A dry-cured ham-derived salty peptide, and a preparation method and application thereof

Abstract

This invention discloses a salty peptide derived from dried cured ham, its preparation method, and its application, belonging to the field of bioprotein peptide technology. The salty peptide has an amino acid sequence of one or more of LDFDP, FDGDGDR, and DWDRDL. By combining peptidomics with virtual screening based on molecular docking, the endogenous peptide profile of traditional dried cured ham is comprehensively characterized, thereby screening for salty peptides derived from dried cured ham. The process is as follows: (1) The endogenous peptide profile of dried cured ham is obtained through peptidomics, and the peptides are sorted by an online prediction model based on machine learning; (2) Molecular docking technology is used to evaluate the binding affinity of endogenous peptides to three salty receptors to obtain candidate salty peptides with high potential; (3) The candidate peptides are chemically synthesized and the salty intensity is objectively quantified through sensory evaluation, electronic tongue analysis, etc.; (4) Finally, the transcriptional response of key salty receptor genes in cells under peptide stimulation is quantified by real-time quantitative polymerase chain reaction.

Description

Technical Field

[0001] This invention belongs to the field of bioprotein peptide technology, and relates to a salty peptide derived from dry-cured ham, its preparation method and application. Background Technology

[0002] Saltiness is one of the five basic taste patterns, playing a crucial role not only in the palatability of food but also as an important physiological regulator, modulating electrolyte balance, neuronal excitability, and humoral homeostasis. Sodium chloride (NaCl) has long been a major source of saltiness in industrial food processing and home cooking. However, mounting epidemiological evidence suggests that excessive dietary sodium intake is associated with an increased risk of hypertension, cardiovascular disease, and cerebrovascular events, making high-salt diets a major modifiable risk factor for the global burden of noncommunicable diseases. The World Health Organization (WHO) recommends that adults consume no more than 5 grams of salt per day, but many populations worldwide consistently exceed this threshold, thus highlighting the urgent need for effective sodium reduction strategies. Currently, finding NaCl alternatives is a primary salt reduction strategy, including: chloride alternatives (such as potassium chloride, magnesium chloride, calcium chloride, etc.), non-chlorinated salt alternatives (such as lactate, phosphate, sodium citrate, calcium ascorbate, etc.), natural plant extracts and their derivatives, and endogenous food saltiness / saltiness-enhancing peptides. However, chloride substitution and non-chlorinated salt substitution often produce unpleasant bitter or metallic tastes and have limits on their food additive content. The diversity of natural plant extracts and their derivatives can easily raise regulatory and safety concerns. Endogenous food saltiness / saltiness-enhancing peptides mainly come from fermented foods. They trigger saltiness perception by binding to saltiness receptor proteins through peptide chains, exhibiting superior flavor fidelity, metabolic compatibility, and technical stability. They are also highly safe and easy to apply, showing broad application prospects.

[0003] Molecular docking is an important method in modern structural biology and rational food composition design, and it can serve as a powerful strategy for discovering endogenous salty / salty-enhancing peptides in the context of sodium reduction. In molecular docking, candidate peptides are computationally localized into the ligand-binding pockets of validated salty receptors, and scored based on predicted binding affinity, conformational stability, and complementarity of electrostatic / hydrophobic interactions. This enables high-throughput sequencing of potential salty peptides before experimental validation. This comprehensive computational workflow combines structural insights with functional predictions, elucidating salty taste perception mechanisms at the atomic level and contributing to the rational design of salty peptide molecules.

[0004] Dry-cured ham is a fermented meat product with a centuries-old traditional process, renowned for its rich, savory flavor and high nutritional value. During the long maturation process of dry-cured ham, endogenous and microbial proteases extensively hydrolyze various muscle proteins, including myofibrillar proteins, releasing a large number of small molecule peptides with unique physiological activities. Increasing evidence suggests that dry-cured ham is an important source of bioactive peptides. Furthermore, in the field of salt reduction and salt enhancement research, based on the high content and diversity of peptide sequences of peptides derived from dry-cured ham, it is also a promising source of salty peptides / salty-enhancing peptides that has not yet been fully explored. Chinese patent CN115028682A isolated three salt-enhancing peptides—DL, FMSALF, and HVRRK—from Jinhua dry-cured ham using chromatographic separation technology; however, this method is complex, and the chromatographic purification process does not meet the requirements of green processing. Salty / salty-enhancing peptides from dry-cured ham have great potential in flavor regulation; however, the systematic mining and annotation of its rich salty bioactive peptide resources remains incomplete, and a rapid and effective mining workflow is lacking. Furthermore, the molecular basis of their interaction with taste receptors has not yet been scientifically characterized. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to provide a novel salty peptide derived from dry-cured ham, its preparation method and application.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] This invention proposes a salty peptide derived from dry-cured ham, the amino acid sequence of which is one or more of LDFDP (SEQ ID No: 1), FDGDGDR (SEQ ID No: 2), and DWDRDL (SEQ ID No: 3).

[0008] LDFDP: Leu Asp Phe Asp Pro; molecular weight is 605.65 Da.

