- Email: [email protected]
Relating ionisation of calcium chloride in saliva to bitterness perception
Physiology & Behavior 81 (2004) 505 – 510
Relating ionisation of calcium chloride in saliva to bitterness perception Eric Neyraud, Eric Dransfield* Station de Recherches sur la Viande, INRA-Theix, 63122 Saint Gene`s Champanelle, France Received 22 October 2003; received in revised form 19 December 2003; accepted 10 February 2004
Abstract Saliva plays a role in the perception of bitter, sour and salty tastes that are presumed to be derived from the concentration of free cations or anions ions dissolved in saliva. The role of ionisation of calcium in bitter taste was studied by determining binding in vitro mixture of saliva and protein solutions and in spit. In vitro, the addition of whey to calcium chloride solutions increased the calcium binding, pH and viscosity. The addition of saliva to these mixtures, the increased calcium binding and the induced small changes in viscosity and pH were thought not to contribute significantly to bitterness perception. Nonstimulated saliva, at pH 7.5, contained about 5 mM calcium, of which about one third was ionised. The bitter threshold of fully ionised calcium chloride in water varied between 1 and 15 mM among individuals. In spit, after tasting whey, ionised calcium was found to have increased at low, but decreased at high, calcium concentrations and varied 30% among individuals. Bitterness was related, on average, to the concentration of ionised calcium and not to the total concentration of calcium in spit. A general explicative model based on the composition of bulk saliva is discussed in relation to perception threshold and the likely importance of saliva from von Ebner’s gland. D 2004 Elsevier Inc. All rights reserved. Keywords: Bitter taste; Saliva; Free calcium; Calcium binding
1. Introduction Little is known of the mechanism of the role of saliva in the perception of physical and chemical properties of foods. The first reports that saliva could affect taste perception came from studies on NaCl and acids that showed that low amounts of saliva reduced the taste thresholds [1 – 3]. Although saliva volume has been studied extensively [4], the importance of its composition has rarely been assessed despite the fact that its buffering capacity [5] is directly implicated in sourness perception. The objective of this study was to determine the extent to which the degree of ionisation of salts in saliva affected the psychophysical taste response. Similar attempts to relate taste to free-ion concentrations have been studied in gums [6]. Sodium ion mobility was shown to be less in ionic gums, which were perceived to be less salty than nonionic gums. However, differences were also perceived in the thickness and saltiness, which were also correlated with bitterness. A * Corresponding author. Tel.: +33-473-624-395; fax: +33-473-624268. E-mail address: [email protected] (E. Dransfield). 0031-9384/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.02.018
better statistical fit to the taste intensity was found when the sodium ion concentration was combined with calcium and/or potassium ion concentrations, suggesting that the endogenous potassium and calcium ions in the gums also contributed to the saltiness. With such interactions, the need is to have a single discrete taste, detectable ions and ion binding, which could be used to alter the free-ion concentration at the same total ion concentration in the psychophysical range. To avoid the complexity of solid foods and the variations in breakdown of the food and release of taste stimuli during mastication [7,26], a liquid system of whey retentate, capable of binding calcium ions, was chosen. Calcium chloride solutions usually taste bitter but may have a salty taste depending on the concentration [8]. In addition, calcium ions interact with milk proteins such as caseins [9], a-lactalbumin and h-lactoglobulin [10], and in cows’ milk, about two thirds of the calcium and half the phosphate are found in casein micelles. Hence, this simple model system could be tested, in which bitterness could be related to free calcium ion concentration in the psychophysical range. Saliva contains more calcium than many other tissues do [11] and is undersaturated with respect to h-tricalcium
506
E. Neyraud, E. Dransfield / Physiology & Behavior 81 (2004) 505–510
phosphate, octacalcium phosphate, calcium carbonate and calcium fluoride [12]. Early work found about 1.5 mM calcium [13], but later work [14] found generally higher concentrations, of which half was ionised. Several proteins, proline-rich proteins and statherin also bind Ca to prevent the precipitation of Ca-phosphates in saliva [15]. Hence, in this work, psychophysical response was related to the binding of calcium ions measured in saliva after evaluating bitterness and in vitro using mixtures of saliva and protein solutions.
2. Materials and methods 2.1. Material An acid whey retentate powder (Toury, France) containing 35% protein, of which about 80% are caseins, was dissolved in ultra pure water (Elgastat UHQ II, USF Elga, UK), giving a stable white colloidal solution. 2.1.1. Ionised calcium in solutions and saliva The ionised calcium (‘mobile’ or ‘free’ calcium ion) concentration was determined [16] with an Orion ion-selective membrane (97-20 ionplus, Orion, UK), connected to an Orion ionic concentration specific reader (model 290A). Spit from five participants was collected between 2 and 3 p.m. and was pooled, and the ionised calcium was determined using the above protocol. The concentration of the ionised calcium was also determined in three whey solutions (0%, 1% and 10% w/v), to which nonstimulated saliva, at a final concentration of 0%, 10% or 50%, had been added. The procedures were done in triplicate. 2.2. Measurement of pH, viscosity and total calcium and magnesium levels The pH was measured directly in the solutions using a portable pH meter (Hanna, HI 931000). The dynamic viscosity was determined on each of three samples by capillary flow and density measurement. All measurements were made at 20 jC. For mineral determinations, 0.25 to 0.5 g of dried whey solutions were dry ashed (10 h at 500 jC) and then extracted at 130 jC in (2:1) HNO3/H2O2 (Suprapur, Merck, Darmstadt, Germany) until discoloration. Saliva was assayed directly. The final dilution was made in 1 g lanthanum chloride/l solution. Mineral concentrations were determined by atomic absorption spectrophotometry (Model 560, Perkin-Elmer Cetus, USA) in an acetylene air flame at 422 nm (Ca) and 285 nm (Mg). 2.3. Sensory evaluation procedures Sensory sessions were conducted in the mornings over a period of 6 months, under conditions set out in ISO 8589
(1998). The tasters were aged between 25 and 50 years. Approximately 10 ml samples at 20 jC were presented in 15-ml disposable plastic cups. The order of sample presentations was determined by a Latin square design. Distilled water and pieces of apple were provided at each session. 2.3.1. Calcium chloride taste in water Thirteen male and 9 female consenting tasters evaluated, under normal lighting, the taste of calcium chloride in water. Tasters were acquainted with the taste of calcium chloride solutions over four sessions. During the first session, the tasters assessed a series of five solutions of 0, 5, 15, 30 and 70 mM CaCl2 and classified them in order of taste intensity. They then tasted two reference solutions, one (water) labelled ‘A’ and the other (100 mM CaCl2) labelled ‘B’, and then rated the five others on a nonstructured 20-cm line, marked ‘A’ at the left end and ‘B’ at the right. In three subsequent sessions, the tasters rated the taste intensity of 1, 5, 10, 15, 20, 30, 50 and 70 mM of calcium chloride solutions on a similar line, limited by the letters ‘A’ and ‘B’. The tasters could assess the two references solutions ‘A’ and ‘B’ and each sample as many times as they wished. This method of not describing the type of taste avoided any variations between the bitter and salty tastes of the solutions of calcium chloride whilst allowing intensity ratings that were later scored as distances (cm) from the left (A, water) end. 2.3.2. Calcium chloride taste in whey protein solution Rating the intensity of calcium in whey solutions was performed under red lighting. In each of two sessions, 16 from the above 22 tasters assessed solutions of 1%, 5%, 10% and 20% of whey without calcium chloride and the same four solutions containing 100 mM calcium chloride. All solutions were rated independently for ‘milky’ and ‘calcium chloride’ tastes. Statistical analyses (ANOVA; 12 Tasters 2 Protein Concentrations 4 Concentrations of Calcium Chloride) showed that protein ( P=.001) but not calcium (.004) concentration affected the milky taste, whilst calcium concentration ( P=.001) but not protein (.079) affected bitter taste. In each of three further sessions, 14 tasters from the 16 previously cited tasted eight samples of 0, 5, 20 and 40 mM CaCl2 in both 1% and 10% whey solutions. One of these tasters also tasted a further seven samples of 0, 10, 20, 30, 40 and 50 mM added calcium chloride in both 1% and 10% whey solutions. Milky and CaCl2 taste intensities were rated as above, with Solutions A and B provided as references. 2.4. Statistical analyses Statistical analyses on taste intensity and taster were performed using a general linear model (GLM, SAS, 8.1 software). Relationships between added calcium chloride and ionised calcium in solutions and mixtures of whey and saliva were found by linear regression. Relationships be-
E. Neyraud, E. Dransfield / Physiology & Behavior 81 (2004) 505–510
507
tween taste intensity and calcium chloride concentration were found by regression using Gompertz-type sigmoid equations (Excel 2000), for which the model, taste intensity = S0+(SM S0)exp{ exp[k(T Ca) + 1]}, was used and where S0 is the predicted score at zero calcium concentration, SM is the maximum predicted intensity score, k is related to the rate of increase of taste with calcium concentration, T is the threshold (mM) for taste and Ca is the ionised calcium concentration (mM).
