Snail-derived umami peptide with salt-enhancing effect, preparation method thereof, and application in flavor-enhancing and salt-reducing foods

Abstract

The present invention belongs to the field of bioactive peptides, and specifically relates to a snail-derived umami peptide with a salt-enhancing effect, a preparation method thereof, and an application thereof in flavor-enhancing and salt-reducing foods. The present invention separates two new snail-derived umami peptides, PIYEGY and TVPIYEG, from snail meat hydrolysates by enzymatic hydrolysis, ultrafiltration, RP-HPLC, and UPLC-QTOF-MS / MS. Sensory evaluation and electronic tongue evaluation found that both umami peptides have obvious umami characteristics and salty taste enhancement effects, and the umami stability of the two peptides is good under different pH and temperature conditions. Both PIYEGY and TVPIYEG can bind to the umami receptor T1R1 / T1R3 and the salty taste receptor TMC 4, indicating that they have umami and salty taste enhancing activity. The snail-derived umami peptides obtained by the present invention are expected to be used as new umami enhancers in the food industry, providing a theoretical reference for the high-value utilization of snail meat.

Description

Technical Field

[0001] The present invention belongs to the field of bioactive peptides, and in particular relates to a snail-derived umami peptide with a salt-increasing effect, a preparation method thereof, and an application thereof in flavor-enhancing and salt-reducing foods. Background Art

[0002] In 1908, Ikeda first proposed the concept of umami, describing it as a delicious, savory taste. Umami is a flavor that enhances the flavor of food and creates a pleasant sensation. With the discovery of umami receptors, umami has become widely recognized as one of the five basic tastes, alongside sour, sweet, bitter, and salty. Umami substances typically include free amino acids, 5'-nucleotides, umami peptides, Maillard reaction products, and organic acids. Umami peptides are a class of small, bioactive peptides with a molecular weight typically below 3 kDa. Umami peptides were first identified from beef hydrolysate treated with papain. Currently, over 100 umami peptides have been discovered from a variety of food ingredients, including high-protein foods such as ham, mushrooms, oysters, chicken, and soybeans. Saltiness plays a crucial role in food seasoning, and salt is the most basic and widely used salting agent. Excessive salt intake increases sodium ion concentration, leading to increased osmotic pressure, vascular tone, and cardiac output, ultimately increasing the risk of cardiovascular disease. Therefore, the search for ways to reduce salt intake without reducing saltiness has garnered widespread attention worldwide. Recent studies have found that umami peptides can enhance saltiness. Xie et al. extracted umami peptides from Ruditapes philippinarum and ham, which have the effect of increasing saltiness and reducing salt content (XIE X, DANG Y, PAND, et al. The enhancement and mechanism of the perception of saltiness by umami peptides from Ruditapes philippinarum and ham [J / OL]. Food Chemistry, 2023, 405: 134886). Most umami peptides isolated and identified by Zhu et al. from sauces have the ability to enhance saltiness while reducing bitterness (ZHU W, LUA NH, BU Y, et al. Flavor characteristics of shrimp sauces with different fermentation and storage time [J / OL]. LWT, 2019, 110: 142-151).Chen et al. used Stropharia rugosoannulata as raw material to isolate five umami peptides, all of which can bind to TMC 4 receptors. Among them, the umami peptide SGCVNEL has a very significant saltiness-enhancing effect when mixed with salt (CHEN W, LI W, WU D, et al. Exploring of multi-functional umami peptides from Stropharia rugosoannulata: Saltiness-enhancing effect and mechanism, antioxidant activity and potential targetsites [J / OL]. Food Chemistry, 2024, 439: 138138.). Umami peptides not only have the effect of increasing freshness and reducing salt, but also exhibit other potential biological activities, such as ACE inhibitory activity, hypoglycemic activity, and antioxidant activity. Therefore, finding umami peptides with high-performance umami quality is a hot topic in current research.

[0003] Taste is generated by the contact of food with taste cells on the tongue and transmitted to the brain through nerves. Different types of receptors are involved in the perception of umami. Currently, there are eight candidate umami receptors, including the heterodimer T1R1 / T1R3, the metabotropic glutamate receptors mGluR1 and mGluR4, the taste-specific metabotropic glutamate receptors taste-mGluR1 and taste-mGluR4, the extracellular calcium-sensing receptor (CaSR), GPRC6A, and GPR92. Among them, T1R1 / T1R3 is considered to be the main receptor for umami (DANGY, HAO L, CAO J, et al. Molecular docking and simulation of the synergistic effect between umami peptides, monosodium glutamate and taste receptor T1R1 / T1R3 [J / OL]. Food Chemistry, 2019, 271: 697-706.). The perception of salty taste depends on specific receptors or ion channels, which serve as the main threshold for recognizing salty substances. TMC4 is a novel chloride channel sensitive to high concentrations of NaCl. This protein contains a single transmembrane domain and is expressed in taste buds located on the back of the tongue. Activation of TMC4 by substances demonstrates its ability to enhance salinity, making it useful for identifying and screening salinity enhancers. Currently, the crystal structures of T1R1 / T1R3 and TMC4 have not been fully resolved, but homology modeling can be used to construct unknown protein structures. Homology modeling is a computer simulation method that predicts protein structure based on known homologous protein structures (ProMod3—A versatile homology modeling toolbox). Molecular docking is an important tool for computer-assisted receptor-ligand binding. Combining these two methods facilitates the study of peptide-receptor interactions. Currently, this research system is relatively mature and has been successfully applied to studies on the mechanisms of umami peptide expression and the screening of salty peptides.

[0004] River snail (Sinotaia quadrata) is a common culinary aquatic product in China. It is widely loved for its unique and delicious cooking flavor. River snails are also one of the essential key ingredients in the Chinese delicacy, river snail noodles, giving the dish its delicious taste. Yao (YAO J, ZHAO W, BAI X, et al. Non-volatile taste active compounds in the meat of river snail (Sinotaia quadrata) determined by 1H NMR, e-tongue and sensory analysis [J / OL]. International Journal of Gastronomy and Food Science, 2023, 34: 100803.) used 1 Qualitative and quantitative analysis of free amino acids, nucleotides, and organic acids in boiled snails by H NMR revealed 11 major taste-active compounds that mimic the characteristic flavor of snail meat. However, there are currently no reports on the extraction of umami peptides from snail meat. Summary of the Invention

[0005] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a snail-derived umami peptide with a salt-enhancing effect.

[0006] Another object of the present invention is to provide a method for preparing the snail-derived umami peptide having a saltiness-enhancing effect.

