Fungus-derived salty peptide ahdqy and application thereof

Abstract

The present application relates to the technical field of small molecule peptides, and particularly relates to a fungus-derived salty peptide AHDQY and application thereof. The amino acid sequence of the salty peptide AHDQY is shown as SEQ ID NO:1. The fungus-derived salty peptide AHDQY with strong salty taste response is successfully screened through a salty peptide prediction model, virtual proteolysis of fungus protein, hierarchical screening and electronic tongue verification. The peptide can be used for improving salty taste of food, enhancing salty taste perception of food, reducing the amount of sodium chloride added in food, and can be applied to the development of low-sodium food. The present application provides a new candidate raw material and technical basis for the development of natural salty peptide resources and the creation of low-sodium food ingredients.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide technology, specifically to a fungal-derived salty peptide AHDQY and its applications. Background Technology

[0002] Salt is one of the most commonly used seasonings in food processing and daily diets. Its main component, sodium chloride, imparts a typical salty taste to food and participates in flavor formation, texture regulation, and maintenance of storage stability. However, long-term excessive sodium intake is closely related to health risks such as hypertension, cardiovascular disease, and vascular endothelial dysfunction. With increasing public health awareness, reducing the sodium content in food while maintaining a good salty taste perception and overall flavor quality has become an important goal in the development of low-sodium and healthy foods.

[0003] Currently, common salt reduction strategies mainly include directly reducing the amount of sodium chloride added, partially using alternative salts such as potassium chloride, and adding umami agents or flavor enhancers. While these methods can reduce sodium intake to some extent, they still have significant shortcomings. Directly reducing the amount of sodium chloride can easily result in insufficient saltiness, bland flavor, and decreased consumer acceptance; alternative salts such as potassium chloride may bring unpleasant flavors such as bitterness or metallic taste; some flavor enhancers have complex sources or limited flavor coordination, making it difficult to meet the needs of developing natural, safe, and high-quality salt-reduced foods. Therefore, developing salt-reducing ingredients that are naturally derived, structurally sound, highly safe, and can improve the saltiness of food is of great significance.

[0004] Food-derived short peptides have attracted widespread attention in recent years for their small molecular weight, well-defined structure, wide availability, easy absorption, and good biocompatibility, particularly in food flavor regulation and functional food development. Some short peptides can directly exhibit a salty taste and are called savory peptides. Compared with inorganic salt substitutes, food-derived savory peptides have advantages such as natural sources, diverse structures, mild flavors, and higher safety, showing promising application prospects in the development of low-sodium foods, low-sodium seasonings, and health food ingredients.

[0005] Fungal protein resources are abundant and diverse in amino acid composition. Furthermore, some edible and medicinal fungi have long been used as food or food ingredients, providing a good safety foundation and significant resource development value. Through enzymatic hydrolysis or processing, fungal proteins can release a large number of short peptides, some of which may possess saltiness-regulating activity or other potential biological activities. Compared to well-studied protein sources such as oysters, yeast, and meat, research on fungal-derived salty peptides and saltiness-enhancing peptides is still relatively insufficient, indicating significant potential for development. However, the sheer number of peptides generated after fungal enzymatic hydrolysis makes traditional screening methods, which rely on separation, purification, and sensory evaluation, time-consuming and low-throughput, hindering efficient discovery.

[0006] To address the problems existing in the aforementioned background technologies, this invention establishes a strategy for discovering salty peptides that combines artificial intelligence virtual screening, electronic tongue verification, cell safety evaluation, and molecular simulation analysis, and successfully obtained a fungal-derived salty peptide, AHDQY. This peptide has a well-defined amino acid sequence, a small molecular weight, good salty response, and low cytotoxicity, and can be used to improve the saltiness of food, reduce the amount of sodium chloride added to food, and prepare reduced-sodium or low-sodium foods. This invention provides new candidate components and technical basis for the development of natural salty peptide resources and the creation of ingredients for healthy reduced-sodium foods. Summary of the Invention

[0007] The purpose of this invention is to provide a fungal-derived salty peptide AHDQY and its applications, in order to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A fungal-derived savory peptide, AHDQY, has the amino acid sequence shown in SEQ ID NO:1.

[0009] The application of the fungal-derived salty peptide AHDQY in the preparation of low-sodium foods.

[0010] The aforementioned low-sodium foods include dairy products, meat products, puffed foods, or condiments.

