Kokumi Substances Research Articles

Kokumi-Enhancing Mechanism of N-l-lactoyl-l-Met Elucidated by Sensory Experiments and Molecular Simulations.

Recent research indicates that N-lactoyl amino acid derivatives have the potential as kokumi substances, with their kokumi profile closely linked to that of amino acids. This study aimed to explore the unexplored effects resulting from the introduction of lactate groups into l-Methional (l-Met), a prevalent flavor compound found in foods, such as tomatoes, known for its ability to activate the monosodium glutamate response. N-l-Lac-l-Met was enzymatically synthesized using food grade, and its taste profile and underlying mechanisms were investigated. The structure of N-l-Lac-l-Met was determined by high-performance liquid chromatography (HPLC)-mass spectrometry (MS)/MS. Sensory evaluation revealed the presence of astringency, kokumi, and bitterness of N-l-Lac-l-Met. In a stimulated broth, N-l-Lac-l-Met exhibited enhanced umami and kokumi taste perception compared to l-Met while demonstrating good stability within pH 5 to 9. A molecular simulation and quantum mechanics analysis indicated that the formation of an amide bond played a crucial role in the kokumi-enhancing effect of N-l-Lac-l-Met, specifically by increasing its affinity with umami receptors T1R1-T1R3 and a kokumi receptor CaSR. These findings established the relationship between amide bond formation and the kokumi-enhancing effect of N-l-Lac-l-Met, presenting its potential application as the kokumi substance in the food industry.

Volatile Short-Chain Aliphatic Aldehydes Act as Taste Modulators through the Orally Expressed Calcium-Sensing Receptor CaSR.

Aldehydes are natural volatile aroma compounds generated by the Maillard reaction of sugars and amino acids in food and affect the flavor of food. They have been reported to exert taste-modifying effects, such as increases in taste intensity at concentrations below the odor detection threshold. The present study examined the taste-enhancing effects of short-chain aliphatic aldehydes, such as isovaleraldehyde (IVAH) and 2-methylbutyraldehyde, thus attempting to identify the taste receptors involved. The results obtained revealed that IVAH enhanced the taste intensity of taste solutions even under the condition of olfactory deprivation by a noseclip. Furthermore, IVAH activated the calcium-sensing receptor CaSR in vitro. Receptor assays on aldehyde analogues showed that C3-C6 aliphatic aldehydes and methional, a C4 sulfur aldehyde, activated CaSR. These aldehydes functioned as a positive allosteric modulator for CaSR. The relationship between the activation of CaSR and taste-modifying effects was investigated by a sensory evaluation. Taste-modifying effects were found to be dependent on the activation state of CaSR. Collectively, these results suggest that short-chain aliphatic aldehydes function as taste modulators that modify sensations by activating orally expressed CaSR. We propose that volatile aroma aldehydes may also partially contribute to the taste-modifying effect via the same molecular mechanism as kokumi substances.

Investigation of free amino acids in lactic acid bacteria fermented milk and their ability to activate the calcium sensing receptor

A fullness in sensorial response is sought in low fat dairy foods to improve palatability, which may be achieved through activation of the calcium sensing receptor (CaSR). Free l-amino acids (FAA) are known kokumi substances that bind to the CaSR and impart attributes of creaminess and mouthfullness. In this study, skim milk (SM) was fermented using five different lactic acid bacteria (LAB) strains and their ability to activate the CaSR was compared. Our results demonstrated that fermented milks (FM) activated the CaSR with varying potency and efficacy in rank order: Leuconostoc pseudomesenteroides (CL3ST), Lactococcus lactis (BL1) > Streptococcus thermophilus (ST), Leuconostoc mesenteroides (CL3) > Lactobacillus helveticus (LH).