3. Results 3.1. Physicochemical measurements 3.1.1. Total calcium and magnesium concentrations Whey retentate powder contained 98.8 F 5.8 mmol Ca/ kg and 37.0 F 0.2 mmol Mg/kg of powder. Resting saliva from three of the tasters contained 5.4 F 1.0 mM calcium and 0.47 F 0.15 mM magnesium. 3.1.2. The pH and viscosity of mixtures of saliva and whey The addition of saliva and whey solutions to water increased their pH (Table 1). Viscosity was similar in all solutions, but there was a tendency for viscosity to decrease with increasing salivary concentration and for viscosity to increase with increasing concentration of whey. 3.1.3. Ionised calcium concentration in mixtures of saliva and whey The calcium ion concentration in aqueous solutions (Table 1) increased in proportion to the amount of added saliva. The addition of CaCl2 increased proportionately (r2>.98) the ionised calcium concentration. Increasing the concentration of whey protein increased the ionised calcium (Table 1), which was lowered when diluted with saliva in both 1% and 10% whey (Table 1).
Table 1 In vitro pH, viscosity, ionised calcium and the effect of CaCl2 addition to saliva, whey solutions and mixtures (n = 3) Saliva Whey pH
Viscosity
Ionised Ca
Slope 0 to 50 mM CaCl2
%
%
Value S.D. cps
S.D. mM
S.D. mM/mM CaCl2
S.D.
0 10 50 0 10 50 0 10 50
0 0 0 1.0 0.9 0.5 10 9 5
5.67 7.17 7.30 6.30 6.77 7.13 6.20 6.50 6.77
0.02 0.02 0.04 0.02 0.01 0.03 0.02 0.02 0.01
0.00 0.21 0.15 0.01 0.10 0.06 0.01 0.17 0.06
0.08 0.04 0.06 0.01 0.02 0.05 0.00 0.01 0.04
0.64 0.01 0.21 0.04 0.07 0.14 0.03 0.01 0.07
1.00 1.00 0.93 1.00 1.00 0.95 1.05 1.03 1.00
0.00 0.17 0.53 0.90 0.40 0.53 3.10 1.10 0.93
1.00 0.91 0.71 0.90 0.87 0.69 0.71 0.60 0.56
The slope is the linear increase in concentration of ionised calcium relative to the added Ca2 + ion concentration.
Fig. 1. Relationship between bitter taste intensity and calcium concentration in water. Values are the means and standard deviations of 22 tasters, and the dotted lines represent the extremes among the individuals.
The ability of the mixtures of whey and saliva to bind added Ca2 + ions (Table 1, slopes) was reduced by the increasing amounts of saliva both in 1% and 10% whey solutions. Considering all the combinations of saliva and whey, the Ca2 + ion concentration was not related to the viscosity of the solutions (Table 1). 3.2. Sensory evaluation 3.2.1. Taste intensity of calcium chloride in water The taste intensity of calcium chloride in water, rated with reference to water and to a 100 mM CaCl2 solution, is represented in Fig. 1 for all 22 tasters. On average (solid line), a taste was detected at about 5 mM, its intensity increased approximately linearly up to about 45 mM CaCl2 and began to plateau with concentrations over about 60 mM. The most sensitive taster (upper dotted line) had an approximate detection threshold around 1 mM CaCl2 and reached a plateau for concentrations over 30 mM, whilst the least sensitive (lower dotted line) began to perceive taste at about 10 mM CaCl2 and its intensity had not reached a plateau even at 100 mM CaCl2 (Fig. 1). 3.2.2. Taste intensity of calcium chloride in whey solutions Without added calcium chloride, the taste intensity increased with increasing concentration of whey (Table 2). The taste derives from the calcium salts present in the whey. The increase corresponds to an increase in ionised calcium, from 0.9 to 3.4 mM (Table 2), which would be just detectable in water by some of the tasters (Fig. 1). With the addition of 100 mM calcium chloride, the taste intensity decreased slightly from 1% to 10% whey and then stayed the same at 20% whey solution (Table 2). This tendency is consistent with the decrease in ionised calcium concentration from 91 to 57 mM (calculated from the data given in Table 1) with increasing whey. This decrease would be expected (from Fig. 1) to reduce the average bitterness
508
E. Neyraud, E. Dransfield / Physiology & Behavior 81 (2004) 505–510
Table 2 Effect of whey concentration (1% to 20%) on the bitterness (means and standard deviations from six tasters), total and ionised calcium concentrations in solution at two levels of CaCl2 addition 1%
5%
10%
0 mM Added calcium Taste intensity (0 – 20) 1.8 F 2.3 2.1 F 2.2 2.8 F 2.0 Total Ca (mM) 1.0 5.0 9.8 Ionised Ca (mM) 0.9 2.4 3.1 100 mM Added calcium Taste intensity (0 – 20) 16.4 F 3.5 15.4 F 3.2 13.7 F 3.3 Total Ca (mM) 100.9 105.0 109.8 Ionised Ca (mM) 90.9 91.4 74.1
Table 3 In situ variations in composition of saliva when testing whey solutions Solution
20% 0 mM CaCl2 4.6 F 3.6 19.6 3.4 14.2 F 3.8 119.6 57.4
5 mM CaCl2
20 mM CaCl2
40 mM CaCl2
score in water from about 19 to 16, similar with the observed scores (Table 2). Comparisons were also made between the taste intensity of 1% and 10% whey solutions over the range 0 to 50 mM added CaCl2, and the effect of whey on the taste of calcium ions was found to depend on the taster. For 10 tasters, there was no significant difference between the taste intensities in 1% and 10% whey at the four CaCl2 concentrations. However, for four other tasters, taste intensity showed a strong dependency on whey concentration (Fig. 2A). At 5 mM added CaCl2, both 1% and 10% whey solutions had similar low taste intensities, but at 20 and 40 mM added CaCl2, the taste intensities were significantly lower in the 10% whey solutions.