[0011] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention improves the efficiency of screening for salty peptides by adopting a technical route that combines virtual enzymatic digestion of fungal proteins, prediction by machine learning models, verification by electronic tongue, cell experiments and molecular simulation. It can quickly identify AHDQY peptide compounds with salty potential from a large number of candidate peptide sequences derived from fungal proteins, thereby reducing the experimental costs and time required for traditional separation, purification and sensory screening, and improving the accuracy and reliability of natural salty peptide discovery. 2. This invention successfully screened and validated a fungal-derived salty peptide, AHDQY. Electronic tongue evaluation results showed that AHDQY has a good salty taste response and can be used to improve the saltiness of food and compensate for the insufficient saltiness caused by the reduction of sodium chloride in low-sodium foods. At the same time, cell safety evaluation results showed that AHDQY had no significant cytotoxicity to HUVEC cells within the tested concentration range and showed a certain protective effect in a high-salt-induced HUVEC cell damage model, indicating that it has a good foundation for food application safety and potential health value. Therefore, AHDQY can be used as a natural salty taste regulator in the development and application of low-salt and low-sodium foods, providing a candidate raw material for novel functional low-sodium foods (dairy products, meat products, puffed foods, or condiments) that have the potential to improve flavor and intervene in the risk of high salt intake. Attached Figure Description

[0012] Figure 1 This is a confusion matrix diagram of the prediction model in this invention; Figure 2 This is the ROC curve of the prediction model in this invention; Figure 3 The saltiness intensity of the candidate samples in this invention. Figure 3 **** indicates a highly significant difference (p<0.0001), and ns indicates no significant difference (p≥0.05). Figure 4 To establish the high-salt-induced HUVEC cell damage model in this invention, Figure 4 ** indicates a highly significant difference (p<0.01), and **** indicates an extremely significant difference (p<0.0001). Figure 5 This invention demonstrates the protective effect of the sample against high-salt-induced damage to HUVEC cells. Figure 5 The numbers #### indicate that the difference is extremely significant (p<0.0001); Figure 6 This refers to the NO content in the sample after high-salt-induced damage to HUVEC cells in this invention. Figure 6 **** indicates extremely significant difference (p<0.0001), # indicates significant difference (p<0.05), and ## indicates very significant difference (p<0.01). Figure 7 This refers to the relative ROS content of the sample after high-salt-induced damage to HUVEC cells in this invention. Figure 7 **** indicates a highly significant difference (p<0.0001), and #### indicates a highly significant difference (p<0.0001). Figure 8 This is a schematic diagram of the molecular docking between the sample and the TMC4 protein in this invention; Figure 9 This is a schematic diagram of the two-dimensional molecular docking interaction between the sample and the TMC4 protein in this invention; Figure 10 This is a schematic diagram of the energy contribution of the sample and the TMC4 complex MM / PBSA in this invention. Detailed Implementation

[0013] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0014] Please see Figures 1 to 10 The present invention provides: Example 1: Data Collection and Cleaning This invention uses keywords such as "salty peptide" to collect reported salty active peptide sequences from published literature and public databases such as PubMed, BIOPEP-UWM, PubChem, TastePeptides-Meta, and ChEMBL. The search period was February 10, 2026. Peptides with experimentally verified salty activity were used as positive samples, and a total of 376 positive samples were collected. Bitter, sweet, and sour peptides with clearly defined non-salty properties were selected as negative samples, with a positive to negative sample ratio of 1:2. The collected peptide sequences were cleaned and standardized to remove repetitive sequences, incomplete sequences, sequences containing non-standard amino acid characters, and sequences with conflicting taste annotations or unclear categories. After processing, a taste-active peptide dataset for salty peptide identification was constructed.

[0015] Example 2: Evaluation of the Predictive Model To improve the screening efficiency of fungal-derived salty peptides, this invention establishes a salty peptide prediction model based on the taste-active peptide dataset constructed in Example 1. Considering that the taste activity of short peptides is closely related to amino acid composition, residue position, and sequence context information, it is difficult to fully characterize their activity differences by simply relying on traditional physicochemical properties. Therefore, this invention uses Uni-Mol, MolCLR, and ESM-3 to encode peptide sequences, and inputs them uniformly into the FT-Transformer model for training and evaluation.