[0009] FDGDGDR: Phe-Asp-Gly-Asp-Gly-Asp-Arg; molecular weight is 780.75 Da.

[0010] DWDRDL: Asp-Trp-Asp-Arg-Asp-Leu; molecular weight is 818.84 Da.

[0011] The salty peptides were screened from polypeptides derived from dry-cured ham and are bound to ENaC, TRPV1, and TMC4 receptor proteins.

[0012] This invention also proposes a method for preparing the above-mentioned salty peptides derived from dry-cured ham, including artificial synthesis; the artificial synthesis includes, but is not limited to, chemical synthesis and biological synthesis; the chemical synthesis includes, but is not limited to, one or more of solid-phase peptide synthesis and liquid-phase peptide synthesis.

[0013] This invention also proposes the application of the above-mentioned salty peptides derived from dry-cured ham in the preparation of umami and / or salty seasonings and foods.

[0014] The application is the use of the above-mentioned dry-cured ham-derived salty peptides in the preparation of seasonings and food products.

[0015] The present invention also proposes a umami and / or saltiness enhancer, the main components of which include the above-mentioned salty peptides derived from dry-cured ham.

[0016] This invention also proposes a method for screening the above-mentioned salty peptides derived from dry-cured ham, comprising the following steps: (1) Extraction of peptides from dry-cured ham using ethanol precipitation method; (2) Peptidomics analysis of dried cured ham: The peptide solution of dried cured ham was subjected to ultrafiltration and desalting; the peptides were analyzed by liquid chromatography-mass spectrometry system and PEAKS Studio software to obtain the peptide profile of dried cured ham. (3) Screening of salty peptides: Screening for peptides with a bioactivity score ≥0.5 and a salty score >0.3, that are hydrophilic and can bind to salty taste receptors ENaC, TRPV1, and TMC4; (4) The peptides screened in step (3) were synthesized by solid-phase synthesis. The selected salty peptides, LDFDP, FDGDGDR, and DWDRDL, were verified by sensory evaluation, electronic tongue analysis and mathematical modeling.

[0017] Preferably, in step (1), the specific steps of the ethanol precipitation method are as follows: take dried cured ham pieces, chop them, homogenize them, filter them, centrifuge them, add 2-3 times the volume of ethanol to the supernatant for precipitation, remove the ethanol by rotary evaporation, and freeze-dry them to obtain dried cured ham polypeptide powder; the volume fraction of ethanol is 40%.

[0018] Further preferred, in step (1), the dry-cured ham is Jinhua ham, Rugao ham, or Xuanwei ham.

[0019] Preferably, in step (2), the dry-cured ham peptide solution is a mixture of dry-cured ham peptide and pure water prepared in step (1).

[0020] Preferably, in step (2), the concentration of the dry-cured ham polypeptide solution is 1-2 mg / mL; more preferably 1 mg / mL.

[0021] Preferably, in step (2), the ultrafiltration method is to perform ultrafiltration through a 10 kDa ultrafiltration centrifuge column (Millipore, Billerica).

[0022] Preferably, in step (2), desalination is performed using a Pierce C18 centrifugal column.

[0023] Preferably, in step (3), the screening target is dry-cured ham peptide with no more than 10 amino acid residues and a relative molecular mass of less than 1000 Da.

[0024] Preferably, in step (3), the specific screening steps are as follows: a. The bioactivity of peptides is predicted using the PeptideRanker online server (https: / / distill-deep.ucd.ie / PeptideRanker / ), and candidate peptides with a score ≥0.5 are selected for taste annotation; b. Taste annotation is performed using the BIOPEP-UWM online database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ), and candidate peptides with a salty score >0.3 are designated as potential salty peptides; c. The water solubility of potential salty peptides is predicted and evaluated using a peptide evaluation tool (https: / / www.genscript.com.cn / tools / peptide-analyzing-tool); d. Potential salty peptides are molecularly docked with three salty-related salty receptors (ENaC (PDB ID: 6WTH), TRPV1 (PDB ID: 7LQY), and TMC4 (UniProt ID: Q7Z404)) using ChemDraw 22.2. The 3D structure of the ligand was subjected to energy minimization. All three receptor protein models were evaluated using the PROCHECK program. Receptor preparation was performed in AutoDock Tools 1.5.7. Molecular docking simulation was performed using AutoDockVina, and the lowest binding energy conformation of each peptide-receptor complex was retained for analysis. More preferably, in step (3), the docking scores of the potential salty peptides with salty-related receptors ENaC, TRPV1, and TMC4 range from -10.5 to -5.3 kcal / mol.

