A tetrapeptide KA4 with anti-fatigue function and its preparation method and application

Abstract

The present invention discloses a tetrapeptide KA4 with anti-fatigue properties, as well as its preparation method and application, belonging to the field of biotechnology. The tetrapeptide KA4, screened from Pacific salmon sperm protease hydrolysate, has an amino acid sequence of KYPA and exhibits strong binding abilities to KEAP1, AMPK, and LDH. It can significantly reduce serum lactate levels and increase the weighted swimming time of fatigue model mice. It exhibits anti-fatigue properties and can be used in the development of functional foods that alleviate fatigue.

Description

Technical Field

[0001] The present invention relates to a small molecule peptide and a preparation method and application thereof, and in particular to a tetrapeptide KA4 with anti-fatigue function and a preparation method thereof and application thereof in fatigue-relieving functional foods, belonging to the field of biotechnology. Background Art

[0002] Fatigue is a common health problem in modern society, manifesting as persistent physical and mental exhaustion and an imbalance in energy metabolism, seriously impacting individual quality of life. Based on its underlying mechanisms, fatigue can be categorized as central fatigue (neurotransmitter imbalances caused by dysfunction of the hypothalamic-pituitary-adrenal axis) and peripheral fatigue (energy crisis triggered by skeletal muscle mitochondrial dysfunction). While adequate rest can alleviate fatigue, chronic, accumulated fatigue can lead to an imbalance in the oxidative-antioxidant system, exacerbating the decline in tissue and organ function.

[0003] Current anti-fatigue research focuses on three main pathways:

[0004] (1) Activate the PGC-1α signaling axis to promote mitochondrial biogenesis;

[0005] (2) Regulate the HO-1 / NQO1 pathway to enhance the antioxidant defense system;

[0006] (3) AMPK energy sensing network is mediated by changes in the AMP / ATP ratio.

[0007] Among them, the dynamic balance of lactate dehydrogenase (LDH) is correlated with the PGC-1α signaling axis, the activation of the Nrf2 / ARE signaling pathway is considered to be related to the antioxidant defense system, and the level of the AMPK energy sensing network can reflect changes in the AMP / ATP ratio. Therefore, these three indicators can serve as important biological targets for predicting anti-fatigue function.

[0008] Chemically synthesized drugs have side effects such as easily causing arrhythmias. Food-derived active peptides have unique advantages in the development of anti-fatigue functional factors due to their multi-target regulatory properties, high bioavailability and good metabolic adaptability.

[0009] Differentiated enzymatic hydrolysis of different proteins can yield a library of active peptides with diverse structures and functions. For example, hydrolysis of marine collagen with 2709 alkaline protease primarily releases short glycine-rich peptides, while hydrolysis of whey protein with a combination of enzymes (trypsin and A.S1398 neutral protease) more readily yields peptides enriched in branched-chain amino acids. Notably, active peptides with identical functions can exhibit completely different amino acid sequence characteristics and mechanisms of action. Studies have shown that the wheat-derived pentapeptide PQLPQ and the codfish-derived tripeptide GPH both prolong the time from onset to exhaustion in mice swimming. These two active peptides, with distinct amino acid sequence characteristics, share the same function (anti-fatigue), but the former exerts its effects by enhancing glycogen stores, while the latter primarily works by improving mitochondrial respiratory chain efficiency.

[0010] Recent studies have revealed a new mechanism of action for anti-fatigue peptides:

[0011] (1) The article "Anti-Fatigue Effect by Peptide Fraction from Protein Hydrolysate of Croceine Croaker (Pseudosciaena crocea) Swim Bladder through Inhibiting the Oxidative Reactions including DNA Damage" (Zhao YQ, Zeng L, Yang ZS, Huang FF, Ding GF, Wang B, Mar Drugs, 2016 Dec 13; 14 (12): 221) pointed out that large yellow croaker protein hydrolysate has anti-fatigue function, which can increase the level of liver glycogen, reduce the levels of blood urea nitrogen, lactic acid and malondialdehyde, and enhance the activity of lactate dehydrogenase.

[0012] (2) The article “Experimental Study on Anti-fatigue and Hypoxia Tolerance of Antarctic Krill Defatted Protein Peptide” (Xu Kai, Liu Yun, Wang Yaen, et al., Food Science, 2011, 32(11): 10-313) pointed out that high doses of Antarctic krill defatted protein peptide can significantly increase the weighted swimming time and serum LDH content, LA clearance rate and liver glycogen content of mice.

