Kokumi Peptides in Salt Reduction: Preparation, Evaluation Methods, Perceptual Mechanisms, and Prospects for Food Applications

ABSTRACT

Excessive dietary salt intake poses significant health risks and is closely associated with the pathogenesis of hypertension and cardiovascular diseases. Developing strategies that achieve salt reduction without compromising saltiness perception has become a major focus in food science and public health research. The study and application of kokumi peptides provide a novel approach to achieving “salt reduction without loss of saltiness.” Although kokumi substances themselves are usually tasteless or only weakly flavored, they can markedly enhance the intensity and persistence of other taste modalities within complex food systems, thereby improving overall flavor perception. Kokumi peptides are widely derived from natural food sources and can effectively reduce sodium intake without sacrificing palatability, showing remarkable potential for food applications. This review systematically summarizes the primary preparation methods, sensory evaluation techniques, and molecular mechanisms underlying the salt‐enhancing properties of kokumi peptides. Particular emphasis is placed on calcium‐sensing receptor (CaSR)‐mediated taste sensation and its potential crosstalk with key salt‐taste transduction pathways, including the epithelial sodium channel (ENaC), transient receptor potential vanilloid 1 (TRPV1), and transmembrane channel‐like protein 4 (TMC4). Moreover, the review discusses the potential applications of kokumi peptides in salt‐reduced foods and seasonings, as well as their roles in flavor enhancement, physiological activity, and multisensory synergy. Finally, the current challenges in kokumi peptide research are identified, and promising prospects for health‐oriented food innovation and salt‐reduction strategy optimization are proposed.

1. Introduction

Excessive dietary sodium intake remains one of the leading modifiable risk factors for hypertension, cardiovascular disease, and stroke, with global average salt consumption estimated at 9–12 g/day, approximately double the World Health Organization's recommended upper limit of < 5 g/day (World Health Organization ²⁰²⁵). Despite decades of public health initiatives, meaningful sodium reduction in processed foods and prepared meals has proven elusive. Conventional strategies, such as direct reduction, partial replacement with potassium, magnesium, or calcium salts, and physical modification of NaCl crystals, frequently introduce bitterness, metallic off‐notes, or a perceptible lack of flavor, mouthfulness, and continuity, resulting in poor consumer acceptance and limited industrial adoption (Hurst et al. 2021; Antúnez et al. ²⁰¹⁸; Pateiro et al. ²⁰²¹; Greer et al. ²⁰²⁰). This persistent sensory barrier underscores the urgent need for a paradigm shift from simple salt substitution toward holistic flavor modulation that preserves, or even enhances, overall palatability at lower sodium levels.

In this context, kokumi peptides offer a novel and effective approach to sodium reduction in foods. Flavor perception is an inherently multisensory phenomenon arising from the integration of taste, aroma, and texture (L. Huang et al. ²⁰²⁴). It is characterized by attributes such as complexity, mouthfulness, and persistence, which together determine consumer liking and acceptance. Taste perception encompasses five basic modalities, namely, sweet, sour, salty, bitter, and umami, elicited by nonvolatile compounds interacting with specific taste receptors in the oral cavity (P. Wang, Ye, et al. ²⁰²⁴). Aroma perception is mediated by volatile compounds that stimulate the olfactory system and can occur either orthonasally before ingestion or retronasally during mastication and swallowing, with retronasal aroma playing a particularly important role in shaping overall flavor perception (Frankin et al. 2023; Gonzalez Viejo et al. ²⁰²⁵). Texture contributes tactile sensations that influence mouthfeel and flavor continuity, thereby enhancing perceptions of richness and mouthfulness within food matrices (Koehler et al. 2024). Within this multisensory framework, the Japanese concept of koku describes the depth, roundness, and lingering richness commonly associated with slow‐cooked, fermented, or fat‐rich foods such as curry, cheese, and coffee. Its perceptual counterpart, kokumi, refers to sensations of enhanced mouthfulness, thickness, and flavor continuity that extend beyond the five basic tastes (Nishimura and Kuroda 2019). Compounds responsible for eliciting these sensations, collectively referred to as kokumi substances, are typically tasteless or only weakly flavored, yet they substantially enhance and prolong other taste modalities through cross‐modal sensory integration. Among these compounds, kokumi peptides, predominantly γ‐glutamyl dipeptides and tripeptides, have emerged as naturally occurring contributors to flavor enhancement with promising potential for sodium reduction applications. Mechanistically, kokumi‐active compounds can allosterically activate the calcium‐sensing receptor (CaSR) expressed in taste bud cells, thereby modulating intracellular signaling pathways involved in saltiness, umami, and sweetness perception (Ohsu et al. 2010; Maruyama et al. ²⁰¹²; Amino et al. ²⁰¹⁶) (Figure 1). Through this mechanism, kokumi peptides act as sensory amplifiers, enhancing the perceived intensity, continuity, and mouthfulness of taste without imparting their own distinct flavor. In addition, they synchronize gustatory and olfactory processing, reinforcing retronasal aroma and textural cues to produce a more harmonious and persistent flavor experience (Ployon et al. 2017; Ai and Han ²⁰²²). This capacity to modulate flavor across multiple sensory channels represents a transition from unimodal taste enhancement toward multisensory flavor engineering.

FIGURE 1.

Kokumi substances enhance overall complexity, mouthfulness, and continuity by activating the calcium‐sensing receptor (CaSR), which influences taste, aroma, mouthfeel, and texture.

Recent advances in enzymatic transpeptidation, microbial fermentation, peptidomics, and in silico modeling have accelerated the discovery and preparation of kokumi peptides with targeted flavor‐modulating properties (Suzuki et al. 2017; Yi et al. ²⁰²⁵; J. Yang et al. ²⁰¹⁹). These peptides have been identified in diverse food‐derived sources and matrices, including edible mushrooms, oysters, fermented dairy products, dry‐cured meat products, soy protein hydrolysates, yeast extracts, and seafood‐derived hydrolysates (Du et al. 2024; Fu et al. ²⁰²⁴; Jia et al. ²⁰²²; Ji et al. ²⁰²⁵; Lao et al. ²⁰²⁴). For example, γ‐glutamyl peptides derived from soybean or yeast proteins, such as γ‐Glu‐Gly‐Pro‐Phe and γ‐Glu‐Ala‐Phe‐Phe, have shown pronounced saltiness‐enhancing activity and CaSR‐associated kokumi activity (J. Liu et al. ²⁰²⁴). However, kokumi‐mediated saltiness enhancement should not be interpreted as a single‐receptor phenomenon. Current evidence supports CaSR as the most established receptor associated with kokumi perception, whereas epithelial sodium channel (ENaC), transient receptor potential vanilloid 1 (TRPV1), and transmembrane channel‐like protein 4 (TMC4) are more closely related to salt‐taste transduction and may contribute to peptide‐mediated saltiness enhancement in a peptide‐, concentration‐, and matrix‐dependent manner. Therefore, a clearer integration of peptide preparation, sensory and instrumental evaluation, receptor‐level mechanisms, and food application is needed to understand how kokumi peptides can be rationally used in sodium‐reduced foods. This review systematically examines the following aspects: (i) the main strategies for kokumi peptide preparation; (ii) complementary approaches for their characterization and evaluation; (iii) mechanisms underlying saltiness enhancement and receptor‐level interactions; and (iv) the applications, prospects, and challenges associated with their use in low‐sodium food development. By integrating recent scientific findings with practical food innovation, this work aims to support the development of healthier, consumer‐preferred sodium‐reduced foods without compromising flavor quality.

2. Preparation of Kokumi Peptides

Kokumi peptides have become increasingly important in the modern food industry, as single‐dimensional flavor profiles can no longer satisfy consumers' expectations for rich and layered taste experiences (Hossain et al. 2025; Kitajima et al. ²⁰²⁶). Extracting kokumi peptides from natural products and fermented foods is an effective approach to obtaining high‐flavor‐impact compounds (Shibata et al. 2017). Previous studies have identified multiple kokumi peptides in yeast extracts, including eleven γ‐glutamyl peptides, such as γ‐Glu‐Val, γ‐Glu‐Leu, and γ‐Glu‐Ile. These compounds significantly enhance the richness, complexity, and persistence of chicken soup flavor (J. Liu et al. ²⁰¹⁵). However, traditional extraction methods are often limited by raw material availability, process complexity, and low yields, restricting industrial‐scale production. To overcome these challenges and improve saltiness perception through kokumi enhancement, researchers are exploring diverse sources and more efficient preparation methods. Kokumi peptides are primarily derived from animal, plant, and microbial origins. Animal‐derived peptides are obtained from protein‐rich meats, fish, and their by‐products; plant‐derived peptides are extracted mainly from legumes; and microbe‐derived peptides arise from fermentation processes involving edible fungi and yeasts. In food applications, kokumi peptides can enhance thickness, mouthfulness, and continuity, suggesting their potential as flavor‐modifying ingredients in reduced‐salt and fermented foods (Yamamoto and Inui‐Yamamoto ²⁰²³). Current preparation methods include extraction, enzymatic synthesis, microbial fermentation, and chemical synthesis (Figure 2). The combined use of these methods provides a solid technical foundation for applying kokumi peptides in flavor enhancement and the development of health‐oriented food products.

FIGURE 2.

Schematic representation of kokumi peptide preparation methods.

2.1. Extraction Method

Extraction remains one of the principal approaches for preparing kokumi peptides from natural and fermented materials. In most cases, however, kokumi peptide preparation does not rely on a single extraction step but rather on a multistage workflow that combines membrane‐based fractionation and chromatographic purification, including reverse osmosis, ultrafiltration, ion‐exchange chromatography, gel filtration, and high‐performance liquid chromatography (HPLC), to enrich low‐abundance peptides from complex food matrices (H. Wang, Suo, et al. ²⁰²²). Representative early work demonstrated that garlic and onion were important sources of kokumi substances, particularly γ‐glutamyl peptides (Ueda et al. 1990; Ueda et al. ¹⁹⁹⁴). More recent studies have expanded kokumi peptide extraction to a broader range of food matrices and increasingly adopted integrated purification workflows. For example, kokumi‐active γ‐glutamyl peptides have been identified in cheese matrices following chromatographic purification (Toelstede and Hofmann 2009), while novel kokumi peptides from yeast extract and Wuding chicken soup have been obtained through workflows combining ultrafiltration and gel chromatography (Lao et al. 2024; C. Yang, Du, et al. ²⁰²⁵).

Overall, these studies suggest a methodological evolution from predominantly sensory‐guided fractionation toward integrated extraction–purification–identification workflows that combine sensory validation with instrumental analysis to discover and characterize kokumi‐active peptides from diverse food matrices. Nevertheless, extraction‐based approaches remain labor‐intensive and time‐consuming because multiple separation and purification steps are usually required to isolate trace peptides from highly complex matrices. In addition, many studies still focus primarily on identifying peptides with general kokumi characteristics, whereas targeted extraction and screening of peptides with clear salt‐enhancing performance in reduced‐sodium food systems remain relatively limited (J. Li et al. ²⁰²⁵). Therefore, future work should place greater emphasis on coupling efficient purification workflows with high‐throughput peptidomics and bioinformatics‐assisted screening to improve discovery efficiency and better support the practical development of kokumi peptides as sodium‐reduction ingredients (C. Yang, Tang, et al. ²⁰²⁶).