[0025] The beneficial effects of this invention are as follows: 1. The salty peptides LDFDP, FDGDGDR, and DWDRDL derived from dried ham provided in this invention exhibit high binding affinity to salty taste receptors ENaC, TRPV1, and TMC4. The binding energies of FDGDGDR to TMC4 are -8.1 kcal / mol, TRPV1 is -8.8 kcal / mol, and ENaC is -9.4 kcal / mol; the binding energies of LDFDP to TMC4 are -8.9 kcal / mol, TRPV1 is -7.7 kcal / mol, and ENaC is -9.2 kcal / mol; the binding energies of DWDRDL to TMC4 are -7.4 kcal / mol, TRPV1 is -8 kcal / mol, and ENaC is -9.9 kcal / mol. Generally, binding energies below -5.0 kcal / mol indicate stable receptor-ligand binding, demonstrating the thermodynamic advantages of the salty peptides in this invention and their significant potential as a primary medium for salty taste perception.

[0026] 2. According to RT-qPCR analysis, DWDRDL significantly upregulated the mRNA expression of all three salt taste receptors ENaC, TMC4, and TRPV1 in HepG2 cells; FDGDGDR selectively enhanced the expression of TMC4, while LDFDP played an important role in increasing the transcriptional level of ENaC.

[0027] 3. This invention establishes an efficient method for screening salty peptides, confirming the potential of peptides derived from dry-cured ham as natural salty enhancers, and providing new scientific insights into the molecular regulatory mechanism of salty peptides and human salty perception.

[0028] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0029] Figure 1 The peptide profile features of traditional dry-cured ham in Example 1 of this invention are shown below; (A) relative molecular mass distribution; (B) peptide chain length distribution; (C) upset diagram of differentially abundant peptides; and (D) length distribution of common peptides. Figure 2 This is a three-dimensional diagram of the docking of three salty peptides with salty receptors in Example 1 of the present invention; Figure 3 This is a diagram showing the binding sites of LDFDP and TMC4 in Embodiment 1 of the present invention; Figure 4 This is a diagram showing the binding sites of FDGDGDR and TRPV1 in Embodiment 1 of the present invention; Figure 5This is a diagram showing the binding sites of DWDRDL and ENaC in Embodiment 1 of the present invention; Figure 6 The taste characteristics of the three synthetic peptides in Example 1 of this invention; Figure 7 The analysis of NaCl solution of equal concentration (1 mg / mL) and peptides LDFDP, FDGDGDR and DWDRDL by electronic tongue in Example 1 of the present invention; Figure 8 Cell viability as determined by the MTT assay in Example 1 of this invention; Figure 9 The mRNA expression levels of TMC4, TRPV1, and ENaC in Example 1 of this invention are shown in Figure 1 (P<0.05). Figure 7 , Figure 8 middle, P < 0.05; P < 0.01; P < 0.001; P < 0.0001. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0031] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0032] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0033] Example 1: 1.1. Sample Information This invention selected three traditional Chinese dry-cured hams as research subjects. Jinhua ham was purchased from Zhejiang Jinhua Jinzi Ham Co., Ltd., Rugao ham from Jiangsu Changshou Group, and Xuanwei ham from Xuanwei Puji Ham Food Co., Ltd. Biceps femoris muscles were extracted from the dry-cured hams and cut into 1 cubic centimeter pieces. The chopped samples were temporarily stored in an ultra-low temperature freezer at -80°C.

[0034] 1.2. Peptidomics Analysis The extraction of peptides from dried ham was performed using the ethanol precipitation method. Dried ham pieces were chopped and homogenized with ultrapure water at a ratio of 1:5 (meat / water, m / V) (22000 r / min, 15 s / time, 3 times). The resulting homogenate was filtered through three layers of gauze (to remove insoluble matter such as connective tissue). The filtrate was then centrifuged (12000 rpm). g (20 min), take the supernatant (containing some large protein molecules and small peptide molecules), add 3 volumes of ethanol (ethanol volume fraction of 40%) for sedimentation (to precipitate large protein molecules), let it stand for 12 h for sedimentation, and then centrifuge (12000) g The supernatant was obtained after 20 min of rotary evaporation (40 °C) to remove the ethanol from the small molecule peptides. The resulting ethanol solution was then freeze-dried to obtain the dried cured ham peptide powder, which was stored at -80 °C. Before peptidomics analysis, the peptide powder was reconstituted in ultrapure water to prepare a 1 mg / mL peptide solution.

[0035] Peptide solutions were ultrafiltered using a 10 kDa ultrafiltration column (Millipore, Billerica) and desalted using a Pierce C18 column. Peptidomics analysis was performed using an UltiMate 3000-Orbitrap Eclipse (Thermo Fisher Scientific, MA, USA) liquid chromatography-mass spectrometry system and PEAKS Studio software. The database used was uniprot-Sus scrofa (version 2025, 22832 entries) and the target sequence, with enzymatic digestion set to None. Search parameters included: fragment ion mass tolerance of 0.02 Da, precursor ion mass tolerance of 10 ppm, variable modifications: Oxidation (M) 15.99, Deamidation (NQ) 0.98, protein card value (containing at least one unique peptide), and peptide card value (-10lgP ≥ 20).

[0036] 1.3. Quick Screening Process (1) The bioactivity of peptides was predicted using the PeptideRanker online server (https: / / distill-deep.ucd.ie / PeptideRanker / ). Candidate peptides with scores ≥0.5 were selected for taste annotation.

