Umami peptide from litopenaeus vannamei and application of umami peptide in preparation of flavor enhancer

Abstract

The invention discloses umami peptides derived from litopenaeus vannamei and application of the umami peptides in preparation of a flavor enhancer, and belongs to the technical field of bioactive peptides. The number of the flavor peptides is five, the amino acid sequences of the flavor peptides are respectively as follows: TGPDPDPTE, LPNFR, SWDVRN, GPDPDPTEY and GPDPDPTEF, and the amino acid sequences of the flavor peptides are as shown in SEQ ID NO.1-5. The umami peptide can be used as or used for preparing a flavor enhancer. According to the method, gel filtration chromatography and liquid chromatography-mass spectrometry are combined with sensory evaluation to separate and identify potential novel umami peptides from shrimp meat enzymatic hydrolysate. Subsequently, the binding mechanism of the umami peptide and the T1R1 / T1R3 taste receptor is studied by using a molecular docking method. The invention provides a theoretical basis for researching the action mechanism of the novel umami peptide of the shrimp meat, and provides a basis for high-value utilization of shrimp meat products.

Description

Technical Field

[0001] The present invention relates to several umami peptides derived from Litopenaeus vannamei and their application in the preparation of umami agents, belonging to the technical field of bioactive peptides. Background Art

[0002] Umami is widely regarded as the fifth taste perception, which can bring a pleasant sensory experience. Research shows that umami peptides are the main and indispensable source of umami substances. Umami peptides have excellent processing performance and nutritional value, as well as advantages in terms of naturalness, safety, and palatability. Most importantly, two remarkable properties of umami peptides are their ability to synergize and interact with various tastes, making them a hot topic in food science research.

[0003] Traditional umami peptide screening procedures require multiple steps of separation and purification processes, such as ultrafiltration, gel filtration chromatography, and reverse-phase high-performance liquid chromatography to obtain umami components, and then amino acid sequence identification. This process is both cumbersome and expensive, and may lead to the loss of highly active peptides. On the contrary, gel filtration chromatography is equipped with simple, easy-to-operate, and reproducible equipment, obtaining all umami fractions in one step, and 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 exhibit umami properties by interacting 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, which is the main receptor for umami peptides. Among them, it has been demonstrated that the T1R3 subunit plays a crucial role in activating umami. However, the crystal structure of T1R1 / T1R3 has not been fully resolved, limiting the study of the umami recognition mechanism mediated by umami peptides. Computer modeling provides a feasible method for identifying proteins with unknown structures, and at the same time, the binding mode between umami peptides and receptors can be explained through molecular docking technology.

[0005] Litopenaeus vannamei is one of the most important commercial shrimp species in China. Its shrimp meat has a strong umami flavor and is rich in various small molecular peptides. Screening 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] The present invention is achieved by the following technical solutions: There are 5 umami peptides derived from Litopenaeus vannamei, and their amino acid sequences are as follows: (1) LPNFR, as shown in SEQ ID NO.1; (2) SWDVRN, as shown in SEQ ID NO.2; (3) TGPDPDPTE, as shown in SEQ ID NO.3; (4) GPDPDPTEY, as shown in SEQ ID NO.4; (5) GPDPDPTEF, as shown in SEQ ID NO.5.

[0008] Use of the above umami peptides as or in the preparation of umami agents.

[0009] The present invention uses gel filtration chromatography and liquid chromatography - mass spectrometry combined with sensory evaluation to isolate and identify potential novel umami peptides from shrimp meat hydrolysate. Subsequently, the binding mechanism of umami peptides with the T1R1 / T1R3 taste receptor was studied by using the molecular docking method. The research of the present invention provides a theoretical basis for studying the action mechanism of novel umami peptides in shrimp meat and provides a basis for the high - value utilization of shrimp meat products.

[0010] Various terms and phrases used in the present invention have the general meanings well - known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Elution curve of Sephadex G - 10 gel filtration chromatography.

[0012] Figure 2 : Schematic diagram of the results of electronic tongue measurement of 5 components obtained by gel column chromatography.

