Litopenaeus vannamei head umami peptide and preparation method thereof

Abstract

The invention provides a litopenaeus vannamei head umami peptide and a preparation method thereof, and the umami peptide is characterized in that the amino acid sequence of the umami peptide is selected from any one of the sequences as shown in SEQ ID NO: 1-5; the delicate flavor threshold value of the delicate flavor peptide is 0.2 to 0.5 mmol / L. According to the method, high-value utilization of the penaeus vannamei head byproducts is achieved, and the problems of wasting of aquatic product processing resources and environmental protection pain are effectively solved; five brand-new umami peptide sequences are obtained for the first time, the umami threshold value is far lower than that of monosodium glutamate, the synergistic fresh-increasing effect is outstanding, and the blank of an aquatic product source umami peptide library is filled.

Description

Technical Field

[0001] This invention belongs to the field of food bioprocessing technology, specifically relating to a umami peptide from the head of Litopenaeus vannamei and its preparation method. Background Technology

[0002] Umami, as the fifth basic taste, is key to enhancing the flavor and quality of food. Umami peptides, due to their natural safety, ability to synergistically enhance flavor with monosodium glutamate and salt, and suitability for low-sodium food development, have become a research hotspot for novel umami agents. However, existing technologies still have significant drawbacks: chemical umami agents have a limited flavor profile and are easily deactivated at high temperatures; natural umami peptides have high raw material costs and insufficient umami intensity, making it difficult to meet the needs of industrial applications.

[0003] In the research of shrimp-derived umami peptides, existing technologies mostly use edible shrimp meat from Antarctic krill and Procambarus clarkii as raw materials (such as Chinese patents CN 202510839988.9 and CN 202510332322.4). This not only results in high raw material costs but also fails to utilize aquatic processing by-products, making it difficult to achieve green and high-value utilization. Meanwhile, existing screening methods mostly rely on a reverse model of "first separation and purification, then sensory / electronic tongue blind screening, and finally mass spectrometry identification," which is highly subjective, has low throughput, and a high false positive rate. Umami activity prediction often uses a single database, usually BIOPEP-UWM, for matching known taste fragments, which cannot predict novel unknown sequences, resulting in generally low prediction accuracy and severely limiting the efficient development of highly active umami peptides.

[0004] Litopenaeus vannamei (whiteleg shrimp) is the most produced farmed shrimp species in my country. The shrimp head accounts for 35%-45% of its body weight and contains 15%-17% protein, making it a potential high-quality protein source. However, current technology generally considers that Litopenaeus vannamei heads are rich in trimethylamine, volatile fishy-smelling substances, and hydrophobic bitter peptides. Conventional enzymatic hydrolysis products have a strong fishy and bitter taste, limiting their use to low-value applications in feed and fertilizer, or as functional additives at ≤1% concentrations. They cannot be used as primary umami enhancers, and this perception has become a long-standing technical bias in the field. Currently published related research (such as Chinese patents CN 202510964747.7 and CN202510964748.1) uses shrimp heads to prepare functional peptides for lowering uric acid and anti-oxidation, but lacks any targeted preparation, sequence screening, or research on flavor mechanisms specifically for umami activity.

[0005] Therefore, there is an urgent need to develop an efficient, precise, and industrially suitable technology for the preparation and screening of shrimp head-derived umami peptides. Summary of the Invention

[0006] In view of this, the present invention provides a flavor peptide from the head of Litopenaeus vannamei and a method for preparing the same. The technical solution of the present invention is as follows: A umami peptide from the head of Litopenaeus vannamei, characterized in that the amino acid sequence of the umami peptide is selected from any one of the sequences shown in SEQ ID NO:1-5; the umami threshold of the umami peptide is 0.2-0.5 mmol / L; wherein, SEQ ID NO:1 is WDEGL, SEQ ID NO:2 is PDPTF, SEQ ID NO:3 is YTVFDR, SEQ ID NO:4 is FSGVPDR, and SEQ ID NO:5 is LTDW.

[0007] In this invention, SEQ ID NO:1-5 are the first fresh umami peptide sequences isolated and identified from the head by-products of Litopenaeus vannamei. After novelty searches and comparisons in domestic and international patent databases, core academic literature databases, and related bioactive peptide libraries, no identical or highly homologous umami peptide sequences were found. This invention enables the high-value utilization of Litopenaeus vannamei head by-products and provides a foundation for solving the industry problem of shrimp heads being used only as feed / waste and protein resources being wasted in the prior art.

[0008] In addition, the umami threshold of the five peptides is much lower than that of most aquatic umami peptides disclosed in the prior art, and all thresholds are much lower than the umami threshold of commercially available monosodium glutamate (L-glutamate) of 1.48±0.52mmol / L, which provides a basis for solving the problems of weak umami ability and large addition amount of existing umami peptides.

[0009] More importantly, all five sequences are small molecule oligopeptides with 4-7 amino acids, which have the characteristics of being small molecules that can easily penetrate taste cell membranes, have high freshness preservation efficiency, are easily absorbed by the human body, and are non-antigenic, thus meeting the safety requirements for food additives.

[0010] Furthermore, when the umami peptide is used in combination with monosodium glutamate (MSG), it has a synergistic umami-enhancing effect, which improves the umami intensity of the MSG solution.

[0011] The umami peptides of this invention can bind together with MSG to the umami receptors T1R1 / T1R3, and through multi-target synergistic activation of the umami signaling pathway, amplify the umami signal transduction, and achieve a "1+1>2" umami enhancement effect, thus solving the problem of insufficient umami after MSG reduction in low-sodium foods.

[0012] Furthermore, the umami peptide has a final concentration of 0.05-0.2 mg / mL, and the L-glutamate has a final concentration of 2-5 mg / mL. The shrimp head umami peptide can increase the umami intensity of L-glutamate in the system by 1.2-1.5 times.

