Physiological Studies on Umami Taste

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Kenzo Kurihara2 and Makoto Kashiwayanagi Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan 060-0812

ABSTRACT

The first electrophysiological studies on umami taste were conducted with rats and cats. Unlike humans, these animals did not show a large synergism between monosodium glutamate (MSG) and disodium guanylate (GMP) or disodium inosinate (IMP). The taste nerve responses of these animals to umami substances were not differentiated from the salt responses. The canine taste system was sensitive to umami substances and showed a large synergism between MSG and GMP or IMP. The umami substances showed no enhancing effects on other basic tastes. Amiloride, an inhibitor for the response to NaCl, did not inhibit the large response induced by the synergism between MSG and the nucleotides, indicating that the response to the umami substances is independent of the response to salt. Single-fiber analysis on the responses of mouse glossopharyngeal nerve and monkey primary taste cortex neurons also showed that the responses to umami substances are independent of other basic tastes. On the basis of these results, it was proposed that the umami taste is a fifth basic taste, and that there is a unique receptor for umami substances. Hence, we compared the taste of agonists for brain glutamate receptors. In humans, the order of intensity of umami taste induced by a mixture of 0.5 mmol/L GMP and 1.5 mmol/L of various agonists was glutamate . ibotenate . L(1)-2-amino-4-phosphonobutyric acid (L-AP4) 5 (6)1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD). Kainate, N-methyl-D-aspartic acid (NMDA) and (RS)–amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), which are agonists for ionotropic receptors, had no umami taste. It was concluded that the umami receptor is not identical to any known glutamate receptors; there seems, therefore, to be a unique receptor for umami. J. Nutr. 130: 931S–934S, 2000. KEY WORDS: c dogs c synergism c taste nerve response c glutamate agonists c amino acids History of physiological studies on umami taste In nature, there are three umami substances as follows: monosodium glutamate (MSG),3 disodium gluanylate (GMP) and disodium inosinate (IMP). The umami substances are contained abundantly in various foods, including vegetables (e.g., tomato, potato, cabbage, mushroom, carrot, soybean and green tea), seafood (e.g., fish, kelp, seaweed, oyster, prawn, crab, sea urchin, clam and scallop), meat (e.g., beef, pork and chicken) and cheese, and contribute greatly to the characteristic tastes of these foods. For example, the characteristic taste of snow crab meat is reproduced by mixing glycine, alanine, arginine, MSG, IMP and salts in a particular ratio (Konosu et al. 1987). When the umami constituents are eliminated, the characteristic taste of the crab meat disappears. Thus, umami substances are essential for producing the unique taste of many natural foods. Europeans and Americans have considered that umami substances potentiate flavor by enhancing the four basic tastes, although no reliable scientific data supporting this idea have been published. Yamaguchi (1987) showed that MSG has no enhancing effect on the four basic tastes in humans, but these data have not been accepted readily by Europeans and Americans. The umami substances are originally acids; hence, at neutral pH, they exist in the salt form. Usually they are sodium salts, i.e., monosodium glutamate, disodium inosinate and disodium guanylate. Thus, the umami substances contain the sodium ion. The first electrophysiologic studies on reception of the umami substances were conducted with rats (Sato et al. 1967) and cats (Adachi et al. 1967) In these animals, single fibers of the chorda tympani nerve responded to MSG, but also to NaCl. Hence the response to MSG was considered to be related to the salt response. In humans, a remarkable synergism exists between MSG and IMP or GMP (Kuninaka 1967). For example, 0.5 mmol/L GMP alone or 1.5 mmol/L MSG alone elicits practically no taste, but a mixture of GMP and MSG at these concentrations 1 Presented at the International Symposium on Glutamate, October 12–14, 1998 at the Clinical Center for Rare Diseases Aldo e Cele Dacco´ , Mario Negri Institute for Pharmacological Research, Bergamo, Italy. The symposium was sponsored jointly by the Baylor College of Medicine, the Center for Nutrition at the University of Pittsburgh School of Medicine, the Monell Chemical Senses Center, the International Union of Food Science and Technology, and the Center for Human Nutrition; financial support was provided by the International Glutamate Technical Committee. The proceedings of the symposium are published as a supplement to The Journal of Nutrition. Editors for the symposium publication were John D. Fernstrom, the University of Pittsburgh School of Medicine, and Silvio Garattini, the Mario Negri Institute for Pharmacological Research. 