Flavor peptide from euphausia superba and application thereof

Abstract

The application provides a Euphausia superba-derived flavor peptide and application thereof, and belongs to the technical field of deep processing of marine aquatic protein. Four kinds of flavor peptides, namely, EQNLDELEDSLER, VELE, LEEL and LEDL, are identified from Euphausia superba enzymatic hydrolysate through liquid chromatography-tandem mass spectrometry. The application takes EQNLDELEDSLER as a core flavor peptide, and VELE, LEEL and LEDL can be compounded, so as to solve the problems of salt reduction, insufficient fresh and sweet taste and weakened thick taste in salt-reduced soy sauce. When the flavor peptide composition of the application is applied to salt-reduced seasonings, the amount of the flavor peptide composition of the application is low, the bitter taste or astringent taste is not obviously increased, the salty taste can be maintained on the basis of 30% reduction of sodium chloride, the fresh and sweet taste is improved, and the thick taste is enhanced, so the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the technical field of food flavoring peptides, low-sodium seasonings, and deep processing of marine aquatic proteins, specifically relating to a flavor peptide derived from Antarctic krill and its applications. Background Technology

[0002] Antarctic krill is rich in protein with a relatively balanced amino acid composition, making it an important raw material for preparing aquatic flavor peptides. Through appropriate enzymatic hydrolysis, Antarctic krill protein can release small-molecule peptides that regulate umami, sweetness, saltiness, or richness. Peptides rich in umami amino acids such as glutamic acid, aspartic acid, glycine, and alanine are particularly abundant after controlled enzymatic hydrolysis. Current technologies primarily focus on the overall flavor of Antarctic krill hydrolysates or crude peptide powders, with limited research on specific functional peptides. Summary of the Invention

[0003] The purpose of this invention is to provide a flavor peptide derived from Antarctic krill, and to provide specific application examples of this flavor peptide, in order to overcome the shortcomings of the prior art.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A flavor peptide derived from Antarctic krill, comprising EQNLDELEDSLER, or VELE, or LEEL, or LEDL.

[0005] Furthermore, the specific sequences of the flavor peptide EQNLDELEDSLER are shown in SEQ No. 1, the specific sequences of the flavor peptide VELE are shown in SEQ No. 2, the specific sequences of the flavor peptide LEEL are shown in SEQ No. 3, and the specific sequences of the flavor peptide LEDL are shown in SEQ No. 4.

[0006] The application of flavor peptides EQNLDELEDSLER, VELE, LEEL, and LEDL in food processing.

[0007] A flavor peptide composition comprising flavor peptides EQNLDELEDSLER, VELE, LEEL, and LEDL.

[0008] Among them, EQNLDELEDSLER is the core flavor peptide; VELE, LEEL and LEDL are combination flavor peptides.

[0009] The application of the flavor peptide composition in the preparation of low-sodium soy sauce, soy sauce-based seasoning liquid, compound seasoning, low-sodium soup base, low-sodium dipping sauce, pre-made dish seasoning liquid, or low-sodium salt-based seasoning base.

[0010] The four polypeptides can be isolated and purified from Antarctic krill enzymatic hydrolysates, or they can be obtained through artificial synthesis. To ensure product stability, it is preferred to use artificially synthesized polypeptides or purified polypeptides as raw materials for the composition.

[0011] The peptide was combined in a specific ratio and applied to a low-sodium soy sauce.

[0012] Furthermore, by weight, EQNLDELEDSLER is 1 part, VELE is 0.05 to 20 parts, LEEL is 0.01 to 10 parts, and LEDL is 0.01 to 10 parts.

[0013] Furthermore, by weight, the recommended amounts are: EQNLDELEDSLER 1 part, VELE 0.1–10 parts, LEEL 0.05–0.5 parts, and LEDL 0.05–0.5 parts. However, adding more than 1 part of LEEL and LEDL will result in a slight bitter taste, so it is recommended to keep the amounts below 0.5 parts in actual production.

[0014] The sodium chloride content in reduced-sodium soy sauce with this peptide composition is reduced by 25% to 35% compared to unreduced-sodium soy sauce.

