Characterization of GshAB of Tetragenococcus halophilus: a two-domain glutathione synthetase.

The γ-glutamyl tripeptide glutathione (γ-Glu-Cys-Gly) is a low molecular thiol that acts as antioxidant in response to oxidative stress in eukaryotes and prokaryotes. γ-Glutamyl dipeptides including γ-Glu-Cys, γ-Glu-Glu, and γ-Glu-Gly also have kokumi activity. Glutathione is synthesized by first ligating Glu with Cys by γ-glutamylcysteine ligase (Gcl/GshA), and then the resulting dipeptide γ-glutamylcysteine is ligated with Gly by glutathione synthetase (Gs/GshB). GshAB/GshF enzymes that contain both Gcl and Gs domains are capable of catalyzing both reactions. The current study aimed to characterize GshAB from Tetragenococcus halophilus after heterologous expression in Escherichia coli. The optimal conditions for GshAB from T. halophilus were pH 8.0 and 25 °C. The substrate specificity of the Gcl reaction of GshAB was also determined. GshAB has a high affinity to Cys. γ-Glu-Cys was the only dipeptide generated when Glu, Cys, Gly, and other amino acids were present in the reaction system. This specificity differentiates GshAB from T. halophilus from Gcl of heterofermentative lactobacilli and GshAB of Streptococcus agalactiae, which also use amino acids other than Cys as glutamyl-acceptor. Quantification of gshAB in cDNA libraries from T. halophilus revealed that gshAB was overexpressed in response to oxidative stress but not in response to acid, osmotic, or cold stress. In conclusion, GshAB in T. halophilus served as part of the oxidative stress response but this study did not provide any evidence for a contribution to the resistance to other stressors.Key points Glutathione synthesis in Tetragenococcus halophilus is carried out by the two-domain enzyme GshAB. GshAB is inhibited by glutathione and is highly specific for Cys as acceptor. T. halophilus synthesizes glutathione in response to oxidative stress.

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.

Supplementation effects of a kokumi substance, γ-Glu-Val-Gly, on the ingestion of basic taste solutions in rats.

In addition to the well-accepted taste receptors corresponding to the 6 basic taste qualities, sweet, salty, sour, bitter, umami, and fatty, another type of taste receptor, calcium-sensing receptor (CaSR), is located in taste bud cells. CaSR is called the kokumi receptor because its agonists induce koku (or kokumi), a Japanese word meaning the enhancement of flavor characteristics, such as thickness, mouthfulness, and continuity. Kokumi is an important factor in enhancing food palatability. γ-Glu-Val-Gly (EVG) is the most potent agonist of CaSR, which induces a strong kokumi flavor. However, no behavioral studies have been documented in animals using EVG. Here, we show that EVG at low concentrations that do not elicit a taste of its own enhances preferences for umami, fat, and sweet taste solutions in rats. An increased preference for inosine monophosphate (IMP) and Intralipos was the most dominant effect. NPS-2143, an antagonist of CaSR, abolished the additive effect of EVG on IMP and Intralipos solutions. These effects of EVG on taste stimuli are thought to occur in the oral cavity, because the effects of EVG were confirmed in a brief exposure test. The additive effects on IMP and Intralipos remained after the transection of the chorda tympani, indicating that these effects also occur in the palate and/or posterior part of the tongue. Moreover, the additive effects of EVG were verified in electrophysiological taste nerve responses. These results may partially provide the underlying mechanisms for EVG to induce kokumi flavor in humans.

Recent insights in flavor-enhancers: Definition, mechanism of action, taste-enhancing ingredients, analytical techniques and the potential of utilization.

The consumers' demand for clean-label food products, lead to the replacement of conventional additives and redesign of the production methods in order to adopt green processes. Many researchers have focused on the identification and isolation of naturally occurring taste and flavor enhancers. The term "taste enhancer" and "flavor enhancer" refer to umami and kokumi components, respectively, and their utilization requires the study of their mechanism of action and the identification of their natural sources. Plants, fungi and dairy products can provide high amounts of naturally occurring taste and flavor enhancers. Thermal or enzymatic treatments of the raw materials intensify taste and flavor properties. Their utilization as taste and flavor enhancers relies on their identification and isolation. All the above-mentioned issues are discussed in this review, from the scope of listing the newest trends and up-to-date technological developments. Additionally, the appropriate sensory analysis protocols of the naturally occurring taste-active components are presented. Moreover, future trends in using such ingredients by the food industry can motivate researchers to study new means for clean-label food production and provide further knowledge to the food industry, in order to respond to consumers' demands.