[0013] (3) In the field of plant protein, the article "Anti-fatigue effects of fermented soybean protein peptides in mice" (Fang L, Zhang RX, Wei Y, Ling K, Lu L, Wang J, Pan XC, Cai MY, JSci Food Agric, 2022 May; 102 (7): 2693-2703) pointed out that fermented soybean protein hydrolysate can increase forced swimming time and significantly prolong oxygen tolerance survival time. At the same time, it reduces the concentrations of lactic acid and blood urea nitrogen. The article "Bioactive Peptides from Marine Organisms" (Wang P, Zhang Y, Hu J, Tan BK, Protein Pept Lett, 2024; 31 (8): 569-585) systematically summarizes active peptides with anti-fatigue function derived from marine organisms, and there is no sequence identical or similar to the structure of the present invention. In addition, if modifications are to be made based on the reported active peptide sequences, since there are 20 common amino acids to choose from at each amino acid position, there is a 20ⁿ (n is the length of the peptide chain) exponential growth screening problem, which is not feasible.

[0014] The salmon belongs to the order Salmoniformes, family Salmonidae, and genus Salmon in biological taxonomy. It is also called Pacific salmon in my country. Its scientific name is Oncorhynchus keta Protamine is a migratory pelagic fish that is high in protein, low in fat, and rich in ω-3 polyunsaturated fatty acids. It is an important aquatic resource with both nutritional and ecological value. Protamine, rich in arginine and histidine, is a high-quality raw material for the preparation of antimicrobial peptides, immunomodulatory peptides, and metabolically active peptides. Summary of the Invention

[0015] The purpose of the present invention is to provide a small molecule peptide screened from Pacific salmon sperm protein hydrolysate, which has anti-fatigue function and can be used in fatigue-relieving functional foods.

[0016] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0017] A tetrapeptide KA4 with anti-fatigue function, wherein the amino acid sequence of the tetrapeptide KA4 is KYPA, as shown in SEQ ID NO: 10 in the sequence table, and has anti-fatigue function.

[0018] The application of the aforementioned tetrapeptide KA4 with anti-fatigue function in fatigue-relieving functional foods.

[0019] The preparation method of the aforementioned tetrapeptide KA4 having anti-fatigue function adopts a solid phase synthesis method, specifically:

[0020] The solid phase synthesis was carried out using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid phase carrier using the Fmoc solid phase synthesis strategy.

[0021] The preparation method of the aforementioned tetrapeptide KA4 with anti-fatigue function adopts an enzymatic hydrolysis method, specifically:

[0022] (1) Take Pacific salmon sperm, remove impurities, add water, and heat to 65±2℃ for 12 hours;

[0023] (2) Remove the fish essence and beat it into a pulp, put it into a reactor, add water, start the stirrer and heat it to 48°C, add papain for enzymatic hydrolysis, continue to heat it to 57°C, add nuclease, deaminase, alkaline protease and neutral protease for enzymatic hydrolysis for 3 hours, then add flavor protease for enzymatic hydrolysis for 4 hours, continue to heat it to 85°C and maintain it for 30 minutes to obtain the enzymatic hydrolyzate;

[0024] (3) Adding the chitosan aqueous solution to the enzymatic hydrolysate while cooling, centrifuging after floccules appear, retaining the supernatant and filtering to obtain a clear and transparent filtrate;

[0025] (4) Adjust the pH value of the filtrate to 5.50±1.50, then add coconut shell activated carbon, decolorize and deodorize at 70±2℃ for 30min, filter again, desalinate and concentrate the filtrate to obtain a concentrate;

[0026] (5) The concentrated liquid is dried to obtain Pacific salmon protamine polypeptide, which contains tetrapeptide KA4.

[0027] The present invention is beneficial in that:

[0028] (1) The tetrapeptide KA4 obtained from the protease hydrolysate of Pacific salmon sperm has strong binding ability with KEAP1, AMPK and LDH, can significantly reduce the serum lactate content of fatigue model mice (P<0.01), and significantly increase the weighted swimming time of fatigue model mice (P<0.01). It has anti-fatigue function and can be used to develop functional foods that relieve fatigue.