[0015] Furthermore, the amount of the flavor peptide composition added to the reduced-sodium soy sauce is 0.0005% to 0.5% (w / w) on a dry basis of peptides.

[0016] Furthermore, the amount of the flavor peptide composition added to the reduced-sodium soy sauce is 0.001% to 0.1% (w / w) on a dry basis of peptides.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: This invention identifies four flavor-related peptides—EQNLDELEDSLER, VELE, LEEL, and LEDL—from Antarctic krill enzymatic hydrolysates using liquid chromatography-tandem mass spectrometry. Further analysis using artificial synthesis, molecular docking, sensory evaluation, and electronic tongue reveals that these four peptides each possess their own flavor properties and can all be used in food seasoning processing, enhancing both sweetness and richness.

[0018] This invention uses EQNLDELEDSLER as the core flavor peptide, which can be combined with VELE, LEEL, and LEDL. It is specifically designed to address the issues of reduced saltiness, insufficient umami and sweetness, and weakened richness in low-sodium soy sauce. Molecular docking results show that EQNLDELEDSLER, VELE, LEEL, and LEDL can all bind to the umami receptor T1R1 / T1R3 and the richness receptor CaSR. Among them, EQNLDELEDSLER has a lower docking energy with T1R1 / T1R3 and CaSR, and more binding sites, making it suitable as the core flavor peptide in the composition.

[0019] When the flavor peptide composition of the present invention is applied to a low-sodium seasoning, the amount of flavor peptide composition added is low. Without significantly increasing bitterness or astringency, it can maintain the perception of saltiness while enhancing the umami and sweetness and strengthening the rich flavor. Attached Figure Description

[0020] Figure 1 Total ion chromatogram of Antarctic krill enzymatic hydrolysate.

[0021] Figure 2 This is the mass spectrum of the EQNLDELEDSLER.

[0022] Figure 3 This is the mass spectrum of VELE.

[0023] Figure 4 This is the mass spectrum of LEDL.

[0024] Figure 5 This is the mass spectrum of LEEL.

[0025] Figure 6 This is a three-dimensional docking conformation diagram of EQNLDELEDSLER with umami receptors T1R1 / T1R3.

[0026] Figure 7 This is a three-dimensional docking conformation diagram of VELE with umami receptors T1R1 / T1R3.

[0027] Figure 8 This is a three-dimensional docking conformation diagram of LEDL with umami receptors T1R1 / T1R3.

[0028] Figure 9 This is a three-dimensional docking conformation diagram of LEEL with umami receptors T1R1 / T1R3.

[0029] Figure 10 This is a three-dimensional docking conformation diagram of EQNLDELEDSLER and the strong taste receptor CaSR.

[0030] Figure 11This is a three-dimensional docking conformation diagram of VELE and the strong taste receptor CaSR.

[0031] Figure 12 This is a three-dimensional docking conformation diagram of LEDL and the strong taste receptor CaSR.

[0032] Figure 13 This is a three-dimensional docking conformation diagram of LEEL and the strong taste receptor CaSR. Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments described below are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0034] Example 1 Preparation of Antarctic krill enzymatic hydrolysate Take Antarctic krill or Antarctic krill protein powder, add deionized water and homogenize to obtain an Antarctic krill protein dispersion. Adjust the pH of the system to 5.0–9.0, and add papain for enzymatic hydrolysis.

[0035] The enzymatic hydrolysis conditions were as follows: hydrolysis temperature 30–60℃, hydrolysis time 0.5–8 h, and enzyme addition amount 500–10000 U / g raw material protein. After hydrolysis, the enzyme was inactivated by heating, cooled, and centrifuged. The supernatant was collected to obtain Antarctic krill hydrolysate.

[0036] Example 2 Identification of target peptides in Antarctic krill enzymatic hydrolysates The Antarctic krill enzymatic hydrolysate obtained in Example 1 was analyzed by liquid chromatography-tandem mass spectrometry. The total ion current of the Antarctic krill enzymatic hydrolysate was as follows: Figure 1 As shown, further fragment ion matching via secondary mass spectrometry is performed, such as... Figure 2 , Figure 3 , Figure 4 , Figure 5 As shown, the following peptides were identified: SEQ ID No.1:EQNLDELEDSLER; SEQ ID No. 2: VELE; SEQ ID No. 3: LEEL; SEQ ID No. 4: LEDL.