Fig. 2. The curves show the increase in taste with increasing concentration of calcium ions. (A) The means and standard deviations from four tasters for sensory scores against the concentration of calcium in 1% (dotted) and 10% whey (solid) solutions are shown. The large points are the scores related to the concentration of ionised calcium in spit for 1% (open) and 10% (filled) whey solutions. Their relationship (thick solid line) is given. (B) Similar with Panel A, except that the data are from one taster.
Saliva
Whey (%)
Ca (mM)
pH
Ca (mM)
pH
0 1 10 0 1 10 0 1 10 0 1 10
0.1 1.3 3.9 7.7 6.3 6.0 22.8 22.0 14.4 43.2 37.6 28.3
4.5 6.7 6.6 5.1 6.4 6.3 5.1 6.1 6.1 4.9 5.9 5.9
0.4 F 0.2 1.9 F 3.0 3.3 F 0.4 4.5 F 1.2 4.8 F 1.0 5.2 F 0.9 17.1 F 4.0 16.2 F 3.2 11.6 F 2.3 33.5 F 7.9 31.6 F 7.1 24.1 F 4.0
6.4 F 0.4 6.5 F 0.3 6.5 F 0.1 6.1 F 0.5 6.3 F 0.2 6.4 F 0.1 6.0 F 0.5 6.1 F 0.2 6.2 F 0.1 5.8 F 0.5 6.0 F 0.2 5.9 F 0.1
The solutions contained 0, 1 or 10% whey with 0, 5, 20 or 40 mM added calcium chloride. Values are for ionised calcium and pH and those in saliva are the mean and standard deviation among 6 tasters and 3 repetitions for the saliva after keeping each solution in the mouth for 10 s.
3.3. Calcium concentrations in saliva and taste intensity The average composition of spit during the tasting of solutions of calcium chloride and whey by six tasters is shown in Table 3. The pH in solution was correlated significantly with that in the saliva for four participants and not correlated for the other two. For Ca2 + ions, the average concentration of ionised calcium (from 0 to 43 mM) in whey solutions were highly correlated (r2>.98) with those (from 1 to 34 mM) in spit. However, there were significant variations among the tasters. The proportion of ionised calcium in spit varied from 0.7 to almost 1.0 among tasters. Hence, at an ionised calcium concentration in solutions of whey of 40 mM, the ionised calcium in spit varied from 27 to 38 mM among individuals. When the two independent curves at 1% and 10% whey, relating the concentrations of added CaCl2 to taste (Fig. 2A), were plotted against the concentration of ionised calcium in spit (Fig. 2A, solid line), a single ‘S’ shaped curve was obtained, showing that the taste intensity was largely independent of the concentration of whey (Fig. 2A). The psychophysical relationship between perceived intensity and ionised calcium (Fig. 2A) was steeper than that for total CaCl2 (Fig. 1). The effect of ion binding on taste may be related to an individual’s threshold, as one taster with a high threshold of 10 to 15 mM showed a marked effect of calcium binding by whey and very low bitterness intensity, even at 50 mM CaCl2 addition (Fig. 2B). In this case, the variations in bitterness were not fully accounted for by the ionised calcium in the spit.
4. Discussion The bitterness thresholds of calcium chloride solutions in water vary from 1 to 15 mM among individuals and were within the 2 to 30 mM (mean 10 mM) given by Pfaffmann
E. Neyraud, E. Dransfield / Physiology & Behavior 81 (2004) 505–510
[17]. However, using an ‘up and down’ method, a very much lower threshold (average log detection threshold of 0.008 mM), but varying four orders of magnitude between individuals, has been reported [8]. Such a low threshold should be interpreted with caution [18] because we confirm that nonstimulated saliva contains about 1 mM ionised calcium. Whey contained about 100 mmol Ca/kg and about 30 mmol Mg/kg. Mg2 + ions in water have a bitter taste and a similar or slightly higher threshold than that of Ca2 + ions, but their concentration was always lower than the taste threshold and, thus, would not have contributed to the taste intensity. These divalent ions interact with milk proteins such as caseins [9], a-lactalbumin and h-lactoglobulin [10], and in cows’ milk, about two thirds of the calcium and half the phosphate are found in casein micelles. The biological significance appears to be that casein micelles may allow the total calcium and phosphate concentrations to exceed the solubility of calcium phosphate without causing the precipitation of calcium phosphate. In this work, up to 20% (w/v) whey protein gave a stable, milky liquid, with quantities of ionised calcium in the psychophysical range. Although calcium binding could have been increased by increasing the pH, as the binding of Ca2 + ions increases 10-fold from pH 6.2 to 7.3 [19], we used solutions in water at around pH 6.2 to avoid adding buffers that may have introduced other tastes. Early work found about 1.5 mM calcium [13], and that 85% of calcium was ionised in saliva, with 8% bound to macromolecules and 6% as ion pairs [20], with levels varying from 0.49 to 0.74 mM depending on the type of saliva. A larger proportion of ionised calcium was found in stimulated saliva. Later work [14] found half-ionised and higher levels in saliva. In this work, spit contained about 1 mM ionised Ca and about 5 mM total calcium. In low-flow participants, total calcium in parotid saliva decreased with pH and flow rate, but in high-flow participants, calcium increased with pH and was not related to salivary flow rate [2]. However, both ionised and total calcium increased with flow rate of parotid saliva [21]. Hence, it appears that the wide range of values reported for Ca2 + ions in saliva is due to the variations in the site of collection of saliva, the salivary gland, the degree, the type of stimulation and the possible role of flow rate, which could be linked to gland size [22]. With no added CaCl2, increasing the whey concentration from 1% to 20% increased slightly the taste intensity and was accompanied by an increase in Ca2 + ion concentration from the whey. With 100 mM added CaCl2, increasing the whey concentration from 1% to 20% decreased the taste intensity from 16.2 to 14.0 and was accompanied by a measured decrease in ionised calcium concentration. From the measured ionised calcium and sensory tests of Ca2 + ions in water, the predicted bitter intensity values were 1, 2, 18 and 16 in good agreement with the observed 1.8 F 2.3, 4.6 F 3.6, 16.2 F 3.5 and 14 F 3.8, respectively. The de-
509