2.2. Enzymatic Synthesis

Enzymatic synthesis is an important strategy for preparing kokumi peptides, especially γ‐glutamyl peptides. This route is mainly based on γ‐glutamyl transfer reactions catalyzed by microbial γ‐glutamyl transpeptidase (GGT; EC 2.3.2.2) or l‐glutaminase (EC 3.5.1.2) with transpeptidation activity. These enzymes can use glutamine, glutathione (GSH), or related γ‐glutamyl compounds as γ‐glutamyl donors and transfer the γ‐glutamyl moiety to amino acid or peptide acceptors, thereby forming new γ‐glutamyl bonds (J. Yang et al. ²⁰¹⁹; Saini et al. ²⁰²¹). Compared with direct extraction from natural materials, enzymatic synthesis offers milder reaction conditions and better controllability for producing selected γ‐glutamyl peptides, although the product profile depends strongly on the enzyme source, donor–acceptor combination, and reaction conditions. In this γ‐glutamyl transfer‐based route, the contribution to flavor improvement is mainly linked to the formation of kokumi‐active γ‐glutamyl peptides. For example, J. Yang et al. (2022) showed that γEL, γEV, and γEγEV significantly enhanced the umami perception of monosodium glutamate and stimulated the umami receptor. In another study, GGT treatment of porcine hemoglobin hydrolysates generated γ‐glutamyl peptides and reduced the bitter sensory score from 7.18 to 5.72 while increasing the umami score from 4.18 to 5.82, indicating that enzymatically generated γ‐glutamyl peptides can improve flavor balance in specific food systems (Cheng et al. 2025). These findings support the potential of enzymatic synthesis for preparing kokumi‐active peptides for flavor enhancement and sodium‐reduction applications. Nevertheless, this approach still faces practical limitations, including enzyme cost, substrate consumption, by‐product formation, and the limited availability of food‐grade biocatalysts suitable for industrial manufacture. Future work should therefore focus on improving catalytic selectivity, simplifying purification, and validating the performance of synthesized kokumi peptides in reduced‐sodium food models.

2.2.1. Major Enzymes and Their Functions

2.2.1.1. GGT

GGT is widely distributed in microorganisms, plants, and animals. It catalyzes the transfer of a γ‐glutamyl group from donors such as glutamine or GSH to amino acid or peptide acceptors, forming new γ‐glutamyl bonds under mild, ATP‐independent conditions. Sayed et al. (2010) studied GGT from Escherichia coli K‐12, which synthesized 110 mM γ‐glutamylglutamine with an 88% conversion rate under optimal conditions (250 mM l‐glutamine, 1.1 U/mL enzyme, pH 10.5, 37°C, 7 h). GGT‐catalyzed reactions not only yield flavor‐enhancing peptides but also mask bitterness from amino acids such as valine. GGTs have been isolated from E. coli (Sayed et al. 2010), Bacillus amyloliquefaciens (L. Cao et al. ²⁰²³), horse kidney (Bruni et al. 2020), maize (Masi et al. 2007), and bovine milk (L. Cao et al. ²⁰²¹), with marked differences in substrate specificity and activity. For instance, the enzyme derived from B. amyloliquefaciens shows higher transpeptidation activity than that from bovine milk, demonstrating its potential advantages for industrial applications.

Immobilizing GGT on suitable carriers enhances its stability and reusability, enabling efficient continuous synthesis of γ‐glutamyl amino acids. However, hydrolytic side reactions can reduce yield, and microbial GGT safety requires assessment. Future work should focus on developing enzyme variants with high transpeptidation and low hydrolytic activity, proven safety, and strong process adaptability.

2.2.1.2. l‐Glutaminase (EC 3.5.1.2)

Traditionally, l‐glutaminase is known for hydrolyzing l‐glutamine to glutamate and ammonia (Suzuki et al. 2017). Recent studies, however, show that some microbial l‐glutaminases also exhibit γ‐glutamyl transpeptidation activity, enabling γ‐glutamyl peptide synthesis. Using beef protein hydrolysates as substrates, l‐glutaminase produced 15 γ‐glutamyl amino acids and 10 γ‐glutamyl tripeptides (140–170 mg/g yield) under mild conditions (pH 10, 37°C) (P. Chen, Zeng, et al. ²⁰²⁴), significantly enhancing umami, saltiness, and mouthfulness while reducing bitterness. l‐Glutaminase exhibits both hydrolytic and transpeptidation activities. Early studies showed that l‐glutaminase from Aspergillus oryzae can catalyze the formation of γ‐glutamyl dipeptides such as γ‐Glu‐Phe, while enzymes from B. amyloliquefaciens demonstrate stronger transpeptidation capacity, enabling the synthesis of polymeric γ‐glutamyl peptides (γ _n ‐Phe/Val/Met, n ≤ 5) under alkaline conditions (pH 10.0, 37°C, 3 h)—a transformation difficult to achieve with conventional GGT‐based systems (Tomita et al. 1989; Suzuki et al. ²⁰¹⁷; J. Yang et al. ²⁰¹⁸). These catalytic properties have practical applications in food protein hydrolysates, including defatted peanut meal and soybean protein hydrolysates, where γ‐glutamylation reduces bitterness and enhances umami and saltiness. The effect can be further amplified by supplementation with l‐glutamine (5%, w/w) as the γ‐glutamyl donor (Tu et al. 2023). Specific γ‐glutamyl peptides, such as γ‐Glu‐Gly‐Pro‐Phe and γ‐Glu‐Ala‐Phe‐Phe, have been structurally characterized and sensorially validated to enhance salty taste (Yi et al. 2025). Optimization of fermentation conditions can effectively enhance the production and activity of l‐glutaminase. However, the optimal reaction pH of this enzyme is typically alkaline (approximately pH 10), which is incompatible with the pH range of most food systems and thus limits its direct application in flavor enhancement. Improving catalytic efficiency under near‐neutral conditions is therefore critical for broader application in the food industry and may be achieved through protein engineering to modify enzymatic properties or by screening novel enzyme sources that balance hydrolytic and transpeptidation activities while being more suitable for food‐related conditions.

2.2.1.3. Other Enzyme Systems

Beyond GGT and l‐glutaminase, other enzymes and microbial pathways contribute to kokumi peptide biosynthesis in fermentation systems. γ‐Glutamylcysteine ligase (GCL; EC 6.3.2.2), a key enzyme in GSH synthesis, catalyzes dipeptide formation (e.g., γ‐Glu‐Ile, γ‐Glu‐Cys) during sourdough fermentation by Lactobacillus reuteri (Yan et al. 2018). Genomic analysis confirms the presence of GCL genes but the absence of GSH synthetase and GGT, underscoring GCL's importance in γ‐glutamyl dipeptide formation. Transglutaminase (TGase) has also been used to introduce γ‐glutamyl linkages in protein hydrolysates. When applied to egg white peptides, TGase catalyzed the formation of multiple γ‐glutamyl dipeptides and tripeptides, reducing bitterness and astringency while enhancing umami, sweetness, saltiness, and kokumi‐related richness (J. Liu, Wang, et al. ²⁰²⁵). Further study of enzyme specificity, mechanism, and function in diverse fermentation systems will facilitate the rational design of new biosynthetic pathways for kokumi peptide production.

2.3. Microbial Fermentation Method

The microbial fermentation method is an important biotechnological approach for synthesizing kokumi‐active compounds under controlled conditions through the endogenous metabolic pathways and enzymatic activities of specific microorganisms. Its main strengths are its mild reaction conditions, compatibility with complex food matrices, and ability to generate kokumi‐active γ‐glutamyl peptides in situ during fermentation; moreover, the γ‐glutamyl peptide profile can be modulated through strain selection and process control, which makes this approach attractive for the development of naturally derived flavor enhancers and reduced‐sodium seasonings (Toelstede and Hofmann 2009; Zhao et al. ²⁰¹⁶; Valerón et al. ²⁰²³). Evidence from cheese, fermented seasonings, sourdough, and yeast systems suggests that microorganisms can actively shape, rather than merely release, kokumi‐related γ‐glutamyl peptides in complex food matrices, although the resulting peptide profile depends strongly on the microbial species and the fermentation substrate. For example, Penicillium roquefortii was shown to produce and secrete γ‐glutamyl peptides in cheese‐related systems, γ‐Glu‐Val‐Gly was identified in commercial fish sauces and soy sauces, sourdough fermented with L. reuteri contained higher concentrations of taste‐active γ‐glutamyl dipeptides than chemically acidified controls, and Saccharomyces cerevisiae was shown to form γ‐glutamyl peptides such as γ‐Glu‐Val and γ‐Glu‐Val‐Gly through more than one biosynthetic route (Zhao and Gänzle 2016; Kuroda et al. ^{2012, 2013}; Sofyanovich et al. ²⁰¹⁹).

Despite these strengths, the current literature also reveals important limitations. Fermentation‐derived kokumi peptide formation is strongly dependent on strain, substrate, and process conditions, and the γ‐glutamyl peptide profile can change substantially with fermentation time, pH, and redox‐related parameters, which makes process standardization difficult (Valerón et al. 2023). More importantly, most published studies in this area remain descriptive, focusing mainly on peptide occurrence and quantification in fermented foods rather than on scalable production or purification. As a result, quantitative information on peptide yield, substrate use, and peptide recovery is still limited. Because direct engineering studies on fermentation‐derived kokumi peptides are still scarce, current process‐level lessons are drawn largely from the broader microbial GSH literature, where low production levels and metabolic constraints remain important barriers, although recent studies in S. cerevisiae also show that strain improvement and metabolic engineering can substantially enhance GSH biosynthesis (Z. Hu et al. ²⁰²⁵; Nho et al. ²⁰²⁵). Therefore, future work should move beyond occurrence‐focused studies and place greater emphasis on precision fermentation, strain engineering, and validation in reduced‐sodium food models so that microbial fermentation can be developed into a more efficient and industrially relevant route for producing kokumi‐active flavoring ingredients.

2.4. Chemical Synthesis Method

Chemical synthesis, predominantly solid‐phase peptide synthesis (SPPS) using Fmoc or Boc strategies, remains indispensable for structure–activity relationship studies and the preparation of well‐defined reference peptides. A key challenge lies in the selective formation of γ‐glutamyl linkages rather than conventional α‐linkages, which is commonly addressed through orthogonal protection of the α‐carboxyl group of glutamic acid and selective activation of the γ‐carboxyl group, followed by coupling and global deprotection (Amino et al. 2016; Hillmann and Hofmann ²⁰¹⁶). Recent advances, including automated microwave‐assisted SPPS and the exploration of greener solvent systems such as γ‐valerolactone as alternatives to DMF, have been reported to reduce synthesis time and solvent‐related environmental burden while enabling high peptide purity under optimized conditions (J. Xie, Zhao, et al. ²⁰²⁴).

Although more labor‐intensive and cost‐intensive than biocatalytic routes, chemical synthesis has been instrumental in elucidating structure–activity relationships governing CaSR activation. Systematic studies have shown that γ‐Glu‐Xaa peptides bearing medium‐sized hydrophobic or sulfur‐containing side chains, such as Val, Met, and Cys, are among the most potent kokumi‐active structures reported to date (Amino et al. 2016; Yamaguchi et al. ²⁰²⁵). Chemical synthesis also enables the rational design of peptide analogs with improved stability or tailored saltiness‐enhancing properties. Consequently, while it is unlikely to serve as the primary route for large‐scale production, chemical synthesis continues to play a critical role in lead optimization, reference standard preparation, and regulatory dossier development for novel kokumi peptide ingredients intended for low‐sodium applications.