[0037] (2) Taste annotation was performed using the BIOPEP-UWM online database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ), and candidate peptides with a salty score >0.3 were identified as potential salty peptides.

[0038] (3) Potential salty peptides were molecularly docked with three salty-related receptors (ENaC (PDB ID: 6WTH), TRPV1 (PDB ID: 7LQY), and TMC4 (UniProt ID: Q7Z404)). The ligand 3D structures were subjected to energy minimization using ChemDraw 22.2. All three receptor protein models were evaluated using the PROCHECK program (https: / / saves.mbi.ucla.edu / ), and receptor preparation was performed in AutoDock Tools 1.5.7. Molecular docking simulations were performed using AutoDockVina, and the lowest binding energy conformation of each peptide-receptor complex was preserved for analysis.

[0039] 1.4. Analysis of the taste characteristics of synthetic peptides 1.4.1 Sensory evaluation Solid-phase synthesis was used to rapidly screen salty peptides, achieving a purity of over 98%. Eight trained sensory evaluators (4 men and 4 women, aged 22-30 years) participated in this study; all participants had extensive sensory evaluation experience and had received systematic training. Sensory evaluation was used to determine the taste characteristics and taste thresholds of potential salty peptides.

[0040] Taste Characteristic Evaluation Method: Sensory testing was conducted in an environment with a temperature of 21–27 °C and a relative humidity of 40%–75%. Prior to sample evaluation, sensory evaluators were trained using reference standard solutions: 0.08% citric acid (representing sourness), 1% sucrose (representing sweetness), 0.002% quinine (representing bitterness), 0.35% sodium chloride (representing saltiness), 0.35% monosodium glutamate (representing umami), and 0.08% tea polyphenols (representing astringency). Each salty peptide sample was prepared into a 1 mg / mL solution using deionized water, randomly coded with a three-letter alphanumeric identifier, and placed in a 30 mL disposable plastic cup. Sensory evaluators rated the perceived intensity of each taste attribute on a 10-category scale (0 = "not perceived", 10 = "extremely strong"), with a perceived intensity of 5.0 for the reference standard solution. A mandatory 5-minute break is scheduled between each sample test by the sensory evaluator, and the patient's mouth is cleaned with deionized water to minimize residual effects and taste fatigue.

[0041] Taste threshold evaluation method: A triangulation method was used. The salty peptides were dissolved in deionized water to prepare a stock solution with an initial concentration of 1 mg / mL, followed by serial 2-fold dilutions to prepare different concentration gradients. For each test concentration, two independent blank control groups (deionized water) were included. In the triangulation, trained sensory evaluators were asked to distinguish samples that differed in taste from the two control groups. The threshold was defined as the average of the highest concentration of the salty peptide solution that could be reliably distinguished from the control group and the lowest concentration that could not be distinguished.

[0042] 1.4.2 Electronic tongue measurement The saltiness intensity of salty peptides was evaluated using an electronic tongue system (cTongue electronic tongue, Shanghai Baosheng Industrial Development Co., Ltd.).

[0043] 1.4.3 Quantitative assessment of the salty intensity of synthetic peptides A sensory evaluation-instrument calibration model was established based on partial least squares regression. Six precisely controlled NaCl standard solutions were prepared: 0, 3, 6, 9, 12, and 15 mg / mL. A trained sensory panel first familiarized themselves with the saltiness intensity of all six standard solutions. Subsequently, samples were assigned blind codes and randomly ranked for sensory evaluation. Sensory panel members ranked the samples according to perceived saltiness intensity and recorded the perceived NaCl concentration (mg / mL). Simultaneously, an electronic tongue was used to detect the samples and collect the detection signals. A partial least squares regression model was established: Y = 0.9343X + 0.3616, where Y is the perceived NaCl concentration and X is the electronic tongue test data. A synthetic peptide (final concentration of 1 mg / mL) was added to NaCl solutions of 6, 9, and 12 mg / mL, respectively. The resulting peptide-sodium chloride mixtures were analyzed using a cTongue system, and their perceived NaCl concentration was predicted using the validated calibration model.

[0044] 1.5. Cell Experiments 1.5.1 Cell viability assay HepG2 cells were cultured in DMEM medium (Gibco, Auckland, New Zealand) containing 10% fetal bovine serum and 100 U / mL penicillin and 100 mg / mL streptomycin. To assess the cytotoxicity of synthetic peptides, HepG2 cells were treated for 4 hours with different concentrations of screened salty peptides (0, 250 µmol / L, 500 µmol / L, and 1000 µmol / L). To assess the effect of salty peptides on the expression of salt protein taste receptors (TMC4, TRPV1, ENaC), a peptide concentration of 1000 µmol / L was selected for cell treatment, with 1000 µmol / L NaCl used as a positive control.

[0045] Cell viability assay: HepG2 cell viability was detected using the MTT assay kit (Beyotime Biotechnology, Shanghai, China).

