Umami peptide derived from litopenaeus vannamei and application thereof in preparing umami agent

Abstract

The application discloses umami peptides derived from Litopenaeus vannamei and application of the umami peptides in preparation of umami agents, and belongs to the technical field of bioactive peptides. The umami peptides are five in number, and the amino acid sequences are respectively TGPDPDPTE, LPNFR, SWDVRN, GPDPDPTEY and GPDPDPTEF, and are sequentially shown as SEQ ID NO. 1-5. The umami peptides are applied in umami agents as or for preparing umami agents. Gel filtration chromatography and liquid chromatography-mass spectrometry are used in combination with sensory evaluation to separate and identify potential novel umami peptides from shrimp meat enzymatic hydrolysate. Subsequently, a molecular docking method is used to study the binding mechanism of the umami peptides and T1R1 / T1R3 taste receptors. The application provides a theoretical basis for studying the action mechanism of novel umami peptides of shrimp meat, and provides a basis for high-value utilization of shrimp meat products.

Description

Technical Field

[0001] This invention relates to several umami peptides derived from Litopenaeus vannamei and their application in the preparation of umami agents, belonging to the field of bioactive peptide technology. Background Technology

[0002] Umami is widely considered the fifth taste sensation, bringing a pleasurable sensory experience. Research indicates that umami peptides are a major and indispensable source of umami substances. Umami peptides possess excellent processing properties and nutritional value, as well as advantages in naturalness, safety, and palatability. Most importantly, two significant properties of umami peptides are their synergistic effects and their ability to interact with various tastes, making them a hot topic in food science research.

[0003] Traditional umami peptide screening procedures require multiple separation and purification steps, such as ultrafiltration, gel filtration chromatography, and reversed-phase high-performance liquid chromatography, to obtain umami components, followed by amino acid sequence identification. This process is cumbersome, expensive, and may result in the loss of highly active peptides. In contrast, gel filtration chromatography, equipped with simple, easy-to-operate, and reproducible equipment, obtains all umami fractions in one step. Combined with the high sensitivity, selectivity, throughput, and accuracy of LC-MS / MS, it can more comprehensively identify the amino acid sequences of potential umami peptides.

[0004] Umami peptides manifest their umami properties through interaction with umami receptors present in human taste bud cells. The heterodimer T1R1 / T1R3 in taste bud cells is a family of G protein-coupled receptors with a taste recognition region called the "Venus flytrap domain," and is the main receptor for umami peptides. The T1R3 subunit has been shown to play a crucial role in activating umami. However, the crystal structure of T1R1 / T1R3 has not been fully resolved, limiting research into the umami recognition mechanism mediated by umami peptides. Computer modeling provides a feasible method for identifying proteins with unknown structures, and molecular docking techniques can be used to explain the binding patterns between umami peptides and receptors.

[0005] Litopenaeus vannamei is one of the most important commercial shrimp species in China. Its meat is rich in umami flavor and contains various small-molecule peptides. Screening for umami peptides from Litopenaeus vannamei is of great significance for the high-value utilization of shrimp meat products. Summary of the Invention

[0006] In view of the above-mentioned prior art, the present invention provides several umami peptides derived from Litopenaeus vannamei and their application in the preparation of umami agents.

[0007] This invention is achieved through the following technical solution:

[0008] Five umami peptides derived from Litopenaeus vannamei are present, with the following amino acid sequences:

[0009] (1) LPNFR, as shown in SEQ ID NO.1;

[0010] (2) SWDVRN, as shown in SEQ ID NO.2;

[0011] (3) TGPDPDPTE, as shown in SEQ ID NO.3;

[0012] (4) GPDPDPTEY, as shown in SEQ ID NO.4;

[0013] (5) GPDPDPTEF, as shown in SEQ ID NO.5.

[0014] The above-mentioned umami peptides are used as or in the preparation of umami agents.