Taken together, these preparation strategies are complementary in scope and function. Extraction allows access to kokumi peptide mixtures that reflect the chemical complexity of real food systems, whereas enzymatic synthesis provides a scalable approach for producing well‐defined peptides at high purity. In contrast, chemical synthesis enables precise sequence control, which is essential for mechanistic studies and the design of peptide analogs. The combined use of these approaches facilitates the systematic development and practical application of kokumi peptides in sodium‐reduction strategies.

3. Evaluation Methods of Kokumi Peptides

With the increasing recognition of the potential applications of kokumi peptides in flavor enhancement, sodium reduction, and the development of functional seasonings, establishing a scientific, systematic, and reproducible evaluation framework is crucial for elucidating their mechanisms of action and promoting their application in the food industry. To date, numerous kokumi peptides have been identified from various food sources, exhibiting notable saltiness‐enhancing and taste‐enriching effects. Their origins, sequences, identification methods, and enhancement intensities are summarized in Table 1. Current research on kokumi peptides and their flavor‐enhancing properties mainly relies on several complementary evaluation approaches, including sensory evaluation, CaSR activation assays, electronic tongue analysis, physiological models, and computational prediction. Their main purposes, advantages, and limitations are summarized in Table 2.

TABLE 1.

Kokumi peptides with saltiness‐enhancing properties.

Source	Sequence	Structural properties	Identification/spectral evidence	Saltiness enhancement	References
Pea protein hydrolysate (Pisum sativum)	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; γ‐Glu‐Gln‐Tyr	γ‐Glu tripeptides; γ‐Glu + hydrophobic/aromatic residues	HILIC–UHPLC–MS/MS	1.65 ± 0.25–2.29 ± 0.45 mmol/L	J. Yang et al. (2024)
Boletus edulis	Tyr‐Arg‐Phe (YRF)	Linear tripeptide; aromatic‐basic‐aromatic motif	LC‒MS/MS; virtual screening/docking; sensory validation	With a taste threshold (0.14 mmol/L) lower than GSH (0.31 mmol/L)	Y. Liu, Shen, et al. (2025)
Shiitake mushroom hydrolysate (Lentinula edodes)	γ‐Glu‐Glu‐Leu‐Lys‐Gly‐Asp‐Phe; γ‐Glu‐Val‐Leu‐Lys‐Ala‐Val‐Asp‐Glu	Long‐chain γ‐Glu peptides; acidic/basic + hydrophobic residues	LC‒MS/MS peptidomics; CaSR docking; sensory validation	Enhanced by 6.0‐/7.0‐fold (sensory score: 1.0 → 6.0/7.0 in 0.35% NaCl solution)	X. Chen, Du et al. (2025)
Wuding chicken soup	LPVPAFN (LN‐7), SAAPAAPVPA (SA‐10), VPAFNVING (VG‐9), AGFAGDDAPR (AR‐10), etc.	Linear oligopeptides rich in hydrophobic residues	UF/GFC + nano‐HPLC–MS/MS; CaSR docking	Significantly better in saltiness than fraction F2 (p < 0.05)	C. Yang, Du, et al. (2025)
Beef protein hydrolysate	γ‐Glu‐Arg; γ‐Glu‐Glu; γ‐Glu‐Thr; γ‐Glu‐Val; γ‐Glu‐Tyr; γ‐Glu‐Leu; γ‐Glu‐Phe; γ‐Glu‐Trp; γ‐Glu‐Glu‐Ala; γ‐Glu‐Glu‐Tyr; γ‐Glu‐Glu‐Met; γ‐Glu‐Glu‐Val; γ‐Glu‐Glu‐Leu; γ‐Glu‐Glu‐Phe; γ‐Glu‐Glu‐Trp	γ‐Glu di‐/tripeptides; acidic/basic/hydrophobic/aromatic residues	HPLC‐Q‐TOF–MS/MS; γ‐Glu peptide assignment	Saltiness score increased from 3.5 (BPH) to 5.38 (γ‐GBPH)	P. Chen, Zeng, et al. (2024)
γ‐Glutamylated beef protein hydrolysate (Maillard reaction products)	γ‐Glu‐Lys‐Lys; γ‐Glu‐Gly‐Tyr; γ‐Glu‐Ala‐Leu; γ‐Glu‐Asn‐Leu; γ‐Glu‐Met‐Trp; γ‐Glu‐Asn‐Phe; γ‐Glu‐Thr‐Phe; γ‐Glu‐Ala‐Phe; γ‐Glu‐Phe‐Met; γ‐Glu‐Leu‐Leu	γ‐Glu tripeptides; γ‐Glu + polar/hydrophobic/aromatic residues	HPLC‐Q‐TOF–MS/MS	Significantly higher saltiness than the control (BH‐MRP)	J. Yang, Zhang, et al. (2025)
Peanut protein	SPDIY, DPSPR, QPGDY, SPPGER	Linear pentapeptides/hexapeptide; acidic/basic + Pro‐rich motifs	Virtual screening/docking; electronic tongue; sensory validation	0.16–0.64 mmol/L (∼56.7% NaCl)	P. Wang, Li, et al. (2025)
Coppa Piacentina PDO (dry‐cured pork neck)	γ‐Glu‐Val; γ‐Glu‐Arg; γ‐Glu‐Pro; γ‐Glu‐S‐Methyl‐Cys; γ‐Glu‐Ser; γ‐Glu‐Tyr; γ‐Glu‐Tau; γ‐Glu‐Glu	γ‐Glu dipeptides/derivatives; kokumi‐related markers	UHPLC–HRMS	—	Leni et al. (2024)
Defatted peanut meal protein hydrolysate	γ‐Glu‐Ile‐Lys; γ‐Glu‐Ala‐Ile; γ‐Glu‐Leu‐Leu; γ‐Glu‐Phe‐Leu; γ‐Glu‐Thr‐Leu; γ‐Glu‐Ile‐Met; γ‐Glu‐Val‐Leu; γ‐Glu‐Ser‐Tyr	γ‐Glu tripeptides; mainly hydrophobic side chains	HPLC	1.65 ± 0.21–2.23 ± 0.20 mM	Tu et al. (2023)
Fish protein hydrolysate/fermented fish sauces	RP, RA, AR, RG, RS, RV, VR, RM	Arginyl dipeptides; short basic peptides	UF/GPC/HILIC; PFP‐ and HILIC‐HPLC–MS/MS	50 mM NaCl → ∼54.3 mM perceived	Schindler et al. (2011)
Casein/Lysozyme hydrolysates	RP, RA, AR, RG, RS, RV, VR, RM	Arginyl dipeptides; short basic peptides	PFP‐LC‒MS/MS + HILIC–MS/MS	Enhanced saltiness in cheese matrix	Harth et al. (2018)
Agaricus bisporus mushroom	Gly‐Leu‐Pro‐Asp; Gly‐His‐Gly‐Asp	Acidic tetrapeptides; C‐terminal Asp.	UPLC‐Q‐TOF–MS	Enhanced the chicken broth model by increasing mouthfulness, complexity, continuity, and perceived saltiness	T. Feng et al. (2019)
Fermented fish (Pla‐ra, Thai)	γ‐Glu‐Val‐Gly	Classical γ‐Glu tripeptide; γ‐Glu‐Val motif	LC‒MS/MS; descriptive sensory analysis	Saltiness‐enhancing attributes clearly perceived	Phuwapraisirisan et al. (2024)

TABLE 2.

Methods for evaluating kokumi peptides.

Method	Main purpose	Advantages	Limitations
Sensory evaluation	To verify perceived kokumi effects, including saltiness/umami enhancement, mouthfulness, continuity, and temporal persistence	Provides the most direct evidence of human perception and captures matrix‐dependent sensory interactions	Labor‐intensive and susceptible to panel variability; consumer validation in finished foods remains limited
CaSR activation assays	To screen kokumi‐active candidates and support CaSR‐related mechanistic interpretation	Quantitative, reproducible, and suitable for receptor‐level screening of peptide activity	CaSR activation alone does not establish kokumi perception; sensory confirmation is needed
Electronic tongue analysis	To characterize instrumental taste‐response patterns in peptide fractions, hydrolysates, or food matrices	Rapid, objective, and useful for preliminary screening and sample classification	Indirect and instrument dependent; limited in capturing mouthfulness, continuity, and temporal perception
Taste‐cell, animal, and organoid models	To evaluate physiological taste responses and receptor/channel‐related pathways	Provides mechanistic evidence closer to biological taste systems than heterologous assays	Technically demanding and lower‐throughput; species and model differences may limit translation to humans
Computational prediction	To predict peptide–receptor interactions and prioritize potential kokumi or salt‐enhancing peptides	Efficient for virtual screening, structure–activity analysis, and hypothesis generation	Dependent on model quality and dataset reliability; experimental and sensory validation remains essential

3.1. Sensory Evaluation

Sensory evaluation is a fundamental approach for the qualitative and quantitative analysis of food flavor through human perception of taste, aroma, and oral texture. In kokumi peptide research, it has mainly been used to determine taste thresholds, quantify the modulation of basic tastes, verify synergistic enhancement of saltiness and umami, and characterize kokumi‐related attributes such as thickness, mouthfulness, and continuity. Early studies, such as Ueda et al. (1990), established the sensory basis of kokumi research by evaluating the taste threshold of GSH and its enhancing effects on umami and saltiness in simple food matrices. Later, Dunkel et al. (2014) further strengthened sensory interpretation by introducing the dose‐over‐threshold concept to assess whether a compound is likely to contribute directly to food flavor. Subsequent studies have expanded sensory evaluation from threshold determination to the assessment of taste‐modulating effects, usually through trained‐panel intensity scaling, triangle tests, paired‐comparison tests, equivalent‐concentration approaches, or quantitative descriptive analysis. Rhyu et al. (2020) evaluated the sensory‐modulating effects of kokumi‐active peptide fractions isolated from mature Korean soy sauce (Ganjang) using human sensory evaluation with previously trained panelists and intensity scales. Saltiness and umami intensities were assessed utilizing a 15‐point scale (calibrated with reference NaCl solutions) and a 10‐point scale (referenced to a standardized fish soup base), respectively. This standardized methodology demonstrated that peptide supplementation significantly enhanced perceived saltiness at concentrations of 0.3%–0.5% and umami at 0.3%. Chen et al. (2021) integrated three‐alternative forced‐choice (3‐AFC) threshold measurements with modified paired comparison and linear scale matching tests. Using a trained sensory panel, they confirmed the saltiness‐enhancing potential of the decapeptide EDEGEQPRPF isolated from fermented soybean curd. Through this quantitative approach, they demonstrated that the addition of 0.4 mg/mL of this peptide to a 50 mmol/L sodium chloride solution increased the perceived saltiness to a level equivalent to a 63 mmol/L sodium chloride reference, highlighting its practical relevance for sodium‐reduction applications. Similar sensory approaches have also been applied to peptide‐rich systems such as fermented fish sauce and yeast extract, where the perceptual expression of saltiness enhancement was shown to depend strongly on the food matrix (Schindler et al. 2011; Zheng et al. ²⁰²¹).