Current progress in kokumi-active peptides, evaluation and preparation methods: a review.

Kokumi is a complex sensation characterized by thickness, mouthfulness and continuity. Kokumi-active peptides, which are distributed in many kinds of food, induce a rich and long-lasting mouthfeel of food. Aimed to provide a comprehensive overview of kokumi peptides, this review covers the aspects of preparation and evaluation methods for kokumi peptides, kokumi receptor calcium-sensing receptor (CaSR), as well as structural features of kokumi peptides and derivatives. Apart from extraction and separation from natural and fermented food, preparation of kokumi peptides can be effectively obtained from enzymatic generation. Kokumi peptides are perceived by CaSR in taste cells and the proposed transduction pathway has been described. The evaluation on kokumi-inducing effect of peptides has employed a combination of sensory assessment and CaSR method. The discovered kokumi peptides mainly comprise glutamyl peptides, leucyl peptides and other peptides without specific features. Derivatives of amino acids and peptides including sulphur-containing amino acids, N-acyl-Tyr derivatives, N-acetylated amino acids and Maillard reaction products (MRPs) also work as kokumi enhancers. Based on the summarized developments, great sensory properties and bioactivities enable kokumi peptides as promising protein ingredients in future application.

Sensory-Guided Identification and Characterization of Kokumi-Tasting Compounds in Green Tea (Camellia sinensis L.).

The chemical substances responsible for the kokumi taste of green tea infusion are still unclear. Here, we isolated the kokumi compound-containing fractions from green tea infusion through ultrafiltration, and the major kokumi compounds were characterized as γ-Glu-Gln and γ-Glu-Cys-Gly (GSH) through ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS). The results indicated that peptides and amino acids were essential compounds in the kokumi-enriched fractions for conducting the sense of kokumi. L-theanine had an enhancing effect on the kokumi taste of green tea infusion, which was confirmed in the sensory reconstitution study. Thus, peptides, especially γ-Glu-Gln and GSH, are the major kokumi compounds in green tea infusion, which has the potential of improving the flavor of tea beverages.

Isolation, identification and sensory evaluation of kokumi peptides from by-products of enzyme-modified butter.

BACKGROUND: Enzyme-modified butter is used as a common raw material to obtain a natural milk flavor. Butter protein is a by-product in butter processing that can be used as substrate to produce taste-active peptides, which can create additional value and new application opportunities, making the method more environmentally friendly. RESULTS: Putative kokumi peptides from hydrolysates of protein by-products were isolated by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The isolated peptide fraction with the most pronounced kokumi taste was screened by sensory evaluation and electronic tongue analysis. Eleven peptides were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Six peptides were synthesized to verify their taste characteristics. Five synthetic peptides (FTKK, CKEVVRNANE, EELNVPG, VPNSAEER and YPVEPFTER) showed different intensity levels of kokumi taste. Of these peptides, the decapeptide CKEVVRNANE had the highest kokumi intensity. CONCLUSION: The newly identified kokumi peptides increased the kokumi taste intensity and showed some synergistic effect with umami taste. Both termini of the peptides seem to play an important role in taste characteristic. Glu residue at both termini can increase the kokumi taste intensity. This work indicated that it was feasible to produce kokumi peptides by enzymatic hydrolysis of the protein by-products of butter. © 2022 Society of Chemical Industry.

Characterization of γ-glutamyl cysteine ligases from Limosilactobacillus reuteri producing kokumi-active γ-glutamyl dipeptides.

