1. Introduction
Salty taste, often regarded as the “king of flavors”, plays a fundamental role in our diet and is the only taste with significant physiological effects; it regulates the osmotic balance between cells and blood and normal water and salt metabolism. However, high-salt diets are strongly associated with hypertension and other cardiovascular diseases [
1]. The World Health Organization (WHO) recommends a daily salt intake of no more than 5 g, but Chinese Dietary Guidelines indicate that about 80% of people exceed this recommendation. However, the addition of salt makes an important contribution to the flavor and edible quality of many foods (e.g., breads, processed soy products, potato chips, biscuits, canned products). In response to increasing consumer demand for healthier food options, the food industry will make more far-reaching efforts to reduce salt. Therefore, researchers have been exploring various strategies to reduce salt intake while maintaining desirable flavor. One promising approach is the development of salt substitutes that ensure a salty taste, to reduce sodium ion intake, and offer health benefits. And many researchers have attempted various methods to salt reduction, including optimizing the structure and distribution of salt [
2], and using metal chlorides [
3] and flavor enhancers to reduce sodium salt intake [
4], with the most attention being focused on the study of salty peptides such as Orn-β-Ala·HCl and Orn-Tau·HCl [
5]. These peptides do not contain Na
+ but have a salty taste comparable to or even stronger than sodium chloride, which has inspired further research into their taste mechanisms and applications. Salty peptides, as novel food additives, have shown potential for replacing table salt without sacrificing flavor, gaining significant attention in recent years [
6].
Salty peptides can be derived from various sources, including animal [
7,
8,
9], microbial [
10,
11,
12], and plant sources. Among these, plant-derived salty peptides are particularly noteworthy, with legumes being a rich source. Legumes, due to their high protein content, and richness in salty and umami amino acids, provide an excellent basis for extracting salty peptides. For instance, enhanced salty effects were achieved by deep enzymatic hydrolysis of pea protein [
13]; salty peptide EDEGEQPRPF was isolated from commercial soy milk, demonstrating its salty taste enhancement equivalent to 63 mmol/L NaCl [
14]. Additionally, several salty dipeptides such as Ile-Gln, Pro-Lys, Ile-Glu, Thr-Phe, and Leu-Gln have been identified in soy sauce [
15]. Despite these advancements, there is still a scarcity of research focused on the enzymatic hydrolysis of a soy protein isolate (SPI) to extract salt-containing peptides. This gap presents a significant opportunity for future investigations aimed at exploring SPI for the development of effective sodium salt substitutes.
Transmembrane channel protein 4 (TMC4) is a significant protein recently identified in the field of taste physiology [
16]. It is expressed in taste bud cells and plays a crucial role in the transmission of taste signals. TMC4 is highly expressed in the fungiform and circumvallate papillae, which project to the glossopharyngeal nerve and mediate responses to high concentrations of NaCl. Electrophysiological analyses using HEK293T cells have shown that TMC4 functions as a voltage-dependent Cl
− channel, with currents completely inhibited by the anion channel blocker NPPB [
17]. The action mechanism of salty peptides primarily involves ionizing cations that enter the cell through transient receptor potential vanilloid (TRPV) channels on the cell membrane, leading to calcium ion polarization. This influx of calcium ions triggers the release of neurotransmitters, activating the next level of neurons. Neural signals are then transmitted to central brain regions, such as the insula and orbitofrontal cortex, where they are encoded as taste signals, producing the salty taste sensation [
18]. Despite existing research on the effects of various peptide substances on taste [
19], there is still less information on the interaction between specific salty peptides and TMC4 [
20]. Therefore, studying the molecular interactions between TMC4 and soybean-derived salty peptides is essential for understanding the functional mechanisms of salty peptides and provides a theoretical foundation for developing novel food seasonings.
The aim of this study is to use SPI as a raw material to prepare novel salty peptides through enzymatic hydrolysis, separation, purification, and identification. Sensory evaluation and electronic tongue analysis techniques were employed to characterize these peptides. Furthermore, molecular docking with the TMC4 receptor protein was used to elucidate the taste mechanism, and basic bioinformatics methods were applied to screen for higher-quality salty peptides. This research not only provides new insights into the taste effects of salty peptides but also offers theoretical references for their application in the food industry, thereby promoting public health.
2. Materials and Methods
2.1. Materials and Chemicals
SPI were obtained from ANYANG BEIJIA FOOD CO., Ltd. (Anyang, Henan, China). Pepsin (30,000 U/g), flavourzyme (30,000 U/g), citric acid, and Sephadex G-10 were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Food-grade monosodium glutamate (MSG) was purchased from Lianhua Holding Company Limited (Xiangcheng, China). Sodium chloride (NaCl) was purchased from China National Salt Industry Group Co., Ltd. (Beijing, China). Caffeine was purchased from Shandong Jukang Biotechnology Co., Ltd. (Jinan, China). HCl was purchased from Liaoning Quanrui Reagent Co., Ltd. (Jinzhou, China). The purity of the above chemicals is analytically pure.