[0007] Another object of the present invention is to provide the use of the snail-derived umami peptide with salt-increasing effect in flavor-enhancing and salt-reducing foods.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A snail-derived umami peptide with a salt-increasing effect, wherein the amino acid sequence is PIYEGY or TVPIYEG;

[0010] The method for preparing the snail-derived umami peptide comprises the following steps:

[0011] The snail umami peptide is prepared directly by solid phase synthesis or snail meat is used as raw material, and the snail-derived umami peptide with saltiness-enhancing effect is obtained by trypsin hydrolysis and purification;

[0012] The specific operation of the trypsin hydrolysis is preferably:

[0013] The homogenized snail meat is mixed with water, and trypsin is added for enzymatic hydrolysis in a water bath; the enzyme is then inactivated in a water bath, cooled, centrifuged, the supernatant is collected, and further freeze-dried;

[0014] The material-liquid ratio of the homogenized snail meat to water is preferably 1:2;

[0015] The conditions for the water bath enzymatic hydrolysis are preferably: hydrolysis at 50°C and pH 8.0 for 3 hours;

[0016] The enzyme-substrate ratio of the water bath enzymatic hydrolysis is preferably 0.19% (m / m);

[0017] The conditions for the enzyme inactivation in a water bath are preferably 90°C for 15 minutes;

[0018] The purification comprises the steps of ultrafiltration separation and liquid chromatography separation;

[0019] The specific operation of the ultrafiltration separation is:

[0020] The snail meat hydrolysate was separated using an ultrafiltration tube, and the fractions with molecular weight <3 kDa were collected;

[0021] The specific operation of the liquid chromatography separation is:

[0022] Ultrafiltration fractions <3 kDa of the snail meat hydrolysate were separated and purified by HPLC using a preparative reverse-phase HPLC system; the active fractions were collected, concentrated by rotary evaporation, and freeze-dried to obtain snail-derived umami peptides with a salt-enhancing effect;

[0023] The conditions of the liquid chromatography are preferably:

[0024] Mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: 0.1% trifluoroacetic acid in methanol; elution program: 0-10 min, 10%-15% B; 10-30 min, 15-35% B; 30-90 min, 35-65% B; 90-100 min, 65-95% B; all percentages are by volume; elution flow rate: 10 mL / min; detection wavelengths: 214 and 280 nm;

[0025] Application of the snail-derived umami peptide in food processing;

[0026] Application of the snail-derived umami peptide in flavor-enhancing and salt-reducing foods;

[0027] The food may be a snail noodle soup package product, etc.

[0028] Application of the snail-derived umami peptide in the field of flavor bases;

[0029] Application of the snail-derived umami peptide in the field of food additives;

[0030] The food additives may be flavor enhancers, etc.

[0031] A freshness-enhancing and salt-reducing food, comprising at least one of the snail-derived umami peptide, a snail meat trypsin hydrolyzate containing the snail-derived umami peptide, and a hydrolyzate containing the snail-derived umami peptide as an active ingredient;

[0032] A flavor base comprising at least one of the snail-derived umami peptide, a snail meat trypsin hydrolyzate containing the snail-derived umami peptide, and a hydrolyzate containing the snail-derived umami peptide as an active ingredient;

[0033] A food additive comprising at least one of the snail-derived umami peptide, a snail meat trypsin hydrolyzate containing the snail-derived umami peptide, and a hydrolyzate containing the snail-derived umami peptide as an active ingredient;

[0034] A flavor enhancer comprising at least one of the snail-derived umami peptide, a snail meat trypsin hydrolyzate containing the snail-derived umami peptide, and a hydrolyzate containing the snail-derived umami peptide as an active ingredient;

[0035] The present invention has the following advantages and effects compared to the prior art:

[0036] (1) The present invention uses enzymatic hydrolysis, ultrafiltration (UF), preparative high-performance liquid chromatography (RP-HPLC), ultra-performance liquid chromatography-tandem time-of-flight mass spectrometry (UPLC-ESI-Q-TOF MS / MS) combined with sensory evaluation to isolate two novel peptides, PIYEGY (molecular weight 740.3381) and TVPIYEG (molecular weight 777.3909), from snail meat hydrolysate. Sensory evaluation and electronic tongue evaluation found that both umami peptides have obvious umami characteristics and salty taste enhancement effects, and the umami stability of the two peptides is good in the complex environment of the food matrix under different pH and temperature conditions.

[0037] (2) The present invention further studied the interactions between umami peptides and the umami receptors T1R1 / T1R3 and the salty receptor TMC 4 through homology modeling and molecular docking to better understand the mechanism by which umami peptides produce umami and salty tastes. The results showed that both PIYEGY and TVPIYEG could bind to umami receptors (T1R1 / T1R3) and salty receptors (TMC 4), indicating that they have umami and salty taste enhancing activities. Among them, Ser 217 and Arg 151 may play a key role in binding to T1R3; Phe 405 and Pro 409 may play a key role in binding to TMC 4.

[0038] (3) The umami peptide obtained by the present invention can be added to food to effectively enhance its overall flavor, and is expected to be used as a new umami enhancer, seasoning or food additive in the food industry.

[0039] (4) The present invention provides a new perspective for improving the high-value utilization of snail resources, and also provides a theoretical reference for the efficient screening of natural umami peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is an analysis chart of the results of manual sensory evaluation (taste characteristics) of SHP and ultrafiltration fractions.

[0041] Figure 2 This is the result analysis chart of electronic tongue analysis of SHP and ultrafiltration components.

[0042] Figure 3 This is the RP-HPLC absorption wavelength spectrum of the U2 component.

[0043] Figure 4 This is an analysis chart of the results of manual sensory evaluation (taste characteristics) of RP-HPLC fractions.

[0044] Figure 5 This is the result analysis chart of the main components of the electronic tongue of the RP-HPLC fraction.

[0045] Figure 6 This is the basepeak diagram of the mass spectrum of the F1 component.

[0046] Figure 7 It is the secondary map of the amino acid sequence of umami peptides, where (A) is the secondary map of PIYEGY; (B) is the secondary map of TVPIYEG.

[0047] Figure 8 This is an analysis chart of the results of manual sensory evaluation of umami peptides (PIYEGY and TVPIYEG) at a concentration of 1 mg / mL.

[0048] Figure 9 This is an analysis chart of the results of umami peptides (PIYEGY and TVPIYEG) regulating the flavor of the broth model.

[0049] Figure 10 This is an analysis chart of the results of electronic tongue principal component analysis of umami peptides (PIYEGY and TVPIYEG).

[0050] Figure 11 This is an analysis chart of the effects of umami peptides (PIYEGY and TVPIYEG) and MSG on umami taste at different pH values.

[0051] Figure 12 This is an analysis chart of the effects of umami peptides (PIYEGY and TVPIYEG) and MSG on umami taste at different temperatures.

[0052] Figure 13 This is a model structure diagram of the umami taste receptor T1R1 / T1R3.

[0053] Figure 14 It is the Ramachandran diagram of the homology model T1R1 / T1R3.

[0054] Figure 15 It is the Errat diagram of the homology model T1R1 / T1R3.