γ-Glutamyl cysteine ligases (Gcls) catalyze the first step of glutathione synthesis in prokaryotes and many eukaryotes. This study aimed to determine the biochemical properties of three different Gcls from strains of Limosilactobacillus reuteri that accumulate γ-glutamyl dipeptides. Gcl1, Gcl2, and Gcl3 were heterologously expressed in Escherichia coli and purified by affinity chromatography. Gcl1, Gcl2, and Gcl2 exhibited biochemical with respect to the requirement for metal ions, the optimum pH and temperature of activity, and the kinetic constants for the substrates cysteine and glutamate. The substrate specificities of the three Gcls to 14 amino acids were assessed by liquid chromatography-mass spectrometry. All three Gcls converted ala, met, glu, and gln into the corresponding γ-glutamyl dipeptides. None of the three were active with val, asp, and his. Gcl1 and Gcl3 but not Gcl2 formed γ-glu-leu, γ-glu-ile, and γ-glu-phe; Gcl3 exhibited stronger activity with gly, pro, and asp when compared to Gcl2. Phylogenetic analysis of Gcl and the Gcl-domain of GshAB in lactobacilli demonstrated that most of Gcls were present in heterofermentative lactobacilli, while GshAB was identified predominantly in homofermentative lactobacilli. This distribution suggests a different ecological role of the enzyme in homofermentative and heterofermentative lactobacilli. In conclusion, three Gcls exhibited similar biochemical properties but differed with respect to their substrate specificity and thus the synthesis of kokumi-active γ-glutamyl dipeptides. KEY POINTS: • Strains of Limosilactobacillus reuteri encode for up to 3 glutamyl cysteine ligases. • Gcl1, Gcl2, and Gcl3 of Lm. reuteri differ in their substrate specificity. • Gcl1 and Gcl3 produce kokumi-active dipeptides.

N-terminal [Glu]3 moiety of γ-glutamyl peptides contributes largely to the activation of human calcium-sensing receptor, a kokumi receptor.

γ-glutamyl peptides have been suggested to impart kokumi properties to foods by activating human calcium-sensing receptor (hCaSR). In this study, the relationship between γ-glutamyl peptide structure and hCaSR activity was systematically analyzed using γ-[Glu](n=0-4)-α-[Glu](n=0-3)-Tyr. Our results suggest that N-terminal [Glu]3 moiety is very important for hCaSR activities of γ-glutamyl peptides.

Quantification of the kokumi peptide, γ-glutamyl-valyl-glycine, in cheese: Comparison between cheese made from cow and ewe milk.

Recent studies have shown that several types of cheese contain kokumi γ-glutamyl dipeptides, and the kokumi tripeptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly), is a component of various fermented foods. The quantification of γ-Glu-Val-Gly in various types of cheese was herein conducted by HPLC-tandem mass spectrometry followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate. The γ-Glu-Val-Gly concentrations were between 0.35 and 0.59 µg/g in cheese made from ewe milk, but were not detected in cheese made from cow milk. The amino acid sequences of major milk proteins showed that the ß-caseins of sheep had the Val-Gly sequence at the 9-10 position, whereas ß-caseins of cows contained a Pro-Gly sequence at the same position. The Val-Gly sequence was absent in other caseins of sheep and cattle. These results suggest that the different γ-Glu-Val-Gly concentrations present in cheese made from cow and ewe milk are due to differences in the amino acid sequences of caseins.

Isolation and characterization of key contributors to the "kokumi" taste in soybean seeds.

The water extract of soybean seeds (Glycine max (L.) Merr.) is nearly tasteless, but "kokumi" taste sensation was confirmed upon addition of a basic umami solution containing glutamic acid, inosine monophosphate, and sodium chloride. To identify the key contributors to the "kokumi" taste sensation in soybean seeds, sensory-guided fractionation, taste sensory analyses, and LC-MS/MS analyses were utilized. γ-glutamyl-tyrosine and γ-glutamyl-phenylalanine were identified as contributors to "kokumi taste"; specifically, these γ-glutamyl peptides imparted the "kokumi" taste sensation at a low taste threshold in a basic umami solution. Raffinose and stachyose, which are sufficiently present in soybean seeds, exhibited a synergistic effect in regard to the enhanced "kokumi" taste sensation of γ-glutamyl peptides. This is the first report that the combined use of γ-glutamyl peptides and oligosaccharides can increase the "kokumi" intensity, which suggests that soybean extracts or soymilk can be used to enhance the "kokumi" taste sensation in food products.