[0013] In this invention, the synergistic flavor-enhancing effect of Litopenaeus vannamei head-derived umami peptides and MSG was quantified. 0.1 mg / mL of umami peptides can increase the umami intensity of a 3 mg / mL MSG solution by up to 1.39 times, which can effectively reduce the amount of MSG added to food and is in line with the development trend of healthy food.

[0014] Furthermore, throughout the 100ns molecular dynamics simulation, the number of hydrogen bonds formed between the umami peptide and the umami receptors T1R1 / T1R3 remained dynamically stable at 2-10. In the later 20-40ns of the simulation, the number of hydrogen bonds tended to stabilize without drastic fluctuations. All systems could reach conformational equilibrium in the later stages of the simulation, and the RMSD curve of the complex backbone after equilibrium showed low fluctuation characteristics.

[0015] In this invention, hydrogen bonds are the core driving force for the binding of peptides to receptors. The number of hydrogen bonds is dynamically stable at 2-10, ensuring the persistence and robustness of the binding between umami peptides and receptors, and preventing instantaneous binding and rapid dissociation. In the later stages of simulation, the hydrogen bonds tend to stabilize in the 20-40 ns period, the complex reaches conformational equilibrium, and the RMSD fluctuates low, proving that the complex structure formed by the umami peptide and receptor is highly stable and can continuously activate the umami signaling pathway. This provides a guarantee for the present invention to achieve the characteristics of low umami threshold and long duration of umami flavor.

[0016] Furthermore, the umami peptides achieve stable umami enhancement by specifically binding to the Asp88, Ala90, and Asn130 key sites of the umami receptors T1R1 / T1R3.

[0017] The umami specificity of umami peptides is directly determined by their binding sites with receptors. The five umami peptides of this invention can all specifically bind to three key sites on the umami receptor T1R1 / T1R3: Asp88, Ala90, and Asn130. Asp88 appears a total of eight times in the binding of the five peptides and is the core recognition site. These three sites are novel binding targets not reported in existing technologies and are the core reason for the umami specificity and excellent stability of the umami peptides of this invention.

[0018] This invention is the first to discover that Asp88, Ala90, and Asn130 of T1R1 / T1R3 are key core sites for the binding of umami peptides, filling a research gap in the field of umami peptide umami mechanisms. It also provides a novel target reference for the molecular design and targeted modification of novel umami peptides, possessing significant industry guidance value.

[0019] The present invention also provides a method for preparing the above-mentioned shrimp head umami peptide, characterized by comprising the following steps: S1. Raw material pretreatment and targeted enzymatic hydrolysis: Using shrimp heads, a by-product of Litopenaeus vannamei processing, as raw material, the mixture was homogenized, water was added and mixed, the pH was adjusted to 7.0, a complex protease was added for enzymatic hydrolysis, the enzyme was inactivated, the supernatant was collected by centrifugation, and the crude polypeptide extract was obtained by freeze drying. S2. Targeted enrichment: The crude peptide extract is sequentially passed through 5kDa, 3kDa, and 1kDa ultrafiltration membranes for fractionation, and the ultrafiltration fraction with a molecular weight <1kDa is collected. S3. Gel purification: The ultrafiltration fraction was separated by Superdex30 gel filtration chromatography, and the elution peak fraction with the strongest umami activity was collected by electronic tongue evaluation. S4. Forward Precision Screening: The peptide sequences of the elution peak components are resolved by LC-MS / MS. The obtained peptides are then subjected to initial screening by three umami prediction models: Umami-MRNN, UMPed-FRL, and Umami-YYDS. They are then subjected to secondary screening based on safety and processing adaptability physicochemical properties to obtain potential umami peptides. S5. Verification and preparation: Molecular docking verification was performed on the potential umami peptides and umami receptors T1R1 / T1R3. Peptides with binding energies that met the requirements were selected for solid-phase synthesis and activity verification to obtain the shrimp head umami peptides.

[0020] In the preparation method of this invention, small molecule umami peptides <1kDa are first precisely enriched through targeted enzymatic hydrolysis, three-stage ultrafiltration, and gel chromatography, while large molecule proteins, bitter peptides, and fishy impurities are removed to reduce interference for subsequent screening. Then, full-sequence analysis by LC-MS / MS is performed, followed by primary screening using three deep learning models combined with physicochemical property screening to identify potential high-activity umami peptides from the source, completely eliminating reliance on artificial sensory evaluation. Finally, target umami peptides are screened step by step through molecular docking verification, solid-phase synthesis, and activity confirmation, significantly improving screening efficiency and accuracy compared to existing technologies.

[0021] This invention constructs a complete process for preparing umami peptides from the head by-products of Litopenaeus vannamei, realizing the high-value utilization of shrimp head protein and solving the industry pain points of environmental pollution and low-value utilization of aquatic processing by-products. Furthermore, this invention combines three deep learning models—Umami-MRNN, UMPred-FRL, and Umami-YYDS—to construct a forward precision screening system for umami peptides. This system achieves several times the screening efficiency compared to existing blind screening methods, significantly reducing the false positive rate and effectively addressing the problems of low screening efficiency and large subjective errors in existing technologies.

[0022] Furthermore, in step S1, the ratio of shrimp head to water after homogenization is 1:5. The complex protease is a mixture of flavor protease and trypsin at a mass ratio of 1:2, and the amount added is 3% of the substrate protein mass. The enzymatic hydrolysis conditions are 50℃ water bath for 4 hours. After the enzymatic hydrolysis is completed, the enzyme is inactivated in a 95℃ water bath for 10 minutes, and the supernatant is collected by centrifugation at 4℃ and 8000r / min for 10-15 minutes. In step S2, the operating conditions for ultrafiltration separation are 4℃, 8000r / min, and a centrifugation time of 20min each time; In step S3, the gel filtration chromatography uses 2×PBS as the elution buffer, a flow rate of 0.5 mL / min, a detection wavelength of 214 nm, a sample concentration of 10 mg / mL, a sample volume of 100 µL, and a column temperature of 30 °C.