[0013] Figure 3 : Schematic diagram of the umami enhancement results of the interaction models of each potential umami peptide with monosodium glutamate, where (a) is GPDPDPTEF; (b) is GPDPDPTEY; (c) is TGPDPDPTE; (d) is SWDVRN; (e) is LPNFR.

[0014] Figure 4 : Schematic diagram of the 2D visualization results of the molecular docking of each potential umami peptide with T1R3, where from top to bottom are: GPDPDPTEF, GPDPDPTEY, TGPDPDPTE, SWDVRN, LPNFR. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be further described below in conjunction with examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0016] For the instruments, reagents, and materials involved in the following examples, unless otherwise specified, they are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, they are all conventional experimental methods and detection methods existing in the prior art.

[0017] Experiment: Screening and Verification of Umami Peptides 1. Materials and Methods 1.1 Materials and Chemicals Litopenaeus vannamei was purchased from a local aquatic product market in Qingdao, Shandong Province, China. Papain and Flavorzyme were purchased from Pangbo Biotech Co., Ltd. (Nanning, China). Peptide segments were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), with a purity greater than 95%, and desalted. HPLC-grade reagents were from Merck Pharmaceutical Biotechnology Company. Food-grade sodium chloride and monosodium glutamate (MSG) were purchased from a local supermarket. All other reagents were analytical-grade reagents and were available on the market.

[0018] 1.2 Preparation of Enzymatic Hydrolysate of Litopenaeus vannamei The heads and shells of Litopenaeus vannamei were removed, and the shrimp meat was mixed with pure water (weight ratio 1:1) and stirred in a blender to obtain a shrimp meat homogenate. The shrimp meat homogenate was enzymatically hydrolyzed under the optimal experimental conditions obtained by prior orthogonal optimization: the shrimp meat homogenate was diluted to 30% of the total mass by the mass of the shrimp meat, and then papain with an enzyme amount of 4000 U / g was added according to the mass of the shrimp meat, and enzymatically hydrolyzed for 2 hours; then Flavorzyme with an enzyme amount of 1000 U / g was added and the enzymatic hydrolysis continued for 2 hours; the whole system was carried out under the conditions of 50 °C and 200 rpm / min. After enzymatic hydrolysis, the sample was boiled for 10 minutes to inactivate the enzyme. The hydrolysate was cooled and centrifuged (8000 r / min, 10 minutes), and the supernatant was collected for standby.

[0019] 1.3 Separation and Purification of Peptides by Ultrafiltration and Gel Filtration Chromatography The above supernatant was fractionated through an ultrafiltration membrane with a molecular weight cut-off of 5,000 Da (Sartorius Group, Germany). Ultrafiltration was carried out at 4 °C. The ultrafiltration fraction with a molecular weight less than 5,000 Da was recovered.

[0020] Sephadex G-10 (Solarbio Science & Technology Co., Ltd.) was used as the packing material for gel filtration chromatography. The packing material was fully washed and swollen and added to a 16 mm × 70 cm chromatography column. The sample concentration was 50 mg / mL, the sample loading volume was 3 mL, ultrapure water was used as the eluent, at 25 °C, and the flow rate was 1 mL / min. The sample was detected at a UV wavelength of 220 nm.

[0021] Results: The elution curve is as Figure 1As shown, five peaks were separated from the fraction with a molecular weight less than 5,000 Da by gel filtration chromatography, named P1, P2, P3, P4, and P5. Each fraction was collected multiple times, mixed, freeze-dried, and stored at -20°C.

[0022] 1.4 Electronic tongue analysis of different fractions A 0.3 mg / mL fraction solution was prepared with ultrapure water. The taste characteristics of the fractions were measured using an SA-402B taste analysis system. Each sample was measured four times, and the first measurement was automatically discarded.