Contribution of crosslinking products in the flavour enhancer processing: the new concept of Maillard peptide in sensory characteristics of Maillard reaction systems.

In this study, the flavour-enhancing properties of the Maillard reaction products (MRPs) for different systems consisted of different peptides (sunflower, SFP; corn, CP and soyabean SP) with, xylose and cysteine were investigated. Maillard systems from peptides of sunflower, corn and soyabean with xylose and cysteine were designated as PXC, MCP and MSP, respectively. The Maillard systems were prepared at pH of 7.4 using temperature of 120C for 2 h. Results showed that all systems were significantly different in all sensory attributes. The highest scores for mouthfulness and continuity were observed for MCP with the lowest peptides distribution between 1000 and 5000 Da, known as Maillard peptide. This revealed that the MCP with the lowest Maillard peptide content had the strongest "Kokumi" effect compared to the other MRPsand demonstrated that "kokumi effect" of MRPs was contributed by not only the "Maillard peptide" defined by the molecular weight (1000-5000 Da). Results on sensory evaluation after fractionation of PXC followed by enzymatic hydrolysis showed no significant differences between PXC, P-PXC and their hydrolysates. This observation therefore confirmed that the presence of other contributors attributed to the "Kokumi" effect rather than the Maillard peptide. It can be deduced that the unhydrolyzed crosslinking products might have contributed to the "Kokumi" effect of MRPs. The structures of four probable crosslinking compounds were proposed and the findings have provided new insights in the sensory characteristics of xylose, cysteine and sunflower peptide MRPs.

Discovery of kokumi peptide from yeast extract by LC-Q-TOF-MS/MS and sensomics approach.

BACKGROUND: Yeast extract can impart thickness, complexity and long-lasting taste impression, coined kokumi taste, to blank chicken broth. In this research, the kokumi-active peptide in yeast extract was discovered by ultrafiltration, liquid chromatographic and quadrupole-time-of-flight-tandem mass spectrometric technologies. Furthermore, the sensory characters of these peptides were evaluated by a sensomics approach. RESULTS: A total of 10 kokumi peptides were identified from yeast extract. They were γ-Glu-Cys-Gly, γ-Glu-Leu, γ-Glu-Val, γ-Glu-Tyr, Leu-Lys, Leu-Gln, Leu-Ala, Leu-Glu, Leu-Thr and Ala-Leu. Apart from the well-known kokumi-active glutathione and γ-glutamyl dipeptides, five leucyl dipeptides were first proposed having kokumi activity. Among them, Ala-Leu was found to have the highest kokumi threshold concentration (1.5 mmol L(-1) ) in the blank chicken broth, while Leu-Glu was the lowest (0.3 mmol L(-1) ). A subsequent dose-response experiment indicated that the bitter-tasting leucyl dipeptides could impart kokumi taste to chicken broth at low concentrations (less than bitter threshold concentrations). Interestingly, the kokumi sensation began to decrease when such peptides exceeded the threshold concentration by approximately 16-fold in the blank chicken broth. CONCLUSION: Key kokumi-active fractions were purified from yeast extract. Among them, ten important kokumi peptides from yeast extract were identified.

The contribution of sturgeon species and processing conditions on caviar texture, metabolomic profile and sensory traits.

A comprehensive evaluation of the attributes that determine caviar quality, including sturgeon species and processing methods, is still lacking in the scientific literature. In this study, eight types of caviar, obtained from six different sturgeon species and produced using either salt or a combination of salt and sodium tetraborate (E285) with pasteurization, were characterized through untargeted metabolomics, sensory analysis, and texture evaluation. The results showed that Huso huso caviar was rich in gamma-glutamyl peptides (contributing to a kokumi taste), while the other sturgeon species were primarily distinguished by lipids (mainly glycerophospholipids) and nucleotides (such as AMP, inosinic acid, and other metabolites driving umami taste). Moreover, untargeted metabolomics revealed the technological effects of salting and pasteurization. Sensory analysis identified crunchiness, color homogeneity, and darkness as the main traits differentiating the products. Butter, hazelnut, and briny flavours were also key characteristics of the caviar.