Oral expressions and functional analyses of the extracellular calcium-sensing receptor (CaSR) in chicken

In vertebrates, the extracellular calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis by sensing slight changes in extracellular Ca2+. CaSR is also expressed in mammals including rodent taste cells and is involved in sensing kokumi, a rich, savory quality that enhances the intensities of salty, sweet, and umami tastes. In this study, we focused on chicken CaSR (cCaSR) since calcium is an essential nutrient that is necessary for making eggshell and for the extremely rapid initial growth of bones. First we confirmed that cCaSR is expressed in taste cells. Next we cloned the cCaSR gene from kidney and transiently transfected human embryonic kidney 293 T (HEK293T) cells with the recombinant cCaSR, or empty vector and looked for the agonists and allosteric modulators (including kokumi substances) of cCaSR by Ca2+ imaging. We found that cCaSR was activated by extracellular Ca2+ and Mg2+ in a dose dependent manner. Several L-amino acids and kokumi substances such as glutathione enhanced the response of cCaSR. In addition, NPS2143 as a negative allosteric modulator of human CaSR negatively modulated the response of cCaSR. These results suggest that cCaSR can sense extracellular Ca2+ and Mg2+ as well as positive and negative allosteric modulators. Taken together, the results imply that CaSR might be a multifunctional receptor for calcium, amino acids, and kokumi substances in chicken. The present finding that functional CaSR is expressed in the chicken oral tissues will allow us to further elucidate the physiological role of CaSR in the chickens' taste sense, and to create new feeds that will contribute to the poultry industry.

Perceptive mechanism and sensory characteristics of kokumi substances

<h2>Abstract</h2> Some foods are known to have flavors such as complexity, mouthfulness and continuity that cannot be explained by the five basic tastes alone. Previously, the compounds responsible for such flavoring effect of garlic extract has been investigated, and sulfur-containing compounds including glutathione were found to be responsible for this effect, although they are tasteless at the concentration tested. Substances with these properties were proposed to be referred to as <i>kokumi</i> substances. From that glutathione was one of the agonists of the calcium-sensing receptor (CaSR), it was hypothesized that CaSR was involved in the perception of <i>kokumi</i> substances. From the results as [1] all CaSR agonists have a property of <i>kokumi</i> substance, [2] the sensory effect of <i>kokumi</i> peptides eliminated by a CaSR specific antagonist, [3] the CaSR activity of γ-glutamyl peptides positively correlated to sensory activity as <i>kokumi</i> substance, CaSR was suggested to be involved in the perception of <i>kokumi</i> substances. Among the γ-glutamyl peptides tested, γ-Glu-Val-Gly was found be as potent <i>kokumi</i> substance. γ-Glu-Val-Gly enhanced the intensity of mouthfulness, and continuity when added to chicken consommé and reduced-fat peanut butter. These results suggest that γ-Glu-Val-Gly is a potent <i>kokumi</i> peptide and would be useful for improving the flavor of food.

Development of Enzymatic Synthesis of γ-Glutamylcarnosine and Its Effects on Taste.

γ-Glutamyl peptides have amide bonds between the γ-carboxy group of glutamic acid and the amino group of amino acids or peptides. Some of these γ-glutamyl peptides are known as kokumi substances. Kokumi substances enhance the taste, mouthfulness, thickness, and continuity of the dish. γ-Glutamylcarnosine (γ-l-glutamyl-β-alanyl-l-histidine) is a γ-glutamyl peptide, and this peptide has been suggested as a kokumi substance; however, its effects on taste have not been evaluated directly. As γ-glutamylcarnosine is not available commercially, the conditions for its enzymatic synthesis using a γ-glutamyltranspeptidation reaction of γ-glutamyltranspeptidase of Escherichia coli was optimized. The synthesized peptide was purified with a Dowex 1 × 8 column, and its structure was identified by mass spectrometry and NMR spectroscopy. This is the first report of the enzymatic synthesis of γ-glutamylcarnosine. Using this purified preparation, its effects on the sense of taste were investigated. However, the effects of γ-glutamylcarnosine on the sense of taste were not detected except for increased bitterness.