[0023] 1.5 Identification of peptide sequences 1.5.1 Polypeptide extraction An appropriate amount of 0.1% trifluoroacetic acid solution (TFA) was added to the sample and mixed evenly. The mixture was centrifuged at 20,000 g for 5 min, and the supernatant was taken and transferred to a 10KDa ultrafiltration centrifugal tube. Then it was centrifuged at 12,000 g for 15 min. 200 μL of 0.1% TFA was added, and it was centrifuged at 12,000 g for 15 min. This was repeated twice. The filtrate was collected, desalted using a C18 StageTip, and the desalted solution was subjected to vacuum freeze-drying. After drying, the peptide segments were redissolved in 0.1% formic acid solution, and the peptide segment concentration was measured at OD 280 Determine the peptide segment concentration.

[0024] 1.5.2 Liquid chromatography-mass spectrometry analysis The peptides were chromatographically separated using a nano-flow Easy nLC 1200 chromatography system. The chromatographic column was equilibrated with 100% 0.1% formic acid solution. After the sample was injected into a C18 Trap Column (100 μm × 20 mm, 5 μm), it was subjected to gradient separation through a C18 analytical column (75 μm × 150 mm, 3 μm). The buffer was a mixed solution of 0.1% formic acid, acetonitrile, and water (where acetonitrile was 80%), and the flow rate was 300 nL / min. The liquid separation linear gradient was as follows: from 2% to 5% from 0 min to 2 min; from 5% to 28% from 2 min to 44 min; from 28% to 40% from 44 min to 51 min; from 40% to 100% from 51 min to 53 min; maintained at 100% from 53 min to 60 min.

[0025] After peptide separation, DDA mass spectrometry analysis was performed using a Q-Exactive HF-X mass spectrometer. The analysis duration was 60 min, the detection mode was positive ion, the parent ion scan range was 350 - 1800 m / z, the resolution of the first-level mass spectrometry was 60,000 @ m / z 200, the AGC target was 3e6, and the first-level Maximum IT was 50 ms. The second-level mass spectrometry analysis of peptides was collected according to the following method: After each full scan, the second-level mass spectrometry spectra of 20 parent ions with the highest intensities were triggered for collection. The resolution of the second-level mass spectrometry was 15,000 @ m / z 200, the AGC target was 1e5, the second-level Maximum IT was 50 ms, the MS2 Activation Type was HCD, the Isolation window was 1.6 m / z, and the Normalized collision energy was 28.

[0026] 1.5.3 Database Search The MaxQuant 2.4.14.0 software was used to search the Uniport protein database, and the analysis parameters of the MaxQuant database search software were set as shown in Table 1.

[0027]

[0028] 1.6 Screening and Synthesis of Peptides After obtaining the umami taste of peptides through the joint screening and identification of two umami prediction models, TastePeptidesDM (http: / / tastepeptides-meta.com / TPDM) and UMPred-FRL (https: / / pmlabstack.pythonanywhere.com / UMPred-FRL), the PeptideRanker (https: / / distilldeep.ucd.ie / PeptideRanker / ) prediction bioactivity tool was used to further shortlist Umami candidate peptides, and peptides with a score exceeding 0.5 were obtained. Finally, ToxinPretool (https: / / crdd.osdd.net / raghava / toxinpred / index.html) was used for toxicity screening. The screening results were compared with the peptides reported in the BIOPEUWM bioactive peptide library. Peptides meeting the above requirements were synthesized using the Fmoc solid-phase method of Sangon Biotech (Shanghai) Co., Ltd., China.

[0029] 1.7 Sensory Evaluation The sensory panel consisted of 8 males and 8 females who were in good health, had regular schedules, had no bad habits, and could accurately distinguish the five basic tastes. The sensory evaluation was carried out in an environment with a temperature of 25±2 °C and a humidity of 50±5%. The panelists were required to take a sip of the peptide solution, swirl it in their mouths for 10 seconds, and then spit it out. The panelists were instructed to clean their mouths with 50 mL of drinking water at least twice between evaluating two different samples to avoid the effects of residual samples and taste fatigue.