[0023] The enzymatic hydrolysis system constructed in this invention employs a combination of flavor protease and trypsin, which can efficiently hydrolyze the protein in shrimp heads while avoiding excessive hydrolysis that produces a large amount of hydrophobic bitter peptides, thus preserving the umami base of the hydrolysate. A three-stage ultrafiltration process ensures precise fractional separation while preventing denaturation of umami peptides during separation, accurately enriching highly active small molecule umami peptides <1kDa. The gel chromatography process used perfectly matches the molecular weight range of the target small molecule peptides, and the matching eluent, flow rate, and loading parameters enable efficient peptide separation and precise collection of the most umami-active components.

[0024] Furthermore, in step S4, the LC-MS / MS adopts a data-dependent acquisition mode, with a first-level full scan resolution of 70,000 and a scan range of 300-1500 m / z. The second-level fragmentation adopts a high-energy collision dissociation method with a normalized collision energy of 28%. The physicochemical property screening includes toxicity, water solubility, stability, and allergenicity detection. In step S5, the molecular docking constructs a homology model of umami receptor T1R1 / T1R3 using the metabolite glutamate receptor mGluR1 as a template. The AutoDockVina semi-flexible docking method is used to select peptides with binding energy ≤ -8.5 kcal / mol for solid-phase synthesis. After synthesis, the activity is verified by sensory evaluation, umami threshold determination and synergistic umami enhancement.

[0025] In this invention, the characteristics of small molecule oligopeptides can be optimized by setting LC-MS / MS parameters, achieving a sequence identification accuracy of ≥95%. Through physicochemical property screening, while screening for umami activity, pre-screening of core food industry attributes such as toxicity, allergenicity, and water solubility is completed simultaneously, solving the problem that peptides screened by existing technologies cannot be directly applied industrially. A molecular docking system is established, and the homology modeling method using mGluR1 as a template is currently the most reliable modeling method for the umami receptors T1R1 / T1R3. Simultaneously, a hard threshold of binding energy ≤-8.5 kcal / mol is set, which can directly eliminate low-affinity false positive peptides, significantly improving the screening success rate. The final activity verification process comprehensively verifies from three dimensions: sensory evaluation, threshold, and synergistic umami enhancement, ensuring that the screened peptides have practical industrial application value.

[0026] This invention constructs a comprehensive forward precision screening system consisting of "three deep learning models for initial screening + physicochemical property secondary screening + molecular docking verification + multi-dimensional activity confirmation," which differs from the commonly used reverse blind screening mode of "physical separation + sensory coarse screening + full peptide identification + post-verification." Furthermore, by setting a binding energy ≤-8.5 kcal / mol as a rigid threshold for umami peptide screening, the success rate is several times higher than existing technologies, while the false positive rate is significantly reduced.

[0027] The above-mentioned shrimp head umami peptide is used in food flavor enhancement, characterized in that the shrimp head umami peptide enhances flavor by specifically binding to the Asp88, Ala90, and Asn130 key sites of the umami receptor T1R1 / T1R3, and the food includes low-salt food, high-temperature sterilized food, condiments, meat products, frozen food, or baked food.

[0028] Furthermore, the shrimp head umami peptide is compounded with L-glutamate and added to food to enhance the umami intensity of the food and reduce the amount of L-glutamate and sodium chloride added; wherein, the amount of shrimp head umami peptide added to the food is 0.1%-1% (w / w), and the food is high-temperature sterilized canned food, low-salt seasoning, meat products or quick-frozen noodle and rice products.

[0029] In particular, based on the three core advantages of the umami peptides of this invention—excellent basic umami performance, synergistic umami enhancement effect with MSG, and highly stable binding characteristics with receptors—they can be applied to various food umami enhancement scenarios, thus fully expanding the application scope of aquatic umami peptides.

[0030] This invention is the first to use the head of Litopenaeus vannamei for the targeted preparation of highly active umami peptides, successfully producing polypeptides that can be directly used as primary umami agents, breaking through the long-held industry belief that shrimp heads can only be used for low-value purposes. Unlike existing technologies that rely on human sensory evaluation and a "needle in a haystack" approach of separation followed by blind screening, this invention constructs a novel forward precision screening system. Through high-precision full-sequence analysis by LC-MS / MS, combined primary screening using three deep learning models—Umami-MRNN, UMPred-FRL, and Umami-YYDS—and preliminary screening using physicochemical properties such as toxicity, allergenicity, water solubility, and stability, and further combined with T1R1 / T1R3 homology modeling molecular docking with a binding energy ≤-8.5 kcal / mol as a hard screening threshold, a complete process of "full-sequence analysis - virtual primary screening - physicochemical secondary screening - mechanism verification - targeted synthesis" is formed. This completely eliminates the reliance on human sensory evaluation and fundamentally solves the core pain points of traditional blind screening, such as high subjectivity, low throughput, and high false positives.

[0031] The screening system of this invention is not a simple aggregation tool, but rather a multi-algorithm cross-validation approach that significantly improves the prediction accuracy of novel, unknown umami sequences compared to existing technologies. Simultaneously, it eliminates unsafe and unsuitable peptides for industrial applications in the early stages of screening, achieving a dual breakthrough in screening efficiency and industrial adaptability. This invention not only obtained five novel fresh umami peptide sequences but also established a complete correlation mechanism of "peptide sequence-binding conformation-binding stability-umami effect," revealing its umami-enhancing mechanism at the molecular level: these peptides specifically bind to three novel key sites—Asp88, Ala90, and Asn130—forming highly stable complexes with the umami receptors T1R1 / T1R3, thereby achieving a low umami threshold and a high synergistic umami enhancement effect, distinct from existing technologies that study known sites. This invention further combines macroscopic sensory evaluation, threshold determination, and microscopic molecular dynamics verification such as RMSD, RMSF, and hydrogen bond quantity, forming a complete closed-loop proof from effect to mechanism, making the technical effects of this invention more conclusive and credible.