[0015] This invention utilizes gel filtration chromatography and liquid chromatography-mass spectrometry combined with sensory evaluation to isolate and identify potential novel umami peptides from shrimp enzymatic hydrolysates. Subsequently, the binding mechanism of the umami peptides to T1R1 / T1R3 taste receptors was investigated using molecular docking methods. This research provides a theoretical basis for studying the mechanism of action of novel umami peptides in shrimp and offers a foundation for the high-value utilization of shrimp products.

[0016] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0017] Figure 1 Elution curves of Sephadex G-10 gel filtration chromatography.

[0018] Figure 2 Schematic diagram of the electronic tongue determination results of the five components obtained by gel column chromatography.

[0019] Figure 3 : Schematic diagram of the umami enhancement results of the interaction models between various potential umami peptides and monosodium glutamate, where (a) is GPDPDPTEF; (b) is GPDPDPTEY; (c) is TGPDPDPTE; (d) is SWDVRN; and (e) is LPNFR.

[0020] Figure 4 : Schematic diagram of 2D visualization results of docking of each potential umami peptide with T1R3 molecule, from top to bottom: GPDPDPTEF, GPDPDPTEY, TGPDPDPTE, SWDVRN, LPNFR. Detailed Implementation

[0021] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0022] 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.

[0023] Screening and validation of experimental umami peptides

[0024] 1. Materials and Methods

[0025] 1.1 Materials and Chemicals

[0026] Litopenaeus vannamei was purchased from a local seafood market in Qingdao, Shandong Province, China. Papain and flavor protease were purchased from Pangbo Biotechnology Co., Ltd. (Nanning, China). Peptides were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) with a purity greater than 95% and underwent desalting. HPLC-grade reagents were from Merck Pharmaceuticals, Inc., and food-grade sodium chloride and monosodium glutamate (MSG) were purchased from a local supermarket. All other reagents were analytical grade and commercially available.

[0027] 1.2 Preparation of enzymatic hydrolysate of Litopenaeus vannamei

[0028] Litopenaeus vannamei was shelled and deheaded, and the shrimp meat was mixed with pure water (1:1 by weight) and stirred in a blender to obtain a shrimp meat homogenate. The shrimp meat homogenate was then enzymatically hydrolyzed under pre-optimized experimental conditions obtained through orthogonal optimization: the shrimp meat homogenate was diluted to 30% of the total shrimp meat mass, and then 4000 U / g papain was added according to the shrimp meat mass, followed by hydrolysis for 2 hours; then 1000 U / g flavor protease was added, and hydrolysis continued for another 2 hours; the entire process was carried out at 50℃ and 200 rpm / min. After hydrolysis, the sample was boiled for 10 minutes to inactivate the enzyme. The hydrolysate was cooled, centrifuged (8000 r / min, 10 minutes), and the supernatant was collected for later use.

[0029] 1.3 Peptides were separated and purified by ultrafiltration and gel filtration chromatography.

[0030] The supernatant was fractionated using an ultrafiltration membrane (Sartorius Group, Germany) with a molecular weight cutoff of 5,000 Da. Ultrafiltration was performed at 4°C. The ultrafiltration fraction with a molecular weight less than 5,000 Da was recovered.

[0031] Sephadex G-10 (Solepro Biotechnology Co., Ltd.) was used as the packing material for gel filtration chromatography. The packing material was thoroughly washed and swollen before being added to a 16 mm × 70 cm chromatography column. The sample concentration was 50 mg / mL, the loading volume was 3 mL, and ultrapure water was used as the eluent. The chromatography was performed at 25 °C and a flow rate of 1 mL / min. The sample was detected at a UV wavelength of 220 nm.

[0032] Results: The elution curve is as follows: Figure 1 As shown, five peaks were separated from the fraction with a molecular weight less than 5,000 Da by gel filtration chromatography and named P1, P2, P3, P4 and P5. Each fraction was collected multiple times, mixed and lyophilized, and stored at −20 °C.