[0030] 1.7.1 Determination of taste threshold Three-alternative forced-choice test (3-AFC) methods were used to examine the recognition threshold of synthetic peptides according to ISO 4120:2004. The initial peptide solution was gradually diluted (1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL) until the panelists could not distinguish the sample from ultrapure water. The average of this concentration and the previous concentration was calculated and used as the individual threshold, and the taste threshold of the peptide was determined by calculating the average of the individual thresholds.

[0031] 1.7.2 Analysis of interactions in the binary solution system of synthetic peptide salts by S-curve The synthetic peptide was mixed with monosodium glutamate in a mass ratio of 1:3, and a 4 g / L mixed solution was prepared with pure water. Subsequently, the mixed solution was gradient-diluted with pure water (volume ratio 1:1), and sensory evaluation was carried out using a triangle test from high concentration to low concentration. The final concentrations of the samples and pure water that the panelists could not correctly distinguish were recorded. The detection probability (P) was corrected using the chance factor as follows: P=(3p - 1) / 2; where P is the corrected value of the correct detection probability, and p is the actually measured correct detection probability value.

[0032] Assuming that the logarithm concentration of the mixture and the probability of correct detection are fitted to an S-curve, the two are fitted as follows: y = a / (1 + be -kx ) ; where x is the logarithm concentration of the mixture, and y is the probability value of the correct detection probability.

[0033] The theoretical probability of correctly detecting the mixture is calculated as follows: p(AB) = p(A) + p(B) - p(A)p(B); where p(AB) is the theoretical probability of correctly detecting the mixture, and p(A) and p(B) are the probabilities of detecting the salt and synthetic peptide.

[0034] The final correct detection probability obtained from the mixing experiment was compared with the theoretical correct detection probability p(AB). The corresponding concentration when P = 0.5 was defined as the detection threshold of the solution.

[0035] 1.8 Molecular Docking of T1R3 and Umami Peptides The three-dimensional structures of human umami receptors T1R1 and T1R3 were generated using computer modeling methods. 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 heterodimer complex was predicted using the multimer of Alphafold 2.3.0. The active pocket center of T1R1 / T1R3 was predicted using DeepSite. The processed receptor T1R1 / T1R3 was docked using the Autodock Vina algorithm in PyRx to simulate the interaction. The ligand and receptor were co-docked 8 times, and the lowest docking binding energy was taken. The interaction forces and binding sites between the peptide ligand and the receptor were analyzed using Discovery Studio 4.5.

[0036] 1.9 Statistical Analysis 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.

[0037] 2. Results and Discussion 2.1 Umami Identification of the Isolated Fractions of Litopenaeus vannamei Enzymatic Hydrolysate Gel filtration chromatography separates and purifies raw materials according to the molecular weight. Components with smaller molecular weights can directly enter the gel particles and continuously flow with the eluent in the gel particles and between the gel gaps. During the elution process, the travel path is longer and the peak elution is later. The results of the electronic tongue measurement of the 5 components obtained by gel column chromatography are as Figure 2 shown, where P0 is the ultrafiltration sample before gel column chromatography separation. As Figure 2 can be seen, the umami intensities of P4 and P5 in the eluted components are relatively obvious, probably because small molecular substances such as umami peptides, free amino acids, and nucleotides in them contribute greatly to the umami of the enzymatic hydrolysate. Liquid chromatography-mass spectrometry analysis was performed on the component with the strongest umami to identify the peptide sequence.

[0038] 2.2 Identification of Umami Peptides from Component P4 by Proteomics Technology As an important branch and continuation of proteomics, peptidomics technology has been effectively applied to the determination of peptide components in foods. The application of mass spectrometry is currently widely deployed in the field of peptidomics analysis because of its significant advantages, including high-resolution and high-throughput analysis capabilities. The mass spectrometry data contained in the original file was analyzed by performing database scans through MaxQuant 2.4.14.0. After analysis, the present invention successfully obtained 107 peptide sequences. Through virtual screening of the target peptides by 1.6, 5 potential umami peptides were obtained. The situations of the 5 potential umami peptides are shown in Table 2, and the amino acid sequences of the 5 potential umami peptides are shown as SEQ ID NO.1 to 5 respectively. These 5 potential umami peptides were synthesized.