2 To whom correspondence should be addressed. 3 Abbreviations used: ACPD, (6)1-aminocyclopentane-trans-1,3-dicarboxylic acid; AMPA, (RS)–amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; L-AP4, L(1)-2-amino-4-phosphonobutyric acid; GMP, disodium guanylate; IMP, disodium inosinate; MSG, monosodium glutamate; NMDA, N-methyl-D-aspartic acid. 0022-3166/00 $3.00 © 2000 American Society for Nutritional Sciences. 931S Downloaded from jn.nutrition.org by guest on May 30, 2017 elicits a strong umami taste. This remarkable synergism was not seen in rats or cats. In rats, the synergism was seen in sucrose-sensitive fibers (Sato et al. 1967). Thus the responses to umami substances were related to responses to both NaCl and sucrose; the umami substances did not elicit unique responses independently of the four basic tastes in rats or cats. Canine taste nerve responses to umami substances In the canine chorda tympani nerve, a large synergism between MSG and GMP or IMP, similar to that found in humans, was observed (Kumazawa and Kurihara 1990b, Kumazawa et al. 1991, Nakamura and Kurihara 1991) (Fig. 1). GMP (0.5 mmol/L) alone induced only a small response, which reverted to a spontaneous level during stimulation. After adaptation of the response, solutions containing MSG of varying concentrations and 0.5 mmol/L GMP were applied to the tongue. Hence, the responses to the mixture of MSG and GMP did not contain the response to GMP itself. The response was observed to rise with increasing concentrations of MSG. In the presence of 0.5 mmol/L GMP, the response was much greater than that observed in the absence of GMP. This increase due to the presence of GMP resulted from a synergism between MSG and GMP. The synergism was remarkable at lower concentrations of MSG and attenuated at high concentrations. The synergism between 100 mmol/L MSG and 0.2 mmol/L of various species of nucleotides was then examined. The synergistic effects were seen with IMP and GMP, whereas CMP, AMP and cAMP showed practically no synergistic effect. This observation was similar to that in humans. The effects of 0.2 mmol/L GMP on responses to various stimuli were also examined (Fig. 2). GMP (0.2 mmol/L) showed practically no effect on responses to 0.1 mol/L NaCl, HCl (pH, 3), 500 mmol/L sucrose, 10 mmol/L quinine and 100 mmol/L glycine. Thus, the umami substance (GMP) had no ability to enhance the responses to the four basic taste stimuli. The synergism between MSG and the nucleotides was ultimately explained in terms of an allosteric effect (Fig. 1) (Kumazawa et al. 1991). Amiloride is known to inhibit the response to NaCl. In dogs, the inhibitory effect appears at a dose between 1 and 10 mol/L of the drug, and 0.3 mmol/L amiloride completely inhibits the response to 100 mmol/L NaCl (Fig. 3) (Nakamura and Kurihara 1990). Amiloride also inhibits the response to MSG. On the other hand, the response to 3 mmol/L GMP alone or the response induced by the synergism between 0.5 mmol/L GMP and 100 mmol/L MSG was practically unaffected by amiloride (over the range of concentrations examined) (Nakamura and Kurihara 1991). These results suggest that the response to MSG alone contains a component derived from the sodium ion, but that the response to GMP alone or to MSG and GMP together is independent of the sodium component. In other words, the response to GMP alone or to MSG and GMP together reflects purely a umami component. Umami taste as a basic taste Taste has been classified into four basic groups, i.e., sweet, sour, salty and bitter. Although there are many chemicals that stimulate these basic tastes, some elicit a unique taste that cannot be classified into any of the four basic tastes. The definition of a basic taste is not absolutely objectively based, but is to some extent based on convention. Nevertheless, the concept of basic taste is very useful and reasonable. A basic taste should include the following properties: 1) A basic taste is a characteristic taste that is clearly different from any other basic taste. 2) A basic taste is not reproduced by mixing the other basic taste stimuli. 