[0032] The beneficial effects of this invention are as follows: 1. To realize the high-value utilization of the shrimp head by-products of Litopenaeus vannamei, and solve the problems of resource waste and environmental protection in aquatic product processing; 2. Compared with the existing technology, the present invention adopts a forward precision screening system using three deep learning models in combination, which is different from the traditional reverse blind screening mode. The screening efficiency of umami peptides is greatly improved and the accuracy of umami prediction of new sequences is significantly improved. 3. Five completely fresh flavor peptide sequences were obtained for the first time. The umami threshold is much lower than that of monosodium glutamate, and the synergistic flavor-enhancing effect is outstanding, filling the gap in the aquatic umami peptide library. 4. Revealing three novel key binding sites—Asp88, Ala90, and Asn130—of the umami receptor T1R1 / T1R3, and elucidating the molecular mechanism of umami presentation through closed-loop analysis; 5. To provide reliable technical support and implementation path for umami peptides to move from laboratory research to industrialization. Attached Figure Description

[0033] Figure 1 The process flow diagram for preparing shrimp head umami peptides according to the present invention is shown below. Figure 2 The electronic tongue radar diagram of the ultrafiltrate components prepared in this invention (U1: >5kDa, U2: 3-5kDa, U3: 1-3kDa, U4: <1kDa); Figure 3 This is the gel chromatography chromatogram of the present invention; Figure 4 This is a umami evaluation diagram of the gel chromatography-separated components (F1-F3) of the present invention; Figure 5 This is a model structural diagram of the umami receptors T1R1 / T1R3 of the present invention; Figure 6 The Ramachandran diagram of the homologous model T1R1 / T1R3 of this invention; Figure 7 This is an electronic tongue radar image for screening peptides according to the present invention; Figure 8 Evaluation of the synergistic flavor enhancement of umami peptides in monosodium glutamate solution; Figure 9 The changes in the detection threshold of the umami peptide and MSG mixture system are shown in the following figures: (A: Change in detection threshold after mixing MSG with FSGVPDR; B: Change in detection threshold after mixing MSG with LTDW; C: Change in detection threshold after mixing MSG with PDPTF; D: Change in detection threshold after mixing MSG with WDEGL; E: Change in detection threshold after mixing MSG with YTVFDR). Figure 10 This is a schematic diagram of the molecular docking of the umami peptide of the present invention with the T1R3 receptor (A: FSGVPDR, B: LTDW, C: PDPTF, D: WDEGL, E: YTVFDR). Figure 11 The results of a 100 ns molecular dynamics simulation of the umami peptide-receptor complex are shown in Figure 1 (A: Root Mean Square Deviation (RMSD); B: Root Mean Square Fluctuation (RMSF); C: Radius of Gyre (Rg); D: Number of Hydrogen Bonds). Detailed Implementation

[0034] The technical solutions will now be clearly and completely described in conjunction with embodiments of the present invention. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art. Example

[0036] The process for preparing umami peptides from Litopenaeus vannamei heads is as follows: Figure 1 As shown, the specific steps include: (a) Preparation of shrimp head enzymatic hydrolysate: The heads of Litopenaeus vannamei were placed in a blender and stirred to obtain a head homogenate. The head homogenate was mixed with ultrapure water at a mass ratio of 1:5 (w / v) and the pH was adjusted to 7.0. Then, 3% protease (a compound protease, flavor protease: trypsin = 1:2) was added according to the protein content of the shrimp meat. The entire system was placed in a constant temperature water bath at 50℃ and 140 rpm / min for 4 hours for enzymatic hydrolysis. After hydrolysis, the sample was placed in a 95℃ water bath for 10 minutes to inactivate the enzyme. The cooled hydrolysate was placed in a high-speed refrigerated centrifuge and centrifuged at 8000 rpm for 20 minutes. The supernatant was collected to obtain the shrimp head hydrolysate.

[0037] (II) Ultrafiltration separation of umami components from enzymatic hydrolysate: (2.1) Ultrafiltration (UF): The shrimp head enzymatic hydrolysate obtained in step (I) was first passed through a 10kDa ultrafiltration membrane and centrifuged for 20 min at 4℃ and 8000 r / min. Then, the filtrate was passed through 5kDa, 3kDa, and 1kDa ultrafiltration membranes in sequence to obtain components U1-U4, corresponding to molecular weights >5kDa, 5~3kDa, 3~1kDa, and <1kDa, respectively. All components obtained by ultrafiltration were collected, freeze-dried into powder, and used for electronic tongue analysis. The component with the strongest umami flavor was selected for the next step of separation and purification.

[0038] (2.2) Electronic tongue analysis: In order to screen out the main umami substances in the shrimp head enzymatic hydrolysate, electronic tongue analysis was performed on the four components (U1-U4) separated by ultrafiltration of the enzymatic hydrolysate. The evaluation results are as follows: Figure 2 As shown. From Figure 2 It can be seen that all the separated components can be perceived as umami, but U3 and U4 not only have a higher umami intensity than U1-U2, but also have lower bitterness and astringency. This suggests that U3 and U4 may contain a large amount of umami peptides among these four separated components.

[0039] (III) Gel chromatography separation of ultrafiltration components: (3.1) Gel chromatography purification: A Superdex 30 pre-packed column (separation range 100-7000 Da) packed with agarose and dextran complex was used to separate and purify the fractions <3 kDa, namely ultrafiltration fractions U3 and U4. First, the ultrafiltration fractions with a molecular weight less than 3 kDa were reconstituted with ultrapure water to a concentration of 10 mg / mL and filtered through a 0.45 μm filter. The mobile phase for liquid chromatography was 2×PBS (0.02 M phosphate buffer, 0.28 M sodium chloride, 0.006 M potassium chloride, pH 7.4), with a loading volume of 100 µL, a column temperature of 30 °C, a flow rate of 0.5 mL / min, and a wavelength of 214 nm. The desired result was as follows: Figure 3 The spectrum shown is divided into three parts, named F1, F2, and F3. Each component was collected multiple times and subjected to concentration, dialysis desalting, and freeze-drying to obtain freeze-dried powder, which was then collected and stored. Electronic tongue analysis was performed to determine the component with the strongest umami flavor.