[0016] The FT-Transformer can learn the nonlinear relationships between high-dimensional embedded features through feature labeling and self-attention mechanisms, making it suitable for classification modeling of peptide sequence features. By comparing the prediction performance of different feature representation methods under the same model framework, this invention determines the optimal feature extraction method suitable for screening fungal-derived salty peptides. Specific results are shown in Table 1. Under the same model conditions, the Micro-average AUC, Macro-average AUC, and AUC (salty peptide category) of ESM-3 are 0.967, 0.887, and 0.946, respectively, which are generally higher than Uni-Mol and MolCLR. Especially in salty peptide category identification, ESM-3 has the highest Salty AUC, indicating that it can more effectively extract sequence semantic features and residue position information related to salty activity. Therefore, this invention selects ESM-3 as the feature extraction method for subsequent screening of fungal-derived candidate salty peptides.

[0017] Table 1: Performance comparison of different feature vectors for salty peptide prediction with FT-Transformer

[0018] This invention validates the classification results of the ESM-3 combined with the FT-Transformer model. The confusion matrix results are as follows: Figure 1 As shown, in the salty peptide category, the model correctly identified 114 salty peptides, with only a small number misclassified as other categories, indicating that the model has good classification accuracy for salty peptides. This result demonstrates that the constructed model can effectively distinguish between salty and non-salty peptides, providing a reliable basis for subsequent candidate peptide selection. The ROC curve results are shown below. Figure 2 As shown, the ESM-3 combined with the FT-Transformer model exhibits good discrimination ability across multiple taste categories. Specifically, the AUC for the salty peptide category is 0.946, indicating that the model has a strong ability to identify salty peptides.

[0019] This invention ultimately determined ESM-3 as the peptide sequence feature extraction method and FT-Transformer as the core classification model for high-throughput screening of fungal candidate peptide libraries.

[0020] Example 3: Graded Screening and Electronic Tongue Verification This invention obtains proteomic sequences of 14 fungi from the UniProt database, constructs a fungal protein library, and performs virtual enzymatic digestion. The virtual enzymatic digestion systems include combined trypsin-pepsin digestion, combined bromelain-papain digestion, and combined alkaline protease-neutral protease digestion. After these three types of virtual digestion, the resulting peptides are merged and repetitive sequences are removed, yielding a total of 3,099,108 non-redundant peptide sequences. Short peptides with a length of 3-7 amino acids are further retained, resulting in 1,485,403 candidate peptides. Subsequently, the candidate peptides are initially filtered based on the physicochemical properties of known positive samples of saline peptides, ultimately retaining 828,477 fungal-derived candidate peptide sequences.

[0021] This invention uses the ESM-3 and FT-Transformer combined model established in Example 2 to predict the salty activity of the above-mentioned candidate peptides. The model outputs the predicted probability of each candidate peptide belonging to the salty peptide category, and combines the model confidence and classification margin to comprehensively evaluate the candidate peptides. Based on the comprehensive score results, the candidate sequences predicted as salty peptides are ranked, and the top three candidate salty peptides are selected for chemical synthesis. All candidate peptides are prepared by solid-phase chemical synthesis, and after purification, the purity is not less than 95%, synthesized by Liaoning Newkin Biochemical Technology Co., Ltd., China.

[0022] To verify the salty taste activity of the aforementioned candidate peptides, this invention employed an electronic tongue system to evaluate the salty taste response of three candidate salty peptides. Each candidate peptide was dissolved in deionized water to prepare a 0.25 mg / mL sample solution. Electronic tongue detection was performed using taste sensors such as CO0, AE1, CA0, CTO, and AAE. Each sample was tested repeatedly for four cycles. After removing the data from the first cycle, subsequent stable response values ​​were analyzed. The electronic tongue results are as follows: Figure 3 As shown, all three candidate peptides exhibited a certain salty taste response, with AHDQY (SEQ ID NO:1, full name Ala-His-Asp-Gln-Tyr) showing the strongest salty taste response. The salty taste intensity of AHDQY was 7.79, significantly higher than that of AGHQR (SEQ ID NO:2, full name Ala-Gly-His-Gln-Arg) (2.89) and SKPGNR (SEQ ID NO:3, full name Ser-Lys-Pro-Gly-Asn-Arg) (1.92). This result indicates that AHDQY is the peptide with the strongest salty taste perception among the candidate peptides selected through comprehensive scoring, possessing a clear salty taste characteristic.

[0023] As a reference to "Study on Salty-Savory Interaction Based on Electronic Tongue Technology and σ-τ Model", the salty response value of 0.5 mg / mL sodium chloride solution was measured to be 0.98 using the same type of electronic tongue (SA-402B, Insent Corporation, Japan). In this invention, the salty intensity (7.79) of AHDQY at a concentration of 0.25 mg / mL is significantly higher than that of sodium chloride, indicating that this peptide has a much stronger salty taste sensing activity than sodium chloride.