3) A basic taste is a universal taste induced by components of many foods. If a taste is not often encountered, we do not call it a basic taste, even if it is a unique taste. For example, inorganic salts such as Na2SO4, LiCl or NH4Cl elicit unique tastes that are quite different from the salty taste of NaCl and are not a mixed taste of FIGURE 1 Left panel: The relative magnitude of canine chorda tympani nerve responses to monosodium glutamate (MSG) of varying concentrations in the absence (M) and presence (f) of 0.5 mmol/L disodium guanylate (GMP). Right panel: A schematic allosteric model for synergism between MSG and GMP. FIGURE 2 The relative magnitude of the canine chorda tympani nerve response (R/Ro) to various stimuli in the presence of 0.2 mmol/L disodium guanylate (GMP), where Ro and R represent the magnitude of the response to each stimulus in the absence and the presence of GMP, respectively. FIGURE 3 The relative magnitude of the canine chorda tympani nerve response (R/Ro) to 100 mmol/L NaCl (M), 300 mmol/L monosodium glutamate (MSG) (f), 3 mmol/L disodium guanylate (GMP) (F) and a mixture of 0.5 mmol/L GMP and 100 mmol/L MSG (E) as a function of the logarithmic concentration of amiloride, where Ro and R represent the magnitude of the response before and after treatment with amiloride, respectively. 932S SUPPLEMENT Downloaded from jn.nutrition.org by guest on May 30, 2017 salty and bitter. Succinate and methionine also elicit unique tastes that are quite different from any of the basic tastes. Although these unique tastes are evidently different from known basic tastes, we do not classify them into basic tastes because these tastes are typically not encountered in foods. 4) A basic taste should be proved electrophysiologically to be independent of other basic tastes. The umami taste fulfills criteria 1, 2 and 3. The fact that the umami taste is independent of the salt response in dogs speaks in part to criterion 4. In addition, Ninomiya and Funakoshi (1989) showed that in the glossopharyngeal nerve of certain species of mice, there were single fibers that were sensitive only to MSG or mixtures of MSG and the nucleotides, and insensitive to the other basic taste stimuli. Baylis and Rolls (1991) showed that in the taste cortex and adjoining orbitofrontal cortex of the macaque monkey, single neurons were observed that were tuned to respond best to MSG. Thus, the umami taste clearly fulfills criterion 4, and we can conclude that the umami taste is a fifth basic taste. Agonist and modulator properties of umami substances In humans, IMP or GMP alone elicits the umami taste, but their taste intensity is rather weak compared with that of MSG. In humans, MSG can be considered an agonist and IMP an enhancer or modulator of the umami taste. In macaque monkeys, MSG also acts as an agonist because MSG alone induces a large neural response (Baylis and Rolls 1991). The synergism between MSG and the nucleotides is not robust in monkeys. In mice, MSG alone induced a large umami response in the glossopharyngeal nerve (Ninomiya and Funakoshi 1989), and therefore MSG is considered an agonist in this species. In dogs, GMP or IMP alone induces a large response (Kumazawa and Kurihara 1990, Kumazawa et al. 1991, Nakamura and Kurihara 1991), indicating that the nucleotides are the primary agonists, whereas MSG is a modulator. Thus the function of MSG as a modulator or agonist varies with the species of animal. Comparison of glutamate receptors in brain with umami receptors Glutamate receptors in the brain are classified as either ionotropic or metabotropic. The agonists of the ionotropic receptors are (RS)–amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartic acid (NMDA). The metabotropic receptors are classified into three groups, i.e., I (mGluR1, mGluR5), II (mGluR2, mGluR3) and III (mGluR4, mGluR6, mGluR7, mGluR8) (Tanabe et al. 1993). Stimulation of group I receptors with agonists activates adenylyl cyclase, protein kinase C and phospholipase C. mGluR1 and mGluR5 are thus excitatory receptors. On the other hand, stimulation of group II or III receptors with agonists inhibits adenylyl cyclase; they are thus classed as inhibitory receptors. Chaudhari et al. (1996) reported that mGluR4 is expressed in rat papillae-bearing taste buds and suggested that mGluR4 may be a chemosensory receptor responsible for umami taste. Bigiani et al. (1997) examined the actions of glutamate on the membrane properties of rat taste cells using whole-cell patchclamp techniques. In the majority of taste cells, application of glutamate induced “sustained” glutamate responses, which consisted of an outward current (reduction of the maintained inward current). L(1)-2-Amino-4-phosphonobutyric acid (LAP4), a specific agonist of mGluR4 receptors, mimicked the sustained glutamate responses. In psychometric studies in humans, we have evaluated the intensity of the umami taste induced by various glutamate agonists (Kurihara and Kashiwayanagi 1999). A solution containing 0.5 mmol/L GMP and 1.5 mmol/L MSG together elicited a umami taste that was much stronger than that of 20 mmol/L MSG alone. The intensity of the umami taste induced by a solution containing 0.5 mmol/L GMP and 1.5 mmol/L of each of several glutamate receptor agonists was also evaluated and compared with the response to MSG alone