[0040] (3.2) Electronic tongue analysis: The results of electronic tongue analysis for F1-F3 are as follows Figure 4 As shown in the results, umami and sweetness are the main characteristic flavors of the F1-F3 fractions. F2 and F3 have similar sensory properties, with F2 exhibiting a higher umami intensity than F1. Therefore, fractions F2 and F3 were selected for further mass spectrometry identification.

[0041] (iv) Identification of peptide amino acid sequences: Dissolve the peptide sample from step (III) in washing buffer (0.1% FA, 2% ACN), desalt it first using a C18 desalting column, and then elute it with elution buffer (0.1% FA, 60% ACN). Centrifuge, concentrate, and dry the eluted sample for mass spectrometry analysis.

[0042] Chromatographic conditions: Separation was performed using a Nano-HPLC system, UltiMate 3000 RSLC nano (Thermo Fisher Scientific, USA). Solution A was 0.1% formic acid-water solution, and solution B was 0.1% formic acid-acetonitrile solution. The chromatographic column was a Trapcolumn, 100 μm × 20 mm (RP-C18, Agilent), equilibrated with 100% solution A at a flow rate of 3 μL / min. The sample was loaded onto the Trapcolumn column via an autosampler and then separated using a PepMap C18 column (2 μm, 75 μm × 250 mm) at a flow rate of 300 nL / min. The gradient program was as follows: 0-20 min, phase B linearly increased from 5% to 38%; 20-22 min, phase B rapidly increased to 95%; 22-30 min, column washing was performed while maintaining 95% phase B.

[0043] Mass spectrometry conditions: After chromatographic separation, the peptides were electrospray ionized and detected using a QExactivePlus mass spectrometer (ThermoFisher Scientific). The mass spectrometer was operated in Data-Dependent Acquisition (DDA) mode. The full scan parameters were: resolution 70,000 (at m / z 200), scan range 300-1500 m / z, automatic gain control target (AGC target) 3e6, and maximum ion implantation time (Max IT) 100 ms. After each full scan, a secondary fragmentation scan (MS / MS) was performed on the top 20 most intense precursor ions with the following parameters: resolution 17,500, AGC target 1e5, and Max IT 50 ms. Fragmentation was performed using high-energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 28%. The dynamic exclusion time was set to 25 s. The raw mass spectrometry data were processed using PEAKS Studio 8.5 software (Bioinformatics Solutions Inc., Waterloo, Canada).

[0044] (v) Screening of potential umami peptides: (5.1) Umami model prediction: Machine learning algorithms were applied to evaluate additional peptide properties, improving the accuracy of umami peptide selection. A total of 820 peptide sequences from step (IV) with ALC > 95% and those matching the database were analyzed for umami activity using three umami prediction models: UMPED-FRL (https: / / pmlabstack.pythonanywhere.com / UMPred-FRL), Umami-MRNN (https: / / umami-mrnn.herokuapp.com / ), and Umami-YYDS (http: / / tastepeptides-meta.com / ca). This analysis yielded 318 potential umami peptides.

[0045] (5.2) Prediction of peptide physicochemical properties: The toxicity, stability, water solubility, and allergenicity of peptides were predicted using online tools ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / ), Expasy-protparam (https: / / web.expasy.org / protparam / ), Innovagen (http: / / www.innovagen.com / proteomics-tools), and AllerTOP (https: / / www.ddg-pharmfac.net / AllerTOP / ). Further screening of potential umami peptides yielded 61 potential umami peptides.

[0046] (vi) Molecular docking of umami peptides: (6.1) Homology modeling of human umami receptors: Homology models were constructed using the SWISS-MODEL server (https: / / swissmodel.expasy.org / ). The amino acid sequences of T1R1 (ID: Q7RTX1) and T1R3 (ID: Q7RTX0) were obtained from UniProtKB, with the metabolic glutamate receptor (PDBID: 1EWK) as the template. To confirm the reliability of the homology models, the obtained models will be evaluated using PROCHECK. The structures of the T1R1 / T1R3 umami receptors are shown below. Figure 5 As shown, the Laplace plot of the T1R1 / T1R3 umami receptor model assessment is as follows: Figure 6 As shown, the optimal allowed region in the T1R1 / T1R3 umami receptor model is 86.1%, and the additional allowed region is 9.6%. More than 90% of the amino acid residues are distributed in the optimal and additional allowed regions, indicating that the model is reasonable.

[0047] (6.2) Molecular docking: The T1R1 / T1R3 receptor model was dehydrated and hydrogen-added using PyMOL, and the 3D structure of the peptides was generated by ChemDraw. The receptors T1R1 / T1R3 and potential umami peptides were semi-flexibly docked using AutoDockVina. From the docking results of 61 peptides, the 10 peptides with the lowest docking binding energies were selected, and the results are shown in Table 1.

[0048] Table 1. Docking energies between peptides and T1R1 / T1R3

[0049] (vii) Synthesis of umami peptides The results of step (VI) were compared with peptides reported in the BIOPEP-UWM active peptide library. None of these 10 peptides had been reported before; they were sequences appearing for the first time. These 10 peptides were then synthesized in solid phase by Nanjing Jietai Biotechnology Co., Ltd., with a purity greater than 98%.