crease in taste was therefore accounted for by the ionised, rather than the total, calcium concentration. Changes also occurred in pH and, at high concentrations of Ca2 + ions, the saliva was less able to buffer, but changes in pH were small (less than 0.5 units) and not thought to have influenced bitterness perception. Subsequent tests were performed in the more sensitive linear part of the psychophysical range of Ca2 + ion concentration. Ten tasters found no differences in taste intensity between the 1% and 10% whey concentrations in the range 10 – 50 mM added CaCl2. It is unlikely that the small differences in viscosity of these dilute aqueous solutions [23] would affect the taste. A group of four other tasters found significantly higher taste intensity for 1% than for 10% whey. The differences were largely accounted for by the differences in Ca2 + ion concentration measured in spit. A similar, but stronger, effect of reduction in taste intensity by calcium binding was shown by one participant. It appears then that this type of relationship is general but cannot be used for any one specific individual. This would be important in explaining the origin of differences among individuals in the psychophysical response to the bitterness of divalent salts. Variations in Ca2 + ion concentration could occur at the site of bitterness perception, which occurs in specialised neuroepithelial receptor cells that are bundled in the taste buds in the lingual epithelium. Small tubulo-alveolar salivary glands, the von Ebner’s glands, secrete into troughs, in direct contact with the taste buds. This saliva secretion is thought to be essential for the concentration and delivery of sapid molecules in the gustatory system and for clearing the tongue surface of taste substances, which would otherwise cause a long-lasting taste sensation [24]. The regulation of the secretion could involve a feedback between the gland and taste buds [25], but little is known of its composition. Hence, it would be expected that a bitter taste would be related to Ca2 + ion concentration in von Ebner’s secretions. In this study, Ca2 + ion concentrations were measured in the spit. Different individual responses would then depend on the equilibrium between von Ebner’s secretion and the bulk (spit, mainly parotid) saliva, which is likely to depend on the morphology of the tongue and the quantities of salivary secretion. All attempts to relate taste to concentration of the stimulus will remain largely circumstantial until the concentration and binding of ions in von Ebner’s saliva has been established.
5. Conclusions A general model that the taste is related to the concentration of free ions was tested using calcium ions and whey solutions. The presence of whey decreased the bitter taste of calcium mostly in relation to its lowering of free Ca2 + ions in bulk saliva (spit), but large variations in binding were found among individuals.
510
E. Neyraud, E. Dransfield / Physiology & Behavior 81 (2004) 505–510
References [1] O’Mahony M, Heintz C. Direct magnitude estimation of salt taste intensity with continuous correction for salivary adaptation. Chem Senses 1981;6:101 – 12. [2] Norris MB, Noble AC, Pangborn RM. Human saliva and taste response to acids varying in anions, titratable acidity and pH. Physiol Behav 1984;32:237 – 44. [3] Christensen CM, Navazesh M, Brightman VJ. Effects of pharmacological reductions in salivary flow on taste thresholds. Arch Oral Biol 1984;29:17 – 23. [4] Humphrey SP, Williamson RT. A review of saliva: normal composition, flow and function. J Prosthet Dent 2001;85:162 – 9. [5] Christensen CM, Brand JG, Malamud D. Salivary changes in solution pH: a source of individual differences in sour taste perception. Physiol Behav 1987;40:221 – 7. [6] Rosett TR, Shirley L, Schmidt SJ, Klein BP. Na + binding as measured by 23Na nuclear magnetic resonance spectroscopy influences the perception of saltiness in gum solutions. J Food Sci 1994;59:206 – 10. [7] Neyraud E, Prinz J, Dransfield E. Sodium chloride and sugar release, salivation and taste during mastication of salted chewing gum. Physiol Behav 2003;79:731 – 7. [8] Tordoff MG. Some basic psychophysics of calcium salt solutions. Chem Senses 1996;21:417 – 24. [9] Thompson MP, Brower DB. Calcium binding proteins in bovine milk. J Dairy Sci 1985;68(Supp1.):59. [10] Baumy JJ, Brule´ G. Binding of bivalent cations to a-lactalbumin and alactoglobulin: effect of pH and ionic strength. Lait 1988;68(1):33 – 48. [11] Schneyer LH, Schneyer CA. Inorganic composition of saliva. In: Code CF, Heidel W, editors. Handbook of physiology section 6: alimentary canal. Secretion. Handbook of Physiology, Volume II. Washington: American Physiological Society, Waverly Press; 1967. p. 497 – 530. [12] Larsen MJ, Pearce EIF. Saturation of human saliva with respect to calcium salts. Arch Oral Biol 2003;48:317 – 22. [13] Dreisbach RH. Calcium binding by normal human saliva. J Dent Res 1960;39:1133 – 40.
[14] Matsuo S, Lagerlo¨f F. Relationship between total and ionised calcium concentrations in human whole saliva and dental plaque fluid. Arch Oral Biol 1991;36:525 – 7. [15] Ferguson DB. Oral bioscience. Edinburgh: Churchill Livingstone; 1999. p. 145. [16] Bertoldo Pacheco MT, Carraro F, Sgarbieri VC. Study of calcium binding to different preparations of yeast (Saccharomyces cerevisiae) protein by using an ion selective electrode. Food Chem 1999;66: 249 – 52. [17] Pfaffmann C. The sense of taste. In: Fiel J, Magoun HW, Hall WE, editors. Handbook of physiology, neurophysiology. Washington: American Physiological Society; 1959. p. 507 – 34. [18] Bartoshuk LM. The psychophysics of taste. Am J Clin Nutr 1978; 31:1068 – 77. [19] Carr CW. Studies on the binding of small ions in protein solution with the use of membrane electrodes: IV. The binding of calcium ions in solutions of various proteins. Arch Biochem Biophys 1953;46: 424 – 31. [20] Gron P. Arch Oral Biol 1973;18:1385 – 92. [21] Lagerlo¨f F, Ekstrand J. The effect of flow rate on the ionised calcium concentration of human parotid saliva. Caries Res 1982;16:123 – 8. [22] Robertson WG, Marshall RW. Ionized calcium in body fluids. CRC Crit Rev Clin Lab Sci 1981;15:85 – 125. [23] Shama F, Sherman P. Variation in stimuli associated with oral evaluation of the viscosities of glucose solutions. J Texture Stud 1973;4: 254 – 62. [24] Schmale H, Holtgreve-Grez H, Christiansen H. Possible role for salivary gland protein in taste reception indicated by homology to lipophilic – ligand carrier proteins. Nature 1990;343:366 – 9. [25] Sbarbati A, Crescimanno C, Osculati F. The anatomy and functional role of the circumvallate papilla/von Ebner gland complex. Med Hypotheses 1999;53:40 – 4. [26] Jack FR, Piggot JR, Paterson A. Cheddar cheese texture related to salt release during chewing, measured by conductivity—preliminary study. J Food Sci 1995;60(2):213 – 7.