[0055] Figure 16 These are the 3D and 2D images of the umami peptide PIYEGY docking with T1R1 / T1R3 molecules.

[0056] Figure 17 These are the 3D and 2D images of the umami peptide TVPIYEG docking with T1R1 / T1R3 molecules.

[0057] Figure 18 This is a model structure diagram of the salty taste receptor TMC 4.

[0058] Figure 19 is the Ramachandran diagram of the homology model TMC 4.

[0059] Figure 20 is the Errat diagram of the homology model TMC 4.

[0060] Figure 21 These are the 3D and 2D images of the umami peptide PIYEGY docking with the TMC 4 molecule.

[0061] Figure 22 These are the 3D and 2D images of the docking of the umami peptide TVPIYEG and TMC 4 molecules.

[0062] Figure 23 This is an analysis chart of the synergistic salt-enhancing effect of umami peptides (PIYEGY and TVPIYEG) and NaCl solution. DETAILED DESCRIPTION

[0063] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0064] Materials used in the examples: snail meat was provided by Dongguan Huaqi Biotechnology Co., Ltd. (Dongguan, China); trypsin (4000 U / g) was purchased from Pangbo Bioengineering Co., Ltd. (Guangxi, China); food-grade taste reference materials used for sensory evaluation were purchased from Beijing Lanjieke Technology Co., Ltd. (Beijing, China); and two peptides (PIYEGY and TVPIYEG) were synthesized with a purity exceeding 95% by Jiepeptide Biotechnology Co., Ltd. (Nanjing, China) via solid-phase synthesis. All reagents were analytical grade, and all solutions were prepared with ultrapure water.

[0065] Statistical analysis: All experimental data are presented as mean ± standard deviation. Statistical analysis was performed using Origin 2019 software for plotting. SPSS 20.0 software was used for one-way ANOVA and Duncan's method to analyze data differences. P < 0.05 was considered significant.

[0066] Example 1

[0067] 1. Experimental Methods

[0068] 1 Preparation of snail meat hydrolysate

[0069] The snail meat protein hydrolysate was prepared based on the enzymatic hydrolysis method. The specific method is as follows: the snail meat was washed and drained, and then crushed into a snail meat homogenate. Ultrapure water was added at a material-liquid ratio of 1:2 (w / v), and the pH was adjusted to 8.0; trypsin was added at an enzyme substrate mass ratio of 0.19% (m / m), and enzymatic hydrolysis was carried out in a water bath at 50°C for 3h; then the enzyme was inactivated in a water bath at 90°C for 15min, cooled to room temperature, centrifuged at 4000r / min for 10min, the supernatant was collected, further freeze-dried, and stored in a -20°C refrigerator for subsequent analysis.

[0070] 2 Isolation, purification and identification of umami peptides from snail meat

[0071] 2.1 Ultrafiltration

[0072] The snail meat hydrolysate prepared in the above steps was initially separated using a 3 kDa ultrafiltration tube (Merck Millipore, Billerica, MA), and the components with molecular weight ≥3 kDa and <3 kDa were collected, vacuum freeze-dried (Alpha 2-4LDplus, Christ Co., Berlin, Germany), and placed in a -20°C refrigerator for subsequent umami taste analysis.

[0073] 2.2RP-HPLC

[0074] Ultrafiltration fractions with high umami intensity were isolated and purified using a preparative reversed-phase high-performance liquid chromatography system (LC-8, Shimadzu, Kyoto, Japan) using a C18 column (20 mm × 450 mm, 10 μm, Shimadzu). Chromatographic conditions were as follows: mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: 0.1% trifluoroacetic acid in methanol; elution schedule: 0-10 min, 10%-15% B; 10-30 min, 15-35% B; 30-90 min, 35-65% B; 90-100 min, 65-95% B; all percentages are by volume. The elution flow rate was 10 mL / min; detection wavelengths were 214 and 280 nm. The eluted peaks were collected, concentrated by rotary evaporation, and the collected fractions were freeze-dried and stored at -20°C for subsequent umami flavor analysis.

[0075] 2.3 Structural identification of snail meat umami peptides

[0076] The entire system is a Q Exactive connected to the EASY-nanoLC 1200 TM Plus mass spectrometer. Purified samples with the highest umami activity were dissolved in ultrapure water containing 0.1% (v / v) formic acid and desalted using a C18 desalting column (Acclaim PepMap 100, 75 μm × 2 cm). The peptide composition and amino acid sequence of the samples were analyzed by LC-MS / MS using an online nanospray ion source. Chromatographic conditions included: column (Acclaim PepMap C18, 75 μm × 25 cm); column flow rate controlled at 300 nL / min; column temperature at 40°C; injection volume: 3 μL; electrospray voltage: 2 kV. Mobile phase A (0.1% formic acid in water), mobile phase B (0.1% formic acid in acetonitrile); elution rates: 0-3 min, 2%-6% B; 3-42 min, 6%-20% B; 42-47 min, 20%-35% B; 47-48 min, 35%-100% B; 48-60 min, 100% B. Percentages are by volume. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) primary mass spectrometry scan range (m / z): 350–1800; resolution: 70,000; automatic gain control target: 3e6; maximum injection time: 50 ms; scan charge: 2–6; (2) HCD-MS / MS: resolution: 17,500; isolation window: 2 m / z; automatic gain control target: 1e5; maximum injection time: 45 ms; collision energy: 28; dynamic exclusion time: 30 s. Tandem mass spectra were analyzed using De Novo software (PEAKS Studio X+). The PEAKSDB protein spectral database was searched, and the enzyme digestion was set to None. Search parameters included fragment ion mass tolerance of 0.02 Da, precursor mass tolerance of 7 ppm, maximum number of missed cleavages of 2, variable modifications of Oxidation (M) 15.99, Deamidation (NQ) 0.98, protein cardinal value of -101gP ≥ 0, containing at least one unique peptide, and peptide cardinal value of -101gP ≥ 15. Peptide sequences were searched against Uniprot's Cipangopaludina cathayensis and Viviparidae protein databases to determine their primary structures.

[0077] 3. Activity prediction of umami peptides

[0078] The potential taste activity of the identified umami peptides was predicted using the BIOPEP database. The BIOPEP database is an open access resource available at (http: / / www.uwm.edu.pl / biochemia / index.php / pl / biopep) that supports the analysis of the relationship between the molecular structure of peptides and their sensory properties and the establishment of potential sensory characteristics of peptides (Biopep database of sensory peptides and amino acids.). The toxicity of the identified peptides was predicted using the toxin prediction method (https: / / webs.iiitd.edu.in / raghava / toxinpred / design.php). The water solubility and stability of the peptides were determined using the peptide property calculator and the ProtParam tool (http: / / www.innovagen.com / proteomics-tools; https: / / web.expasy.org / protparam / ), respectively.