Despite these advances, direct consumer testing of purified kokumi peptides in finished foods remains limited. Most available studies have relied on trained panels and simplified systems, including water, NaCl solutions, umami solutions, or simple broth models. Nevertheless, some product‐oriented studies indicate that kokumi‐ or savory‐related flavor enhancement can be evaluated in more realistic food matrices. An earlier product‐matrix study by Jung et al. (2010) evaluated beef soup supplemented with GSH and/or MSG using trained‐panel descriptive analysis and consumer testing. Separate consumer groups assessed perceived flavor intensities and overall liking, and the samples containing both GSH and MSG showed higher consumer acceptability and stronger perceived flavor intensities than the control. Mitchell et al. (2011) reported that reduced‐salt chili con carne ready meals containing a commercial nucleotide yeast extract showed sensory acceptability comparable to standard‐salt controls. Although this study did not investigate purified kokumi peptides, it provides product‐level evidence that savory flavor enhancers can support sodium reduction in finished foods. More recently, Hwang and Kim (2025) investigated consumer kokumi liking in Doenjang soup using 308 consumers, CATA profiling, and emotional response measures, showing that kokumi perception can be meaningfully evaluated in a real broth‐based food matrix. These findings suggest that future kokumi peptide studies should move beyond trained‐panel validation in model systems and incorporate consumer acceptance tests in finished or semifinished reduced‐sodium foods. Most current kokumi peptide studies still rely mainly on static intensity ratings, although key kokumi‐related attributes, such as continuity, flavor duration, mouthfulness, and lingering taste complexity, are inherently temporal. Therefore, dynamic sensory methodologies such as temporal dominance of sensations (TDS) and temporal check‐all‐that‐apply (TCATA) should be considered in future studies, particularly when kokumi peptides are evaluated in reduced‐sodium foods and complex savory matrices. Direct applications of these methods to purified kokumi peptides remain scarce; however, related studies support their methodological relevance. For instance, TDS combined with dynamic quantitative descriptive analysis has been used to characterize the evolving umami and saltiness of chicken soup containing taste peptides (Liang et al. 2022). Similarly, TCATA profiling of beef broth showed that yeast extract combined with MSG‐IMP enhanced temporal savory attributes, including intensity, richness, body, and duration, indicating that dynamic methods are useful for capturing kokumi‐like sensations that may not be fully described by static profiling alone (Brouwer et al. 2024).

3.2. CaSR Detection Method

The CaSR is a G protein‐coupled receptor (GPCR) widely expressed in taste bud cells that is capable of activation by various ligands, including polyvalent cations (Brown and MacLeod ²⁰⁰¹), l‐amino acids (Conigrave et al. 2007), and peptides (Brown et al. 1991). CaSR consists of 1078 amino acids and forms a heterodimeric structure composed of an extracellular domain (ECD), a seven‐transmembrane domain (7TMD), and an intracellular domain (Magno et al. 2011). Within the ECD, the Venus flytrap (VFT) domain serves as the main binding site for kokumi peptides. When a kokumi peptide binds to the VFT region, it induces conformational changes that activate the PLCβ2/IP₃ signaling pathway, leading to membrane depolarization and neural transmission (Sundararaman and van der Vorst ²⁰²¹). Thus, CaSR is considered the key molecular target underlying kokumi perception. Taste modalities such as kokumi, umami, bitter, and sweet are primarily mediated by GPCRs. Specifically, T1R heterodimers recognize umami (T1R1/T1R3) and sweet (T1R2/T1R3) tastes, while T2Rs detect bitter compounds (Spaggiari et al. 2020). CaSR expression is independent of the T1R3 subunit (Maruyama et al. 2012) but may be coexpressed with certain T2Rs (Brennan et al. 2014). These signaling pathways converge on the PLCβ2/IP₃ pathway, which is shared among kokumi, umami, and bitter transduction. In contrast, saltiness perception depends primarily on ENaC and TRPV1 channels, while sour taste is mediated by OTOP1 and specific TRP channels (Le et al. 2022).

San Gabriel et al. (2009) first demonstrated the expression of the CaSR in taste buds of rats and mice and highlighted its involvement in gustatory signal transduction. Building on this finding, Ohsu et al. (2010) synthesized a library of γ‐glutamyl peptides and evaluated their ability to activate CaSR using HEK‐293 cells expressing the receptor. The half maximal effective concentrations were subsequently compared with sensory data, revealing a strong association between CaSR activation and kokumi intensity. These results provide early functional evidence supporting CaSR as a molecular mediator of kokumi perception. The cellular localization of CaSR was further clarified by Maruyama et al. (2012), who showed through transgenic and immunohistochemical analyses that CaSR is selectively expressed in Type II and Type III taste cells of the circumvallate papillae. Structural activity relationship studies by Amino et al. (2016) subsequently demonstrated that the γ‐glutamyl moiety is essential for CaSR activation. Peptides containing a moderately sized hydrophobic residue at the second position, such as valine or methionine, exhibited higher activity, while the presence of glycine at the third position appeared to favor receptor binding. More recent studies have proposed γ‐glutamyl peptides as positive allosteric modulators of CaSR, thereby indirectly enhancing salt perception through the amplification of umami signaling. Le et al. (2022) advanced this concept and J. Yang et al. (2024) further supported it using molecular docking and homology modeling, showing that γ‐glutamyl tripeptides can stably associate with CaSR in the presence of Ca²⁺ and promote cooperative receptor activation. Notably, low concentrations of Ca²⁺ further potentiated CaSR responses, suggesting that the mineral composition of food matrices may modulate kokumi‐related flavor enhancement.

Currently, CaSR activity is primarily assessed using HEK‐293 cell systems and Xenopus oocyte models (H. Wang, Suo, et al. ²⁰²²). In the HEK‐293 system, human CaSR cDNA is cloned and inserted into a pcDNA3.1 vector and transfected into cells. After exposure to test samples, intracellular Ca²⁺ changes are detected using calcium‐sensitive fluorescent dyes, with fluorescence intensity reflecting receptor activation. In the oocyte system, CaSR cRNA is microinjected into oocytes; subsequent activation triggers calcium‐dependent chloride channels, producing measurable inward currents. CaSR assays are highly suitable for high‐throughput screening of kokumi‐active compounds and require relatively low safety constraints. However, CaSR activation alone does not confirm kokumi taste, since some non‐kokumi compounds can also activate the receptor. Therefore, CaSR assays should be used as a rapid prescreening tool and complemented by trained sensory evaluation, including threshold determination in water and/or model broth, discrimination tests in salty/umami model solutions, and descriptive analysis in real food matrices to confirm kokumi‐related attributes. Recent studies have strengthened the utility of CaSR‐based methods. Kim et al. (2022) used targeted peptidomics on dry‐cured mutton by‐products to identify 15 γ‐glutamyl dipeptides and evaluated their EC₅₀ and efficacy using a CaSR‐expressing cell model. The l‐glutaminase‐treated hydrolysates exhibited the strongest activation (EC₅₀ = 0.14 mg/mL), confirming their superior flavor‐enhancing potential. Such findings demonstrate that CaSR‐based in vitro assays provide an efficient prescreening tool for sensory evaluation, enabling rapid, reproducible, and cost‐effective assessment of kokumi activity under diverse processing conditions.

3.3. Electronic Tongue

Electronic tongues are biomimetic analytical systems designed to emulate key aspects of human taste perception. By employing multisensor arrays coupled with pattern recognition algorithms, they capture holistic taste profiles of samples and have been widely applied in food flavor analysis, seasoning development, and the screening of bioactive peptides. Compared with conventional sensory evaluation, electronic tongues provide higher objectivity, sensitivity, and reproducibility while avoiding panelist fatigue, which makes them particularly suitable for high‐throughput screening. They are also advantageous for the preliminary evaluation of nonedible or unapproved samples, offering a safe and efficient analytical approach. In saltiness analysis, defined signal ranges have been reported to correspond to perceptual thresholds within specific electronic tongue systems, enabling comparative assessment of salt‐enhancing effects (X. Chen, Luo, et al. ²⁰²⁴). Despite these advantages, electronic tongues are inherently limited in their ability to characterize complex flavor compounds, such as umami or kokumi peptides, that act through multiple receptors and synergistic signaling pathways. As a result, several recent studies have combined electronic tongue measurements with targeted sensory validation, particularly trained‐panel saltiness/umami intensity scaling and equivalent‐concentration or scale‐matching tests, to assess saltiness enhancement in model salt solutions and broth‐based systems. The electronic tongue provides rapid and reproducible instrumental evidence of taste‐related changes, whereas sensory evaluation is still needed to confirm whether these changes translate into perceivable saltiness enhancement. For example, X. Xie et al. (2023) combined electronic tongue analysis with a sensory intensity ranking test in NaCl‐containing systems and reported that ten peptides enhanced the salty taste of 0.3% NaCl to the equivalent of 0.4%–0.6% NaCl. In contrast, Zheng et al. (2022) applied electronic tongue analysis to enzymatic hydrolysates of bovine bone and demonstrated that ultrasound‐assisted hydrolysis significantly increased saltiness‐ and umami‐related responses. Furthermore, Xia et al. (2022) integrated electronic tongue analysis with trained sensory evaluation to quantify the saltiness‐enhancing effects of γ‐glutamyl peptides in a chicken broth model. The study employed 10 professional assessors with at least 6 months of standardized training and a 15‐point intensity scale ranging from 0 (no taste) to 15 (maximum intensity) and reported saltiness enhancement rates of up to 57.69%. More recently, molecular docking approaches have been integrated with electronic tongue analysis to validate the salt‐enhancing effects of peptides such as QIF and IPVM, providing additional insight into peptide–receptor interactions (Bu et al. 2024). Within this context, electronic tongue analysis serves as a robust high‐throughput screening tool for identifying flavor‐enhancing peptides. When used alongside sensory evaluation and receptor‐based assays such as CaSR activation, it contributes to a multidimensional framework for the objective characterization of umami‐ and salt‐enhancing peptides. Such integrated strategies are increasingly adopted to support the rational evaluation of peptide functionality and their development for flavor enhancement and sodium reduction applications.

3.4. Others

3.4.1. Cell, Animal, and Organoid Models

The evaluation of kokumi peptides has expanded beyond conventional sensory analysis and receptor‐based assays to encompass cell‐based models, animal studies, and organoid systems. Using models that closely mimic in vivo taste physiology, Xu et al. (2017) demonstrated that arginine dipeptides (AR, RA, and RP) significantly enhanced NaCl‐evoked responses in human fungiform taste cells, indicating a specific salt‐modulatory mechanism. Similarly, Bigiani and Rhyu (2023) reported that CaSR expressed in rat fungiform taste cells responds to umami stimuli and that γ‐glutamyl peptides enhance salt perception through an ENaC‐independent pathway. Consistent results were obtained by Y. P. Chen et al. (2021), who found that bioactive components from fermented tofu activated calcium signaling in primary taste cells isolated from Sprague–Dawley rats, thereby amplifying sodium chloride‐induced physiological responses. With advances in bioengineering, research has increasingly shifted from primary taste cell cultures to organoid‐based systems. Fan et al. (2023) established long‐term taste bud organoids derived from mouse circumvallate papilla stem cells using three‐dimensional (3D) culture techniques. These organoids exhibited concentration‐dependent intracellular calcium responses to monosodium glutamate stimulation, closely replicating in vivo taste signaling. By reconstructing the native architecture and cellular heterogeneity of taste buds, organoid models offer a stable and physiologically relevant platform that enhances cross‐species comparability.