[0046] 1.5.2 Real-time quantitative PCR (RT-qPCR) RT-qPCR detection: Total RNA was extracted from HepG2 cells using Trizol reagent (Takara, Osaka, Japan) and reverse transcribed into cDNA using a reverse transcription kit (Takara, Osaka, Japan). RT-qPCR was performed using the MyiQ 2 real-time PCR detection system. GAPDH was used as the reference gene, and data were processed using the ΔΔCT method.

[0047] The following are the synthesized specific primers: TMC4: (F)5′-CAGGCCCATCGCTGTCTTC-3′ (SEQ ID No: 4) and (R) 5′-GCTGGCATTTCTTTGCCTGT-3′ (SEQ ID No: 5); TRPV1: (F)5′-AGCTCTCCCTTCGAGTAGCA-3′ (SEQ ID No: 6) and (R) 5′-CCAGTGTGCAACCAGCTAGA-3′ (SEQ ID No: 7); ENaC: (F) 5′-CTGCACCTGTCAGGGGAACA-3′ (SEQ ID No: 8) and (R) 5′-CTTCATGAGCCCTGGAGTGG-3′ (SEQ ID No: 9); GAPDH: (F) 5′-AATGGGCAGCCGTTAGGAAA-3′ (SEQ ID No: 10) and (R) 5′-GCCCAATACGACCAAATCAGA-3′ (SEQ ID No: 11).

[0048] All primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0049] 2. Results and Analysis 2.1. Distribution characteristics of peptides in dry-cured ham Figure 1 The peptide profiles of three traditional dry-cured hams were shown: Figure 1 The letter A indicates that the molecular weight of these peptides is mainly between 1000 and 2000 Da; for example... Figure 1 The peptide chain length distribution shown in Figure B indicates that most of the identified peptides consist of 7-20 amino acid residues; for example... Figure 1Figures C and D show that the three types of cured hams share 1360 common peptides, with the lengths of these shared peptides mainly concentrated in the ranges of 3-4 and 8-17 residues. Typically, peptides with a molecular weight less than 1000 Da and a length of 4-10 amino acid residues have been reported to have superior antioxidant capacity, usually mediated by hydrogen atom transfer or single-electron transfer mechanisms. Furthermore, many experimentally validated salty peptides or salt-enhancing peptides are also concentrated in this low molecular weight and long peptide chain region. The structural similarity between antioxidant peptides and salty peptides suggests that they share a set of physicochemical mechanisms, such as conformational flexibility, solvent exposure of hydrophobic / aromatic side chains, and favorable charge distribution, which may facilitate their dual binding to redox-sensitive targets (such as Keap1) and taste receptors (such as ENaC). Previous studies have shown that the salty pentapeptide QPGDY exerts dose-dependent antioxidant activity through hydrogen atom transfer (HAT), effectively scavenging free radicals such as DPPH• and ABTS•. While the functional overlap between bioactivity and taste modulation requires rigorous mechanistic validation, our data highlight the value of targeting peptides smaller than 1000 Da from conventionally cured hams. These peptides not only serve as natural flavor enhancers but also possess the potential to act as multifunctional nutritional and health-promoting compounds. Overall, this peptide-centric analytical approach advances our understanding of the molecular basis of regional ham quality and lays the foundation for the rational design of next-generation functional meat components.

[0050] 2.2. Results of rapid screening of salty peptides in dry-cured ham This invention focuses on a rapid screening procedure for peptides with no more than 10 amino acid residues, typically with a molecular weight of around 1000 Da or lower. Using PeptideRanker online prediction, 562 candidate peptides were screened from Jinhua ham, 543 from Rugao ham, and 487 from Xuanwei ham. Flavor characteristics of the candidate peptides were annotated using the BIOPEP-UWM online database. Among the annotated peptides, 39 were from Jinhua ham, 18 from Rugao ham, and 33 from Xuanwei ham, with a saltiness score ≥ 0.3. After removing common peptides, a total of 60 peptides were identified. These 60 peptides were designated as potential salty peptides and subsequently subjected to molecular docking analysis.

[0051] Table 1 summarizes the predicted binding energies (kcal / mol) of 60 candidate salty peptides after docking with each salty taste receptor. All potential salty peptide docking scores ranged from -10.5 to -5.3 kcal / mol, with binding energies generally below -5.0 kcal / mol indicating stable receptor-ligand binding. This demonstrates the thermodynamic advantages of the salty peptides in this invention and their significant potential as a primary mediator of salty taste perception. While energy-based scoring is an important indicator for assessing the reliability and biocompatibility of molecular docking, it is inherently receptor-dependent. Therefore, comprehensive multi-receptor evaluation is crucial to avoid target bias. This invention systematically integrates three functionally distinct salty taste receptors (ENaC, TRPV1, and TMC4) into a unified virtual screening framework. This holistic approach enables a detailed assessment of the physiological basis and mechanisms of peptide-receptor interactions in traditional cured ham.