Relating ionisation of calcium chloride in saliva to bitterness perception Eric Neyraud, Eric Dransfield* Station de Recherches sur la Viande, INRA-Theix, 63122 Saint Gene`s Champanelle, France Received 22 October 2003; received in revised form 19 December 2003; accepted 10 February 2004
Abstract Saliva plays a role in the perception of bitter, sour and salty tastes that are presumed to be derived from the concentration of free cations or anions ions dissolved in saliva. The role of ionisation of calcium in bitter taste was studied by determining binding in vitro mixture of saliva and protein solutions and in spit. In vitro, the addition of whey to calcium chloride solutions increased the calcium binding, pH and viscosity. The addition of saliva to these mixtures, the increased calcium binding and the induced small changes in viscosity and pH were thought not to contribute significantly to bitterness perception. Nonstimulated saliva, at pH 7.5, contained about 5 mM calcium, of which about one third was ionised. The bitter threshold of fully ionised calcium chloride in water varied between 1 and 15 mM among individuals. In spit, after tasting whey, ionised calcium was found to have increased at low, but decreased at high, calcium concentrations and varied 30% among individuals. Bitterness was related, on average, to the concentration of ionised calcium and not to the total concentration of calcium in spit. A general explicative model based on the composition of bulk saliva is discussed in relation to perception threshold and the likely importance of saliva from von Ebner’s gland. D 2004 Elsevier Inc. All rights reserved. Keywords: Bitter taste; Saliva; Free calcium; Calcium binding
1. Introduction Little is known of the mechanism of the role of saliva in the perception of physical and chemical properties of foods. The first reports that saliva could affect taste perception came from studies on NaCl and acids that showed that low amounts of saliva reduced the taste thresholds [1 – 3]. Although saliva volume has been studied extensively [4], the importance of its composition has rarely been assessed despite the fact that its buffering capacity [5] is directly implicated in sourness perception. The objective of this study was to determine the extent to which the degree of ionisation of salts in saliva affected the psychophysical taste response. Similar attempts to relate taste to free-ion concentrations have been studied in gums [6]. Sodium ion mobility was shown to be less in ionic gums, which were perceived to be less salty than nonionic gums. However, differences were also perceived in the thickness and saltiness, which were also correlated with bitterness. A * Corresponding author. Tel.: +33-473-624-395; fax: +33-473-624268. E-mail address: [email protected] (E. Dransfield). 0031-9384/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.02.018
better statistical fit to the taste intensity was found when the sodium ion concentration was combined with calcium and/or potassium ion concentrations, suggesting that the endogenous potassium and calcium ions in the gums also contributed to the saltiness. With such interactions, the need is to have a single discrete taste, detectable ions and ion binding, which could be used to alter the free-ion concentration at the same total ion concentration in the psychophysical range. To avoid the complexity of solid foods and the variations in breakdown of the food and release of taste stimuli during mastication [7,26], a liquid system of whey retentate, capable of binding calcium ions, was chosen. Calcium chloride solutions usually taste bitter but may have a salty taste depending on the concentration [8]. In addition, calcium ions interact with milk proteins such as caseins [9], a-lactalbumin and h-lactoglobulin [10], and in cows’ milk, about two thirds of the calcium and half the phosphate are found in casein micelles. Hence, this simple model system could be tested, in which bitterness could be related to free calcium ion concentration in the psychophysical range. Saliva contains more calcium than many other tissues do [11] and is undersaturated with respect to h-tricalcium
506
E. Neyraud, E. Dransfield / Physiology & Behavior 81 (2004) 505–510
phosphate, octacalcium phosphate, calcium carbonate and calcium fluoride [12]. Early work found about 1.5 mM calcium [13], but later work [14] found generally higher concentrations, of which half was ionised. Several proteins, proline-rich proteins and statherin also bind Ca to prevent the precipitation of Ca-phosphates in saliva [15]. Hence, in this work, psychophysical response was related to the binding of calcium ions measured in saliva after evaluating bitterness and in vitro using mixtures of saliva and protein solutions.
2. Materials and methods 2.1. Material An acid whey retentate powder (Toury, France) containing 35% protein, of which about 80% are caseins, was dissolved in ultra pure water (Elgastat UHQ II, USF Elga, UK), giving a stable white colloidal solution. 2.1.1. Ionised calcium in solutions and saliva The ionised calcium (‘mobile’ or ‘free’ calcium ion) concentration was determined [16] with an Orion ion-selective membrane (97-20 ionplus, Orion, UK), connected to an Orion ionic concentration specific reader (model 290A). Spit from five participants was collected between 2 and 3 p.m. and was pooled, and the ionised calcium was determined using the above protocol. The concentration of the ionised calcium was also determined in three whey solutions (0%, 1% and 10% w/v), to which nonstimulated saliva, at a final concentration of 0%, 10% or 50%, had been added. The procedures were done in triplicate. 2.2. Measurement of pH, viscosity and total calcium and magnesium levels The pH was measured directly in the solutions using a portable pH meter (Hanna, HI 931000). The dynamic viscosity was determined on each of three samples by capillary flow and density measurement. All measurements were made at 20 jC. For mineral determinations, 0.25 to 0.5 g of dried whey solutions were dry ashed (10 h at 500 jC) and then extracted at 130 jC in (2:1) HNO3/H2O2 (Suprapur, Merck, Darmstadt, Germany) until discoloration. Saliva was assayed directly. The final dilution was made in 1 g lanthanum chloride/l solution. Mineral concentrations were determined by atomic absorption spectrophotometry (Model 560, Perkin-Elmer Cetus, USA) in an acetylene air flame at 422 nm (Ca) and 285 nm (Mg). 2.3. Sensory evaluation procedures Sensory sessions were conducted in the mornings over a period of 6 months, under conditions set out in ISO 8589
(1998). The tasters were aged between 25 and 50 years. Approximately 10 ml samples at 20 jC were presented in 15-ml disposable plastic cups. The order of sample presentations was determined by a Latin square design. Distilled water and pieces of apple were provided at each session. 2.3.1. Calcium chloride taste in water Thirteen male and 9 female consenting tasters evaluated, under normal lighting, the taste of calcium chloride in water. Tasters were acquainted with the taste of calcium chloride solutions over four sessions. During the first session, the tasters assessed a series of five solutions of 0, 5, 15, 30 and 70 mM CaCl2 and classified them in order of taste intensity. They then tasted two reference solutions, one (water) labelled ‘A’ and the other (100 mM CaCl2) labelled ‘B’, and then rated the five others on a nonstructured 20-cm line, marked ‘A’ at the left end and ‘B’ at the right. In three subsequent sessions, the tasters rated the taste intensity of 1, 5, 10, 15, 20, 30, 50 and 70 mM of calcium chloride solutions on a similar line, limited by the letters ‘A’ and ‘B’. The tasters could assess the two references solutions ‘A’ and ‘B’ and each sample as many times as they wished. This method of not describing the type of taste avoided any variations between the bitter and salty tastes of the solutions of calcium chloride whilst allowing intensity ratings that were later scored as distances (cm) from the left (A, water) end. 2.3.2. Calcium chloride taste in whey protein solution Rating the intensity of calcium in whey solutions was performed under red lighting. In each of two sessions, 16 from the above 22 tasters assessed solutions of 1%, 5%, 10% and 20% of whey without calcium chloride and the same four solutions containing 100 mM calcium chloride. All solutions were rated independently for ‘milky’ and ‘calcium chloride’ tastes. Statistical analyses (ANOVA; 12 Tasters 2 Protein Concentrations 4 Concentrations of Calcium Chloride) showed that protein ( P=.001) but not calcium (.004) concentration affected the milky taste, whilst calcium concentration ( P=.001) but not protein (.079) affected bitter taste. In each of three further sessions, 14 tasters from the 16 previously cited tasted eight samples of 0, 5, 20 and 40 mM CaCl2 in both 1% and 10% whey solutions. One of these tasters also tasted a further seven samples of 0, 10, 20, 30, 40 and 50 mM added calcium chloride in both 1% and 10% whey solutions. Milky and CaCl2 taste intensities were rated as above, with Solutions A and B provided as references. 2.4. Statistical analyses Statistical analyses on taste intensity and taster were performed using a general linear model (GLM, SAS, 8.1 software). Relationships between added calcium chloride and ionised calcium in solutions and mixtures of whey and saliva were found by linear regression. Relationships be-
E. Neyraud, E. Dransfield / Physiology & Behavior 81 (2004) 505–510
507
tween taste intensity and calcium chloride concentration were found by regression using Gompertz-type sigmoid equations (Excel 2000), for which the model, taste intensity = S0+(SM S0)exp{ exp[k(T Ca) + 1]}, was used and where S0 is the predicted score at zero calcium concentration, SM is the maximum predicted intensity score, k is related to the rate of increase of taste with calcium concentration, T is the threshold (mM) for taste and Ca is the ionised calcium concentration (mM).