3.4.2. Computer Simulations

Computer simulations, including molecular docking and molecular dynamics simulations, have become a valuable tool for investigating the receptor recognition and flavor‐enhancing mechanisms of kokumi peptides. These approaches can predict, at the atomic level, peptide binding sites, affinities, and conformational changes with taste‐related receptors such as CaSR, ENaC, and TMC4, providing structural insights that complement experimental data. Yi et al. (2025) applied docking and MD simulations to γ‐glutamyl peptides derived from soy protein isolate and found that these peptides form stable interactions with the VFT domain of CaSR through hydrogen bonds and hydrophobic contacts, involving key residues Ser301 and Asp410, and potentially modulate saltiness, umami, and overall taste perception. X. Xie, Dang, et al. (2024) reported similar binding patterns and highlighted the role of γ‐glutamyl peptides in enhancing perceived flavor mouthfulness. Beyond CaSR, other taste‐related receptors have been investigated. Peptides from hydrolyzed Philippine clams were predicted to form stable hydrogen bonds with TMC4 residues Lys568, Trp145, Tyr565, Arg151, and Gln155, with binding characteristics of LEDKVE closely associated with saltiness enhancement. J. Yang et al. (2021) constructed a T1R3 and MSG complex model, showing that γ‐glutamyl peptides may enhance MSG‐induced umami responses primarily through van der Waals interactions, supported by hydrogen bonds and hydrophobic contacts. These computational findings provide molecular‐level evidence for the synergistic flavor‐enhancing mechanisms of umami and kokumi peptides.

3.4.3. Machine Learning

Beyond experimental and conventional computational approaches, machine learning has emerged as a complementary tool for predicting functional flavor peptides. Machine learning models can learn relationships between peptide sequences, amino acid composition, and sensory attributes from large datasets, enabling efficient screening and facilitating the identification of target peptides. Recent work has demonstrated the feasibility of applying machine learning to kokumi‐related flavor prediction. He et al. (2024) developed a systematic modeling framework for kokumi/non‐kokumi classification and multi‐flavor prediction, in which saltiness was included as one of the target sensory attributes. Among the evaluated models, the WeaveGNN model achieved the highest performance in kokumi/non‐kokumi prediction with an area under the receiver operating characteristic curve (AUC) of 0.94, while the MLP‐E3FP model excelled in multi‐flavor classification (AUC = 0.94, MCC = 0.74), demonstrating the capability of 3D structural descriptors in capturing taste‐relevant features. The integration of multiple molecular representations, such as 2D and 3D fingerprints, protein–ligand interaction features, and graph‐based descriptors, enabled deep learning models to effectively capture structural characteristics associated with kokumi perception. Despite these advances, current machine learning applications in kokumi peptide research remain constrained by limited dataset size, heterogeneous compound types, and the challenge of distinguishing peptide‐specific sequence effects from general small‐molecule features. As larger, peptide‐focused datasets become available, the integration of sequence information, physicochemical properties, and receptor interaction data within machine learning and deep learning frameworks is expected to accelerate the identification of saltiness‐enhancing kokumi peptides.

4. Receptor‐Mediated Mechanisms of Taste Perception and Saltiness Enhancement

4.1. CaSR‐Mediated Mechanisms of Taste Perception

Accumulating evidence indicates that kokumi substances modulate multiple taste qualities primarily through activation of the CaSR, contributing to enhanced umami, sweet, and pungent sensations, while also being implicated in bitterness suppression (Ohsu et al. 2010; Kitajima et al. ²⁰²⁵; Ahmad et al. ²⁰²⁵; Viana ²⁰¹¹). Umami perception is mainly mediated by the T1R1/T1R3 heterodimer (Zhu et al. 2025), and CaSR activation by kokumi compounds has been shown to potentiate downstream signaling within this pathway, thereby amplifying umami intensity.

Among the various taste modalities, the mechanism underlying sweet taste enhancement by kokumi substances has been characterized in detail. A representative CaSR agonist, γ‐EVG, was shown to enhance sweet perception through an intra–taste bud cholinergic signaling cascade (Maruyama 2024). Specifically, activation of CaSR‐positive taste bud cells induces acetylcholine release, which then acts in a paracrine manner on M3 muscarinic receptors expressed by adjacent sweet receptor cells. This signaling amplifies ATP release and ultimately enhances sweet taste perception. While this pathway provides a coherent molecular framework, whether similar signaling logic applies broadly to other kokumi compounds remains to be clarified. Bitter taste is primarily mediated by members of the TAS2R receptor family (Sternini and Rozengurt 2024). Several kokumi substances, including CaSR agonists such as protamine, have been reported to suppress bitterness (Nishioka et al. 2006), suggesting a negative modulatory influence of CaSR activation on bitter signaling. Notably, TAS2Rs are capable of forming heteromeric receptor complexes (Kuhn and Meyerhof 2013), and CaSR itself has been shown to heterodimerize with other GPCRs, including GABAB receptors (Goolam et al. 2022). In addition, receptor–receptor interactions between olfactory receptors and muscarinic M3 receptors have been demonstrated to alter ligand responsiveness (Jiang et al. 2015). These observations support the possibility that CaSR and TAS2Rs participate in receptor‐level or cell–cell interactions within taste buds. It is plausible that activation of CaSR‐expressing bitter taste cells by kokumi substances leads to the release of neurotransmitters such as acetylcholine, which may indirectly potentiate signaling in neighboring umami, sweet, or salty taste receptor cells, thereby reshaping overall taste perception (Figure 3a). With respect to pungency, although compounds such as capsaicin directly elicit burning sensations through activation of the TRPV1 receptor, available evidence suggests that kokumi substances may modulate pungent intensity indirectly via CaSR‐mediated signaling pathways (Ohno et al. 2025). Such modulation likely occurs through interactions with gustatory or trigeminal signaling components rather than direct activation of nociceptive receptors, providing a plausible explanation for kokumi‐associated enhancement of pungency reported in complex food systems (Figure 3b).

FIGURE 3.

Proposed mechanisms of kokumi substance‐induced sensory modulation. (a) In taste cells, CaSR activation by kokumi substances influences other taste receptors, contributing to the suppression of bitterness and the enhancement of umami and sweetness. (b) Activation of the calcium‐sensing receptor (CaSR) by kokumi substances triggers Ca²⁺ influx and neuronal excitation in trigeminal nerve fibers, contributing to the perception of pungency.

The link between CaSR activation and saltiness enhancement remains incompletely understood. Although CaSR‐mediated signaling has been implicated in the modulation of several kokumi‐related sensory attributes, a direct mechanistic pathway connecting CaSR activation to sodium‐specific salt taste transduction has not yet been established. Electrophysiological studies have shown that the kokumi‐active γ‐glutamyl peptides GSH and γ‐Glu‐Val‐Gly did not potentiate amiloride‐sensitive ENaC activity in rat fungiform sodium‐taste cells but slightly inhibited ENaC‐mediated currents (Bigiani and Rhyu 2023). These findings suggest that the saltiness‐enhancing effects of these peptides may not be primarily attributed to potentiation of the canonical ENaC pathway. Instead, CaSR activation may contribute to saltiness enhancement by modulating taste‐bud responsiveness, intercellular signaling, or the integration of salty stimuli within a given food matrix. Therefore, kokumi‐mediated saltiness enhancement should be considered a broader modulatory process involving CaSR‐related signaling and salt taste pathways. Such effects are likely to vary with peptide structure, salt concentration, ionic composition, and food matrix composition.

4.2. Salt Taste Pathways in Peptide‐Mediated Saltiness Enhancement

Salt taste is one of the five basic taste modalities in humans and is primarily elicited by sodium ions (Na⁺). Upon dissolution of dietary salt (NaCl) in the oral cavity, released Na⁺ ions interact with specific receptors or ion channels on taste bud cells, triggering neural signals that are subsequently processed in the cerebral cortex to generate the perception of saltiness (Chaudhari and Roper 2010; Nomura et al. ²⁰²⁰). At the molecular level, salt taste transduction involves three principal receptors or channels (Figure 4), each contributing to Na⁺ detection and collectively forming the basis of salt taste signaling. In addition to Na⁺, other cations can influence salt perception through distinct channel‐dependent mechanisms, adding further complexity to this sensory modality (Delompré et al. 2019; Sood et al. ²⁰²⁵). Although kokumi peptides do not evoke a salty taste per se, they can markedly enhance perceived saltiness in food systems. This enhancement is thought to arise from the modulation of specific intracellular signaling processes, notably through CaSR pathways, thereby increasing the sensitivity of taste buds to existing salty stimuli. Through this indirect mechanism, kokumi peptides offer a means to intensify saltiness perception without increasing sodium content (Ohsu et al. 2010).

FIGURE 4.

Proposed mechanism by which kokumi peptides enhance saltiness perception through the activation of calcium‐sensing receptor (CaSR) and modulation of ion channels such as epithelial sodium channel (ENaC), transient receptor potential vanilloid 1 (TRPV1), and transmembrane channel‐like protein 4 (TMC4). AI, amiloride‐insensitive; AS, amiloride‐sensitive; DAG, diacylglycerol; ER, endoplasmic reticulum; IP₃, inositol 1,4,5‐trisphosphate; IP₃R, inositol 1,4,5‐trisphosphate receptor; PIP₂, phosphatidylinositol 4,5‐bisphosphate; PLC, phospholipase C.

4.2.1. ENaC

Salt taste perception is initiated when sodium ions (Na⁺) released from dissolved salt enter taste buds. Based on their sensitivity to the inhibitor amiloride, salt taste transduction pathways are broadly classified into amiloride‐sensitive and amiloride‐insensitive mechanisms. The amiloride‐sensitive pathway is primarily mediated by the ENaC, which is highly selective for Na⁺ and plays a dominant role in the detection of low salt concentrations and the generation of salt preference responses (Liman et al. 2014; Stolle et al. ²⁰¹⁸; Lewandowski et al. ²⁰¹⁶; Chamoun et al. ²⁰¹⁸). In contrast, amiloride‐insensitive pathways, involving ion channels such as TRPV1, contribute predominantly to the perception of higher salt levels.

ENaC is a heteromeric ion channel composed of α, β, and γ subunits, with an additional δ subunit expressed in specific cell populations in humans (Lossow et al. 2020). These subunits are localized in taste receptor cells within the circumvallate, foliate, and fungiform papillae of the tongue (Taruno and Gordon 2023). Sodium entry through ENaC leads to membrane depolarization and activation of downstream signaling cascades. At the downstream signaling level, bridging receptor activation with neural transmission and sodium‐induced depolarization triggers neurotransmitter release via ATP‐permeable channels such as CALHM1 and CALHM3. The released ATP subsequently activates P2×2 and P2×3 receptors on gustatory nerve fibers, initiating signal propagation to central taste‐processing regions (Y. Hu et al. ²⁰²⁴; Taruno and Gordon ²⁰²³; Bigiani ²⁰²¹). Importantly, ENaC activity is dynamically regulated by extracellular sodium concentration: under low‐salt conditions, ENaC remains highly active and enhances sensitivity to weak salt stimuli, whereas at higher salt concentrations, its contribution diminishes, and salt perception is increasingly mediated by amiloride‐insensitive pathways such as TRPV1 (Le et al. 2022). This dynamic regulation enables discrimination across a broad range of salt intensities.

Within the context of peptide‐mediated saltiness enhancement, ENaC has been most clearly implicated in studies of specific salt‐enhancing dipeptides. Xu et al. (2017) showed that the arginyl dipeptides Ala‐Arg, Arg‐Ala, and Arg‐Pro increased the proportion of cultured human fungiform taste cells responding to NaCl, whereas non‐arginyl dipeptides did not produce comparable effects. Pharmacological inhibition and siRNA‐mediated knockdown further indicated that Ala‐Arg enhanced NaCl responses mainly in amiloride‐sensitive cells, with ENaCα and ENaCδ subunits contributing to this response. These findings suggest that certain arginine‐containing short peptides can modulate sodium‐entry pathways and thereby increase cellular responsiveness to NaCl. More recently, peptide fractions derived from walnut meal protein were reported to exhibit strong predicted binding affinity toward ENaC, and molecular docking and molecular dynamics simulations supported stable peptide–channel interactions, suggesting a possible contribution of ENaC to their saltiness‐enhancing effects (P. Huang et al. ²⁰²⁵). These studies indicate that ENaC may serve as an important molecular target for selected salt‐enhancing peptides, particularly those with structural features that favor sodium channel modulation. For kokumi‐active γ‐glutamyl peptides, however, saltiness enhancement may be more closely associated with CaSR‐related signaling, intercellular communication within taste buds, and matrix‐dependent integration of salty stimuli. Thus, peptide‐induced saltiness enhancement in reduced‐sodium food systems appears to involve multiple levels of taste modulation, rather than ENaC‐mediated sodium detection alone.