[0050] (viii) Flavor characteristics of synthetic peptides (8.1) Sensory evaluation of synthetic peptides: The synthetic peptides were dissolved in ultrapure water at a concentration of 1 mg / mL. The sensory panel repeatedly evaluated representative sample solutions corresponding to ten different tastes. Each sample was placed in the mouth for 2 minutes to comprehensively assess taste characteristics. In the sensory evaluation, the following solutions were used to represent specific tastes: monosodium glutamate solution (3 mg / mL) for umami, sucrose solution (5 mg / mL) for sweetness, quinine solution (0.03 mg / mL) for bitterness, citric acid solution (0.4 mg / mL) for acidity, and NaCl solution (2 mg / mL) for saltiness. Sensory evaluations were scored from 0 to 10, with 0 indicating no taste and 10 indicating a strong taste. The reference solution was scored out of 5.

[0051] (8.2) Taste dilution analysis of synthetic peptides: A triangulation test was used to assess the taste threshold. The peptide was initially diluted to a concentration of 1 mg / mL in ultrapure water, followed by a series of gradient dilutions by mixing equal volumes of ultrapure water with the peptide solution. The recognition threshold was set as the maximum dilution at which sensory evaluation members could consistently distinguish the synthetic peptide. Team members simultaneously obtained three solutions (one sample solution and two aqueous solutions) and tested them sequentially by sensory tasting, from low to high concentration, until team members could no longer distinguish between the sample and ultrapure water. This concentration, averaged with the previous concentration, was used as the taste threshold. The experiment was repeated three times for each sample group.

[0052] Table 2. Taste descriptions and taste thresholds of synthetic peptides

[0053] Sensory analysis results showed that the synthetic peptides all possessed complex flavor properties, primarily exhibiting umami, saltiness, and sourness. Among them, peptides FSGVPDR, PDPTF, LTDW, WDEGL, and YTVFDR showed a distinct umami flavor, while the remaining peptides did not. Electronic tongue detection results showed (e.g.) Figure 7Except for VGSSNFR, the other nine peptides were all identified as having umami signals, and generally exhibited a certain intensity of sweetness and bitterness. VGSSNFR also had a certain astringency. This may be because the concentration of the sensory evaluation samples was low, below the umami threshold that humans can perceive, and therefore could not be perceived by sensory members; it does not affect their research value and development potential. Next, we will only verify the five peptides with excellent activity: FSGVPDR, PDPTF, LTDW, WDEGL, and YTVFDR. Finally, the taste activity test (TDA) was used to evaluate the taste recognition threshold of the umami peptides. As shown in Table 2, the taste recognition thresholds of FSGVPDR, PDPTF, LTDW, WDEGL, and YTVFDR were 0.40±0.11 mmol / L, 0.22±0.08, 0.47±0.17, 0.20±0.07, and 0.21±0.11, respectively, all lower than the umami threshold of monosodium glutamate (1.48±0.52).

[0054] (8.3) Synergistic flavor-enhancing effect of synthetic peptides: To further evaluate the umami-enhancing potential of the synthesized peptides, ten peptides were added to 3 mg / mL monosodium glutamate (MSG) solutions for sensory evaluation. The umami peptides were dissolved in 3 mg / mL MSG solutions for QDA testing, with a final peptide concentration of 0.1 mg / mL. 3.00 and 6.00 mg / mL MSG solutions were designated as umami benchmarks, corresponding to scores of 5 and 10, respectively. Evaluators quantified the umami enhancement effect based on perceived umami intensity after tasting the samples. Results are as follows: Figure 8 As shown, six peptides, WDEGL, FSGVPDR, PDPTF, YTVFDR, LTDW, and ENAANNYAR, can significantly enhance the umami intensity of MSG solutions. p <0.05), while other peptides had no significant effect on enhancing umami flavor. p >0.05). Among them, WDEGL showed the most significant enhancement effect, increasing the umami intensity of the MSG solution by 1.39 times. This enhancement effect stems from the synergistic action of the peptide and MSG on umami receptors, thereby amplifying the umami signal. It is worth noting that although ENAANNYAR did not show significant umami intensity in previous sensory evaluations and electronic tongue analyses, it still demonstrated an umami enhancement effect.

[0055] To deeply explore the umami enhancement mechanism, we selected five peptides, namely WDEGL, FSGVPDR, PDPTF, YTVFDR, and LTDW, which have both umami and umami enhancement effects, for further research. The recognition probability of the peptide-MSG mixed system was measured using the three-point forced-choice method, and its S-shaped curve was fitted. The detection threshold and the theoretical threshold were determined at the point where the correct recognition probability (P) = 0.5 on the experimental and theoretical S-curves, respectively. The R value was obtained by comparing the ratio of the two, which can objectively evaluate the interaction type between the peptide and MSG. It is reported that when R > 1, the two taste compounds show a masking effect, R = 1 indicates no interaction, 0.5 < R < 1 is an additive effect, and R ≤ 0.5 is a synergistic effect. The results showed (as Figure 9 shown), the measured thresholds of all five peptides were lower than the theoretical thresholds. Specifically, the R values of FSGVPDR and YTVFDR were 0.82 and 0.66, respectively, indicating an additive effect between these two peptides and MSG. The R values of the three peptides LTDW, PDPTF, and WDEGL were 0.48, 0.34, and 0.39, respectively, all showing a synergistic effect. This indicates that these peptides can effectively enhance umami through different mechanisms, and the peptides with synergistic effects (especially WDEGL) have a more significant enhancement potential. The above results further confirmed the potential of these synthetic peptides to replace or partially replace monosodium glutamate, which helps to reduce sodium ion intake while maintaining the umami intensity.

[0056] (IX) Analysis of the taste characteristics of umami peptides: The five peptides, namely WDEGL, FSGVPDR, PDPTF, YTVFDR, and LTDW, which have both umami and umami enhancement effects in step (VIII), were selected for further research. The interactions and binding sites between the ligand and the receptor were analyzed using PyMOL and Maestro. The visualization analysis results of the docking results are as Figure 10 shown, and the figure shows the interaction forces between T1R1 / T1R3 and the polypeptide. As Figure 10 can be seen, the peptide ligands can all be well embedded inside the T1R1 / T1R3 molecule, and the interaction forces between the receptor and the polypeptide are mainly non-covalent, such as hydrogen bonds, hydrophobic interactions, and salt bridges. Figure 10 Statistics of the key binding residues found that Asp88, Ala 90, Asn130, Asp198, and Gln230 are the main binding sites of the peptide and the umami receptor. The distances between the binding sites of the five umami peptides and the receptor are all between 1.8 - 2.8 Å, which conforms to the characteristics of strong interactions and helps to stabilize the conformation of the complex.