A Novel Compound with Kokumi Properties: Enzymatic Preparation and Taste Presentation Evaluation of N-Lauroyl Phenylalanine.

To satisfy the demands of the food industry, innovative flavor enhancers need to be developed urgently to increase the food flavor. In our work, N-lauroyl phenylalanine (LP) was prepared from phenylalanine (l-Phe) and lauric acid (Lau) in water through the use of commercial enzymes (Promatex, Sumizyme FP-G, and Trypsin), and its flavor-presenting properties and mechanism were investigated. The highest LP yields obtained under one-factor optimized conditions were 61.28, 63.43, and 77.58%, respectively. Sensory assessment and an e-tongue test revealed that 1 mg/L LP enhanced the kokumi, saltiness, and umami of the simulated chicken broth solution and attenuated the bitterness of the l-isoleucine solution. The molecular simulation results suggested that the mechanisms of LP enhancement of kokumi and umami were related to hCaSR and hT1R1-hT1R3, and that hydrophobic forces and hydrogen bonds were involved in the binding of LP to taste receptors. The results implied that LP is a potential flavor enhancer for food applications.

Enzymatic synthesis and sensory evaluation of the novel kokumi compound N-butyryl phenylalanine.

In this study, food-grade glutamine transaminase (TGase) was utilized for the green-catalyzed preparation of N-butyryl amino acids. For improving the reusability of the enzyme preparation, immobilized TG enzyme (94.23% immobilization rate) was prepared. Furthermore, the yield of N-butyryl phenylalanine (BP) synthesized by TGase was obtained as 20.73% by one-factor experiment. The BP synthesis yield of immobilized TGase was 95.03% of that of TGase and remained above 60% of the initial enzyme activity after five runs. The sensory evaluation and E-tongue results showed that the addition of BP significantly increased the umami, saltiness, and richness intensities of the samples, and decreased the intensities of sourness, bitterness, and aftertaste-B. The molecular docking results indicated that hydrogen bonding dominated the binding of BP to taste receptors in the taste presentation mechanism of BP. These results confirmed the potential of BP as a flavor enhancer with promising applications in the food industry.

Exploring the Relationships Between Bacterial Community, Taste-Enhancing Peptides and Aroma in Thai Fermented Fish (Pla-ra).

As a traditional Thai condiment, Pla-ra is used to add flavor and richness to dishes. Nine treatment combinations of Pla-ra formulations created from 3 types of fish (Mor fish, Kradee fish, and Mor + Kradee fish) and 4 different carbohydrate sources (none, rice bran, roasted rice, and rice bran─roasted rice mixture) were studied through a 12 month fermentation period (1, 3, 5, 7, 8, 9, 10, 11, and 12 months). 16S rRNA Next Generation Sequencing (NGS) and LC-MS/MS techniques were used to analyze the microbial diversity and identify taste-enhancing peptides. Descriptive sensory analysis was performed on the extracts of the 108 Pla-ra samples mixed in a model broth. Koku perception and saltiness-enhancing attributes were clearly perceived and dominant in all samples, even though glutamyl peptides, including γ-Glu-Val-Gly, were found at subthreshold levels. The samples from mixed fish and Mor fish fermented with roasted ground rice and rice bran for 12 months had the most typical Pla-ra odors and tastes and had high taste-enhancing activities. NGS analysis revealed the presence of bacteria containing a large number of protease and aminopeptidase genes in the samples. Bacillus spp., Gallicola spp., and Proteiniclasticum spp. correlated well with the generation of glutamyl and arginyl peptides and typical odors in the samples. These results confirmed the typical sensory quality of Pla-ra depended on protein sources, carbohydrate sources, and bacteria communities. Further optimization of the microbial composition found could lead to the development of starter cultures to control and promote flavor development in fermented fish products.

Sensoproteomic Characterization of Lactobacillus Johnsonii-Fermented Pea Protein-Based Beverage: A Promising Strategy for Enhancing Umami and Kokumi Sensations while Mitigating Bitterness.