[0079] 4 Sensory evaluation

[0080] 4.1 Sensory Evaluation Panel Training

[0081] The method described by Feng et al. (FENG X, WANG R, LU J, et al. Taste properties and mechanism of umami peptides from fermented goose bones based on molecular docking and molecular dynamics simulation using umami receptor T1R1 / T1R3 [J / OL]. Food Chemistry, 2024, 443:138570) was slightly modified as follows: Ten panelists (four men and six women, aged 18–28 years) were selected from the College of Food Science and Engineering at South China Agricultural University for sensory evaluation. These panelists were in good health, had no taste disorders, and had no history of smoking. After sensory training, the panelists were able to accurately identify the five basic tastes of sour, sweet, bitter, salty, and umami. The sensory evaluation was conducted in a sensory laboratory (25 ± 2°C) with good ventilation and proper lighting. Panelists were trained to evaluate the following standard taste solutions: sucrose solution (1%, w / v) for sweetness, monosodium glutamate (MSG) solution (0.35%, w / v) for umami, NaCl solution (0.35%, w / v) for saltiness, L-isoleucine solution (0.25%, w / v) for bitterness, and citric acid solution (0.08%, w / v) for sourness. Samples were scored on a scale of 0 to 10, with 0 indicating no taste and 10 indicating a very pronounced taste. A standard score of 5 was set for all standard taste solutions.

[0082] 4.2 Flavoring properties of umami peptides

[0083] ① Taste characteristics: The enzymatic hydrolysate, ultrafiltration fraction, and RP-HPLC fraction were each prepared into a solution with a mass concentration of 10 mg / mL, and the umami peptide was prepared into a solution with a mass concentration of 1 mg / mL. Sensory panelists placed 1-2 mL of the sample on the central area of ​​the tongue to record the intensity of each taste attribute. The bottles were randomly numbered and assigned to each panelist at room temperature. During the evaluation process, panelists were not allowed to communicate and were required to rest for 1 minute after tasting each sample to reduce taste fatigue. The evaluation test was conducted three times, and the panelists' scores were averaged.

[0084] ② Taste threshold: According to the literature (OTTINGER H, HOFMANN T. Identification of the Taste Enhancer Alapyridaine in Beef Broth and Evaluation of Its Sensory Impact by Taste Reconstitution Experiments [J / OL]. Journal of Agricultural and Food Chemistry, 2003, 51 (23): 6791-6796.), the taste threshold of umami peptides was determined by the triangular test method (TDA). The specific method is as follows: the umami peptide solution was diluted with ultrapure water at a ratio of 1:1 (v / v). When the panelists could distinguish between the previous sample and the blank control (ultrapure water), but could not distinguish between the next level of sample and the blank control, the average of the two levels was the taste threshold. All diluted samples were presented in order of increasing concentration. At the same time, the panelists' descriptions of taste attributes were recorded.

[0085] ③ To explore the taste synergy of umami peptides, the method of Shen et al. (SHEN Q, SUN L, HE Z, et al. Isolation, taste characterization and molecular docking study of novel umami peptides from Lactarius volemus (Fr.) [J / OL]. Food Chemistry, 2023, 401: 134-137.) was slightly modified as follows: A 0.2 mg / mL umami peptide solution was prepared in ultrapure water and added to a model broth containing 2 mg / mL MSG and 2 mg / mL NaCl to obtain an umami peptide complex solution. The model broth was used as a blank control, and the model broth score was set to 5 points. The umami and salty taste of the umami peptide complex solution were scored on a scale of 0-10, with 0 indicating no taste and 10 indicating an extremely strong taste.

[0086] 4.3 Analysis of umami taste stability of umami peptides

[0087] The method of Li Wanqiu (Li Wanqiu, Li Rui, Zhao Xiaoyi, et al. The mechanism of umami presentation and umami stability of sunflower seed peptide ALEPIER) was slightly modified. The specific method is as follows:

[0088] To investigate the effect of pH on the umami taste of umami peptides: umami peptide solutions with a mass concentration of 0.2 mg / mL were prepared, and the pH of the solutions were adjusted to 4.0, 5.0, 6.0, 7.0, and 8.0 at room temperature using 0.5 mol / L HCl or NaOH, respectively. The umami intensity of the solutions at different pH values ​​was measured.

[0089] To investigate the effect of temperature on the umami taste of umami peptides: umami peptide solutions with a mass concentration of 0.2 mg / mL were prepared, treated at 40, 60, 80, 100, and 120°C for 30 min, cooled to room temperature, and the umami intensity of the solutions was measured.

[0090] The umami intensity of MSG solution with a mass concentration of 0.2 mg / mL under the same conditions was compared, and the scoring range was between 0 and 5 points, with 0 indicating no umami and 5 indicating extremely significant umami.

[0091] Salt-enhancing effect of 5 umami peptides in NaCl system

[0092] According to the method of Dang et al. (DANG Y, HAO L, ZHOU T, et al. Establishment of new assessment method for the synergistic effect between umami peptides and monosodium glutamate using electronic tongue [J / OL]. Food Research International, 2019, 121: 20-27.) and Hayabuchi et al. (HAYABUCHI H, MORITA R, OHTA M, et al. Validation of preferred salt concentration in soup based on a randomized blinded experiment in multiple regions in Japan—influence of umami (l-glutamate) on saltiness and palatability of low-salt solutions [J / OL]. Hypertension Research, 2020, 43 (6): 525-533.) with slight modifications. When the NaCl concentration was 0.3%, the saltiness of the added MSG was significant. Therefore, 0.3% NaCl was selected as the blank control. We selected a mixed solution of 0.1 mg / mL umami peptide and 0.3% (w / v) NaCl to evaluate the salty taste enhancement effect of umami peptide. The specific method is as follows:

[0093] The saltiness enhancement sensory experiment was conducted based on the sensory panel training in Example 1. Panelists were asked to taste NaCl solutions ranging from 0.1% (w / v) to 1% (w / v) based on their perceived salt intensity. The saltiness of a 0.3% (w / v) NaCl solution was rated as 5. The score ranged from 0 to 10, with each 0.1% increase in saltiness resulting in a 1-point increase and a 0.1% decrease in saltiness resulting in a 1-point decrease.

[0094] 6 Electronic tongue measurement

[0095] The determination method was slightly modified according to the method of Li et al. (LI X, XIE X, WANG J, et al. Identification, taste characteristics and molecular docking study of novel umami peptides derived from the aqueous extract of the clam meretrix meretrix Linnaeus [J / OL]. Food Chemistry, 2020, 312: 126053.), and the enzymatic hydrolysate, ultrafiltration component, and RP-HPLC component were configured into a solution with a mass concentration of 1 mg / mL, and the umami peptide was configured into a solution with a mass concentration of 0.1 mg / mL, and measured using the taste sensing system Smart Tonge (Shanghai Ruifen Biotechnology Co., Ltd., Shanghai, China). According to the system's pre-set program, a blank model (tasteless model) containing 30mM KCL and 0.3mM tartaric acid was used. The system's built-in program was used to convert the potential value of the test sample into a taste value and then perform taste characteristic analysis. Data were collected 4 times for each sample, and the first data was removed. Principal component analysis (PCA) was used to analyze the relationship between the variables.