[0057] This invention identified five umami peptides from shrimp head enzymatic hydrolysate using separation, purification, and virtual screening techniques, and verified their sensory characteristics. Molecular docking and sensory evaluation results showed that these five monomeric peptides possess distinct umami characteristics. The umami recognition thresholds of all five peptides were lower than those of monosodium glutamate (MSG). All five umami peptides could be well embedded within the T1R1 / T1R3 molecule. Asp88, Ala90, Asn130, Asp198, and Gln230 were the main binding sites between the peptides and umami receptors. The interactions between the receptors and peptides were primarily non-covalent, including hydrogen bonds, van der Waals forces, and anionic π bonds. This research not only provides a reference for studying shrimp-derived umami peptides, helping to understand their umami formation mechanism and potential development as flavor enhancers and salt-reducing food additives, but also improves the high-value utilization of by-products.

[0058] (x) Molecular dynamics (MD) simulations: Molecular dynamics simulations were performed using Gromacs 2023.3 software. Prior to the simulations, the system underwent energy optimization. A 100 ps NVT (isothermal and isochoric) ensemble simulation was conducted at a fixed volume and constant heating rate, allowing the system temperature to slowly increase from 0 K to 310.15 K, further homogenizing the solvent molecules within the solvent box. Subsequently, a 100 ps NPT (isothermal and isobaric) ensemble simulation was performed using a Berendsen constant pressure apparatus to achieve pressure equilibrium between the solvent and the complex system, raising the system pressure to 1 bar. During the molecular dynamics (MD) simulations, the LINCS algorithm was used to constrain the motion of all hydrogen-bonded atoms, with an integration step size of 2 fs. Electrostatic interactions were calculated using the Particle-mesh Ewald (PME) method, with a cutoff value set at 1.2 nm. The cutoff value for non-bonded interactions was set at 10 Å, updated every 10 steps. The simulated trajectories were periodically eliminated, and subsequent analyses of RMSD, RMSF, Rg, and the number of hydrogen bonds were performed.

[0059] To elucidate the binding stability and mechanism of peptides and proteins at the atomic level, we performed 100 ns molecular dynamics simulations on the complexes of five screened peptides (FSGVPDR, LTDW, PDPTF, WDEGL, and YTVFDR) with receptor proteins. Figure 11As shown in Figure A, the structural stability of the complexes was evaluated using the root mean square deviation (RMSD). All systems reached equilibrium in the later stages of the simulation. The FSGVPDR and YTVFDR complexes rapidly reached a stable plateau within approximately 10 ns (RMSD ~0.65-0.70 nm), indicating high initial fit of their binding conformation. The LTDW and WDEGL complexes, however, experienced a longer conformational relaxation period (20-40 ns), reflecting more thorough conformational sampling and optimization of the peptides within the binding pocket. Notably, the RMSD of the PDPTF complex rose sharply to approximately 0.8 nm within the initial 15 ns, after which the rise gradually slowed, attributed to conformational rearrangement and shifting of the peptides from the predicted initial docking site. Ultimately, all systems exhibited stable, low-fluctuation characteristics at the end of the simulation, confirming the formation of stable complexes.

[0060] RMSF is used to characterize the degree of fluctuation of each amino acid residue in a protein during the simulation process, reflecting the flexibility of the local structure. Figure 11 B). The RMSF curves of the five complex systems generally showed a similar trend: the N-terminal and C-terminal residues of the protein exhibited higher volatility due to the terminal effect, while the RMSF values ​​of the core domain and binding pocket-related regions were generally low (mostly below 0.5 nm). This indicates that peptide binding did not cause drastic perturbation of the overall protein structure, and the core framework remained rigid. In some systems (such as PDPTF), local residue segments that directly interacted with the peptide or formed flexible binding loops (such as random coils) showed slightly higher RMSF peaks. This is consistent with the adaptive conformational adjustment of such regions under peptide-induced conditions, further confirming the specificity of peptide binding and its interaction with local flexible regions.

[0061] The radius of gyration (Rg) characterizes the compactness and folding state of the overall protein structure. For example... Figure 11 As shown in Figure C, the Rg values ​​of all five complex systems remained relatively stable throughout the simulation, with the final equilibrium values ​​fluctuating within a narrow range of 3.10 nm to 3.20 nm. The Rg curves of the FSGVPDR and LTDW systems showed a slow decrease in the early to mid-stages of the simulation before stabilizing, suggesting that peptide binding may have caused slight compaction of the protein structure, resulting in a more compact conformation. The Rg values ​​of PDPTF and WDEGL were the highest among the five peptides (around 3.20 nm), which is related to local structural adjustments caused by protein chain folding, but without significant loosening, ensuring the structural rationality of the complexes.

[0062] Hydrogen bonds are crucial polar interactions between peptides and proteins, significantly contributing to binding specificity and stability. We monitored the number of interfacial hydrogen bonds throughout the simulation. All five systems exhibited a persistent hydrogen bond network; there were almost no moments during the simulation when the number of hydrogen bonds was zero. Figure 11D). The FSGVPDR system forms a relatively large and stable number of hydrogen bonds (maintained at 3-10). The rich hydrogen bond network, in conjunction with electrostatic interactions, significantly enhances the stability of the binding interface. In contrast, the LTDW, WDEGL, and PDPTF systems have slightly fewer hydrogen bonds and exhibit greater fluctuations (mostly dynamically forming and breaking within the range of 2-6), which is related to the characteristics of their binding interface and the dynamic adjustment of the peptide conformation. The YTVFDR system also maintains a dynamically stable number of hydrogen bonds (3-7). In the later stages of the simulation (typically the last 20-40 ns), the number of hydrogen bonds in all systems tends to stabilize, with reduced fluctuations. This is consistent with the convergence behavior of RMSD and Rg, indicating that in the optimized stable conformation, the peptide forms a persistent and dynamically balanced hydrogen bond interaction with protein residues, consolidating the stability of the complex.

