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J. Agric. Food Chem. 2007, 55, 6712−6719
Molecular and Sensory Characterization of γ-Glutamyl Peptides as Key Contributors to the Kokumi Taste of Edible Beans (Phaseolus vulgaris L.) ANDREAS DUNKEL,† JESSICA KO¨ STER,†
AND
THOMAS HOFMANN*,#
Institut fu¨r Lebensmittelchemie, Universita¨t Mu¨nster, Corrensstrasse 45, D-48149 Mu¨nster, Germany, and Lehrstuhl fu¨r Lebensmittelchemie und Molekulare Sensorik, Technische Universita¨t Mu¨nchen, Lise-Meitner-Strasse 34, D-85350 Freising-Weihenstephan, Germany
Addition of a nearly tasteless aqueous extract isolated from beans (Phaseolus vulgaris L.) to a model chicken broth enhanced its mouthfulness and complexity and induced a much more long-lasting savory taste sensation on the tongue. Gel permeation chromatography and hydrophilic interaction liquid chromatography/comparative taste dilution analysis (HILIC/cTDA), followed by LC-MS/MS and 1D/ 2D-NMR experiments, led to the identification of γ-L-glutamyl-L-leucine, γ-L-glutamyl-L-valine, and γ-L-glutamyl-L-cysteinyl-β-alanine as key molecules inducing this taste-modifying effect. Sensory analysis of aqueous solutions of these peptides showed threshold concentrations between 3.3 and 9.4 mmol/L for an unspecific, slightly astringent sensation. More interestingly, when added to a savory matrix such as sodium chloride and monosodium glutamate solutions or chicken broth, the detection thresholds of these γ-glutamyl peptides decreased significantly and remarkably enhanced mouthfulness, complexity, and long-lastingness of the savory taste were observed; for example, the threshold of γ-glutamyl-cysteinyl-β-alanine decreased by a factor of 32 in a binary mixture of glutamic acid and sodium chloride. As tasteless molecules inducing mouthfulness, thickness, and increasing continuity of savory foods were coined about 10 years ago as “kokumi” flavor compounds, the peptides identified in raw as well as thermally treated beans have to be considered as kokumi compounds. KEYWORDS: Kokumi; taste; taste enhancer; mouthfullness; taste dilution analysis; beans; homoglutathione; γ-glutamyl peptides
INTRODUCTION
In many countries all over the world, legumes play an important role in consumer diet and account for approximately one-third of the world’s primary crop production (1). In particular, peas, beans, lentils, and other podded plants are key ingredients in the typical cuisines of India, South America, the Middle East, and Mexico, respectively. When combined with meat, beans are long-known to enhance the flavor of famous dishes such as the French cassoulet, the Brazilian feijoada, and Mexican chilis. Many investigations have focused on the chemical composition of pulses. Although the protein quantity and quality were found to vary between different legumes, they contain high levels of proteins when compared with other plant-derived foods such as cereals (2). Among the carbohydrates, beans are rich in starch, oligosaccharides, and dietary fiber, accounting for about 50% of the total weight of dried beans. Whereas the nondigestible oligosaccharides have been identified as prebiotic agents * Author to whom correspondence should be addressed (telephone +498161/71-3381; fax +49-8161/71-4216; e-mail
[email protected]). † Universita ¨ t Mu¨nster. # Technische Universita ¨ t Mu¨nchen.