This study investigated the mechanism underlying the flavor improvement observed during fermentation of a pea protein-based beverage using Lactobacillus johnsonii NCC533. A combination of sensomics and sensoproteomics approach revealed that the fermentation process enriched or generated well-known basic taste ingredients, such as amino acids, nucleotides, organic acids, and dipeptides, besides six new taste-active peptide sequences that enhance kokumi and umami notes. The six new umami and kokumi enhancing peptides, with human recognition thresholds ranging from 0.046 to 0.555 mM, are produced through the degradation of Pisum sativum's storage protein. Our findings suggest that compounds derived from fermentation enhance umami and kokumi sensations and reduce bitterness, thus improving the overall flavor perception of pea proteins. In addition, the analysis of intraspecific variations in the proteolytic activity of L. johnsonii and the genome-peptidome correlation analysis performed in this study point at cell-wall-bound proteinases such as PrtP and PrtM as the key genes necessary to initiate the flavor improving proteolytic cascade. This study provides valuable insights into the molecular mechanisms underlying the flavor improvement of pea protein during fermentation and identifies potential future research directions. The results highlight the importance of combining fermentation and senso(proteo)mics techniques in developing tastier and more palatable plant-based protein products.

New Implications of Metabolites and Free Fatty Acids in Quality Control of Crossbred Wagyu Beef during Wet Aging Cold Storage.

Efficient cold-chain delivery is essential for maintaining a sustainable global food supply. This study used metabolomic analysis to examine meat quality changes during the "wet aging" of crossbred Wagyu beef during cold storage. The longissimus thoracic (Loin) and adductor muscles (Round) of hybrid Wagyu beef, a cross between the Japanese Black and Holstein-Friesian breeds, were packaged in vacuum film and refrigerated for up to 40 days. Sensory evaluation indicated an increase in the umami and kokumi taste owing to wet aging. Comprehensive analysis using gas chromatography-mass spectrometry identified metabolite changes during wet aging. In the Loin, 94 metabolites increased, and 24 decreased; in the Round, 91 increased and 18 decreased. Metabolites contributing to the umami taste of the meat showed different profiles during wet aging. Glutamic acid increased in a cold storage-dependent manner, whereas creatinine and inosinic acid degraded rapidly even during cold storage. In terms of lipids, wet aging led to an increase in free fatty acids. In particular, linoleic acid, a polyunsaturated fatty acid, increased significantly among the free fatty acids. These results provide new insight into the effects of wet aging on Wagyu-type beef, emphasizing the role of free amino acids, organic acids, and free fatty acids generated during cold storage.

Synthesis of taste active γ-glutamyl peptides with pea protein hydrolysate and their taste mechanism via in silico study.

Pea (Pisum sativum L.) protein hydrolysate (PPH) has a bitter taste, which has limited its use in food industry. γ-Glutamylation is used to debitter PPH. Results showed that the bitterness of PPH was decreased significantly due to the formation of γ-glutamyl peptides, including 16 γ-[Glu](n=1/2)-amino acids (AAs) and 8 newly discovered γ-glutamyl tripeptides (γ-Glu-Asn-Phe, γ-Glu-Leu-Val, γ-Glu-Leu-Tyr, γ-Glu-Gly-Leu, γ-Glu-Gly-Phe, γ-Glu-Gly-Tyr, γ-Glu-Val-Val, and γ-Glu-Gln-Tyr). Their total production concentrations were 27.25 µmol/L and 77.76 µmol/L, respectively. The γ-Glu-AA-AAs presented an umami-enhancing, salty-enhancing, and kokumi taste when their concentration reached 1.67 ± 0.20 âˆ¼ 2.07 ± 0.20, 1.65 ± 0.25 âˆ¼ 2.29 ± 0.45 and 0.68 ± 0.19 âˆ¼ 1.03 ± 0.22 mmol/L, respectively. The γ-Glu-AA-AAs exhibited a kokumi taste by entering the Venus flytrap (VFT) of the calcium-sensing receptor and interacting with Ser147, Ala168, and Ser170. γ-Glu-AA-AAs can enhance the umaminess of Monosodium Glutamate (MSG) as they can enter the binding pocket of the taste receptor type 1 subunit 3 (T1R3)-MSG complex.