4.2.2. TRPV1

TRPV1 is a nonselective cation channel that is traditionally known for its roles in nociception, thermosensation, and the detection of chemical irritants such as capsaicin (K. Zhang et al. ²⁰²¹). However, emerging research suggests that TRPV1 may also play a pivotal role in salt taste perception, particularly in complex environments where sodium‐selective mechanisms alone cannot fully account for the sensory experience. Beyond these canonical functions, TRPV1 may also contribute to salt taste perception and to the flavor‐enhancing effects of kokumi peptides, particularly under conditions of high ionic strength or chemical complexity (Masenga et al. 2023; Zhong et al. ²⁰²²). Owing to its nonselective permeability to Na⁺, K⁺, and NH₄ ⁺, TRPV1 has been proposed to play a more prominent role in high‐salt or non‐sodium salt environments, as well as under acidic or hyperosmotic conditions.

TRPV1 is a homotetrameric channel composed of four identical subunits. Each subunit includes six transmembrane helices (S1–S6) and cytoplasmic N‐ and C‐terminal domains, which together facilitate its function as an integrator of both chemical and physical stimuli. In particular, the pore‐forming S5–S6 helices and the S4–S5 linker are crucial for channel gating and ligand‐mediated modulation, while the cytoplasmic domains enable activity‐dependent regulation (E. Cao et al. ²⁰¹³; Liao et al. ²⁰¹³; F. Yang et al. ²⁰¹⁵). This structural architecture enables TRPV1 to respond to salt‐enhancing peptides and other modulatory ligands, highlighting its versatile role in salt taste perception. As a result, when activated by high concentrations of NaCl or other cations, TRPV1 initiates intracellular signaling cascades within taste‐related and trigeminal‐associated sensory cells, promoting the release of neurotransmitters such as ATP or serotonin (K. Zhang et al. ²⁰²¹). These signals, in turn, engage trigeminal afferents and interact with gustatory pathways, which contribute to the perception of saltiness through cross‐modal integration. The involvement of TRPV1 is particularly critical under high‐salt conditions or in complex ionic environments, where sodium‐selective channels alone may not adequately account for the perception of saltiness.

Recent studies have expanded the discussion of TRPV1 from conventional chemesthetic signaling to peptide‐associated saltiness enhancement. For example, W. Li, Sun, et al. (2024) investigated salty peptides from Stropharia rugosoannulata and reported that their interaction with TRPV1 was associated with saltiness perception, with acidic residues and N‐terminal recognition sites contributing to peptide–receptor binding. Askar et al. (2025) further identified WGFGDD from S. rugosoannulata protein hydrolysates as a saltiness‐enhancing peptide selected through TRPV1‐based molecular docking and sensory‐related evaluation. In addition, oyster‐derived synthetic peptides and their zinc complexes were reported to enhance saltiness and umami while reducing bitterness, with molecular docking suggesting interactions with several taste‐related targets, including T1R1/T1R3, TMC4, and TRPV1 (Yu et al. 2025). These studies indicate that TRPV1 may participate in the perception of some peptide‐induced saltiness‐enhancing effects, particularly under conditions involving complex ionic composition, peptide–mineral interactions, or chemesthetic–gustatory integration. However, much of the current evidence remains based on molecular docking, molecular dynamics simulation, electronic tongue analysis, or sensory correlation. Direct evidence from receptor‐specific antagonists, electrophysiology, human taste‐cell models, or TRPV1 knockdown/knockout systems is still limited for most food‐derived peptides. Although TRPV1‐related signaling has been implicated in amiloride‐insensitive salt taste responses, its precise role in salt perception remains incompletely defined. Current evidence suggests that this pathway may contribute to nonselective cationic responses under high‐salt or chemically complex conditions rather than functioning as a sodium‐specific detector. Moreover, the functional relationship between TRPV1‐related signaling, ENaC‐mediated sodium detection, and other salt‐associated pathways requires further clarification.

4.2.3. TMC4

Chloride ions (Cl⁻) have been increasingly recognized as contributors to salt taste perception, particularly under high‐salt conditions. In addition to the Na⁺‐dependent pathway mediated by ENaCs, chloride‐associated mechanisms also participate in salt taste signal transduction. TMC4 has been proposed as a molecular component involved in Cl⁻‐dependent salt taste responses (J. Zhang et al. ²⁰²⁴). As a member of the transmembrane channel‐like protein family, TMC4 contains a conserved TMC domain and multiple predicted transmembrane regions, with several transmembrane segments clustered near the C‐terminal region (Hahn et al. 2009; Yue et al. ²⁰¹⁹). TMC4 is predominantly expressed in taste bud cells of the posterior tongue and has been reported to function as an anion‐permeable channel that mediates Cl⁻ influx in taste receptor cells, thereby contributing to salt‐evoked signaling in mammals (Kasahara et al. 2021). Subsequent neurophysiological and behavioral evidence further supports the involvement of TMC4 in salt taste perception under high ionic strength conditions. In particular, loss of TMC4 reduces neural and behavioral responses to high concentrations of salts such as NaCl and KCl, while responses to other basic taste modalities are largely preserved (Kasahara et al. 2024). These findings suggest that TMC4 contributes to a chloride‐associated salt taste pathway that becomes more functionally relevant at high salt concentrations and operates in parallel with ENaC‐mediated sodium detection.

Electrophysiological and modeling studies suggest that TMC4 participates in the recovery phase of salt‐evoked electrical activity in taste cells. Upon stimulation with high concentrations of NaCl, Na⁺ influx through sodium‐permeable cation channels may depolarize salt‐responsive taste cells and activate voltage‐dependent TMC4. Subsequent Cl⁻ influx through TMC4 is proposed to facilitate membrane repolarization and shorten the interval between action potentials, thereby supporting repeated salt‐evoked signaling (Kasahara et al. 2021). This mechanism is consistent with the finding that 3‐guanidinyl propanol hydrochloride, a derivative of l‐arginine hydrochloride, enhances salt taste by increasing hTMC4‐mediated currents (Kasahara et al. 2022). Nevertheless, this model has been developed mainly from heterologous expression systems, knockout mouse experiments, and computational simulations; therefore, its relative contribution in food systems is likely to depend on salt concentration, chloride availability, ionic composition, and the distribution of TMC4‐expressing taste cells. Several studies have further examined whether naturally derived salty or saltiness‐enhancing peptides interact with TMC4. Computational analyses, including molecular docking and molecular dynamics simulations, have suggested that some saltiness‐enhancing peptides can form stable interactions with TMC4, indicating a possible contribution of this channel to peptide‐mediated salt taste modulation (Shen et al. 2022; H. Wang, Chen, et al. ²⁰²⁴). H. Wang, Lu, et al. (2025) identified salty dipeptides, including DP, DR, and RE, from bovine bone protein hydrolysates and reported their saltiness‐enhancing effects in low‐sodium model systems. Molecular simulation analyses suggested that these peptides interact with specific regions of TMC4 and may influence salt‐related ion‐channel activity. Similar TMC4‐related interaction patterns have also been proposed for peptides derived from porcine bone collagen and oyster enzymatic hydrolysates (X. Chen, Guo, et al. ²⁰²⁵; Xiao et al. ²⁰²⁴). In addition, J. Zhang et al. (2024) constructed a 3D structural model of human TMC4 containing ten predicted transmembrane domains, which has been used for structure–function analysis and in silico investigation of peptide–TMC4 interactions. These studies provide useful structural hypotheses for understanding peptide‐induced saltiness enhancement, although most current evidence is still based on sensory evaluation combined with computational prediction rather than direct functional validation of TMC4 activity.

Overall, current evidence supports TMC4 as a chloride‐associated component of salt taste transduction, particularly under high‐salt or amiloride‐insensitive conditions (Kasahara et al. 2024). In studies of kokumi and salt‐enhancing peptides, TMC4 may be considered a potential complementary target involved in salt taste modulation, rather than a primary kokumi receptor. The relative involvement of TMC4‐related mechanisms may vary with peptide sequence, charge distribution, NaCl concentration, chloride availability, ionic composition, and matrix conditions (Ji et al. 2025; M. Yang, Wang, et al. ²⁰²⁶).

5. Future Perspectives and Multisensory Strategies

5.1. Physiological Activities

Beyond their sensory functions, some kokumi‐active or kokumi‐related peptides have also been investigated for potential physiological activities, including angiotensin‐converting enzyme (ACE) inhibition, antioxidant activity, anti‐inflammatory effects, and metabolic modulation. These findings may broaden the functional relevance of kokumi‐related peptides. Hypertension remains one of the most prevalent metabolic disorders worldwide, with its pathophysiology driven by a variety of interconnected mechanisms, including dysregulation of the renin–angiotensin–aldosterone system (RAAS), endothelial dysfunction, and oxidative stress. Among the most significant contributors is excessive sodium intake, which exacerbates vascular inflammation, arterial stiffness, and cardiovascular complications (Müller et al. 2023). In light of these concerns, kokumi‐active peptides have garnered attention for their potential involvement in blood pressure regulation, particularly through their capacity to inhibit ACE, a key enzyme in the RAAS pathway. ACE plays a central role in regulating vascular tone by catalyzing the conversion of angiotensin I into angiotensin II, a potent vasoconstrictor, while also degrading bradykinin, a vasodilator (Althnaibat et al. 2024). By inhibiting ACE, selected food‐derived bioactive peptides may reduce angiotensin II formation and preserve bradykinin, thereby contributing to vasodilation. To provide a quantitative comparison, milk‐derived lactoferrin peptides have been reported to inhibit ACE‐1 with IC₅₀ values of 52.3 ± 0.8–79 ± 9 µg/mL, whereas the reference ACE inhibitor captopril showed an IC₅₀ of 0.0070 ± 0.0004 µg/mL under the same assay conditions (Borges et al. 2025). Fish protein‐derived peptides also exhibit variable ACE‐inhibitory activity, with representative IC₅₀ values ranging from 7.90 to 105.8 µM (Prakash Nirmal et al. 2023). Beyond their role in vascular regulation, γ‐glutamyl peptides recognized in sensory studies as prototypical kokumi‐active structures have also been implicated in the modulation of inflammatory responses. Specifically, activation of the CaSR by these peptides has been shown to alter the balance between pro‐inflammatory and anti‐inflammatory cytokines, with reductions in IL‐6, IL‐8, and IL‐1β and a concomitant increase in IL‐10 levels. Furthermore, γ‐glutamyl peptides have been observed to suppress NF‐κB signaling, inhibiting the phosphorylation of JNK and IκBα (H. Zhang et al. ²⁰¹⁵).