2.2. Preparation of SPI Enzymatic Hydrolysate
The enzymatic hydrolysis of SPI was performed as reported with slight modifications [
21]. SPI was sequentially hydrolyzed by pepsin and flavourzyme. The SPI was dissolved in 98 mL of deionized water at a substrate concentration of 2% (
w/
w) and adjusted to pH 2.0 with 2 mol/L HCl. Pepsin was then added to the SPI, and the mixture was incubated at 37 °C for 6 h with an enzyme-to-SPI ratio of 4%. Subsequently, the reaction temperature was raised to 50 °C, and the pH was adjusted to 7.0 with 3 mol/L NaOH. Flavourzyme was added, and the protein was further incubated for 4 h with an enzyme-to-SPI ratio of 1.5%. After enzymatic hydrolysis, the enzyme was inactivated by heating in a water bath at 95 °C for 20 min. The SPI enzymatic hydrolysate (SPIEH) was then centrifuged at 4000 rpm for 15 min, freeze-dried, and stored at −20 °C for a further analysis.
2.3. Molecular Weight Distribution of SPIEH
The SPIEH was determined using an integrated gel permeation chromatography (GPC) system (LC20, Shimadzu, Jingdu, Japan). SPIEH (5 mg/mL) was absorbed with a syringe and filtered with a 0.45 μm molecular membrane. A 100 μL filtrate was absorbed by a microsampler and injected into the GPC column.
2.4. Determination of Free Amino Acid Content in SPIEH
The free amino acid contents were determined using a Biochrom30+ amino acid analyzer (Biochrom Ltd., Cambridge, UK) following the reported method with some modifications [
22]. A 1 mL aliquot of the supernatant was mixed with 1 mL of 5% sulfosalicylic acid. The separation was performed using a Na-type cation resin chromatography column (200 mm × 4.6 mm, 3 µm particles) with UV detection at a wavelength of 440 nm. The column temperature was programmed to increase from 55 °C to 65 °C to 77 °C, and the injection volume was 20 µL with a flow rate of 10 mL/h.
2.5. Evaluation of Salt Taste
2.5.1. Sensory Evaluation
The sensory evaluation method was adapted with slight modifications [
23]. The sensory panel consisted of 14 trained individuals (7 males and 7 females, ages 22 to 32, with no history of abnormal taste perception). Following sensory training [
14], evaluations were conducted in a controlled room at 25 ± 1 °C with no communication allowed between participants. The lyophilized powders of enzymolysis from different fractions obtained from multiple collections were prepared into 1% (
w/
v) solutions. Aqueous solutions of 0.08% citric acid, 0.08% caffeine, 0.35% NaCl, and 0.35% MSG were served as the standard references for sour, bitter, salty, and umami tastes, respectively. The taste characteristics of each solution were assessed, and the intensity of the salty taste was rated on a scale from 0 to 10, where 0 indicated no taste and 10 indicated the strongest taste [
24].
2.5.2. Electronic Tongue Analysis
The sample solutions of the same concentration were prepared using a 600 mg/L NaCl solution as the control. The prepared solutions were then poured into the special measuring cup of an electronic tongue (ALPHA MOS (Shanghai) Instrument Trading Co., Ltd., Shanghai, China) and measured at room temperature. Each sample was measured in triplicate, with the instrument stabilizing after the first measurement. The average value of the last three signal data points was used as the sample’s taste signal intensity.
2.6. Separation and Purification Salty Peptides from SPIEH
2.6.1. Separation by UF
Three hundred grams of SPIEH was dissolved in 15 L of ultrapure water and completely dissolved using a KQ 5200 V ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China). A UF system (Jilin Haipu Technology Development Co., Changchun, China) was used to separate proteins based on molecular weight. The SPIEH solution was first passed through a 10 kDa UF membrane, followed by a 5 kDa UF membrane, and then desalted using a desalting column. Further separation was performed using 3 kDa and 1 kDa UF membranes (Millipore Corporation, Shanghai, China). After multiple filtration steps, three peptide sample solutions with molecular weights of 3–5 kDa, 1–3 kDa, and <1 kDa were obtained. These samples were collected, and freeze-dried after spin evaporation, and the group with the highest salt content was selected for further separation and purification based on the evaluation of salt taste.
2.6.2. Purification by GFC
The UF group (MW < 1 kDa) was selected for GFC. The freeze-dried samples were dissolved in ultrapure water to a concentration of 50 mg/mL and then passed through a 0.45 μm filtration membrane. The sample solution was separated using an AKTA protein purifier (GE Healthcare, Chicago, IL, USA) equipped with a Sephadex G-10 gel chromatography column (2.6 × 80 cm, Smart-Lifesciences Corp., Changzhou, China). A 1 mL aliquot of the filtered solution was injected into the gel column, and separation was performed using ultrapure water as the eluent at a flow rate of 0.60 mL/min, with UV detection set to 220 nm. The fractions were collected from multiple injections, concentrated, and freeze-dried for the evaluation of salt taste.