[0052] Based on the molecular docking results of these three salty taste receptors, among 60 potential salty peptides, PFDQDDW exhibited the most favorable binding (i.e., lowest binding energy) to each salty taste receptor: -9.4 kcal / mol with TMC4, -9.2 kcal / mol with TRPV1, and -10.5 kcal / mol with ENaC, indicating that PFDQDDW, as a ligand, forms a more thermodynamically stable complex with the salty taste receptor protein. Meanwhile, among the peptides binding to TMC4, LDFDP ranked second with a binding energy of -8.9 kcal / mol; among the peptides binding to TRPV1, FDGDGDR ranked second with a binding energy of -8.8 kcal / mol; and among the peptides binding to ENaC, DWDRDL ranked second with a binding energy of -9.9 kcal / mol. Therefore, PFDQDDW, LDFDP, FDGDGDR, and DWDRDL were selected as salty peptides. Prior to solid-phase synthesis, the water solubility of these four sequences was predicted and evaluated using a peptide evaluation tool (https: / / www.genscript.com.cn / tools / peptide-analyzing-tool). This website provides a solubility prediction result (Good or Poor) after inputting the peptide sequence. However, experimental evaluation after solid-phase synthesis showed that PFDQDDW had poor solubility in aqueous solution, while LDFDP, FDGDGDR, and DWDRDL exhibited good hydrophilicity. Therefore, only LDFDP, FDGDGDR, and DWDRDL were evaluated for their salty characteristics.

[0053] Table 1. Binding energies of 60 peptides to TMC4, TRPV1, and ENaC

[0054] 2.3. Characteristics and binding sites of salty peptides in dry-cured ham Table 2. Taste characteristics and receptor-ligand binding sites of three synthetic salty peptides

[0055] Table 2 and Figure 2-5 The structural information, taste characteristics, and binding of three synthetic salty peptides (LDFDP-TMC4, FDGDGDR-TRPV1, and DWDRDL-ENaC) are presented. The relative molecular weights of all three peptides are below 1000 Da. LDFDP, FDGDGDR, and DWDRDL all produced a pronounced salty taste, primarily attributed to the presence of aspartic acid (Asp, D) in the peptide sequence. Furthermore, LDFDP and DWDRDL exhibited pronounced bitterness and sourness, while FDGDGDR showed a pronounced umami taste. Astringency was detected in all three peptides, due to the effects of various residue types, including acidic residues (Asp, D), basic residues (Arg, R), and aromatic / hydrophobic residues (Phe, F). Regarding detection thresholds, DWDRDL had the lowest value (0.2344 mg / mL), followed by FDGDGDR (0.4688 mg / mL) and LDFDP (0.9375 mg / mL).

[0056] Binding forces and binding sites indicate that peptide binding to receptor proteins is mainly mediated by hydrogen bonds, electrostatic interactions, and hydrophobic interactions, with hydrogen bonds being the main stabilizing force. A total of 29 receptor-side amino acid residues were identified in all three complexes that directly participate in peptide binding. In the LDFDP-TMC4 complex, LDFDP forms hydrogen bonds with TMC4 residues Gln524, Gln527, and Glu525; in the FDGDGDR-TRPV1 complex, FDGDGDR forms hydrogen bonds with TRPV1 residues Tyr629, Lys641, Asn606, Asp627, Asn654, Gly622, Asp648, Glu650, Phe651, and Arg619; and in the DWDRDL–ENaC complex, DWDRDL forms hydrogen bonds with ENaC residues Tyr140, His298, His299, Ile434, Ala360, Tyr407, Ser261, Leu263, Pro264, and Thr266. All residue numbers are based on the UniProt reference sequences of human TMC4 (UniProt ID: Q7Z404), TRPV1 (PDB ID: 7LQY), and ENaCα (PDB ID: 6WTH). Hydrogen bonds constitute the main molecular interaction mediating peptide binding to the salty taste receptor.

[0057] 2.4. Saltiness value of salty peptides in dry-cured ham The taste characteristics of synthetic peptides were quantitatively characterized using an electronic tongue system. The taste characteristics of three synthetic peptides are as follows: Figure 6 As shown, NaCl, as a positive control, showed the highest salty response value (2.15), with the response values ​​of the three salty peptides decreasing sequentially: LDFDP (1.73), FDGDGDR (1.33), and DWDRDL (1.11). Although the saltiness of these three peptides was lower than that of NaCl, each peptide produced a detectable salty signal. Notably, significant umami response values ​​were observed in all salty peptide samples, with DWDRDL (2.57) exceeding that of NaCl (2.32), while LDFDP (2.19) and FDGDGDR (1.92) also exceeded the levels of NaCl. Given the recurring key residues in these peptide sequences, including Asp (D), Phe (F), and Gly (G), which can simultaneously activate salty and umami receptors, the salty and umami characteristics of the three peptides are mechanistically plausible.

[0058] Sensory evaluation data of standard NaCl solutions were combined with electronic tongue measurement data to construct a partial least squares regression (PLSR) model. This model can predict the perceptible NaCl concentration in peptide solutions based on electronic tongue response values ​​and assess their salinizing effect in NaCl solutions. In the PLSR regression model (Y = 0.9343X + 0.3616), X represents the standardized electronic tongue response value of the test sample, and Y represents the perceptible NaCl concentration (mg / mL) predicted by the regression model. Figure 7 As shown, the perceived NaCl concentrations of the three salt-enhancing peptides after dissolving in deionized water were 0.88 mg / mL (DWDRDL), 0.87 mg / mL (FDGDGDR), and 1.00 mg / mL (LDFDP), respectively, indicating that their inherent saltiness was comparable to, but slightly lower than, that of NaCl. This result is consistent with... Figure 6 Consistent.