3. Results 3.1. Physicochemical measurements 3.1.1. Total calcium and magnesium concentrations Whey retentate powder contained 98.8 F 5.8 mmol Ca/ kg and 37.0 F 0.2 mmol Mg/kg of powder. Resting saliva from three of the tasters contained 5.4 F 1.0 mM calcium and 0.47 F 0.15 mM magnesium. 3.1.2. The pH and viscosity of mixtures of saliva and whey The addition of saliva and whey solutions to water increased their pH (Table 1). Viscosity was similar in all solutions, but there was a tendency for viscosity to decrease with increasing salivary concentration and for viscosity to increase with increasing concentration of whey. 3.1.3. Ionised calcium concentration in mixtures of saliva and whey The calcium ion concentration in aqueous solutions (Table 1) increased in proportion to the amount of added saliva. The addition of CaCl2 increased proportionately (r2>.98) the ionised calcium concentration. Increasing the concentration of whey protein increased the ionised calcium (Table 1), which was lowered when diluted with saliva in both 1% and 10% whey (Table 1).
Table 1 In vitro pH, viscosity, ionised calcium and the effect of CaCl2 addition to saliva, whey solutions and mixtures (n = 3) Saliva Whey pH
Viscosity
Ionised Ca
Slope 0 to 50 mM CaCl2
%
%
Value S.D. cps
S.D. mM
S.D. mM/mM CaCl2
S.D.
0 10 50 0 10 50 0 10 50
0 0 0 1.0 0.9 0.5 10 9 5
5.67 7.17 7.30 6.30 6.77 7.13 6.20 6.50 6.77
0.02 0.02 0.04 0.02 0.01 0.03 0.02 0.02 0.01
0.00 0.21 0.15 0.01 0.10 0.06 0.01 0.17 0.06
0.08 0.04 0.06 0.01 0.02 0.05 0.00 0.01 0.04
0.64 0.01 0.21 0.04 0.07 0.14 0.03 0.01 0.07
1.00 1.00 0.93 1.00 1.00 0.95 1.05 1.03 1.00
0.00 0.17 0.53 0.90 0.40 0.53 3.10 1.10 0.93
1.00 0.91 0.71 0.90 0.87 0.69 0.71 0.60 0.56
The slope is the linear increase in concentration of ionised calcium relative to the added Ca2 + ion concentration.
Fig. 1. Relationship between bitter taste intensity and calcium concentration in water. Values are the means and standard deviations of 22 tasters, and the dotted lines represent the extremes among the individuals.
The ability of the mixtures of whey and saliva to bind added Ca2 + ions (Table 1, slopes) was reduced by the increasing amounts of saliva both in 1% and 10% whey solutions. Considering all the combinations of saliva and whey, the Ca2 + ion concentration was not related to the viscosity of the solutions (Table 1). 3.2. Sensory evaluation 3.2.1. Taste intensity of calcium chloride in water The taste intensity of calcium chloride in water, rated with reference to water and to a 100 mM CaCl2 solution, is represented in Fig. 1 for all 22 tasters. On average (solid line), a taste was detected at about 5 mM, its intensity increased approximately linearly up to about 45 mM CaCl2 and began to plateau with concentrations over about 60 mM. The most sensitive taster (upper dotted line) had an approximate detection threshold around 1 mM CaCl2 and reached a plateau for concentrations over 30 mM, whilst the least sensitive (lower dotted line) began to perceive taste at about 10 mM CaCl2 and its intensity had not reached a plateau even at 100 mM CaCl2 (Fig. 1). 3.2.2. Taste intensity of calcium chloride in whey solutions Without added calcium chloride, the taste intensity increased with increasing concentration of whey (Table 2). The taste derives from the calcium salts present in the whey. The increase corresponds to an increase in ionised calcium, from 0.9 to 3.4 mM (Table 2), which would be just detectable in water by some of the tasters (Fig. 1). With the addition of 100 mM calcium chloride, the taste intensity decreased slightly from 1% to 10% whey and then stayed the same at 20% whey solution (Table 2). This tendency is consistent with the decrease in ionised calcium concentration from 91 to 57 mM (calculated from the data given in Table 1) with increasing whey. This decrease would be expected (from Fig. 1) to reduce the average bitterness
508
E. Neyraud, E. Dransfield / Physiology & Behavior 81 (2004) 505–510
Table 2 Effect of whey concentration (1% to 20%) on the bitterness (means and standard deviations from six tasters), total and ionised calcium concentrations in solution at two levels of CaCl2 addition 1%
5%
10%
0 mM Added calcium Taste intensity (0 – 20) 1.8 F 2.3 2.1 F 2.2 2.8 F 2.0 Total Ca (mM) 1.0 5.0 9.8 Ionised Ca (mM) 0.9 2.4 3.1 100 mM Added calcium Taste intensity (0 – 20) 16.4 F 3.5 15.4 F 3.2 13.7 F 3.3 Total Ca (mM) 100.9 105.0 109.8 Ionised Ca (mM) 90.9 91.4 74.1
Table 3 In situ variations in composition of saliva when testing whey solutions Solution
20% 0 mM CaCl2 4.6 F 3.6 19.6 3.4 14.2 F 3.8 119.6 57.4
5 mM CaCl2
20 mM CaCl2
40 mM CaCl2
score in water from about 19 to 16, similar with the observed scores (Table 2). Comparisons were also made between the taste intensity of 1% and 10% whey solutions over the range 0 to 50 mM added CaCl2, and the effect of whey on the taste of calcium ions was found to depend on the taster. For 10 tasters, there was no significant difference between the taste intensities in 1% and 10% whey at the four CaCl2 concentrations. However, for four other tasters, taste intensity showed a strong dependency on whey concentration (Fig. 2A). At 5 mM added CaCl2, both 1% and 10% whey solutions had similar low taste intensities, but at 20 and 40 mM added CaCl2, the taste intensities were significantly lower in the 10% whey solutions.