(3), a lowering of serum total cholesterol in rats was reported for starches of different bean varieties (4). Furthermore, saponins present in edible beans were reported to induce a decrease of blood lipids and cholesterol levels and to exhibit anticarcinogenic activity (5). Although the mineral content of legumes is rather high, their bioavailability is poor due to the presence of phytate, inhibiting iron absorption by complexation (6). Nutritionally beneficial, beans are an excellent source of folates, which in addition to being essential micronutrients are suggested to reduce the risk of neural tube effects (7). Among the legumes, soybeans seem to play a unique role due to their bioactive phytochemicals such as isoflavones (8). Although multiple studies were aimed at increasing the knowledge on nutritional benefits and health-promoting effects of legumes, no data are available on the bean ingredients that are responsible for the taste-improving effect when beans such as Phaseolus Vulgaris L. are used as part of savory-tasting dishes. To discover such taste modulators in complex food products, we have recently developed so-called “sensomics” techniques by combining analytical natural product chemistry and human psychophysical tools such as the taste dilution analysis (TDA) and the comparative taste dilution analysis (cTDA), respectively (9, 10). This approach led to the structural
10.1021/jf071276u CCC: $37.00 © 2007 American Chemical Society Published on Web 07/07/2007
Kokumi Taste Compounds in Beans and functional characterization of various previously unknown taste compounds such as thermally generated bitter compounds (9), astringent taste compounds in black tea infusions (11) and roasted cocoa nibs (12), the multivalent taste-modulating Maillard reaction product alapyridaine in beef broth (10, 13), umami-enhancing glucosides in morel mushrooms (14), and, most recently, bitterness-suppressing molecules such as 1-carboxymethyl-5-hydroxy-2-hydroxymethylpyridinium inner salt (15). To bridge the gap between pure structural chemistry and sensory perception, the aim of the present investigation was to screen for the key compounds inducing the taste-modulating activity of common beans (P. Vulgaris L.), to isolate and identify the chemical structures, and to determine the sensory thresholds of the compounds found with the highest gustatory response. MATERIALS AND METHODS Chemicals. The following compounds were obtained commercially: trifluoroacetic acid (Riedel de Haen, Taufkirchen, Germany); sodium chloride and L-glutamic acid (Fluka, Taufkirchen, Germany). All of the reference peptides used within this study were from Bachem (Bubendorf, Switzerland). Acetonitrile was of HPLC grade (Fisher Scientific, Schwerte, Germany). Deuterated solvents were supplied by Euriso-Top (Gif-Sur-Yvette, France). A model chicken broth used as savory matrix for the sensory analyses was freshly prepared by diluting a Gourmetbouillon Huhn type concentrate (Nestle´, Singen, Germany) with bottled water (Evian; Danone, Wiesbaden, Germany) in a concentration of 3.0 g/L. Dried bean seeds (P. Vulgaris L.) were obtained from a local market. Preparation of Aqueous Bean Extracts. Dried bean seeds (P. Vulgaris L., 10 g) were cooked in water (100 mL) for 60 min, minced, and centrifuged. The residue was extracted with bottled water (100 mL) with stirring for 12 h at room temperature. The aqueous layers were combined and, after centrifugation, the clear supernatant was freeze-dried to give the water-soluble extract of the cooked beans (4.45 g, yield ) 44.5%). In parallel, dried bean seeds (10 g) were ground in a coffee grinder and then extracted with water (100 mL) for 12 h at room temperature. After centrifugation, the clear supernatant was freezedried to give the water-soluble extract of the dried beans (2.78 g, yield ) 27.8%). Gel Permeation Chromatography (GPC). An aliquot (250 mg) of the dry material of the lyophilized extract isolated from the dried beans was taken up in water (10 mL) and, then, applied onto the top of a water-cooled 100 × 5 cm × K 50/100 glass column (Amersham Bioscience, Uppsala, Sweden) filled with a slurry of Sephadex G-10 (Amersham Bioscience) conditioned with water adjusted to pH 4.0 with aqueous formic acid (1 g/100 g). Chromatographic separation was performed using the same solvent at a flow rate of 2 mL/min for 20 h. Monitoring the effluent by means of an L-7490 type RI detector (Merck, Darmstadt, Gemany) produced five fractions (fractions I-V) collected by means of a fraction collector, and the individual fractions were freeze-dried. The residue obtained for each GPC fraction was used for the sensory analysis as well as for chromatographic subfractionation. Sensory Analyses. Training of the Sensory Panel. Nine assessors (five males, four females, ages 22-39 years), who gave informed consent to participate in the sensory tests of the present investigation and had no history of known taste disorders, were trained in sensory experiments at regular intervals for at least 2 years and were, therefore, familiar with the techniques applied. For the training of the individual gustatory modalities, aqueous solutions (2 mL each) of the following reference taste compounds dissolved in bottled water (pH 6.0) were used: sucrose (50 mmol/L) for sweet taste, lactic acid (20 mmol/L) for sour taste, NaCl (30 mmol/L) for salty taste, caffeine (1 mmol/L) for bitter taste, sodium L-glutamate (3 mmol/L) for umami taste, tannic acid (0.05%) for puckering astringency, and quercetin-3-O-β-Dglucopyranoside (0.01 mmol/L) for a velvety astringent, mouth-drying oral sensation. For the training of viscosity, a gelatin solution (0.5% in water) was used; for the training of the activity of mouthfulness enhancement and complexity increase, coined kokumi activity (16-
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18), the panel was asked to compare the gustatory impact of the blank model chicken broth (control) with a solution of reduced glutathione (5 mmol/L) in chicken broth (both at pH 6.5). Sensory analyses were performed in a sensory panel room at 19-22 °C in three different sessions using nose clamps. Pretreatment of Fractions. Prior to sensory analysis, the fractions or compounds isolated were suspended in water, and, after removal of the volatiles in high vacuum (