[0096] 7. Homology modeling of umami taste receptors (T1R1 / T1R3) and salty taste receptors (TMC 4) and their docking with umami peptide molecules

[0097] 7.1 Homology Modeling

[0098] The homology modeling process typically involves four steps: T1R1 / T1R3 amino acid sequence retrieval, selection of a suitable template, sequence alignment of the target template, and homology model construction, refinement, and validation. The amino acid sequence of the umami taste receptor (T1R1 / T1R3) was retrieved from UniProtKB (https: / / www.UniProt.org / ), and the salty taste receptor (TMC 4) (accession No.: NP_001138775) was obtained from the National Center for Biotechnology Information (NCBI). In this study, a homology model was constructed using the online server SWISS-MODEL (https: / / swissmodel.expasy.org / ). The umami taste receptor was modeled using the metabotropic glutamate receptor (PDB ID: 1EWK) (DANG Y, HAO L, CAO J, et al. Molecular docking and simulation of the synergistic effect between umami peptides, monosodium glutamate and taste receptor T1R1 / T1R3 [J / OL]. Food Chemistry, 2019, 271: 697-706). A preliminary homology model was then imported into Discovery Studio 4.5 and optimized using a minimization protocol. Model evaluation was performed using the residual percentage of the Ramachandran plot and the ERRAT score in SAVESv6.0 (https: / / saves.mbi.ucla.edu / ).

[0099] 7.2 Molecular docking

[0100] First, the 3D structure of the newly constructed homology model was preprocessed and optimized, and then exported as a mol 2 format file for subsequent molecular docking. PyMol software was used to remove duplicate chains, water molecules, and original ligands from the receptor protein molecule and save it as a PDB format file. Autodock Tools 1.5.6 software was then used to perform hydrogenation, charge calculation, and other processing on the receptor protein and ligand, and the files were saved as PDBQT format files. The 2D structure of the ligand was drawn using MarvinSketch software, optimized based on energy minimization, and a 3D structure was constructed and saved as a PDBQT format file. AutoDock Vina 1.1.2 was used to perform molecular simulation docking on the above receptor and ligand, and the docking results were expressed as binding energy values. Finally, Discovery Studio 4.5 software was used to visualize and analyze the molecular docking results.

[0101] 2. Results and Analysis

[0102] 1 Ultrafiltration separation and flavor characteristics analysis of snail meat hydrolysate

[0103] In order to explore the taste characteristics of different molecular weight components, the snail meat hydrolysate was initially separated using a 3kDa ultrafiltration membrane to obtain U1 (≥3kDa) and U2 (<3kDa) ultrafiltration fractions. Subsequently, the freeze-dried U1 (≥3kDa), U2 (<3kDa) and SHP (snail meat hydrolysate) were subjected to manual sensory evaluation analysis. Figure 1 As shown, the most prominent taste of both the hydrolysate and the ultrafiltration fraction was umami, with umami intensities of 5.53, 6.75, and 6.36 for U1, U2, and SHP, respectively. Among them, U2 had the highest umami intensity (6.75), and its taste characteristics were closest to those of the hydrolysate.

[0104] By principal component analysis ( Figure 2 ) It can be seen that the first principal component (PC-1) represents 76.0% of the total information, and the second principal component (PC-2) contributes 16.8% of the information. The closer the samples are in the principal component analysis diagram, the more similar their tastes are. U2 is in the same quadrant as SHP, indicating that the taste characteristics of the <3kDa component are close to SHP. In addition, using 0.1%-0.5% (w / v) MSG solution as a positive control, it was found that the <3kDa component was closer to the 0.2% (w / v) MSG solution, indicating that the <3kDa component has a significant umami taste. The electronic tongue results are consistent with the manual sensory evaluation analysis. Therefore, U2 is an ideal component for further separation.

[0105] 2 RP-HPLC separation and flavor characteristics analysis of different components

[0106] We further separated the U2 component by RP-HPLC and obtained five peaks, which were named F1, F2, F3, F4 and F5 components ( Figure 3 ). Figure 4 Manual sensory evaluation of each RP-HPLC fraction revealed that fractions F1, F2, F4, and F5 all exhibited distinct umami characteristics. Fraction F1, in particular, achieved the highest umami score of 7.35. Compared to ultrafiltration fraction U2 (6.36), the umami intensity increased by 15.57%, indicating enrichment of key umami active ingredients during the isolation and purification process. Fractions F2, F4, and F5 also exhibited distinct umami characteristics compared to the blank control (0.35% (w / v) MSG solution), scoring 6.24, 6.35, and 6.27, respectively.

[0107] Principal component analysis ( Figure 5) further revealed the differences between samples, where the first principal component (PC-1) explained 66.0% of the total information and the second principal component (PC-2) explained 11.38% of the information. In the PCA diagram, the F1 component and the 0.2% (w / v) MSG solution were on the same side ( Figure 5 The F1 fraction was closer to that of a 0.2% (w / v) MSG solution, further demonstrating that the F1 fraction possessed the most pronounced umami intensity. The electronic tongue results were consistent with the manual sensory evaluation analysis, so we subsequently collected the F1 fraction for subsequent peptide composition and amino acid sequence analysis.

[0108] 3 Identification and screening of umami peptides

[0109] The peptide composition and amino acid sequence of F1 were characterized by LC-MS / MS, yielding 402 peptides of varying molecular weights. These peptides ranged from 3 to 20 amino acid residues, with molecular weights ranging from 259.1532 to 2160.1062. Based on the general rule that umami peptides have a molecular weight <1000, the number of sweet amino acids, and the ratio of hydrophobic amino acids (LIU Z, ZHU Y, WANG W, et al. Seven novel umami peptides from Takifugu rubripes and their taste characteristics [J / OL]. Food Chemistry, 2020, 330:127-204), 50 peptides were selected for molecular docking experiments to investigate their interactions with the umami receptors T1R1 / T1R3. Some of the results are shown in Table 1. Among them, we selected the two peptides with the strongest affinity, PIYEGY and TVPIYEG, for synthesis. Their primary and secondary spectra are shown in Figure 2. Figure 6 and 7 And according to the current report, they were identified for the first time, indicating that these two peptides may be new members of the umami peptide family.