Fig. 2. The curves show the increase in taste with increasing concentration of calcium ions. (A) The means and standard deviations from four tasters for sensory scores against the concentration of calcium in 1% (dotted) and 10% whey (solid) solutions are shown. The large points are the scores related to the concentration of ionised calcium in spit for 1% (open) and 10% (filled) whey solutions. Their relationship (thick solid line) is given. (B) Similar with Panel A, except that the data are from one taster.
Saliva
Whey (%)
Ca (mM)
pH
Ca (mM)
pH
0 1 10 0 1 10 0 1 10 0 1 10
0.1 1.3 3.9 7.7 6.3 6.0 22.8 22.0 14.4 43.2 37.6 28.3
4.5 6.7 6.6 5.1 6.4 6.3 5.1 6.1 6.1 4.9 5.9 5.9
0.4 F 0.2 1.9 F 3.0 3.3 F 0.4 4.5 F 1.2 4.8 F 1.0 5.2 F 0.9 17.1 F 4.0 16.2 F 3.2 11.6 F 2.3 33.5 F 7.9 31.6 F 7.1 24.1 F 4.0
6.4 F 0.4 6.5 F 0.3 6.5 F 0.1 6.1 F 0.5 6.3 F 0.2 6.4 F 0.1 6.0 F 0.5 6.1 F 0.2 6.2 F 0.1 5.8 F 0.5 6.0 F 0.2 5.9 F 0.1
The solutions contained 0, 1 or 10% whey with 0, 5, 20 or 40 mM added calcium chloride. Values are for ionised calcium and pH and those in saliva are the mean and standard deviation among 6 tasters and 3 repetitions for the saliva after keeping each solution in the mouth for 10 s.
3.3. Calcium concentrations in saliva and taste intensity The average composition of spit during the tasting of solutions of calcium chloride and whey by six tasters is shown in Table 3. The pH in solution was correlated significantly with that in the saliva for four participants and not correlated for the other two. For Ca2 + ions, the average concentration of ionised calcium (from 0 to 43 mM) in whey solutions were highly correlated (r2>.98) with those (from 1 to 34 mM) in spit. However, there were significant variations among the tasters. The proportion of ionised calcium in spit varied from 0.7 to almost 1.0 among tasters. Hence, at an ionised calcium concentration in solutions of whey of 40 mM, the ionised calcium in spit varied from 27 to 38 mM among individuals. When the two independent curves at 1% and 10% whey, relating the concentrations of added CaCl2 to taste (Fig. 2A), were plotted against the concentration of ionised calcium in spit (Fig. 2A, solid line), a single ‘S’ shaped curve was obtained, showing that the taste intensity was largely independent of the concentration of whey (Fig. 2A). The psychophysical relationship between perceived intensity and ionised calcium (Fig. 2A) was steeper than that for total CaCl2 (Fig. 1). The effect of ion binding on taste may be related to an individual’s threshold, as one taster with a high threshold of 10 to 15 mM showed a marked effect of calcium binding by whey and very low bitterness intensity, even at 50 mM CaCl2 addition (Fig. 2B). In this case, the variations in bitterness were not fully accounted for by the ionised calcium in the spit.
4. Discussion The bitterness thresholds of calcium chloride solutions in water vary from 1 to 15 mM among individuals and were within the 2 to 30 mM (mean 10 mM) given by Pfaffmann
E. Neyraud, E. Dransfield / Physiology & Behavior 81 (2004) 505–510
[17]. However, using an ‘up and down’ method, a very much lower threshold (average log detection threshold of 0.008 mM), but varying four orders of magnitude between individuals, has been reported [8]. Such a low threshold should be interpreted with caution [18] because we confirm that nonstimulated saliva contains about 1 mM ionised calcium. Whey contained about 100 mmol Ca/kg and about 30 mmol Mg/kg. Mg2 + ions in water have a bitter taste and a similar or slightly higher threshold than that of Ca2 + ions, but their concentration was always lower than the taste threshold and, thus, would not have contributed to the taste intensity. These divalent ions interact with milk proteins such as caseins [9], a-lactalbumin and h-lactoglobulin [10], and in cows’ milk, about two thirds of the calcium and half the phosphate are found in casein micelles. The biological significance appears to be that casein micelles may allow the total calcium and phosphate concentrations to exceed the solubility of calcium phosphate without causing the precipitation of calcium phosphate. In this work, up to 20% (w/v) whey protein gave a stable, milky liquid, with quantities of ionised calcium in the psychophysical range. Although calcium binding could have been increased by increasing the pH, as the binding of Ca2 + ions increases 10-fold from pH 6.2 to 7.3 [19], we used solutions in water at around pH 6.2 to avoid adding buffers that may have introduced other tastes. Early work found about 1.5 mM calcium [13], and that 85% of calcium was ionised in saliva, with 8% bound to macromolecules and 6% as ion pairs [20], with levels varying from 0.49 to 0.74 mM depending on the type of saliva. A larger proportion of ionised calcium was found in stimulated saliva. Later work [14] found half-ionised and higher levels in saliva. In this work, spit contained about 1 mM ionised Ca and about 5 mM total calcium. In low-flow participants, total calcium in parotid saliva decreased with pH and flow rate, but in high-flow participants, calcium increased with pH and was not related to salivary flow rate [2]. However, both ionised and total calcium increased with flow rate of parotid saliva [21]. Hence, it appears that the wide range of values reported for Ca2 + ions in saliva is due to the variations in the site of collection of saliva, the salivary gland, the degree, the type of stimulation and the possible role of flow rate, which could be linked to gland size [22]. With no added CaCl2, increasing the whey concentration from 1% to 20% increased slightly the taste intensity and was accompanied by an increase in Ca2 + ion concentration from the whey. With 100 mM added CaCl2, increasing the whey concentration from 1% to 20% decreased the taste intensity from 16.2 to 14.0 and was accompanied by a measured decrease in ionised calcium concentration. From the measured ionised calcium and sensory tests of Ca2 + ions in water, the predicted bitter intensity values were 1, 2, 18 and 16 in good agreement with the observed 1.8 F 2.3, 4.6 F 3.6, 16.2 F 3.5 and 14 F 3.8, respectively. The de-
509