[0037] Mass spectrometry results showed that the above four peptides were present in Antarctic krill hydrolysate. Among them, EQNLDELEDSLER was identified as the core flavor peptide, while VELE, LEEL, and LEDL were identified as combinatorial flavor peptides.

[0038] Example 3 Preparation of synthetic peptides EQNLDELEDSLER, VELE, LEEL, and LEDL were synthesized using a solid-phase synthesis method. The synthesized products were purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and their molecular weight and sequence were confirmed by mass spectrometry (MS). The purity of the synthesized peptides was not less than 90%, preferably not less than 95%.

[0039] The synthetic peptides were prepared into aqueous solutions for subsequent molecular docking, sensory evaluation, and electronic tongue analysis.

[0040] Example 4: Molecular docking of four flavor peptides with taste-related receptors To explain the molecular basis of the flavor peptide composition of the present invention in improving the salty, sweet and savory taste and rich flavor of reduced-sodium soy sauce, the interaction between EQNLDELEDSLER, VELE, LEEL and LEDL and umami receptors T1R1 / T1R3 and rich flavor receptor CaSR was investigated using molecular docking method.

[0041] The conformations of the umami receptors T1R1 / T1R3 and the strong flavor receptor CaSR were optimized using MOE software. EQNLDELEDSLER, VELE, LEEL, and LEDL were used as ligands for semi-flexible molecular docking with these receptors. The same ligand was docked five times, and the result with the lowest docking energy was taken as the final docking result. A docking energy ≤ -6.0 kcal / mol is generally considered to indicate a good binding potential between the ligand and the receptor.

[0042] 1. Docking energies of four flavor peptides with T1R1 / T1R3 and CaSR The docking energies of the four flavor peptides with the umami receptors T1R1 / T1R3 and the strong flavor receptor CaSR are shown in Table 1.

[0043] Table 1. Molecular docking energies of four flavor peptides with taste-related receptors

[0044] As shown in Table 1, the docking energies of EQNLDELEDSLER, LEDL, LEEL, and VELE with T1R1 / T1R3 are all below -8.7 kcal / mol, and their docking energies with CaSR are all below -7.1 kcal / mol and below -6.0 kcal / mol, indicating that all four flavor peptides have the potential to bind to umami receptors and strong flavor receptors.

[0045] On the other hand, the binding energy of EQNLDELEDSLER with T1R1 / T1R3 is -9.256036 kcal / mol, and with CaSR is -11.225498 kcal / mol, both of which are among the lowest binding energies of the four peptides. This suggests that EQNLDELEDSLER has a strong interaction with both umami and savory taste receptors, and can serve as the core flavor peptide in the flavor peptide composition of this invention. VELE, LEEL, and LEDL also show good binding potential with both types of receptors and can participate in overall flavor regulation as composite flavor peptides.

[0046] 2. Interactions between four flavor peptides and umami receptors T1R1 / T1R3 Table 2 shows the docking sites and main interactions between the four flavor peptides and T1R1 / T1R3. Figures 6-9 As shown.

[0047] Table 2. Docking sites and interactions between four flavor peptides and T1R1 / T1R3

[0048] Table 2 shows that EQNLDELEDSLER can interact with sites such as ASP441, VAL474, PHE439, ARG180, and ARG56 on T1R1 / T1R3, forming 5 hydrogen bond donor interactions, 3 hydrogen bond acceptor interactions, and 1 ion interaction. These results indicate that EQNLDELEDSLER exhibits multi-site, multi-force binding characteristics with T1R1 / T1R3, which is beneficial for its umami and sweetness-related effects.