These findings suggest that γ‐glutamyl peptides may play a significant role in modulating chronic low‐grade inflammation. Beyond their anti‐inflammatory properties, certain γ‐glutamyl peptides also exhibit bioactivity in metabolic regulation. For example, these peptides have demonstrated inhibitory effects against dipeptidyl peptidase‐IV (DPP‐IV), an enzyme involved in incretin degradation, thereby contributing to enhanced glycemic control in experimental models (Nagahisa et al. 2022). In addition, the γ‐Glu‐Trp peptide (SCV‐07) has been reported to exert immunomodulatory effects, including immune stimulation and activity against infection and tumor models (Petrov et al. 2006). This further substantiates the potential of γ‐glutamyl peptides to engage immune‐related pathways, supporting their broader functional role. In addition to their roles in inflammation and metabolic regulation, kokumi‐related peptides also contribute to antioxidant defense, operating through both GSH‐dependent and GSH‐independent mechanisms. GSH, a prototypical γ‐glutamyl tripeptide with kokumi activity, plays a pivotal role in maintaining cellular redox homeostasis and supporting metabolic functions (Aquilano et al. 2014). Its cysteine thiol group effectively scavenges reactive oxygen and nitrogen species, converting them into oxidized GSH (GSSG), which is then recycled by GSH reductase in an NADPH‐dependent process. While dietary polyphenols have been shown to enhance endogenous GSH synthesis through modulation of enzymatic pathways (Minich and Brown 2019), the use of GSH itself is limited by its poor oral bioavailability and instability. As a result, recent studies have explored microencapsulation and advanced delivery methods to improve its gastrointestinal stability and systemic bioavailability (Dziadek et al. 2025).

Despite the growing body of evidence supporting the antioxidant, anti‐inflammatory, and immunomodulatory effects of kokumi peptides, the precise structure–function relationships remain inadequately understood. Future progress in this field will require deeper mechanistic insights, a more precise identification of bioactive peptide sequences, and the development of scalable, food‐compatible production methods. Achieving these advancements is essential for positioning kokumi peptides as scientifically validated components within functional food systems.

5.2. Multisensory Synergy in Saltiness Enhancement

Multisensory synergistic salt enhancement strategies aim to enhance overall salt perception by modulating the integration of inputs from different sensory channels without increasing the actual sodium content. Current evidence indicates that taste, olfaction, and trigeminal‐related oral somatosensory information are processed cooperatively at the central nervous system level, which is critical for determining salt intensity and overall flavor perception. Building on this understanding, leveraging cross‐modal sensory interactions associated with kokumi peptides to enhance the integrated perception of salt‐related signals has gradually emerged as a promising approach for sodium reduction (Forde and de Graaf ²⁰²²). Figure 5 illustrates how cross‐modal multisensory interactions can contribute to enhanced salt perception.

FIGURE 5.

Cross‐modal multisensory interactions contributing to enhanced saltiness perception.

5.2.1. Cross‐Modal Enhancement Between Olfaction and Taste

Odor‐induced taste enhancement (OITE) is one of the most well‐documented forms of cross‐modal interaction, arising from the neural integration of olfactory and gustatory cues (Syarifuddin et al. 2016). The enhancement effect depends on the consistency and familiarity between odor and taste: congruent combinations, such as strawberry‐sweet or soy sauce‐salty, can amplify perceived taste intensity, whereas incongruent odors, such as carrot, may attenuate it (Djordjevic et al. 2004). Neuroimaging studies indicate that congruent odor–taste pairings evoke stronger activation in key brain regions involved in flavor integration, including the insula, orbitofrontal cortex, and anterior cingulate cortex (Ohno et al. 2025). The magnitude of OITE is influenced by several factors: the odor pathway, with retronasal olfaction more strongly associated with taste perception than orthonasal routes; odor concentration, with suprathreshold odors producing more pronounced enhancement; and cognitive congruency, where prior experience and associative learning determine the effectiveness of odor–taste combinations (Ai and Han 2022; Lawrence et al. ²⁰⁰⁹). Psychophysical studies further confirm that incorporating saltiness‐congruent odors into low‐sodium systems can significantly enhance perceived saltiness (Seo et al. 2013). Evidence from experimental and sensory studies demonstrates that aromas from cheese, soy sauce, ham, herbs, and spices can enhance salt perception, providing opportunities to formulate cross‐modal flavor solutions for low‐sodium or plant‐based products, thereby improving sensory quality and supporting clean‐label development (Zhou et al. 2021; Syarifuddin et al. ²⁰²²; Y. P. Chen et al. ²⁰²³; Lopes et al. ²⁰¹⁴; Rosa et al. ²⁰²²). It is also conceivable that kokumi peptides may influence saltiness perception not only through potential interactions with olfactory receptors but also by modifying the abundance and profile of aroma compounds within the food matrix.

5.2.2. Taste–Taste Synergistic Interactions

Taste–taste synergistic interactions occur when multiple basic taste stimuli are present simultaneously in the oral cavity and mutually influence perceived intensity, temporal characteristics, or qualitative attributes. These interactions are not simply additive; instead, they can generate integrated perceptual effects that cannot be explained by any single tastant alone. Psychophysical and neurophysiological studies suggest that such synergy may arise from both peripheral mechanisms, including receptor coactivation and ion‐channel modulation, and central integration within gustatory neural circuits (Deepankumar et al. 2019). This concept is particularly relevant to kokumi peptides because kokumi‐active compounds generally do not act as independent taste stimuli at effective concentrations. Rather, they modulate the intensity, continuity, and overall balance of existing taste mixtures, especially those containing salty and umami components. Among known taste interactions, umami–salt synergy is one of the most important for sodium‐reduced food formulation. Umami substances such as monosodium glutamate, inosinate, and guanylate can enhance perceived saltiness when combined with NaCl, thereby increasing the savory impression without relying solely on sodium (Breslin 1996; J. Li, Zhong, et al. ²⁰²⁴). Kokumi substances may further reinforce this integrated perception by enhancing the continuity and mouthfulness of salt–umami mixtures. For example, Goto et al. (2016) reported that adding GSH to an MSG–NaCl solution significantly increased perceived taste intensity, with the mixture perceived as approximately 1.38 times stronger than the same solution without GSH. Similarly, Rhyu et al. (2020) showed that a kokumi‐active peptide fraction from mature Ganjang enhanced salt taste intensity in human subjects and modulated both salt and umami taste responses. These findings indicate that kokumi peptides function as modulators of taste–taste interactions, particularly umami–salt interactions, and may therefore help maintain perceptual balance, continuity, and overall flavor quality in reduced‐sodium foods.

5.2.3. Auditory, Visual, and Textural Modalities

Saltiness perception is not solely determined by chemical stimuli; it is also modulated by auditory, visual, and textural cues. Auditory input, such as the crunching sound of crisp foods, can enhance perceived saltiness through associative learning and attentional mechanisms, whereas excessive background noise may reduce taste sensitivity (Swahn and Nilsen 2023; Guedes et al. ²⁰²³). Visual cues influence taste expectations: deeper‐colored tableware, high‐contrast colors, or rough surface textures can increase perceived saltiness and intensity (Motoki et al. 2023; Spence ²⁰¹⁸; Spence ²⁰²²). Textural properties, including roughness, crispness, and moderate viscosity, affect both the kinetics of sodium release and cognitive associations. For instance, rougher textures increase surface contact between sodium ions and taste buds, enhancing salt perception, while moderate viscosity (e.g., 0.5% starch) has been shown to optimize umami and palatability in fermented soybean soups (Barbosa Escobar and Wang ²⁰²³; Miles et al. ²⁰²²; Chon and Kim ²⁰²⁵).

Together, these findings indicate that multimodal optimization of auditory, visual, and textural attributes can significantly enhance salt perception in low‐sodium formulations. For the development of kokumi peptide–based products, carefully designing auditory characteristics, visual presentation, and textural parameters can complement chemical salt‐reduction strategies, thereby maintaining consumer acceptability.

5.2.4. Trigeminal and Cognitive Modulation

The trigeminal system mediates the perception of pungency, heat, and oral irritation, contributing to the overall complexity of taste (Braud and Boucher 2020). Chemical stimuli such as capsaicin, piperine, and allyl isothiocyanate activate TRPV1 and TRPA1 channels, inducing mild irritation and enhancing sensory alertness (Y. Wang, Zhong, et al. ²⁰²²; Moss et al. ²⁰²³; Amyoony et al. ²⁰²⁴). Recent evidence suggests that γ‐glutamyl peptides can potentiate these sensations via CaSR activation and that the effect is abolished by the selective CaSR antagonist NPS‐2143 (Kitajima et al. 2022). The expression of CaSR in trigeminal ganglia implies that kokumi‐active peptides may modulate TRP channel responsiveness, thereby influencing the perception of spiciness and oral complexity. Moderate trigeminal stimulation can enhance the perception of saltiness and aroma, whereas excessive stimulation may divert attention to discomfort, reducing hedonic appeal. Interindividual variability in trigeminal sensitivity, which is influenced by age, diet, and cultural background, underscores the importance of precise dosing and delivery strategies (Q.‐B. Zhang et al. ²⁰²⁰). Importantly, flavor perception is shaped by top‐down cognitive processes, including prior experience, learned expectations, and cross‐modal associations. Integrating kokumi peptides with congruent aroma, texture, and mild trigeminal stimulation represents a promising multisensory strategy to enhance perceived saltiness in low‐sodium products.

6. Applications

6.1. Reduction of Sodium Content in Foods

The search for alternative methods to reduce sodium content in food products without compromising sensory qualities has led to the exploration of peptides that enhance specific taste perceptions (X. Chen, Luo, et al. ²⁰²⁴). Among these, kokumi peptides have gained attention for their potential to improve the flavor profile of sodium‐reduced formulations by modulating taste perception. In particular, γ‐glutamyl peptides, which can be derived from various protein sources, are being studied for their ability to boost saltiness perception, offering a viable strategy for reducing sodium without sacrificing flavor (Tu et al. 2023).

Several γ‐glutamyl peptides derived from soy protein isolate hydrolysates, such as γ‐Glu‐Gly‐Pro‐Phe and γ‐Glu‐Ala‐Phe‐Phe, have been reported to enhance saltiness perception in NaCl model solutions and in a basic chicken broth (BCB) model, supporting their potential for use in sodium‐reduced formulations (Yi et al. 2025). Similar effects have been observed for seafood‐derived peptides. X. Xie, Dang, et al. (2024) identified eight short peptides from Philippine clam hydrolysates, and their saltiness‐enhancing capacity was confirmed by combining quantitative sensory scoring and S‐shaped curve threshold modeling with principal component analysis (PCA) of electronic tongue sensor data. Notably, when 1 g/L of LEDKVE was evaluated in an aqueous solution containing 3 g/L NaCl, it produced a perceived saltiness equivalent to that of a 5 g/L NaCl solution, demonstrating a significant synergistic saltiness‐enhancing effect. Further application‐oriented evidence was provided by Ren et al. (2024), who screened peptides from tilapia processing by‐products using molecular docking followed by sensory validation. Among the identified peptides, HLDDALR exhibited the highest saltiness intensity in soup‐based model systems, supporting the feasibility of peptide‐assisted sodium reduction in complex food matrices. In addition to γ‐glutamyl peptides, other peptide classes contribute to sodium reduction by improving the overall flavor balance. Arginine‐containing dipeptides and selected Maillard reaction‐derived peptides have been shown to alleviate bitterness associated with potassium‐based salt substitutes and to help maintain flavor continuity in low‐sodium formulations (Niu et al. 2024; Bu et al. ²⁰²⁴). Although not all of these peptides are strictly classified as kokumi compounds, their practical sensory benefits complement kokumi‐based strategies. Accumulating application‐driven studies indicate that the incorporation of kokumi‐active and saltiness‐enhancing peptides at low levels can partially compensate for NaCl reduction while maintaining sensory acceptability. These findings support the potential of kokumi peptides as functional ingredients for the development of next‐generation sodium‐reduced foods.