Perceptive mechanism and sensory characteristics of kokumi substances: Effect on the sensory characteristics of low-fat foods

Certain foods are known to have flavor characteristics such as continuity and mouthfulness which cannot be explained by the five basic tastes alone. It has been demonstrated that these sensations are evoked by the addition of kokumi substances, flavor modifiers with no taste of their own, which are considered to be one of“ Koku” -imparting factors. Recent studies have demonstrated that kokumi substances such as glutathione are perceived via the calcium-sensing receptor （CaSR）. Screening with CaSR assay and sensory evaluations have shown that γ-glutamyl-valyl-glycine （γ-Glu-Val-Gly） is a potent kokumi peptide. In the present study, the sensory effects of γ-Glu-Val-Gly on chicken soup, reduced-fat peanuts butter, and reduced-fat custard cream were investigated by descriptive analysis. Chicken soup containing γ-Glu-Val-Gly had significantly stronger thickness, mouthfulness, and continuity than those of the control. In addition, γ-Glu-Val-Gly significantly increased the oiliness, thick flavor, and aftertaste of reduced-fat peanut butter, significantly increased the thickness of taste of reduced-fat custard cream. As a kokumi substance, γ-Glu-Val-Gly has no taste of it own, it can therefore be used to improve the flavor of both savory and sweet foods including reduced-fat foods.

Investigation of Kokumi Substances and Bacteria in Thai Fermented Freshwater Fish (Pla-ra).

A 16S rRNA next-generation sequencing technique was applied to investigate the microbial diversity and liquid chromatography-tandem mass spectrometry was used to identify glutamyl peptide profiles of 10 Thai fermented freshwater fish (Pla-ra) samples. A total of 12 genera of bacteria were able to be detected, with Tetragenococcus spp., Staphylococcus spp., and Lactobacillus spp. dominating. Of the 18 glutamyl peptides analyzed, 17 were found, even though the amounts detected were lower than the taste threshold. Despite this, an increase in mouthfulness sensation, reflecting kokumi activity, was clearly identified in most of the samples, which might be because of a synergistic effect of different sub-threshold compounds present in the samples. In principle component analysis, the relationship between microorganisms and glutamyl peptide generation was observed, especially between Tetragenococcus spp. and Lentibacillus spp. and the generation of γ-Glu-Val-Gly. Correlations between microbial diversity and the generation of taste enhancers were identified in this study.

Structure-CaSR-Activity Relation of Kokumi γ-Glutamyl Peptides.

Modulation of the calcium sensing receptor (CaSR) is one of the physiological activities of γ-glutamyl peptides such as glutathione (γ-glutamylcysteinylglycine). γ-Glutamyl peptides also possess a flavoring effect, i.e., sensory activity of kokumi substances, which modifies the five basic tastes when added to food. These activities have been shown to be positively correlated, suggesting that kokumi γ-glutamyl peptides are perceived through CaSRs in humans. Our research is based on the hypothesis that the discovery of highly active CaSR agonist peptides will lead to the creation of practical kokumi peptides. Through continuous study of the structure-CaSR-activity relation of a large number of γ-glutamyl peptides, we have determined that the structural requirements for intense CaSR activity of γ-glutamyl peptides are as follows: existence of an N-terminal γ-L-glutamyl residue; existence of a moderately sized, aliphatic, neutral substituent at the second residue in an L-configuration; and existence of a C-terminal carboxylic acid, preferably with the existence of glycine as the third constituent. By the sensory analysis of γ-glutamyl peptides selected by screening using the CaSR activity assay, γ-glutamylvalylglycine was found to be a potent kokumi peptide. Furthermore, norvaline-containing γ-glutamyl peptides, i.e., γ-glutamylnorvalylglycine and γ-glutamylnorvaline, possessed excellent sensory activity of kokumi substances. A novel, practical industrial synthesis of regiospecific γ-glutamyl peptides is also required for their commercialization, which was achieved through the ring opening reaction of N-α-carbobenzoxy-L-glutamic anhydride and amino acids or peptides in the presence of N-hydroxysuccinimide.