[0110] To further evaluate the relationship between peptide characteristics and umami, we used the BIOPEP-UWM database to analyze the umami segments within the identified peptides and calculated the frequency of occurrence of the flavor profile segments. Table 2 lists the predicted evaluation of the peptides' flavor properties. Both peptides were predicted to have umami properties and were predicted to be non-toxic. PIYEGY had the highest umami activity frequency (0.3333). Nevertheless, the flavor properties of these umami peptides require further verification using electronic tongue and manual sensory evaluation.

[0111] Table 1 Docking results of peptides identified from F1 fraction with T1R1 / T1R3 receptor molecules

[0112]

[0113] Table 2 Taste and toxicity prediction of umami peptides (PIYEGY and TVPIYEG)

[0114]

[0115] 4 Taste Analysis of Umami Peptides

[0116] In order to further explore the taste characteristics of umami peptides, the taste detection activity test (TDA) was used to evaluate the taste recognition thresholds of the two umami peptides and define their taste abilities. As shown in Table 3, the taste recognition thresholds of PIYEGY and TVPIYEG were 0.125 mg / mL and 0.15 mg / mL, respectively. These findings are consistent with the frequencies of taste characteristic fragments calculated using the BIOPEP-UWM database. The umami thresholds of both umami peptides are lower than that of MSG (0.3 mg / mL). The lower the umami threshold, the easier it is to perceive umami, indicating that umami peptides are more easily recognized as umami than MSG. The two peptides have a strong umami taste, with umami intensities of 6.63 and 5.42 for PIYEGY and TVPIYEG, respectively ( Figure 8 Meanwhile, sensory analysis revealed that umami peptides have a milder flavor than MSG. Compared to monosodium glutamate, these peptides have a more complex flavor profile, composed of a variety of flavorful amino acids. When the amino acids in the peptides react with enzymes in the mouth, distinct flavors are produced, potentially creating diverse taste experiences during food processing.

[0117] To further explore the role of umami peptides in flavor regulation, we used sensory evaluation to explore their effects on the flavor of the broth model. Figure 9 As shown in the results, compared to the blank control, the broth model supplemented with PIYEG increased its umami intensity to 8.6 points and its saltiness to 6.6 points. In contrast, the broth model supplemented with TVPIYEG had an umami intensity of 6.7 points and a saltiness intensity of 7.61 points. This indicates that PIYEG is more effective in enhancing umami, while TVPIYEG is more effective in enhancing saltiness. Furthermore, both umami peptides enhanced both umami and saltiness in the simulated broth. This suggests that umami peptides can produce a synergistic effect, effectively enhancing the umami and saltiness of food.

[0118] The umami intensity of umami peptides is closely related to the length of the peptide chain, the composition of amino acids, and the position of amino acids. The amino acid skeleton structure of umami peptides is usually -O-(C) nO-, where n values ​​range from 3 to 9. Studies have found that umami is most pronounced when n values ​​are between 4 and 6. In this study, the umami peptides PIYEGY and TVPIYEG identified from snail meat had n values ​​of 6 and 7, respectively. In sensory evaluation, we found that PIYEGY had a higher umami intensity than TVPIYEG, consistent with the inferences of previous studies.

[0119] The formation of umami is closely related to the presence of specific amino acids. Aspartic acid (D), glutamic acid (E), serine (S), alanine (A), glycine (G), threonine (T), and tyrosine (Y) are generally considered key amino acids associated with umami. Among them, aspartic acid (D) and glutamic acid (E) are considered typical representatives of umami. Histidine (H) and valine (V) may also influence the umami taste of peptides. Furthermore, serine (S) and threonine (T), which are generally considered to have sweet properties, may also contribute to the formation of umami. In this study, PIYEGY (Pro-Ile-Tyr-Glu-Gly-Tyr) and TVPIYEG (Thr-Val-Pro-Ile-Tyr-Glu-Gly) both contain amino acid residues associated with umami. The umami-sweet amino acid content of PIYEGY was 67%, while that of TVPIYEG was 57%. Furthermore, hydrophobic amino acids also contribute to the umami expression of umami peptides. Research has shown that most umami peptides contain a certain proportion of hydrophobic amino acids. Furthermore, many umami peptides contain bitter amino acids, and studies have shown that proline (P), histidine (H), valine (V), isoleucine (I), and leucine (L) also contribute to umami. In this study, the hydrophobic amino acid content of the peptides PIYEGY and TVPIYEG we identified was 50% and 57%, respectively, consistent with previous research.

[0120] By principal component analysis (PCA) Figure 10 ), the first principal component (PC-1) represents 66.18% of the total information, and the second principal component (PC-2) contributes 11.15%. In the PCA plot, PIYEGY is closer to a 0.1% (w / v) MSG solution, indicating that PIYEGY has a stronger umami taste. This is consistent with the results of manual sensory evaluation.

[0121] Therefore, subsequent studies will explore the binding of PIYEGY and TVPIYEG to the umami receptors T1R1 / T1R3, respectively, to help reveal the mechanism of action of umami peptides.

[0122] Table 3 Taste thresholds and sensory descriptions of umami peptides (PIYEGY and TVPIYEG)

[0123]

[0124] 5. Effects of different conditions on the umami taste of umami peptides

[0125] 5.1 Effects of different pH values ​​on the umami taste of umami peptides

[0126] pH is an important factor affecting the taste properties of substances. Therefore, this study analyzed the relationship between pH and taste properties of PIYEGY and TVPIYEG to explore the stability of umami peptides.

[0127] Figure 11 The effect of different pH values ​​on the umami intensity of PIYEGY and TVPIYEG was demonstrated, with MSG used as a control experiment. The umami intensity of TVPIYEG and MSG gradually increased with increasing pH value (pH 4.0-8.0), which is similar to the taste characteristics of BMP (Beefy Meaty Peptide) at different pH values. This enhancement may be due to the ionization of TVPIYEG and the interaction of inorganic ions (Na) with increasing pH. + ) interact with each other, thereby enhancing the overall umami flavor. TVPIYEG has the highest umami intensity at pH 8. PIYEG's umami intensity decreases with decreasing pH. However, at pH 4, the umami intensity is strongest. Within the pH range of 4.0-8.0, the umami intensity of both PIYEGY and TVPIYEG is higher than that of MSG. These results not only reveal the taste properties of umami peptides at different pH environments, but also allow the pH value of foods to be adjusted to optimize their taste properties. This has important guiding significance for the future application of umami peptides as umami enhancers.

[0128] 5.2 Effects of different temperatures on the umami taste of umami peptides

[0129] Effect of temperature on the umami taste of PIYEGY and TVPIYEG Figure 12The umami intensity of the umami peptides PIYEGY and TVPIYEG remained relatively stable when treated at 40-120°C. In contrast, the umami intensity of MSG remained constant with increasing temperature between 40-80°C, but decreased with increasing temperature between 100-120°C. These results indicate that the umami peptides PIYEGY and TVPIYEG exhibited greater thermal stability than MSG. This difference in thermal stability may be related to their smaller molecular weight. Furthermore, MSG may undergo dehydration during heating, resulting in a decrease in its content and, consequently, a reduction in umami. The thermal stability of umami peptides is an important consideration during food processing and cooking, particularly in applications involving high-temperature treatment. Future research could further investigate the stability of different types of umami peptides over a wider temperature range and how these changes affect the overall flavor of food. Furthermore, studying the thermal stability mechanisms of umami peptides and the molecular structural changes at high temperatures will provide deeper insights into the field of food science and may facilitate the development and application of novel umami enhancers.