[0039]

[0040] 2.3 Taste characteristics of synthetic peptides 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 sorted according to the primary and secondary tastes. The results showed that all 5 synthetic peptides exhibited umami taste, and 3 synthetic peptides exhibited a certain degree of salty taste. The presence of acidic taste might be due to acetic acid and free amino acid residues during the synthesis process. At the same time, the members of the sensory evaluation panel analyzed the taste thresholds of the peptides, and the peptide TGPDPDPTE had the lowest taste threshold (0.129 ± 0.057 mg / mL).

[0041]

[0042] The interaction between umami peptides and salt solutions was evaluated according to the ratio (R) between the experimental and theoretical taste thresholds, such as masking effect (R > 1), no interaction (R = 1), additive effect (0.5 < R < 1), and synergistic effect (R ≤ 0.5). In the binary mixture system, the actual threshold of SWDVRN-MSG (186.21 mg / L) was reduced by more than half compared with the theoretical threshold (416.87 mg / L), and its R value was only 0.45. The results showed that there was a freshness synergistic effect between peptide SWDVRN and 0.3% MSG solution. The actual taste thresholds of the other 4 peptides were all reduced, and the R values were between 0.5 and 1, indicating that these 4 umami peptides had an additive effect on the umami perception of MSG.

[0043] 2.4 Molecular docking of T1R3 and umami peptides The interaction mode between potential umami peptides and T1R3 was studied and analyzed by means of molecular docking, and the results are as Figure 4 shown. The research results showed that each potential umami peptide could bind well to the active cavity of T1R3. From Figure 4It can be seen that hydrogen bond is the most important binding force between umami peptides and T1R3, and each peptide forms 6 - 13 hydrogen bonds. Other favorable interaction forces include electrostatic force, hydrophobic force, etc. It is worth noting that Asn380, Arg247, Gly168, Asn68, and Glu45 form 2 hydrogen bonds with umami peptides respectively, and these amino acid residues may play an important role in umami perception. These results can provide a basis for the study of the binding sites between other umami peptides and umami receptors.

[0044] 3. Conclusions Through enzymatic hydrolysis reaction and peptidomics technology, 5 umami peptides in the shrimp meat of Litopenaeus vannamei were discovered and their sensory properties were verified. The sensory evaluation results showed that peptide LPNFR had strong umami, and its taste threshold was 0.152 ± 0.096 mg / mL. Molecular docking indicated that conventional hydrogen force was the main interaction force, and umami peptides could bind well to the active cavity of umami receptor T1R3. The research of the present invention enriches the marine - sourced umami peptide library, improves the research value of Litopenaeus vannamei, and provides a reference for studying the umami presentation mechanism of umami peptides with different structures.

[0045] The above - mentioned embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

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

1.

2. Use of the umami peptide according to claim 1 as or in the preparation of an umami agent.

3. An umami peptide derived from Litopenaeus vannamei, characterized in that: The amino acid sequence is SWDVRN, as shown in SEQ ID NO.

2.

4. Use of the umami peptide according to claim 3 as or in the preparation of an umami agent.

5. An umami peptide derived from Litopenaeus vannamei, characterized in that: The amino acid sequence is TGPDPDPTE, as shown in SEQ ID NO.

3.

6. Use of the umami peptide according to claim 5 as or in the preparation of an umami agent.

7. An umami peptide derived from Litopenaeus vannamei, characterized in that: The amino acid sequence is GPDPDPTEY, as shown in SEQ ID NO.

4.

8. Use of the umami peptide according to claim 7 as or in the preparation of an umami agent.

9. An umami peptide derived from Litopenaeus vannamei, characterized in that: The amino acid sequence is GPDPDPTEF, shown in SEQ ID NO.

5.

10. Use of the umami peptide according to claim 9 as or in the preparation of an umami agent.

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