Determination and quantification of kokumi peptide, γ-glutamyl-valyl-glycine, in brewed alcoholic beverages

Recently, it has been demonstrated that kokumi substances such as glutathione are perceived through the calcium-sensing receptor (CaSR), and screening by CaSR assay and sensory evaluation has shown that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) is a potent kokumi peptide. In this study, γ-Glu-Val-Gly contents in various brewed alcoholic beverages were investigated. Contents of γ-Glu-Val-Gly in four brands of wine, four brands of rice wine (sake) and eight brands of beer were analyzed by high performance liquid chromatography-tandem mass spectrometry followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate. The analyses indicated that γ-Glu-Val-Gly was present in all of eight beer samples at concentrations in the range of 0.08-0.18mg/L, although the peptide was not detected in any wine or rice wine samples. These results suggest that amongst the brewed beverages tested, beer contains γ-Glu-Val-Gly, and that γ-Glu-Val-Gly is widely distributed in beer.

Mechanism of the perception of “kokumi” substances and the sensory characteristics of the “kokumi” peptide, γ-Glu-Val-Gly

Some foods are known to have flavours that cannot be explained by the five basic tastes alone, such as continuity, mouthfulness and thick flavour. It was demonstrated that these sensations are evoked by the addition of kokumi substances, flavour modifiers that have no taste themselves. However, their mode of action has been poorly understood. During a study on the perception of amino acids and peptides, it was found that glutathione (GSH) was one of the agonists of the calcium-sensing receptor (CaSR). We have hypothesized that CaSR is involved in the perception of kokumi substances. We found that all CaSR agonists tested act as kokumi substances and that a positive correlation exists between the CaSR activity of γ-glutamyl peptides and kokumi intensity. Furthermore, the kokumi intensities of GSH and γ-Glu-Val-Gly, a potent kokumi peptide, were significantly reduced by the CaSR-specific antagonist, NPS-2143. These results suggest that CaSR is involved in the perception of kokumi substances. A potent kokumi peptide, γ-Glu-Val-Gly, enhanced sweetness, saltiness and umami when added to 3.3% sucrose, 0.9% NaCl and 0.5% MSG solutions, respectively. In addition, γ-Glu-Val-Gly enhanced the intensity of continuity, mouthfulness and thick flavour when added to chicken soup and reduced-fat cream. These results suggest that γ-Glu-Val-Gly is a potent kokumi peptide and would be useful for improving the flavour of food.

Effect of a kokumi peptide, γ-glutamyl-valyl-glycine, on the sensory characteristics of chicken consommé

BackgroundRecent studies have demonstrated that kokumi substances such as glutathione are perceived through the calcium-sensing receptor (CaSR). Screening by a CaSR assay and sensory evaluation have shown that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) is a potent kokumi peptide. In the present study, the sensory characteristics of chicken consommé with added γ-Glu-Val-Gly were investigated using descriptive analysis.ResultsChicken consommé containing γ-Glu-Val-Gly had significantly stronger “umami” and “mouthfulness” (mouth-filling sensation) characteristics than the control sample at a 99% confidence level and significantly stronger “mouth-coating” characteristic than controls at a 95% confidence level.ConclusionsThese data suggest that a kokumi peptide, γ-Glu-Val-Gly, can enhance umami, mouthfulness, and mouth coating, implying that the application of this peptide could contribute to improving the flavor of chicken consommé.