[0049] Furthermore, LEDL can interact with the ASP147 and ASN69 sites of T1R1 / T1R3, forming hydrogen bond donors, hydrogen bond acceptors, and ion interactions; LEEL can interact with the GLU423, ARG52, ASP441, and HIS426 sites, forming hydrogen bond donors and ion interactions; VELE can interact with the GLU423 site, forming a hydrogen bond donor interaction. These results indicate that VELE, LEEL, and LEDL can all bind to umami receptors, but their binding sites and interaction types differ, suggesting that all three, along with EQNLDELEDSLER, may participate in the regulation of umami and salty-umami sensations.

[0050] 3. Interactions between four flavor peptides and the strong flavor receptor CaSR Table 3 shows the docking sites and main interactions between the four flavor peptides and CaSR. Figures 10-13 As shown.

[0051] Table 3. Binding sites and interactions between four flavor peptides and CaSR

[0052] Table 3 shows that the docking energy between EQNLDELEDSLER and CaSR is -11.2255 kcal / mol. EQNLDELEDSLER can interact with multiple sites, including ASP248, GLU481, GLU250, ALA46, GLU354, GLU59, GLU350, GLN309, and ARG62, forming 10 hydrogen bond donor interactions, 4 hydrogen bond acceptor interactions, and 6 ionic interactions. These results indicate that EQNLDELEDSLER has strong binding potential with CaSR, providing a molecular-level explanation for its ability to enhance rich flavors.

[0053] LEDL interacts with GLU241, GLU228, GLU229, GLU232, and GLU191 sites of CaSR, forming 9 hydrogen bond donor interactions and 5 ion interactions; LEEL interacts with GLN309, GLU405, ASP410, ALA306, and ILE503 sites, forming hydrogen bond donor, hydrogen bond acceptor, and ion interactions; VELE interacts with GLU405, ASP410, ARG415, ARG69, and ASP500 sites, forming hydrogen bond donor, hydrogen bond acceptor, and ion interactions. These results indicate that VELE, LEEL, and LEDL all have the potential to bind to CaSR and can participate in the regulation of richness and overall flavor fullness of the flavor peptide composition of this invention.

[0054] 4. Analysis of Molecular Docking Results Based on the docking results of T1R1 / T1R3 and CaSR, all four flavor peptides exhibited low docking energies and were able to form interactions such as hydrogen bonds and ionic interactions. Among them, EQNLDELEDSLER showed low docking energies with both T1R1 / T1R3 and CaSR, and had multiple interaction sites, indicating that it can serve as the core flavor peptide in the composition of this invention. Although VELE, LEEL, and LEDL have shorter peptide chains, they can all interact with two types of taste-related receptors, indicating that they can participate in overall flavor regulation as composite flavor peptides.

[0055] The above molecular docking results provide molecular-level support for the flavor peptide composition of the present invention in food processing, especially in the application of seasonings.

[0056] Example 5 Preparation of Flavor Peptide Composition A flavor peptide composition was prepared by mixing EQNLDELEDSLER, VELE, LEEL, and LEDL in parts by weight.

[0057] Based on EQNLDELEDSLER as 1 part by weight, the dosage of VELE is 0.05 to 20 parts by weight, the dosage of LEEL is 0.01 to 10 parts by weight, and the dosage of LEDL is 0.01 to 10 parts by weight.

[0058] Preferably, based on EQNLDELEDSLER as 1 part by weight, the amount of VELE is 0.1 to 10 parts by weight, the amount of LEEL is 0.05 to 1 part by weight, and the amount of LEDL is 0.05 to 1 part by weight.

[0059] The above-mentioned flavor peptide composition can be made into powder, granules, freeze-dried powder, concentrate or aqueous solution, and can be directly used in soy sauce or other seasoning foods.

[0060] Example 6 Application of flavor peptide composition in reduced-sodium soy sauce Using regular soy sauce as the control group without salt reduction, and soy sauce with a 30% reduction in sodium chloride content as the control group with reduced salt, the flavor peptide composition prepared in Example 5 was added to the reduced salt soy sauce to obtain flavor peptide-enhanced reduced salt soy sauce.