at each of several concentrations. In combination with GMP, ibotenate induced a rather strong umami taste, whereas L-AP4 and (6)1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) induced a weak umami taste; kainate, NMDA and AMPA induced no umami taste (Table 1). The results suggest that the umami receptor is not a subtype of an ionotropic receptor. L-AP4 in the presence of GMP elicited umami taste, although AP4 is an agonist at mGluR4, an inhibitory receptor. Because MSG induces a large response in the taste nerve, a receptor for umami taste should be an excitatory receptor. Thus, it is unlikely that the mGluR4 receptor is a receptor for umami taste, although AP4 has an umami taste. The agonist specificity of the mGluR1 and mGluR5 receptors, which are excitatory, is different from that of the umami receptor. It is concluded that the receptor for umami taste is not identical to any known glutamate receptors, and thus there seems to be a unique receptor mediating the umami taste. Great contribution of amino acids, umami substances and salts to taste of foods Foods contain various species of chemical components, but only a limited number of components contribute to the characteristic taste of the foods. Konosu et al. (1987) showed that the characteristic tastes of many natural foods are reproduced by mixing amino acids, umami substances and salts in appropriate ratios. Regarding amino acids, although various species are contained in foods, only a small number are required to produce the characteristic tastes of foods. As described above, umami substances are also essential to produce the characteristic tastes of many foods. Salts also are essential for producing the characteristic tastes of many foods. Thus, a mixture of amino acids and umami substances in the absence of salts can impart a weak taste, and one that is quite different from that of the parent foods. To confirm the effects of salts on the taste of amino acids, psychometric TABLE 1 Umami taste of various glutamate agonists in humans1,2 Glutamate agonists Umami intensity Glutamate .20 mmol/L Ibotenate 15 mmol/L DL-AP43 7 mmol/L trans-ACPD 5 mmol/L kainate No umami NMDA No umami AMPA No umami 1 The intensity of umami taste induced by a mixture containing 0.5 mM GMP and 1.5 mM agonist was compared with that of standard glutamate solutions. 2 Concentration of MSG whose intensity of umami is equivalent to that of agonists tested. 3 Abbreviations: DL-AP4, DL(1)-2-amino-4-phosphono-butyric acid; ACPD, (6)-1-aminocyclopentone-trans-1,3-dicarboxylic acid; NMDA, N-methyl-D-aspartic acid; AMPA, (RS)–amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid. UMAMI TASTE 933S Downloaded from jn.nutrition.org by guest on May 30, 2017 studies were conducted using a solution containing alanine, glycine or serine (which have a sweet taste) (Ugawa et al. 1992). The sweetness of these amino acids was greatly enhanced by the addition of NaCl. The addition of sodium phosphate also enhanced the sweetness of the amino acids, but its enhancing effect was much less than that of NaCl. The effects of salts on the taste of amino acids were also examined in animals. The enhancing effects of salts were not observed in rats, but were observed in dogs (Ugawa and Kurihara 1993). The responses to most amino acids examined were greatly enhanced by the presence of salts, but the degree of enhancement varied with the species of amino acids. The enhancing effects were dependent on the species of both cations and anions. That is, the responses to most amino acids were enhanced by sodium, potassium and calcium salts, but not magnesium salts. NaCl was much more effective than sodium phosphate. The canine taste nerve responses to umami substances were also enhanced by the presence of salts (Ugawa and Kurihara 1994). That is, the responses to MSG, GMP or the combination of MSG and GMP were enhanced by salts. Similar to the effects of salts on the responses to amino acids, the effects of the salts on the umami responses depended on the species of both cation and anion. It should be noted that the canine taste nerve responses to sugars were also enhanced by the presence of salts (Kumazawa and Kurihara 1990a). In humans as well, the sweetness of sugars is enhanced by NaCl. Salts carrying organic cations such as choline andN-methyl- D-glucosamine (which are impermeable to taste cell membranes) were also found to be effective in enhancing the responses of chemical stimuli. Hence, the enhancing effects of salts were not explained simply in terms of ion permeability at the apical membranes of taste cells. We hypothesize that the binding of both cations and anions to taste receptor membranes may induce exposure of the receptor sites for amino acids, umami substances and sugars. 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