the attributes. Partof the reason for this difficulty may have been due to the differences that exist betweenindividuals. Simply put, no two humans are expected to react with identical sensorymechanisms. This observation has been particularly true among the oral and nasalchemical sensations, such as the irritation produced by pepper-derived compounds suchas capsaicin and pipeline (Stevens and Lawless, 1986). Genetic conditions of taste-blindness and specific anosmia have explained some of the variance in inter-individualreactions to flavorous substances (Wysocki and Beauchamp, 1984; Hall et al., 1975;Amoore, 1977). In the case of astringency, however, the most obvious variable causingindividual differences is salivary flow. Wide individual differences exist in both restingand stimulated salivary flow (Martin and Pangbom, 1971; Froehlich et al., 1987). Giventhat the most prominent theory of astringent reactions concerns precipitation of salivaryproteins, individuals who generate higher levels of salivary protein might be expectedto show lower levels of astringent responses.

Our qualitative and quantitative exploration of astringent compounds needs to besupplemented by structure—activity studies with systematic physico-chemical variables.The chemical reactions underlying astringent sensations are poorly understood, althoughseveral mechanisms have been proposed, and some comparisons have been made ofindividual compounds from ciders. This literature will be briefly reviewed for thoseinterested in potential physical mechanisms.

The word astringent is derived from the Latin adstringere, meaning 'to bind', whichrelates to the ability of astringent materials to bind and precipitate proteins. The mostcommon astringent compounds are the vegetable tannins (Joslyn and Goldstein, 1964).Tannins are water-soluble polyphenolic compounds which have the ability to precipitatealkaloids, gelatin and other proteins (Swain and Bate-Smith, 1962). They are classified

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as hydrolyzable tannins and condensed tannins according to structural type (Haslam,1966). Hydrolyzable tannins consist of a carbohydrate core, the hydroxyl groups ofwhich are esterified by gallic acid or one of its derivatives; the condensed tannins areformed by the condensation of hydroxyflavans (Deshpande et al., 1985). The tanninsare unique among astringent substances in that they are known to precipitate proteinsat their sensory thresholds (Clifford, 1986).

A popular theory concerning the mechanism of astringent sensations is that attributedto Bate-Smith (1973). In his view, polyphenolic compounds such as tannins formcomplexes with salivary proteins and/or mucopolysaccharides, either precipitating themor causing sufficient confonnational changes so that they lose their lubricating power,thus making the mouth feel rough and dry. Others, however, have not ruled out thedirect action of polyphenols on the oral tissues themselves. For instance, the ASTMdefinition that defines astringency as 'sensations due to shrinking, drawing or puckeringof the epithelium' captures this idea of a direct, rather than salivary, action. Furthermore,McManus et al. (1981) proposed that a constriction of the mucosal epithelium causedby the ability of phenols to cross-link protein chains is in part responsible for generatingthe astringent sensation. Guinard et al. (1986) allowed for both possibilities and suggestedthat reactions with the epithelial proteins themselves may take place after the salivaryproteins have been complexed and the mucus layers covering the epithelium have beenstripped away. This idea is supported by the way in which astringency is enhancedby multiple repeated stimulations as well as the inverse relationship between themagnitude of astringency and the interstimulus interval duration (Guinard et al., 1986).

Four types of interactions have been proposed for the formation of phenol-proteincomplexes: hydrogen bonding, hydrophobic interactions, ionic interactions and covalentbonding. Hydrogen bonding and hydrophobic interactions, however, are the most likelyforms of interactions under physiological conditions (Clifford, 1986). The ortho-dihydroxyphenolic groups of tannin molecules are the proposed hydrogen-bonding siteswith the keto-imide groups on the proteins (McManus et al., 1981). The generalcharacteristics of proteins with high affinity for astringent phenols, such as tannins,are a high molecular weight, an open, loose structure (random coil or collagen-likehelices as opposed to globular proteins), and a moderate to high proline content. Theimportance of proline is presumably due to its inability to fit into the alpha-helix. Thisleads to an open, loose structure that is readily accessible to hydrogen bond formationwith the phenolic groups of tannins. In addition, because the proline peptide bondcontains a substituted nitrogen adjacent to a carbonyl, proline rich proteins (PRP) arevery strong hydrogen bond acceptors, which increases their affinity for tannins(Hagerman and Butler, 1981). Two series of PRPs are secreted by the parotid gland,an acidic series of four PRPs which make up as much as 30% of the proteins secretedby the parotid, and a basic series of six PRPs which have a high affinity for astringentphenols (Oppenheim et al., 1971; Belford et al., 1984).

Due to the numerous phenolic groups and aromatic rings in their structures, tanninshave many potential binding sites with which to hydrogen bond to proteins. The numberof phenolic hydroxyl groups available for hydrogen bonding is thought to influencethe degree to which tannins may cross link salivary proteins, causing aggregation andprecipitation and thus delubricating the oral cavity (Beart etal., 1985; Clifford, 1986).In apple cider for instance, it has been shown (Lea and Arnold, 1978) that the polymeric

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condensed tannins are more astringent than the oligomeric ones, but only up to thepoint where they are too large to be soluble. Among the hydrolyzable tannins, the numberof gallic acid residues is hypothesized to influence their ability to cross-link andprecipitate proteins. In vitro studies by Beart et al. (1985) indicate that the interactionsof bovine serum albumin with the monomeric galloyl-d-glucose series increases withaddition of each gallic acid residue (tri< terra <penta). An in vivo sensory parallel tothis observation, however, has not been established.

For future structure—activity studies it would be useful to have an index of activityfor suprathreshold responses to astringent materials. Because of the problem of build-upand persistence with repeated stimulations, astringency is not well-suited to thresholdmeasures, which often require long sequences of stimuli to determine a functionalminimum stimulus. Suprathreshold measures have more relevance to food stimuli, butsuffer from a certain amount of subjectivity as well as arguments concerning the validityof different scaling methods. These problems can be circumvented by specifying activityin terms of responses relative to a standard astringent stimulus. On the basis of ourresults we propose an alum concentration of about 1 g/1 as a citable reference standard.This concentration is relatively low in bitterness and sourness, and the compound isan inexpensive and stable salt, readily obtainable in known purities. A precedent forthe substitution of suprathreshold scaled responses for threshold measures of activitycan be found in recent industrial methods for evalution of pepper heat (Gillette et al.,1984; American Society for Testing and Materials, 1989b). In these procedures, thetraditional Scoville procedure, a dilution-to-threshold measure of activity, was supplantedby a rating procedure, cross-calibrated to standard concentrations of N-vanillyl-n-nonamide. In conclusion, we propose that a procedure which combines the use of aphysical reference with multi-attribute discrete-point time—intensity scaling be usedin future studies to provide a useful and qualitatively rich characterization of astringentstimuli.

Acknowledgements

The authors thank Richard A.Tucciarone and Sandy S.Glatter for gathering part of thedata, and Dr Ann Noble for helpful discussions.

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Received on August 16, 1990; accepted on March 27, 1991

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