crease in taste was therefore accounted for by the ionised, rather than the total, calcium concentration. Changes also occurred in pH and, at high concentrations of Ca2 + ions, the saliva was less able to buffer, but changes in pH were small (less than 0.5 units) and not thought to have influenced bitterness perception. Subsequent tests were performed in the more sensitive linear part of the psychophysical range of Ca2 + ion concentration. Ten tasters found no differences in taste intensity between the 1% and 10% whey concentrations in the range 10 – 50 mM added CaCl2. It is unlikely that the small differences in viscosity of these dilute aqueous solutions [23] would affect the taste. A group of four other tasters found significantly higher taste intensity for 1% than for 10% whey. The differences were largely accounted for by the differences in Ca2 + ion concentration measured in spit. A similar, but stronger, effect of reduction in taste intensity by calcium binding was shown by one participant. It appears then that this type of relationship is general but cannot be used for any one specific individual. This would be important in explaining the origin of differences among individuals in the psychophysical response to the bitterness of divalent salts. Variations in Ca2 + ion concentration could occur at the site of bitterness perception, which occurs in specialised neuroepithelial receptor cells that are bundled in the taste buds in the lingual epithelium. Small tubulo-alveolar salivary glands, the von Ebner’s glands, secrete into troughs, in direct contact with the taste buds. This saliva secretion is thought to be essential for the concentration and delivery of sapid molecules in the gustatory system and for clearing the tongue surface of taste substances, which would otherwise cause a long-lasting taste sensation [24]. The regulation of the secretion could involve a feedback between the gland and taste buds [25], but little is known of its composition. Hence, it would be expected that a bitter taste would be related to Ca2 + ion concentration in von Ebner’s secretions. In this study, Ca2 + ion concentrations were measured in the spit. Different individual responses would then depend on the equilibrium between von Ebner’s secretion and the bulk (spit, mainly parotid) saliva, which is likely to depend on the morphology of the tongue and the quantities of salivary secretion. All attempts to relate taste to concentration of the stimulus will remain largely circumstantial until the concentration and binding of ions in von Ebner’s saliva has been established.
5. Conclusions A general model that the taste is related to the concentration of free ions was tested using calcium ions and whey solutions. The presence of whey decreased the bitter taste of calcium mostly in relation to its lowering of free Ca2 + ions in bulk saliva (spit), but large variations in binding were found among individuals.
510
E. Neyraud, E. Dransfield / Physiology & Behavior 81 (2004) 505–510
References [1] O’Mahony M, Heintz C. Direct magnitude estimation of salt taste intensity with continuous correction for salivary adaptation. Chem Senses 1981;6:101 – 12. [2] Norris MB, Noble AC, Pangborn RM. Human saliva and taste response to acids varying in anions, titratable acidity and pH. Physiol Behav 1984;32:237 – 44. [3] Christensen CM, Navazesh M, Brightman VJ. Effects of pharmacological reductions in salivary flow on taste thresholds. Arch Oral Biol 1984;29:17 – 23. [4] Humphrey SP, Williamson RT. A review of saliva: normal composition, flow and function. J Prosthet Dent 2001;85:162 – 9. [5] Christensen CM, Brand JG, Malamud D. Salivary changes in solution pH: a source of individual differences in sour taste perception. Physiol Behav 1987;40:221 – 7. [6] Rosett TR, Shirley L, Schmidt SJ, Klein BP. Na + binding as measured by 23Na nuclear magnetic resonance spectroscopy influences the perception of saltiness in gum solutions. J Food Sci 1994;59:206 – 10. [7] Neyraud E, Prinz J, Dransfield E. Sodium chloride and sugar release, salivation and taste during mastication of salted chewing gum. Physiol Behav 2003;79:731 – 7. [8] Tordoff MG. Some basic psychophysics of calcium salt solutions. Chem Senses 1996;21:417 – 24. [9] Thompson MP, Brower DB. Calcium binding proteins in bovine milk. J Dairy Sci 1985;68(Supp1.):59. [10] Baumy JJ, Brule´ G. Binding of bivalent cations to a-lactalbumin and alactoglobulin: effect of pH and ionic strength. Lait 1988;68(1):33 – 48. [11] Schneyer LH, Schneyer CA. Inorganic composition of saliva. In: Code CF, Heidel W, editors. Handbook of physiology section 6: alimentary canal. Secretion. Handbook of Physiology, Volume II. Washington: American Physiological Society, Waverly Press; 1967. p. 497 – 530. [12] Larsen MJ, Pearce EIF. Saturation of human saliva with respect to calcium salts. Arch Oral Biol 2003;48:317 – 22. [13] Dreisbach RH. Calcium binding by normal human saliva. J Dent Res 1960;39:1133 – 40.
[14] Matsuo S, Lagerlo¨f F. Relationship between total and ionised calcium concentrations in human whole saliva and dental plaque fluid. Arch Oral Biol 1991;36:525 – 7. [15] Ferguson DB. Oral bioscience. Edinburgh: Churchill Livingstone; 1999. p. 145. [16] Bertoldo Pacheco MT, Carraro F, Sgarbieri VC. Study of calcium binding to different preparations of yeast (Saccharomyces cerevisiae) protein by using an ion selective electrode. Food Chem 1999;66: 249 – 52. [17] Pfaffmann C. The sense of taste. In: Fiel J, Magoun HW, Hall WE, editors. Handbook of physiology, neurophysiology. Washington: American Physiological Society; 1959. p. 507 – 34. [18] Bartoshuk LM. The psychophysics of taste. Am J Clin Nutr 1978; 31:1068 – 77. [19] Carr CW. Studies on the binding of small ions in protein solution with the use of membrane electrodes: IV. The binding of calcium ions in solutions of various proteins. Arch Biochem Biophys 1953;46: 424 – 31. [20] Gron P. Arch Oral Biol 1973;18:1385 – 92. [21] Lagerlo¨f F, Ekstrand J. The effect of flow rate on the ionised calcium concentration of human parotid saliva. Caries Res 1982;16:123 – 8. [22] Robertson WG, Marshall RW. Ionized calcium in body fluids. CRC Crit Rev Clin Lab Sci 1981;15:85 – 125. [23] Shama F, Sherman P. Variation in stimuli associated with oral evaluation of the viscosities of glucose solutions. J Texture Stud 1973;4: 254 – 62. [24] Schmale H, Holtgreve-Grez H, Christiansen H. Possible role for salivary gland protein in taste reception indicated by homology to lipophilic – ligand carrier proteins. Nature 1990;343:366 – 9. [25] Sbarbati A, Crescimanno C, Osculati F. The anatomy and functional role of the circumvallate papilla/von Ebner gland complex. Med Hypotheses 1999;53:40 – 4. [26] Jack FR, Piggot JR, Paterson A. Cheddar cheese texture related to salt release during chewing, measured by conductivity—preliminary study. J Food Sci 1995;60(2):213 – 7.