[0061] The experimental groups are as follows: Group A: Unsalted soy sauce; Group B: Soy sauce with 30% less salt; Group C: Soy sauce with 30% less salt + EQNLDELEDSLER; Group D: Soy sauce with 30% less salt + VELE; Group E: 30% reduced-sodium soy sauce + EQNLDELEDSLER, VELE, LEEL and LEDL flavor peptide composition.

[0062] A trained sensory evaluation team was used to evaluate the saltiness, umami, sweet and savory, salty and umami sensation, richness, bitterness, astringency, and overall acceptability of each group of samples.

[0063] Table 4. Sensory evaluation results of flavor peptide compositions in reduced-sodium soy sauce

[0064] The evaluation results showed that compared with group A, group B had a decrease in saltiness, umami, and richness, and the overall flavor was thinner. Group C showed improvement in umami and richness compared with group B, and bitterness was reduced. Group D showed improvement in saltiness and umami compared with group B. Compared with group B, group E showed a recovery in saltiness perception, a significant increase in umami and richness, and improved overall acceptability; compared with group A, group E did not show a significant decrease in saltiness under the condition of a 30% reduction in salt, and umami and richness were more prominent.

[0065] The above results demonstrate that the flavor peptide composition of the present invention can maintain the saltiness of soy sauce with a 30% reduction in salt content, and improve the problems of insufficient sweetness and reduced richness in reduced-salt soy sauce.

[0066] Example 7 Electronic Tongue Analysis An electronic tongue was used to test each group of samples in Example 6, and the sensor response values ​​related to umami, saltiness, bitterness, astringency and richness were recorded.

[0067] Table 5. Electronic tongue response results of flavor peptide compositions in reduced-sodium soy sauce

[0068] Electronic tongue analysis showed that, compared with the 30% salt-reduced soy sauce group, the addition of the flavor peptide composition of this invention improved the umami-related responses, saltiness-related responses, and richness-related responses, while no significant increase was observed in the bitterness and astringency-related responses. The trends of the electronic tongue analysis results and sensory evaluation results were largely consistent, further demonstrating that the flavor peptide composition of this invention can improve the overall flavor quality of reduced-salt soy sauce. In other words, this invention provides a flavor peptide composition with a clearly defined source, clear sequence, and controllable quality, used to improve the problems of decreased saltiness, insufficient umami and sweetness, and weakened richness in reduced-salt soy sauce.

[0069] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A flavor peptide derived from Antarctic krill, characterized in that, The flavor peptides include EQNLDELEDSLER, VELE, LEEL, or LEDL; the specific sequence of the flavor peptide EQNLDELEDSLER is shown in SEQ No. 1, the specific sequence of the flavor peptide VELE is shown in SEQ No. 2, the specific sequence of the flavor peptide LEEL is shown in SEQ No. 3, and the specific sequence of the flavor peptide LEDL is shown in SEQ No.

4.

2. The application of the flavor peptide EQNLDELEDSLER as described in claim 1 in food processing.

3. The application of the flavor peptide VELE as described in claim 1 in food processing.

4. The application of the flavor peptide LEEL as described in claim 1 in food processing.

5. The application of the flavor peptide LEDL as described in claim 1 in food processing.

6. A flavor peptide composition, characterized in that, The flavor peptide composition includes flavor peptides EQNLDELEDSLER, VELE, LEEL, and LEDL.

7. The use of the flavor peptide composition of claim 6 in the preparation of low-sodium soy sauce, soy sauce-based seasoning liquid, compound seasoning, low-sodium soup base, low-sodium dipping sauce, pre-prepared dish seasoning liquid, or low-sodium salt-based seasoning base.

8. A reduced-sodium soy sauce, characterized in that, The reduced-sodium soy sauce comprises the flavor peptide composition of claim 6.

9. The reduced-sodium soy sauce as described in claim 8, characterized in that, By weight, the flavor peptide EQNLDELEDSLER is 1 part, the flavor peptide VELE is 0.05-20 parts, the flavor peptide LEEL is 0.01-10 parts, and the flavor peptide LEDL is 0.01-10 parts; the amount of the flavor peptide composition added to the reduced-sodium soy sauce is 0.0005wt%-0.5wt% on a dry basis of polypeptides.

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