[0059] To evaluate the saltiness-enhancing effect of salty peptides in NaCl solution, standard NaCl solutions of 6 mg / mL, 9 mg / mL, and 12 mg / mL were prepared, and salty peptides were added to the NaCl solutions (to a concentration of 1 mg / mL). In the 6 mg / mL NaCl standard solution, the perceived NaCl concentration was 6.736 mg / mL. After adding DWDRDL or LDFDP, this value significantly increased to 8.20 mg / mL and 8.02 mg / mL, respectively (P<0.05). This indicates that after deducting the inherent contribution of each peptide (the perceived NaCl concentration of DWDRDL was 0.88 mg / mL, and the perceived NaCl concentration of LDFDP was 1.00 mg / mL), the saltiness enhancement effect was +0.58 mg / mL and +0.28 mg / mL, respectively. In a 9 mg / mL NaCl standard solution, DWDRDL (1 mg / mL) increased the perceived NaCl concentration from 9.09 mg / mL to 10.76 mg / mL (net enhancement: +0.79 mg / mL), while FDGDGDR increased the perceived NaCl concentration to 9.69 mg / mL, but its saltiness enhancement was less than that of DWDRDL. LDFDP showed a slightly decreasing trend under the same conditions. A similar trend was observed in a 12 mg / mL NaCl standard solution: DWDRDL still had a measurable enhancement effect (net increase of +0.11 mg / mL), and this enhancement effect was better than that of FDGDGDR and LDFDP, but it was weaker compared to its saltiness enhancement effect at lower NaCl concentrations (6 mg / mL and 9 mg / mL). In summary, the saltiness values ​​of the three salty peptides are comparable to those of NaCl, with DWDRDL exhibiting the strongest saltiness enhancement effect in different concentrations of NaCl standard solutions.

[0060] 2.5. Cell Experiments The cytotoxicity of the synthetic peptides was assessed using cell viability assays, and RT-qPCR analysis was performed to verify the activation effect of the salty peptides on specific salty taste receptors. Figure 8As shown, cell viability, as determined by the MTT assay, did not change significantly at any of the tested concentrations (250, 500, and 1000 μmol / L). Compared with the untreated control group (0 μmol / L; P>0.05), neither the synthetic peptides nor NaCl showed cytotoxic effects. Notably, compared with the control group (NC) and the equimolar NaCl group, the cell viability of the three peptide treatment groups showed a moderate but consistent increase, suggesting that these peptides may have intrinsic cytoprotective effects. Cured ham is an important source of natural bioactive peptides. In recent years, many studies have found that bioactive peptides from cured ham possess various physiological functions, including antioxidant, anti-inflammatory, blood pressure-lowering, and blood sugar-lowering effects. Therefore, the salty peptides screened by this method may also possess beneficial biological activities.

[0061] TRPV1 (Transient receptor potential vanilloid-1) is a ligand-gated, non-selective cation channel belonging to the transient receptor potential channel family. It can be activated by various physical and chemical stimuli. TRPV1 is evolutionarily conserved in mammals and contributes to cellular perception of hyperosmolarity and certain taste patterns. Functional expression of TRPV1 in the human cell line HepG2 has been experimentally confirmed. Epithelial sodium channels (ENaC) function as a key osmotic-sensitive cation transduction pathway in HepG2 cells. Transmembrane channel-like 4 (TMC4), a chloride-permeable anion channel activated by increased extracellular NaCl concentration, is widely expressed in mammalian cell types. This experiment verified the transcriptional regulation of salty taste receptor expression in HepG2 cells by salty peptides. Figure 9As shown (using GAPDH as an internal reference gene), NaCl, as a positive control, significantly upregulated the mRNA expression of three key salty taste genes (TMC4, TRPV1, and ENaC) (P<0.05), confirming their typical role in activating the cellular salty taste signaling pathway. Among the salty taste peptides tested, DWDRDL significantly upregulated the mRNA expression of TMC4, TRPV1, and ENaC (P<0.05), with upregulation levels exceeding those of an equimolar concentration (1000 µmol / L) of NaCl. FDGDGDR selectively enhanced the mRNA expression of TMC4, with a significantly higher enhancement than sodium chloride (P<0.05), while the mRNA expression levels of TRPV1 and ENaC increased accordingly, but not significantly. LDFDP significantly upregulated the mRNA of ENaC (P<0.05), with an effect exceeding that of the same concentration of NaCl. Overall, RT-qPCR showed that different salty peptides have specific activation modes during their binding to salty receptors.