[0024] This invention successfully obtained the salty peptide AHDQY through virtual enzymatic hydrolysis of fungal proteins, model comprehensive scoring screening, and electronic tongue verification. It can be used as a natural salty flavor regulator to improve the problem of insufficient saltiness caused by the reduction of sodium chloride in low-sodium foods.

[0025] Example 4: Evaluation of cell safety and protective effect against high salt damage To evaluate the cellular safety and high-salt damage protection of the fungal-derived salt peptide AHDQY, this invention used HUVEC cells as an in vitro evaluation model to detect cell viability. HUVEC cells were treated with 50, 125, 250, 500, and 1000 μg / mL AHDQY for 24 h. The results showed that the cell viability remained above 80% after each concentration treatment, and no significant cytotoxicity was observed. The cell viability of the 250 μg / mL AHDQY treatment group was 89.57%, indicating that AHDQY has good cell compatibility.

[0026] HUVEC cells were treated with different concentrations of NaCl for 24 h to establish a high-salt injury model. The results are as follows: Figure 4 As shown, cell viability gradually decreased with increasing NaCl concentration; among them, the cell viability dropped to 64.06% after treatment with 50 mM NaCl, which could form a stable high-salt injury model. Therefore, 50 mM NaCl was selected as the induction concentration for subsequent experiments. Figure 4 The median values ​​represent relative cell viability compared to independent parallel control groups, and do not represent a normalized 100% for the con group (which is the control group without sodium chloride treatment). Due to normal experimental fluctuations in parallel culture and CCK-8 assays, the average absorbance of the con group is slightly higher than that of the reference control group, and the calculated relative cell viability may exceed 100%. The protective effect of AHDQY was further evaluated under 50 mM NaCl injury conditions. Results are as follows... Figure 5 As shown ( Figure 5 The damage condition in the study was 50 mM NaCl. The cell viability in the high-salt damage group was 64.53%, while treatment with 250 μg / mL AHDQY increased the cell viability to 92.02%. Further increases in AHDQY concentration did not significantly increase cell viability, indicating that AHDQY at 250 μg / mL already exhibited superior cell protection. These results demonstrate that AHDQY can significantly improve the high-salt-induced decline in HUVEC cell viability.

[0027] To further evaluate the regulatory effect of AHDQY on high-salt-induced endothelial dysfunction, intracellular NO and ROS levels were measured. The results for NO levels are as follows: Figure 6 As shown, the NO content in the normal control group was 10.85 μM, while it decreased to 4.34 μM in the high-salt injury group. NO levels recovered after AHDQY treatment, with NO contents in the 250 μg / mL and 1000 μg / mL AHDQY treatment groups being 6.94 μM and 8.85 μM, respectively. ROS quantification results are shown below. Figure 7 As shown, the relative ROS level in the high-salt damage group increased to 1.34, while it decreased to 0.64 after treatment with 250 μg / mL AHDQY, indicating that AHDQY can inhibit high-salt-induced ROS accumulation. Figure 6 and Figure 7 In this context, the "con" group was the control group that did not experience high-salt damage, while the "damage" group was the group that experienced high-salt damage.

[0028] AHDQY showed no significant toxicity to HUVEC cells within the tested concentration range and was able to improve cell survival under high salt damage conditions, restore NO production, and reduce ROS accumulation, indicating that it has certain potential for protecting against high salt damage. It can be used as a natural salty peptide with both saltiness improvement and cell protection effects in the development of ingredients for low-sodium foods or health foods.

[0029] Example 5: Molecular dynamics simulation and binding free energy analysis This invention uses the salt-taste-sensing protein TMC4 (UniProt ID: Q7Z404) as the receptor protein for molecular simulation analysis. AlphaFold3 was used to predict the initial binding conformation of AHDQY and TMC4, and the top-ranked complex structure was selected as the initial conformation for subsequent molecular dynamics simulations.

[0030] Molecular dynamics simulations of the AHDQY-TMC4 complex were performed using AMBER 24 software. Both the protein and peptide were simulated using the ff19SB force field, and the solvent was the OPC water model. After energy minimization using a 5000-step steepest descent method and a 5000-step conjugate gradient method, the system was gradually heated to 310 K in the NVT ensemble, followed by a 50 ns equilibrium simulation in the NPT ensemble at 310 K. Subsequently, a 400 ns dynamics simulation was performed in the unconstrained NPT ensemble, and one frame was extracted every 10 frames from the stable conformational range, resulting in 100 representative conformations for MM / PBSA binding free energy analysis. This method reflects the average binding characteristics of AHDQY and TMC4 proteins in a stable state, reducing the randomness of single conformational analysis.