[0130] Molecular docking of 6 umami peptides with umami receptors T1R1 / T1R3

[0131] The final homology model of the umami taste receptor T1R1 / T1R3 is shown in Figure 2. Figure 13 As shown in the figure, the left subunit is the T1R1 portion, and the right subunit is the T1R3 portion. The umami taste receptor T1R1 / T1R3 is a heterodimer of the first taste receptor family within the G protein-coupled receptor family. Its receptor binding domain has a "VFT" structure. The two leaves are connected by a hinge, which together form a cavity. This cavity is the primary site for interaction between ligands and taste receptors.

[0132] Figure 14Ramachandran diagram that can be used to evaluate the calculation of the model. The Ramachandran diagram shows different regions of conformations that are allowed and not allowed due to interactions between groups. It is mainly divided into three regions: allowed region (represented by red), maximum allowed region (represented by yellow) and not allowed region (represented by white). Only when more than 90% of the residues are within the allowed or maximum allowed range, the model is considered reasonable and reliable (JIANG L, HE Y, LUO G, et al. Discovery of potential novel microsomal triglyceride transfer protein inhibitors via virtual screening of pharmacophore modelling and molecular docking [J / OL]. Molecular Simulation, 2016, 42 (15): 1223-1232.) (SHRIVASTAV A, SRIVASTAVA S. Human sweet taste receptor: Complete structure prediction and evaluation [J / OL]. International Journal of Chemical and Analytical Science, 2013, 4 (1): 24-32..). The results showed that 99.9% of the amino acid residues were within the reasonable range, of which 88.1% were in the optimal region, 11.1% were in the acceptable region, and 0.8% were in the generally allowed region. The amino acid residues located in the disallowed region accounted for 0.1%. Figure 15) produced an overall quality factor of 87.161%. The number of non-bonded interactions between different atom types was calculated to within 0.35nm (BU Y, ZHOU Y, SUN C, et al. Identification Novel Salty-Enhancing Peptides from Sea Cucumber Collagen: AlphaFold2 Modeling and Molecular Simulation [J / OL]. Food and Bioprocess Technology, 2023). This means that the calculation error of 87.161% of the residues is below the 95% rejection limit. These data indicate that the model established by homology simulation is reasonable in terms of dihedral angle distribution and three-dimensional collision. In order to further study the taste mechanism of umami peptides with umami receptors T1R1 / T1R3, molecular docking was performed using the AutoDock Vina 1.1.2 tool.

[0133] In this study, both PIYEGY and TVPIYEG interacted with T1R1 / T1R3. Both peptides inserted into the VFT binding pocket of the T1R3 subunit. This is likely because T1R1 is closed, while the binding domain of T1R3 is in an open conformation, leaving the cavity large enough to accommodate and bind to long-chain umami peptides. The docking energies of PIYEGY and TVPIYEG with T1R3 were -9.0 kcal / mol and -8.8 kcal / mol, respectively (Table 4). PIYEGY and TVPIYEG formed seven and six hydrogen bonds, respectively. Hydrogen bonds, as one of the strongest intermolecular interactions, help reduce the binding energy between peptides and receptors. A higher standard free energy charge indicates a stronger binding affinity between the ligand and receptor. PIYEGY exhibited the lowest docking binding energy with T1R3. This is consistent with the sensory evaluation and electronic tongue results. Furthermore, some studies have shown that umami peptides containing one or two acidic amino acids (such as Asp and Glu) are more likely to bind to the umami receptors T1R1 / T1R3. Other studies suggest that peptides with bitter amino acid residues at the C-terminus may facilitate binding to the T1R1 / T1R3 umami receptors. This may be the reason why PIYEGY exhibits a stronger umami taste.

[0134] like Figure 16As shown in the figure, the interaction between PIYEGY and T1R3. PIYEGY interacts with T1R3 and forms hydrogen bonds with Val105, Ser 148, Leu 51, Ser 217, Asn 150, Asp 218, and Glu 178. The binding sites Leu 173, Ala 153, Ala 176, Ala 249, Phe 180, and Met 151 form hydrophobic interactions in the ligand PIYEGY. Figure 17 As shown in the figure, the interaction between TVPIYEG and T1R3, Asp 147, Ser 148, Ser 217, Thr 179, Ser248, and Leu 51 in T1R3 interact with TVPIYEG through hydrogen bonds, and the binding sites Leu 279, Arg 151, and Pro 45 form hydrophobic interactions with TVPIYEG.

[0135] In this study, we found that both PIYEGY and TVPIYEG bind to Ser 217 and Arg 151. Ser 217 and Arg 151 may play a key role in the binding of umami peptides to umami receptors. Furthermore, studies have shown that bitter amino acid residues in umami peptides, such as Pro, Leu, and Ile, can also bind to amino acid residues in T1R3 through interactions. This result suggests that bitter amino acid residues in umami peptides may contribute to umami activity. The two umami peptides bind to T1R3 primarily through hydrogen bonds and electrostatic interactions. The differences in these binding sites may be due to structural differences in umami peptides and the allosteric effects of long-chain peptides.

[0136] Table 4 Molecular docking results of umami peptides (PIYEGY and TVPIYEG) with T1R1 / T1R3

[0137]

[0138] Molecular docking of 7 umami peptides with salty taste receptor TMC 4

[0139] The final homology model of the salty taste receptor TMC 4 is shown in Figure 4. Figure 18As shown. Kasahara et al. (KASAHARA Y, NARUKAWA M, ISHIMARU Y, et al. TMC4 is a novel chloride channel involved in high-concentration salt taste sensation [J / OL]. The Journal of Physiological Sciences, 2021, 71 (1): 23.) found that TMC 4 allows organic anions to penetrate. When TMC 4 is described as a salt receptor screening tool, a structure-activity relationship simulation based on the amino acid sequence structure model of the TMC 4 protein can be used to determine whether the test substance is a compound that can upregulate the functional expression of the TMC 4 protein. For example, a 3D structural model of the TMC 4 protein is constructed, so that ligands that activate the pore region can be screened based on docking simulations with the 3D structural model.

[0140] The TMC 4 salty taste receptor model was evaluated using SEVES v5.0. Ramachandran plots were obtained after evaluation and analysis using the Procheck program ( Figure 19 The evaluation showed that 93.0% of the residues were located in the most favorable region, 6.0% were located in the allowed region, 0.7% were located in the loosely allowed region, and 0.3% were located in the disallowed region. The overall quality factor generated by ERRAT was 96.714% ( Figure 20 ). These data confirm that the model can be used.