[0033] 1.4 Electronic tongue analysis of different fractions

[0034] A 0.3 mg / mL fraction solution was prepared using ultrapure water. The taste characteristics of the fraction were determined using an SA-402B taste analysis system. Each sample was measured four times, with the first measurement automatically discarded.

[0035] 1.5 Identification of peptide sequences

[0036] 1.5.1 Peptide Extraction

[0037] Add an appropriate amount of 0.1% trifluoroacetic acid (TFA) solution to the sample, mix well, centrifuge at 20000 g for 5 min, collect the supernatant, transfer to a 10 kDa ultrafiltration centrifuge tube, and centrifuge at 12000 g for 15 min. Add 200 μL of 0.1% TFA, centrifuge at 12000 g for 15 min, repeat twice, collect the filtrate, desalt using C18 StageTip, and then freeze-dry the desalted solution under vacuum. After drying, reconstitute the peptides with 0.1% formic acid solution, and then... 280 Determine peptide concentration.

[0038] 1.5.2 Liquid Chromatography-Mass Spectrometry Analysis

[0039] Peptides were separated using an Easy nLC 1200 nano-flow rate chromatography system. The column was equilibrated with 100% 0.1% formic acid solution. Samples were injected into a C18 Trap Column (100 μm × 20 mm, 5 μm) and then subjected to gradient separation through a C18 analytical column (75 μm × 150 mm, 3 μm) with a buffer solution of 0.1% formic acid, acetonitrile, and water (acetonitrile comprising 80%) at a flow rate of 300 nL / min. The linear gradient of the liquid chromatography separation was as follows: 0 min–2 min from 2% to 5%; 2 min–44 min from 5% to 28%; 44 min–51 min from 28% to 40%; 51 min–53 min from 40% to 100%; and 53 min–60 min maintained at 100%.

[0040] After peptide separation, DDA mass spectrometry analysis was performed using a Q-Exactive HF-X mass spectrometer. The analysis time was 60 min, detection mode: positive ion, precursor ion scan range: 350–1800 m / z, primary mass spectrometry resolution: 60,000 m / z 200, AGC target: 3e6, primary maximum IT: 50 ms. Secondary mass spectrometry analysis of the peptides was performed using the following method: after each full scan, the secondary mass spectra of the 20 highest intensity precursor ions were acquired. Secondary mass spectrometry resolution: 15,000 m / z 200, AGC target: 1e5, secondary maximum IT: 50 ms, MS2 Activation Type: HCD, Isolation window: 1.6 m / z, Normalized collision energy: 28.

[0041] 1.5.3 Database Retrieval

[0042] MaxQuant 2.4.14.0 software was used to search the Uniport protein database. The analysis parameters of MaxQuant search software are shown in Table 1.

[0043]

[0044] 1.6 Screening and Synthesis of Peptides

[0045] After identifying peptides with umami flavor through joint screening using two umami prediction models, TastePeptidesDM (http: / / tastepeptides-meta.com / TPDM) and UMPRD-FRL (https: / / pmlabstack.pythonanywhere.com / UMPred-FRL), the Umami candidate peptides were further shortlisted using the bioactivity prediction tool PeptideRanker (https: / / distilldeep.ucd.ie / PeptideRanker / ), obtaining peptides with scores higher than 0.5. Finally, toxicity screening was performed using ToxinPretool (https: / / crdd.osdd.net / raghava / toxinpred / index.html). The screening results were compared with peptides reported in the BIOPEUWM bioactive peptide library. Peptides meeting the above requirements were synthesized using the Fmoc solid-phase method at Shanghai Sangon Biotech Co., Ltd., China.

[0046] 1.7 Sensory Evaluation

[0047] The sensory panel consisted of eight men and eight women who were in good health, maintained regular sleep patterns, had no unhealthy habits, and could accurately distinguish the five basic tastes. Sensory evaluations were conducted at a temperature of 25±2℃ and a humidity of 50±5%. Panel members were asked to take a small sip of the peptide solution, swirl it in their mouths for 10 seconds, and then spit it out. Panel members were instructed to clean their mouths with 50 ml of drinking water at least twice between evaluations of two different samples to avoid the influence of residual sample and taste fatigue.