[0031] Molecular docking results as follows Figure 8 As shown, AHDQY can enter the local binding region of the TMC4 protein and interact with multiple surrounding amino acid residues. The binding site mainly involves residues such as ARG317, ARG145, GLU291, ALA163, PHE587, and ALA589. A two-dimensional interaction diagram is shown below. Figure 9 As shown, AHDQY can bind to TMC4 through hydrogen bonds, electrostatic interactions, and hydrophobic interactions, indicating that there is a good spatial matching and interaction basis between AHDQY and TMC4.

[0032] The energy decomposition results of MM / PBSA are as follows: Figure 10As shown, multiple residues in the TMC4 binding region contribute energy to the binding of AHDQY. Among them, ARG312 contributes the most significantly to the binding energy, followed by LYS685, ARG688, and ARG294. All of these residues contribute negative energy values, indicating that they are conducive to the stable formation of the AHDQY-TMC4 complex. The molecular docking results show that the binding of AHDQY to TMC4 is mainly influenced by the interactions involving charged and polar residues. These results provide molecular-level support for the salty response of AHDQY and also suggest that AHDQY can be used as a naturally occurring salty peptide from fungi for salt improvement and flavor regulation in low-sodium foods.

[0033] This invention designs a method for screening salty peptides AHDQY from fungal protein resources and its application in low-sodium foods. It aims to solve the problems of insufficient saltiness, bland flavor, and decreased consumer acceptance caused by the reduction of sodium chloride in existing low-sodium foods, as well as the bitterness, metallic taste, or poor flavor coordination of some salt substitutes. It also provides new ideas for the development of natural salty flavor regulating components.

[0034] This invention establishes an efficient screening and verification process for fungal-derived salty peptides by combining dataset construction, pre-trained protein language model feature extraction, FT-Transformer prediction model, virtual enzymatic digestion of fungal proteins, hierarchical screening, electronic tongue verification, cell experiments, and molecular simulation analysis. Starting with the proteomes of 14 fungal species, this invention selected top-ranked candidate salty peptides for chemical synthesis after hierarchical screening, and verified their salty activity using an electronic tongue. The results showed that AHDQY exhibited the strongest salty response among the candidate peptides, with a salty intensity of 7.79, indicating that AHDQY possesses a clear and strong salty perception characteristic and can be used as a natural salty regulatory peptide for the development of low-sodium foods. Further research showed that AHDQY exhibited good cell compatibility in HUVEC cell experiments, and no significant cytotoxicity was observed within the test concentration range of 50-1000 μg / mL. In a 50 mM NaCl-induced high-salt injury model, 250 μg / mL AHDQY increased cell viability from 64.53% to 92.02% in the high-salt injury group, indicating that it can significantly improve the high-salt-induced decline in cell viability. Simultaneously, AHDQY can restore NO levels in high-salt-injured cells to some extent and reduce excessive ROS accumulation, suggesting that in addition to its salt-taste regulation function, it also has a certain protective potential against high-salt injury. Furthermore, this invention predicted the initial binding conformation of AHDQY with the salt-taste-sensing-related protein TMC4 using AlphaFold3, and performed molecular dynamics simulations and MM / PBSA binding free energy analysis using AMBER 24. The results showed that AHDQY can form a relatively stable binding conformation with TMC4 and interact with multiple key amino acid residues, providing molecular-level support for its salt-taste response.

[0035] This invention successfully screened and verified a fungal-derived salty peptide, AHDQY. This peptide has a well-defined sequence, small molecular weight, and is easy to synthesize and control in quality. It also has a strong salty response, good cell compatibility, and certain potential for protection against high salt damage. It can be used to improve the problem of insufficient saltiness in low-sodium foods due to reduced sodium chloride content, and can also be used as a natural salty flavor regulator in the development of low-sodium foods.

[0036] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A fungal-derived savory peptide AHDQY, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The application of the fungal-derived savory peptide AHDQY as described in claim 1 in the preparation of low-sodium foods.

3. The application according to claim 2, characterized in that, The low-sodium foods include dairy products, meat products, puffed foods, or condiments.

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