[0141] Molecular docking helps to gain a deeper understanding of the nature of the interaction between umami peptides and TMC 4 receptors and their mechanism of salty taste enhancement. The binding energies of PIYEGY and TVPIYEG to TMC 4 are -7.1 Kcal / mol and -7.3 Kcal / mol, respectively. The lower the free energy value, the stronger the binding ability between the peptide and the TMC 4 receptor, indicating that the conformation is more stable and promotes the formation of salty taste. The interaction between PIYEGY and the surrounding residues of the receptor protein TMC 4 is shown in Figure 2. Figure 21 The interaction between TVPIYEG and the surrounding residues of the receptor protein TMC 4 is shown in Figure 22 shown.

[0142] The interactions between the umami peptides PIYEGY and TVPIYEG and TMC-4 are primarily hydrogen bonding and hydrophobic interactions. The active residues Ala 99 and Trp 95 of TMC-4 interact with PIYEGY through hydrophobic interactions, while Glu 83, His 103, Gln 98, Arg 106, Trp 59, and Tyr 51 interact through hydrogen bonds. TVPIYEG interacts primarily with Ala 521, Thr 523, Leu 520, Asn 404, Arg 506, Gln 503, Glu 531, Gln 524, and Glu 525 of TMC-4 through hydrogen bonds. TVPIYEG interacts with the active residues Phe 405 and Pro 409 of TMC-4 through hydrophobic interactions, potentially playing a key role in the salty taste enhancement effect. Shen et al. (SHEN DY, PAN F, YANG ZC, et al. Identification of novel saltiness-enhancing peptides from yeast extract and their mechanism of action for transmembrane channel-like 4 (TMC4) protein through experimental and integrated computational modeling [J / OL]. Food Chemistry, 2022, 388: 132993.) found that Arg, Thr, and Tyr are believed to play an important role in the binding of salty taste-enhancing peptides to TMC 4 receptors. This is consistent with our results. Arg may have a promoting effect on salty taste enhancement. Subsequently, PIYEGY and TVPIYEG were subjected to sensory evaluation to determine their salty taste enhancement effect.

[0143] Table 5 Molecular docking results of umami peptides (PIYEGY and TVPIYEG) and TMC 4

[0144]

[0145] Sensory evaluation of the saltiness-enhancing effect of 8 umami peptides

[0146] Figure 23The results of the saltiness enhancement effect of PIYEGY and TVPIYEG are presented. Using a 0.3% NaCl solution as a blank control, the saltiness intensity was rated as 5 points. In a 0.3% (w / v) NaCl solution, both peptides showed a saltiness enhancement effect. The NaCl solution with PIYEG added was equivalent to the saltiness of 0.6% (w / v) NaCl, while TVPIYEG reached the saltiness of 0.7% (w / v) NaCl. TVPIYEG, in particular, was the most effective in increasing saltiness. Although these two peptides themselves have almost no saltiness, they still showed a saltiness enhancement effect. Related studies have found that the presence of polar amino acids with uncharged side chains (such as Thr and Tyr) at both ends of the peptide chain may produce a saltiness enhancement effect. This may be the reason why TVPIYEG has a better saltiness enhancement effect. These findings are of great significance for the development of low-salt foods and help reduce the use of salt without sacrificing flavor.

[0147] The present invention isolates, purifies, and identifies umami peptides from snail meat hydrolysates using ultrafiltration, RP-HPLC, and UPLC-QTOF-MS / MS. Molecular docking and calculation of potential umami activity identified two novel umami peptides, PIYEGY and TVPIYEG. Sensory evaluation and electronic tongue analysis revealed that these two monomeric peptides exhibit umami characteristics and a salty enhancement effect. Umami recognition thresholds were 0.125 mg / mL and 0.150 mg / mL, respectively. The two umami peptides can integrate into the VFT binding pocket of the umami receptor T1R3 cavity. Ser 217 and Arg 151 are key sites for binding to the T1R3 subunit, with hydrogen bonds and hydrophobic interactions being the primary interactions. Docking of the TVPIYEG peptide with TMC 4 revealed novel binding sites: Phe 405 and Pro 409, which may play a key role in the salty enhancement effect. This study provides a reference for studying snail umami peptides, helping to understand the mechanism of umami formation and its potential for development into food additives for enhancing flavor and reducing salt.

[0148] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A snail-derived umami peptide with a salt-increasing effect, characterized in that Its amino acid sequence is PIYEGY.

2. The method for preparing the snail-derived umami peptide according to claim 1, characterized in that The following steps are included: The snail-derived umami peptide with saltiness-enhancing effect is prepared directly through solid-phase synthesis or snail meat is used as raw material, and the snail-derived umami peptide with saltiness-enhancing effect is obtained through trypsin hydrolysis and purification.

3. The method for preparing the snail-derived umami peptide according to claim 2, wherein: The specific operation of the trypsin hydrolysis is: The homogenized snail meat is mixed with water, and trypsin is added for enzymatic hydrolysis in a water bath; the enzyme is then inactivated in a water bath, the product is cooled, centrifuged, the supernatant is collected, and the product is further freeze-dried.

4. The method for preparing snail-derived umami peptide according to claim 2, characterized in that: The purification comprises ultrafiltration separation and liquid chromatography separation steps.

5. The method for preparing snail-derived umami peptide according to claim 4, characterized in that: The specific operation of the ultrafiltration separation is: Ultrafiltration tubes were used to separate the snail meat hydrolysate and fractions with molecular weight <3 kDa were collected; The specific operation of the liquid chromatography separation is: The ultrafiltration fraction of snail meat hydrolysate <3 kDa was separated and purified by HPLC using a preparative reversed-phase HPLC system. The active components were collected, concentrated by rotary evaporation and freeze-dried to obtain snail-derived umami peptides with salt-enhancing effect.

6. Use of the snail-derived umami peptide according to claim 1 in the field of food processing.

7. Use of the snail-derived umami peptide according to claim 1 in flavor-enhancing and salt-reducing foods.

8. Use of the snail-derived umami peptide according to claim 1 in the field of flavor bases or food additives.

9. A flavor-enhancing and salt-reducing food, comprising at least one of the snail-derived umami peptide according to claim 1, a snail meat trypsin hydrolyzate containing the snail-derived umami peptide according to claim 1, and a hydrolyzate containing the snail-derived umami peptide according to claim 1 as an active ingredient.

10. A flavor base or food additive comprising at least one of the snail-derived umami peptide according to claim 1, a snail meat trypsin hydrolyzate containing the snail-derived umami peptide according to claim 1, and a hydrolyzate containing the snail-derived umami peptide according to claim 1 as an active ingredient.

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