[0048] 1.7.1 Determination of Flavor Threshold

[0049] The recognition threshold of synthetic peptides was examined using a three-alternative forced selection (3-AFC) test according to ISO 4120:2004. Initial peptide solutions were progressively diluted (1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL) until the panel members could no longer distinguish the sample from ultrapure water. The average of this concentration and the previous concentration was calculated as an individual threshold, and the taste threshold of the peptide was determined by calculating the average of these individual thresholds.

[0050] 1.7.2 Analysis of interactions in the synthetic peptide-salt binary solution system using S-curves

[0051] The synthetic peptide was mixed with monosodium glutamate (MSG) at a mass ratio of 1:3, and a 4 g / L solution was prepared using purified water. The solution was then serially diluted with purified water (volume ratio 1:1), and sensory evaluation was performed using a high-to-low concentration triangle test. Samples that the panel members could not correctly distinguish and the final concentrations of purified water were recorded. The probability of detection (P) was corrected using a chance factor, as shown below:

[0052] P = (3p - 1) / 2;

[0053] Where P is the correction value for the correct detection probability, and p is the actual measured correct detection probability value.

[0054] Assuming the logarithmic concentration of the mixture and the probability of correct detection are fitted to the S-curve, the fit is as follows:

[0055] y=a / (1+be -kx );

[0056] Where x is the logarithmic concentration of the mixture, and y is the probability of correctly detecting the probability value.

[0057] The theoretical probability of correctly detecting a mixture is calculated using the following formula:

[0058] p(AB) = p(A) + p(B) - p(A)p(B);

[0059] Where p(AB) is the theoretical probability of correctly detecting the mixture, and p(A) and p(B) are the probabilities of detecting salt and synthetic peptide.

[0060] The final correct detection probability obtained from the mixed experiment is compared with the theoretical correct detection probability p(AB). The corresponding concentration at P = 0.5 is defined as the detection threshold of the solution.

[0061] 1.8 Molecular docking of T1R3 with umami peptides

[0062] The three-dimensional structures of human umami receptors T1R1 and T1R3 were generated using computer modeling. The amino acid sequences of T1R1 (UniProt ID: Q7RTX1) and T1R3 (UniProt ID: Q7RTX0) were obtained from the UniProt database (https: / / www.uniprot.org / ). The T1R1 / T1R3 heterodimeric complex was predicted using multimers in Alphafold 2.3.0. The active pocket center of T1R1 / T1R3 was predicted using DeepSite. The processed receptors T1R1 / T1R3 were docked using the Autodock Vina algorithm in PyRx to simulate interactions. The ligand and receptor were docked eight times, and the lowest docking binding energy was used. Discovery Studio 4.5 was used to analyze the interaction forces and binding sites between the peptide ligand and receptor.

[0063] 1.9 Statistical Analysis

[0064] Data are expressed as mean ± standard deviation. One-way ANOVA and Duncan's multiple comparison analysis were performed using SPSS 26 and Origin 2021. P < 0.05 was considered statistically significant.

[0065] 2. Results and Discussion

[0066] 2.1 Umami Identification of Separated Fractions from Litopenaeus vannamei Enzymatic Hydrolysate

[0067] Gel filtration chromatography separates and purifies raw materials based on their relative molecular mass. Components with smaller relative molecular masses can directly enter the gel particles and continuously flow with the eluent within the gel particles and between gel gaps. They travel a longer path during elution and elute later. The electronic tongue determination results for the five components obtained by gel column chromatography are shown below. Figure 2 As shown, P0 is the ultrafiltration sample before gel column chromatography separation. Figure 2 It was found that P4 and P5 in the elution fraction exhibited more pronounced umami intensity, possibly due to the significant contribution of small molecules such as umami peptides, free amino acids, and nucleotides to the umami flavor of the enzymatic hydrolysate. Liquid chromatography-mass spectrometry (LC-MS) was performed on the fraction with the strongest umami flavor to identify its peptide sequences.