2.7. Identification of the Peptide Sequence
The sample from GFC was desalted using ZipTip C18, vacuum-dried, and analyzed by LC-MS/MS (Thermo, Waltham, MA, USA). The chromatographic separation was performed on a PepMap RSLC C18 (75 μm × 150 mm, 2 μm, 100 A) column with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile (ACN) as mobile phase B. A 4 µL sample was injected, with UV detection at 214 nm and a flow rate of 0.25 μL/min. The elution gradient was as follows: 0–10% B from 0 to 8 min, 10–15% B from 8 to 33 min, 15–28% B from 33 to 43 min, 28–40% B from 43 to 50 min, 40–95% B from 50 to 65 min, and 95–5% B from 65 to 70 min.
Electrospray ionization (ESI) was used as the ionization source for a Mass Spectrometry analysis. The mass spectrum was scanned in the
m/
z range of 300–1400. Nitrogen was used as the collision gas with a flow rate of 250 μL/min. The resolution of the primary mass spectrum was 7 × 10
4 (for
m/
z ≤ 200), with an automatic gain control (AGC) target set to 5 × 10
4, and the collision energy for the secondary mass spectrum was set between 12 and 14 eV. The Mass Spectrometry error tolerance was set to 0.05 Da, and the resolution for the secondary mass spectrum was 1.75 × 10
4 (for
m/
z ≤ 200). A data analysis was performed using the PEAKS 12.0 software with the Uniprot proteome database (
www.uniprot.org; proteome UP000008827_20240125.fasta) for peptide identification and a semi-quantitative analysis.
2.8. Construction of Saltiness Receptor Model TMC4
The TMC4 (transmembrane channel 4-like) protein amino acid sequence was obtained from the NCBI database (
https://www.ncbi.nlm.nih.gov/, accessed on 23 April 2024; accession number: NP_001138775). Initial modeling using SWISS-MODEL (
https://swissmodel.expasy.org/, accessed on 23 April 2024) indicated that the homologous protein model had a low consistency of 22.94%, which is below the 30% threshold for reliable models. Consequently, the TMC4 protein model was created from scratch using the deep learning model AlphaFold2, and the model with the highest predicted accuracy was selected. The overall quality of the model was assessed using the Ramachandran plot and the ERRAT method (
https://services.mbi.ucla.edu/errat/) (accessed on 23 April 2024), which evaluated the rationality and reliability of the protein structure.
2.9. Molecular Docking of Potential Peptides with TMC4
The 3D structure of the polypeptide was constructed using Discover Studio and saved as a PDB file with minimal energy configuration. The receptor protein was prepared by adding polar hydrogen atoms and assigning equilibrium charges using AutoDockTools 1.5.6 software. Both the receptor protein and the ligand molecules were converted into PDBQT format for molecular docking. AutoDock Vina 1.1.2 was employed to perform global molecular docking simulations between the receptor protein and the ligand molecules. The docking results were analyzed visually using PyMOL 3.0 and Discover Studio 2023. AR, RG, and RS peptides were used as positive controls. The top seven peptides with the most favorable free energy of binding were selected for a detailed visual analysis using Discovery Studio.
2.10. In Silico Screening of Premium Salty Peptides
2.11. Statistical Analysis
All the data were analyzed using IBM SPSS Statistics 27.0 software (SPSS Inc., Chicago, IL, USA), and the experiment was repeated in triplicate. The chart was prepared using Origin 2024 (OriginLab Inc., Northampton, MA, USA).
4. Conclusions
In this study, a soybean protein isolate was used as raw material. After hydrolysis by pepsin and flavourzyme, the molecular weight distribution of the hydrolysates was mainly <1 kDa (53%), rich in various hydrophilic amino acids (51.87%) and umami amino acids (38%). The soybean isolate protein hydrolysate (SPIEH) was purified through ultrafiltration (UF) and gel filtration chromatography (GFC), resulting in fractions with a sensory evaluation score of 7 and an electronic tongue score of 10.36. LC-MS/MS identified 84 potential salty peptides. A 3D model of the salty receptor TMC4 was constructed and docked with the potential salty peptides, identifying seven peptides (FPPP, GGPW, IPHF, IPKF, IPRR, LPRR, and LPHF) with lower binding energies. Molecular simulation results indicate that Glu531, Asp491, Val495, Ala401, and Phe405 play key roles in peptide–receptor interactions, contributing significantly to the salty taste of the peptides. The further basic bioinformatics analysis identified IPHF, LPHF, GGPW, and IPKF as four non-toxic, non-sensitizing, and stable salty peptides. In the future, we will study the synthesis of four kinds of salty peptides, and use an electronic tongue to identify the salty intensity.