Flavour improvement of reduced-fat peanut butter by addition of a kokumi peptide, γ-glutamyl-valyl-glycine

Recent studies have demonstrated that kokumi substances, which enhance basic tastes and modify mouthfulness and continuity although they have no taste themselves, are perceived through the calcium-sensing receptor (CaSR). Screening by a CaSR assay and sensory evaluation have shown that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) is a potent kokumi peptide. In our previous study, it was reported that the addition of γ-Glu-Val-Gly to chicken consomme significantly enhanced mouthfulness, continuity and thickness. In this study, the effect of γ-Glu-Val-Gly on reduced-fat peanut butter was investigated. Prior to the evaluation of the effect of γ-Glu-Val-Gly, a comparison test was conducted between full-fat model peanut butter and reduced-fat peanut butter. The sensory attributes in which the score of the full-fat model was significantly higher than that of the reduced-fat sample were used for the evaluation of the effect of γ-Glu-Val-Gly. The addition of γ-Glu-Val-Gly significantly enhanced thick flavour, aftertaste, and oiliness in the reduced-fat peanut butter. A kokumi peptide, γ-Glu-Val-Gly, can enhance thick flavour, aftertaste and oiliness in reduced-fat peanut butter. This suggests that addition of γ-Glu-Val-Gly can improve the flavour of low-fat foods.

Determination and quantification of the kokumi peptide, γ-glutamyl-valyl-glycine, in commercial soy sauces

Recent studies have demonstrated that kokumi substances, such as glutathione, are perceived through the calcium-sensing receptor (CaSR), and screening by CaSR assay and sensory evaluation has shown that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) is a potent kokumi peptide. In this study, the contents of γ-Glu-Val-Gly in six commercial brands of dark-coloured soy sauces, two brands of light-coloured soy sauce, and one brand of white soy sauce, were investigated by high performance liquid chromatography–tandem mass spectrometry (LC/MS/MS), followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate (AQC). The analyses indicated that γ-Glu-Val-Gly was present in all investigated soy sauces at concentrations ranging from 0.15 to 0.61mg/dl, demonstrating that it is widely distributed in soy sauces.

Determination and Quantification of γ-Glutamyl-valyl-glycine in Commercial Fish Sauces

It was recently reported that kokumi substances such as glutathione are perceived through the calcium-sensing receptor (CaSR). In addition, screening by the CaSR assay and sensory evaluation revealed that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) was a potent kokumi peptide. In this study, the quantities of γ-Glu-Val-Gly in various commercial fish sauces originating from Vietnam (Nuoc Mum), Thailand (Nampra), China (Yu-lu), Korea, Japan (Shottsuru and Ikanago-shoyu), and Italy (Garum) were investigated using a LC/MS/MS method followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate (AQC). The analyses revealed γ-Glu-Val-Gly at concentrations ranging from 0.04 to 1.26 mg/dL, indicating that γ-Glu-Val-Gly is widely distributed among various commercial fish sauces.

Kokumi substances, enhancers of basic tastes, induce responses in calcium-sensing receptor expressing taste cells.

Recently, we reported that calcium-sensing receptor (CaSR) is a receptor for kokumi substances, which enhance the intensities of salty, sweet and umami tastes. Furthermore, we found that several γ-glutamyl peptides, which are CaSR agonists, are kokumi substances. In this study, we elucidated the receptor cells for kokumi substances, and their physiological properties. For this purpose, we used Calcium Green-1 loaded mouse taste cells in lingual tissue slices and confocal microscopy. Kokumi substances, applied focally around taste pores, induced an increase in the intracellular Ca2+ concentration ([Ca2+]i) in a subset of taste cells. These responses were inhibited by pretreatment with the CaSR inhibitor, NPS2143. However, the kokumi substance-induced responses did not require extracellular Ca2+. CaSR-expressing taste cells are a different subset of cells from the T1R3-expressing umami or sweet taste receptor cells. These observations indicate that CaSR-expressing taste cells are the primary detectors of kokumi substances, and that they are an independent population from the influenced basic taste receptor cells, at least in the case of sweet and umami.