[0063] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0064] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that can be understood by those skilled in the art. It should be noted that any technical features not described in detail in this invention can be implemented using any existing technology.

Claims

1. A umami peptide from the head of Litopenaeus vannamei, characterized in that, The amino acid sequence of the umami peptide is selected from any one of the sequences shown in SEQ ID NO:1-5; the umami threshold of the umami peptide is 0.2-0.5 mmol / L.

2. The shrimp head umami peptide according to claim 1, characterized in that, When the umami peptides are used in combination with monosodium glutamate (MSG), they have a synergistic umami-enhancing effect, which improves the umami intensity of the MSG solution.

3. The shrimp head umami peptide according to claim 2, characterized in that, The umami peptide has a final concentration of 0.05-0.2 mg / mL, and the final concentration of L-glutamate is 2-5 mg / mL. The shrimp head umami peptide can increase the umami intensity of L-glutamate in the system by 1.2-1.5 times.

4. The shrimp head umami peptide according to claim 1, characterized in that, Throughout the 100ns molecular dynamics simulation, the number of hydrogen bonds formed between the umami peptide and the umami receptors T1R1 / T1R3 remained dynamically stable between 2 and 10. In the later 20-40ns of the simulation, the number of hydrogen bonds tended to stabilize without drastic fluctuations. All systems could reach conformational equilibrium in the later stages of the simulation, and the RMSD curve of the complex backbone after equilibrium showed low fluctuation characteristics.

5. The shrimp head umami peptide according to claim 1, characterized in that, The umami peptides achieve stable umami enhancement by specifically binding to the Asp88, Ala90, and Asn130 key sites of the umami receptors T1R1 / T1R3.

6. A method for preparing the shrimp head umami peptide according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Raw material pretreatment and targeted enzymatic hydrolysis: Using shrimp heads, a by-product of Litopenaeus vannamei processing, as raw material, the mixture was homogenized, water was added and mixed, the pH was adjusted to 7.0, a complex protease was added for enzymatic hydrolysis, the enzyme was inactivated, the supernatant was collected by centrifugation, and the crude polypeptide extract was obtained by freeze drying. S2. Targeted enrichment: The crude peptide extract is sequentially passed through 5kDa, 3kDa, and 1kDa ultrafiltration membranes for fractionation, and the ultrafiltration fraction with a molecular weight <1kDa is collected. S3. Gel purification: The ultrafiltration fraction was separated by Superdex30 gel filtration chromatography, and the elution peak fraction with the strongest umami activity was collected by electronic tongue evaluation. S4. Forward Precision Screening: The peptide sequences of the elution peak components are resolved by LC-MS / MS. The obtained peptides are then subjected to initial screening by three umami prediction models: Umami-MRNN, UMPed-FRL, and Umami-YYDS. They are then subjected to secondary screening based on safety and processing adaptability physicochemical properties to obtain potential umami peptides. S5. Verification and preparation: Molecular docking verification was performed on the potential umami peptides and umami receptors T1R1 / T1R3. Peptides with binding energies that met the requirements were selected for solid-phase synthesis and activity verification to obtain the shrimp head umami peptides.

7. The preparation method according to claim 6, characterized in that, In step S1, the ratio of shrimp head to water after homogenization is 1:

5. The complex protease is a mixture of flavor protease and trypsin in a mass ratio of 1:2, and the amount added is 3% of the substrate protein mass. The enzymatic hydrolysis conditions are 50℃ water bath for 4 hours. After the enzymatic hydrolysis is completed, the enzyme is inactivated in a 95℃ water bath for 10 minutes, and the supernatant is collected by centrifugation at 4℃ and 8000r / min for 10-15 minutes. In step S2, the operating conditions for ultrafiltration separation are 4℃, 8000r / min, and a centrifugation time of 20min each time; In step S3, the gel filtration chromatography uses 2×PBS as the elution buffer, a flow rate of 0.5 mL / min, a detection wavelength of 214 nm, a sample concentration of 10 mg / mL, a sample volume of 100 µL, and a column temperature of 30 °C.

8. The preparation method according to claim 6, characterized in that, In step S4, the LC-MS / MS adopts a data-dependent acquisition mode, with a first-level full scan resolution of 70,000 and a scan range of 300-1500 m / z. The second-level fragmentation adopts a high-energy collision dissociation method with a normalized collision energy of 28%. The physicochemical property screening includes toxicity, water solubility, stability, and allergenicity detection. In step S5, the molecular docking constructs a homology model of umami receptor T1R1 / T1R3 using the metabolite glutamate receptor mGluR1 as a template. The AutoDockVina semi-flexible docking method is used to select peptides with binding energy ≤ -8.5 kcal / mol for solid-phase synthesis. After synthesis, the activity is verified by sensory evaluation, umami threshold determination and synergistic umami enhancement.

9. The use of the shrimp head umami peptide according to any one of claims 1-5 in food flavor enhancement, characterized in that, The shrimp head umami peptide enhances flavor by specifically binding to the Asp88, Ala90, and Asn130 key sites of the umami receptor T1R1 / T1R3. The food includes low-salt food, high-temperature sterilized food, condiments, meat products, frozen food, or baked goods.

10. The use according to claim 9, characterized in that, The shrimp head umami peptide is added to food in combination with L-glutamate to enhance the umami flavor of the food and reduce the amount of L-glutamate and sodium chloride added. The amount of shrimp head umami peptide added to the food is 0.1%-1% (w / w), and the food is high-temperature sterilized canned food, low-salt seasoning, meat products or quick-frozen noodle and rice products.

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