[0068] 2.2 Identification of umami peptides from P4 fraction using peptidomics technology

[0069] Peptidomics, as an important branch and extension of proteomics, has been effectively applied to the determination of peptide components in food. Mass spectrometry is currently widely used in peptidomics analysis due to its significant advantages, including high resolution and high-throughput analysis capabilities. A database scan was performed using MaxQuant 2.4.14.0 to analyze the mass spectrometry data contained in the raw files. After analysis, this invention successfully obtained 107 peptide sequences. Through virtual screening of target peptides (Section 1.6), five potential umami peptides were obtained. The details of these five potential umami peptides are shown in Table 2, and their amino acid sequences are shown in SEQ ID NO. 1–5, respectively. These five potential umami peptides were then synthesized.

[0070]

[0071] 2.3 Flavor Properties of Synthetic Peptides

[0072] The taste characteristics of umami peptides were determined through sensory evaluation. The sensory evaluation results of the synthetic peptides are shown in Table 3. The taste descriptions were ordered according to the degree of flavor. The results showed that all five synthetic peptides exhibited umami, and three synthetic peptides exhibited a certain degree of saltiness. The presence of acidic taste may be due to acetic acid and free amino acid residues during the synthesis process. Simultaneously, the sensory evaluation team analyzed the taste thresholds of the peptides, with peptide TGPDPDPTE showing the lowest taste threshold (0.129 ± 0.057 mg / mL).

[0073]

[0074] The interaction between umami peptides and salt solutions was assessed based on the ratio (R) between experimental and theoretical taste thresholds, including masking effect (R > 1), no interaction (R = 1), additive effect (0.5 < R < 1), and synergistic effect (R ≤ 0.5). In the binary mixture, the actual taste threshold of SWDVRN-MSG (186.21 mg / L) was reduced by more than half compared to the theoretical threshold (416.87 mg / L), with an R value of only 0.45. This indicates a synergistic effect of umami perception between the peptide SWDVRN and the 0.3% MSG solution. The actual taste thresholds of the other four peptides were also reduced, with R values ​​ranging from 0.5 to 1, suggesting an additive effect between these four umami peptides and MSG on umami perception.

[0075] 2.4 Molecular docking of T1R3 with umami peptides

[0076] The interaction between potential umami peptides and T1R3 was studied and analyzed using molecular docking. The results are as follows: Figure 4 As shown. The results indicate that each potential umami peptide can bind well to the active cavity of T1R3. Figure 4Hydrogen bonds are the most important binding force between umami peptides and T1R3, with each peptide forming 6–13 hydrogen bonds. Other beneficial forces include electrostatic and hydrophobic forces. Notably, Asn380, Arg247, Gly168, Asn68, and Glu45 each form two hydrogen bonds with umami peptides, suggesting that these amino acid residues may play an important role in umami perception. These results can provide a basis for research on the binding sites of other umami peptides and umami receptors.

[0077] 3. Conclusion

[0078] Five umami peptides from Litopenaeus vannamei meat were identified using enzymatic hydrolysis and peptidomics techniques, and their sensory properties were verified. Sensory evaluation results showed that peptide LPNFR has a strong umami flavor, with a taste threshold of 0.152 ± 0.096 mg / mL. Molecular docking indicated that conventional hydrogen forces are the main interaction force, and the umami peptide can bind well to the active cavity of the umami receptor T1R3. This research enriches the marine umami peptide library, enhances the research value of Litopenaeus vannamei, and provides a reference for studying the umami presentation mechanism of umami peptides with different structures.

[0079] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A flavor peptide derived from Litopenaeus vannamei, characterized in that: The amino acid sequence is LPNFR, as shown in SEQ ID NO.

1.

2. The use of the umami peptide according to claim 1 as or in the preparation of umami agents.

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