6.2. Improvement of Food Flavor and Mouthfeel

Kokumi peptides have demonstrated clear potential in enhancing the taste complexity and mouthfeel of various foods. For instance, T. Feng et al. (2019) reported that kokumi peptides isolated from Agaricus bisporus enhanced both umami and saltiness in chicken broth, contributing to a richer and more rounded sensory profile consistent with the strategy of reducing sodium without compromising perceived saltiness. Beyond peptides, certain amino acid derivatives also exhibit flavor‐modulating effects. N‐Lactoyl leucine (Lac‐Leu) and N‐lactoyl isoleucine (Lac‐Ile) have been shown to enhance both umami and saltiness, with Lac‐Leu‐containing mixtures being particularly effective in improving the overall flavor intensity of soy sauce (J. Feng, Huang, et al. ²⁰²⁴). Non‐peptidic Maillard reaction products (MRPs) represent another important class of kokumi‐active compounds. MRPs, generated via the reaction of reducing sugars with amino acids under heat, can significantly improve food flavor while also providing antioxidant properties. Du et al. (2024)demonstrated that the Maillard reaction of single‐ring sea urchin hydrolysates reduced bitter‐tasting amino acids (e.g., leucine, lysine) and degraded off‐flavor aldehydes and ketones while generating acids, alcohols, and phenolic compounds that enhanced aroma and masked undesirable odors. Mechanistically, MRPs are formed through initial glycation to generate Amadori products, which subsequently undergo rearrangements, dehydration, and cleavage reactions to produce diverse carbonyl compounds. These compounds can further react with amino acids via Strecker degradation to generate volatile flavor molecules (Shi et al. 2024; Urango et al. ²⁰²⁴). Specific MRPs formed from sulfur‐containing precursors also contribute to kokumi perception. X. Chen, Li, et al. (2024)showed that the Maillard‐type reaction between thiamine and cysteine produced S‐((4‐amino‐2‐methylpyrimidin‐5‐yl)methyl)‐cysteine, a compound capable of eliciting kokumi sensations at low concentrations. Importantly, kokumi effects are not limited to peptides of a specific molecular weight; various non‐peptidic MRPs and sulfur‐containing compounds can act as complementary flavor enhancers, likely through interactions with the CaSR or other sensory pathways. This multicomponent synergy contributes to a richer flavor profile and sustained taste, highlighting the potential of both peptide and non‐peptide kokumi compounds in food formulation. The combined use of kokumi peptides, amino acid derivatives, and select MRPs offers a versatile approach to improve flavor complexity, mouthfeel, and sensory persistence in diverse food systems.

6.3. Novel Seasonings and Matrix‐Specific Food Applications

Kokumi peptides and related kokumi substances have attracted increasing interest as flavor‐modulating ingredients for novel seasonings and health‐oriented food formulations, particularly in sodium‐reduced and reduced‐fat products. Published food‐matrix applications remain relatively limited, and reported applications have mainly focused on savory liquid or semisolid foods, broth‐like systems, selected meat products, sweet beverages, and reduced‐fat matrices (Kitajima et al. 2026). Kokumi‐active hydrolysates and peptide‐rich extracts from yeast, mushrooms, chicken, and other protein sources have recently been explored mainly in broth‐like matrices, soup seasonings, and meat‐flavor systems, where they enhance attributes such as thickness, continuity, kokumi intensity, and overall flavor quality (T. Feng, Ma, et al. ²⁰²⁴; P. Chen, Zeng, et al. ²⁰²⁴; C. Yang, Du, et al. ²⁰²⁵). For example, the addition of 5 ppm γ‐glutamyl‐valyl‐glycine (γ‐Glu‐Val‐Gly, γ‐EVG) to chicken consommé significantly enhanced umami, mouthfulness, and mouth coating, indicating its potential to improve flavor and mouthfeel in savory soup systems (Miyaki et al. 2015). In reduced‐fat peanut butter, 40 ppm γ‐EVG significantly enhanced thick flavor, aftertaste, and oiliness, suggesting that kokumi peptides may help compensate for sensory losses caused by fat reduction (Miyamura et al. 2015). Additional γ‐EVG applications reported by Kuroda (2024) include hamburger steak, orange‐flavored beverages, low‐fat French dressing, and low‐fat custard cream, where this peptide modified sensory attributes such as mouthfulness, thickness of taste, continuity, aftertaste, oiliness, sweetness, juiciness, and meaty flavor, depending on the food matrix.

These examples broaden the reported application scope of γ‐EVG beyond simple model systems and suggest that its sensory effects are strongly matrix dependent. For example, γ‐glutamyl tripeptides showed lower kokumi‐related thresholds in BCB than in MSG solution or NaCl solution (Tu et al. 2023), respectively. This suggests that kokumi peptides may be more effectively perceived in savory matrices containing salt, glutamate, peptides, amino acids, and aroma‐active compounds than in single‐tastant solutions. In practical applications, γ‐Glu‐Val‐Gly at 5 ppm significantly enhanced umami and mouthfulness in chicken consommé, whereas excessive addition may cause sourness, bitterness, or excessive lingering sensations, thereby reducing taste balance (Miyaki et al. 2015). Processing conditions further influence kokumi activity. γ‐Glutamylated beef hydrolysates retained CaSR activity after heating at 80°C, 120°C, and 150°C, but GSH‐derived systems may show reduced or transformed activity under high‐temperature Maillard reactions, depending on the reducing sugar and reaction conditions (Ahmad et al. 2025). pH is another important matrix factor affecting kokumi perception. Recent retronasal threshold testing of nine kokumi peptides commonly found in fermented soybean foods showed that their best‐estimate threshold values decreased significantly as pH increased from 5.0 and 5.3 to 5.8, suggesting that kokumi peptides may be more readily perceived under moderately higher pH conditions (Lee et al. 2025). Fermentation may further promote the generation of kokumi‐related peptides, but the final peptide profile depends strongly on microbial enzymes, substrate composition, salt concentration, and fermentation time (Van Ho and Suzuki ²⁰¹³). Industry reports from Ajinomoto also suggest the commercial translation of kokumi‐related ingredients within seasoning and food‐solution platforms (Ajinomoto Group ²⁰²⁶). In meat systems, γ‐glutamylated hydrolysates from porcine hemoglobin and meat have been incorporated into emulsion‐type pork sausages at 2% and 4%, where they enhanced kokumi sensation and overall flavor intensity and slowed lipid peroxidation during chilled storage (Q. Li et al. ²⁰²²). In addition, solid‐phase synthesis and structural modification of identified peptides may further support the development of stable and potent flavor enhancers with defined sensory functions (Lao et al. 2024). Nevertheless, important limitations remain. Most published studies rely on trained or descriptive sensory panels rather than consumer acceptance testing, and public evidence in complex finished foods is still limited and not always consistent. For instance, Labrador (2024) evaluated commercial kokumi‐containing ingredients in low‐fat cream of mushroom soup and macaroni and cheese but found no significant improvement in creaminess, preference, or overall liking and suggested that processing or reheating may have reduced the effectiveness of kokumi peptides. Therefore, industrial applications require not only stable peptide production but also matrix‐specific sensory validation, control of bitter co‐peptides, storage stability assessment, and process optimization.

6.4. Safety and Regulatory Considerations

Safety and regulatory considerations are important for the practical application of kokumi peptides as flavor‐modifying ingredients. Among chemically defined kokumi peptides, γ‐glutamyl‐valyl‐glycine (γ‐EVG; γ‐Glu‐Val‐Gly) is one of the best‐documented examples. It is included in the FDA Substances Added to Food inventory as a flavoring agent or adjuvant and is identified by FEMA No. 4709 and JECFA No. 2123 (U.S. Food and Drug Administration n.d.; Joint FAO/WHO Expert Committee on Food Additives n.d.; Cohen et al. ²⁰²⁰). JECFA concluded that γ‐EVG presents no safety concern at current levels of intake when used as a flavoring agent, without establishing a numerical ADI (Joint FAO/WHO Expert Committee on Food Additives n.d.; World Health Organization ²⁰¹²). In the JECFA toxicological monograph, the SPET‐based exposure estimate was 4000 µg/person/day, corresponding to approximately 67 µg/kg bw/day. Using a NOAEL of 3130 mg/kg bw/day from a 91‐day rat study on the structurally related peptide l‐alanyl‐l‐glutamine, JECFA estimated a margin of exposure of approximately 47,000. Additional FEMA information reported a NOAEL of 1000 mg/kg bw/day for γ‐EVG itself in a 28‐day dietary study in rats. EFSA also concluded that γ‐EVG is not of safety concern at its estimated level of intake as a flavoring substance (EFSA Panel on Food Contact Materials and Aids ²⁰¹⁴). These evaluations support the feasibility of using selected kokumi peptides as flavor modifiers, but broader application will require attention to peptide purity, production method, use level, food matrix, dietary exposure, and compliance with regional regulations.

7. Conclusion and Perspectives

This review summarizes recent progress in the preparation and identification of kokumi substances, their sensory evaluation, and the characterization of salt‐related receptors and partial underlying mechanisms. Despite these advances, the regulatory principles by which kokumi substances modulate flavor perception remain insufficiently understood, particularly with respect to the links between molecular recognition, sensory enhancement, and cross‐modal flavor integration. At present, only a limited number of compounds have been confirmed to contribute to kokumi‐related sensory attributes such as mouthfulness, complexity, and persistence, and the biological basis of many reported kokumi candidates has yet to be established. Although synergistic interactions among umami compounds, kokumi peptides, and certain aroma‐active molecules have been observed, how these signals are integrated to construct the kokumi experience at the sensory and neural levels remains unclear. Beyond γ‐glutamyl peptides, studies addressing receptor recognition and signal transduction of other potential kokumi molecules, including α‐peptides, N‐acyl amino acids, and lipid‐related compounds, are still scarce. Emerging evidence suggests that kokumi perception is not generated by a single modality but arises from coordinated interactions among gustatory, olfactory, textural, and trigeminal pathways. Within the gustatory system, kokumi peptides do not elicit distinct taste qualities on their own but enhance saltiness and umami perception by modulating intracellular signaling in taste receptor cells. This modulatory effect is consistent with the cooperative involvement of multiple ion channels, such as ENaC, TRPV1, and TMC4, in salt taste transduction. At the level of flavor integration, retronasal olfaction and textural cues further shape the perception of richness, continuity, and aftertaste, highlighting the importance of cross‐modal sensory interactions in kokumi‐related saltiness enhancement. Together, these findings underscore that kokumi peptides act primarily as signal amplifiers within a complex sensory network rather than as direct taste stimuli. A deeper understanding of the molecular, neural, and sensory mechanisms underlying kokumi‐mediated saltiness enhancement will provide a theoretical basis for the rational design of sodium‐reduction strategies. In parallel, systematic evaluation of flavor compatibility, processing stability, and long‐term safety across diverse food matrices will be essential for translating kokumi peptides into practical applications within the food industry.

Author Contributions

Tingting Sun: conceptualization, investigation, formal analysis, writing – original draft, software. Tao Feng: conceptualization, resources. Tao Jiang: conceptualization, resources, formal analysis. Mati Ullah Khan: investigation, writing – review and editing. Huatian Wang: investigation, formal analysis. Lingyun Yao: investigation, formal analysis. Shiqing Song: investigation, formal analysis. Min Sun: funding acquisition, supervision. Chuang Yu: supervision, funding acquisition.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 32402281 and No. 32102123).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The authors have nothing to report.