[0062] 2.6. Summary This invention elucidates the peptidommic characteristics of three traditional dry-cured hams using peptidomics and identifies three peptides with potential saltiness / saltiness-enhancing properties through a virtual screening process. These three peptides, DWDRDL (SEQ ID No: 3), LDFDP (SEQ ID No: 1), and FDGDGDR (SEQ ID No: 2), exhibit high binding affinity to typical salty taste receptors ENaC, TMC4, and TRPV1, respectively. Hydrogen bonds constitute the main molecular interaction mechanism, primarily mediated by hydrophilic amino acid residues located on the salty taste receptors. Electron tongue combined with PLSR modeling analysis shows that the saltiness intensity induced by the synthetic peptides at a concentration of 1 mg / mL is comparable to that of NaCl. Furthermore, DWDRDL and LDFDP significantly enhance the perceived saltiness of low-concentration NaCl standard solutions. Furthermore, RT-qPCR analysis showed that DWDRDL significantly upregulated the mRNA expression of all three salt taste receptors—ENaC, TMC4, and TRPV1—in HepG2 cells; FDGDGDR selectively enhanced TMC4 expression, while LDFDP played an important role in increasing the transcriptional level of ENaC. Overall, this invention establishes an efficient method for screening salty peptides, confirms the potential of peptides derived from cured ham as natural salty taste enhancers, and provides new scientific insights into the molecular regulatory mechanisms of salty peptides and human salty taste perception.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A salty peptide derived from dry-cured ham, characterized in that, Its amino acid sequence is one or more of LDFDP, FDGDGDR, and DWDRDL.

2. The method for preparing the salty peptide derived from dry-cured ham according to claim 1, characterized in that, This includes artificial synthesis; the artificial synthesis includes chemical synthesis and biological synthesis; the chemical synthesis includes one or more of solid-phase peptide synthesis and liquid-phase peptide synthesis.

3. The application of the salty peptide derived from dry-cured ham as described in claim 1 in the preparation of umami and / or salty seasonings and foods.

4. A flavor enhancer for umami and / or saltiness, characterized in that, Its main components include the salty peptide derived from dry-cured ham as described in claim 1.

5. The screening method for salty peptides derived from dry-cured ham according to claim 1, characterized in that, Includes the following steps: (1) Extraction of peptides from dry-cured ham using ethanol precipitation method; (2) Peptidomics analysis of dried cured ham: The peptide solution of dried cured ham was subjected to ultrafiltration and desalting; the peptides were analyzed by liquid chromatography-mass spectrometry system and PEAKS Studio software to obtain the peptide profile of dried cured ham. (3) Screening of salty peptides: Screening for peptides with a bioactivity score ≥0.5 and a salty score >0.3, that are hydrophilic and can bind to salty taste receptors ENaC, TRPV1, and TMC4; (4) The peptides screened in step (3) were synthesized by solid-phase synthesis. The selected salty peptides, LDFDP, FDGDGDR, and DWDRDL, were verified by sensory evaluation, electronic tongue analysis and mathematical modeling.

6. The screening method according to claim 5, characterized in that, In step (1), the specific steps of the ethanol precipitation method are as follows: take dried ham pieces, chop them, homogenize them, filter them, centrifuge them, add 2-3 times the volume of ethanol to the supernatant for precipitation, remove the ethanol by rotary evaporation, and freeze dry to obtain dried ham polypeptide powder; the volume fraction of ethanol is 40%; the dried ham is Jinhua ham, Rugao ham, or Xuanwei ham.

7. The screening method according to claim 5, characterized in that, In step (2), the dry-cured ham peptide solution is a mixture of dry-cured ham peptide and pure water prepared in step (1); the concentration of the dry-cured ham peptide solution is 1-2 mg / mL.

8. The screening method according to claim 5, characterized in that, In step (2), the ultrafiltration method is to use a 10 kDa ultrafiltration centrifuge column for ultrafiltration and to use a Pierce C18 centrifuge column for desalination.

9. The screening method according to claim 5, characterized in that, In step (3), the screening targets are dry-cured ham peptides with no more than 10 amino acid residues and a relative molecular mass of less than 1000 Da.

10. The screening method according to claim 5, characterized in that, In step (3), the specific screening steps are as follows: a. The bioactivity of peptides is predicted using the PeptideRanker online server, and candidate peptides with a score ≥0.5 are selected for taste annotation; b. Taste annotation is performed using the BIOPEP-UWM online database, and candidate peptides with a salty score >0.3 are designated as potential salty peptides; c. The water solubility of potential salty peptides is predicted and evaluated using peptide evaluation tools; d. Potential salty peptides are molecularly docked with three salty-related salty receptors (ENaC, TRPV1, and TMC4), and the 3D structure of the ligands is minimized using ChemDraw 22.

2. All three receptor protein models are evaluated using the PROCHECK program, and receptor preparation is performed in AutoDock Tools 1.5.

7. Molecular docking simulation is performed using AutoDock Vina, and the lowest binding energy conformation of each peptide-receptor complex is retained for analysis; the docking scores of potential salty peptides with salty-related salty receptors ENaC, TRPV1, and TMC4 range from -10.

5. Between -5.3 kcal / mol.

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