[0029] (2) The tetrapeptide KA4 provided by the present invention is obtained by screening the protease hydrolysate of Pacific salmon sperm. Compared with traditional drugs (such as the Chinese medicine Polygonum multiflorum), it has the advantages of less toxic side effects and higher safety in use;

[0030] (3) The tetrapeptide KA4 provided by the present invention has comparable effects on increasing weighted swimming time and reducing blood lactate content compared with the positive control American ginseng. Considering that the dosage of the positive control American ginseng is 100 mg / kg, while the dosage of the tetrapeptide KA4 is only one tenth of that (10 mg / kg), the tetrapeptide KA4 has a significant improvement in anti-fatigue. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 3D schematic diagram of the binding mode of tetrapeptide KA4 and KEAP1;

[0032] Figure 2 2D schematic diagram of the binding mode of tetrapeptide KA4 and KEAP1;

[0033] Figure 3 3D schematic diagram of the binding mode of tetrapeptide KA4 and AMPK;

[0034] Figure 4 2D schematic diagram of the binding mode of tetrapeptide KA4 and AMPK;

[0035] Figure 5 3D schematic diagram of the binding mode of tetrapeptide KA4 and LDH;

[0036] Figure 6 2D schematic diagram of the binding mode of tetrapeptide KA4 and LDH;

[0037] Figure 7 This is the effect of tetrapeptide KA4 on the weighted swimming time of fatigue model mice, ** indicates P < 0.01, *** indicates P < 0.001;

[0038] Figure 8 This is the effect of tetrapeptide KA4 on serum lactate content in fatigue model mice, ** indicates P < 0.01, *** indicates P < 0.001;

[0039] Figure 9 This is the effect of tetrapeptide KA4 on serum urea nitrogen content in fatigue model mice, *** indicates P < 0.001;

[0040] Figure 10 This is a graph showing the effect of tetrapeptide KA4 on liver glycogen content in fatigue model mice. *** indicates P < 0.001. DETAILED DESCRIPTION

[0041] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0042] 1. Preparation of Pacific salmon protamine polypeptide

[0043] Take Pacific salmon sperm, remove impurities, add appropriate amount of purified water, heat to 65±2℃, and maintain for 12 hours (to allow the testicular tissue to fully swell, facilitating extraction and subsequent impurity removal), and stir every 4 hours.

[0044] The fish essence was removed and slurried twice with a bone mud machine, then placed in a reactor. Purified water was added at a solid-liquid ratio of 1:5. The stirrer was started (speed 100 rpm) and the temperature was raised to 48°C. Papain (enzyme substrate ratio 1.0%) was added for enzymatic hydrolysis for 2 h. The temperature was continued to be raised to 57°C. Nuclease (enzyme substrate ratio 0.5%), deaminase (enzyme substrate ratio 0.3%), alkaline protease (Alcalase® 2.4 L, enzyme substrate ratio 1.0%), and neutral protease (Neutrase® 0.8 L, enzyme substrate ratio 0.5%) were added for enzymatic hydrolysis for 3 h. Subsequently, flavor protease (Flavourzyme® 500 MG, enzyme substrate ratio 1.0%) was added for enzymatic hydrolysis for 4 h. The temperature was continued to be raised to 85°C and maintained for 30 min (enzyme inactivation) to obtain the enzymatic hydrolyzate.

[0045] While cooling, a 1 wt% chitosan aqueous solution (food grade, to promote the sedimentation of impurities) was added to the enzymatic hydrolyzate. After floccules appeared, the solution was centrifuged (5000 rpm, 3 min) to retain the supernatant, which was filtered using a plate and frame filter press at a pressure of 0.3 MPa to obtain a clear and transparent filtrate.

[0046] The filtrate was placed in a reactor, and the pH value of the filtrate was adjusted to 5.50±1.50. 1% of the total volume of the liquid was then added with coconut shell activated carbon. The mixture was decolorized and deodorized at 70±2°C for 30 minutes. The mixture was filtered again using a plate and frame filter press. The obtained filtrate was desalted using a nanofiltration system. When salt was no longer detected in the discharged liquid, the mixture was transferred to a double-effect concentrator for further concentration to obtain a concentrated liquid.

[0047] The concentrate was spray dried using a spray dryer with a feed pressure of 18.0 MPa, a flow rate of 300 L / h, an air inlet temperature controlled at 130°C, and an air outlet temperature controlled at 95°C to obtain Pacific salmon protamine polypeptide (beige powder).

[0048] 2. Obtaining the Sequence of the Pacific Salmon Protamine Polypeptide

[0049] The Pacific salmon protamine polypeptide obtained above was detected by LC-MS / MS, and the detection results were analyzed by mass spectrometry analysis software to obtain several polypeptide sequences.

[0050] LC-MS / MS detection conditions are:

[0051] (1) In the liquid phase method: the chromatographic column is C18, 3 μm, 250 mm × 75 μm (Eksigent), phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300 nL / min, the injection volume is 1 μL, and the chromatographic gradient is 70 min. The specific elution gradient is: 0-55 min, phase A is uniformly reduced from 95% to 65%; 55-63 min, phase A is uniformly reduced from 65% to 50%; 63-64 min, phase A is uniformly reduced from 50% to 0; 64-70 min, maintain 0% phase A;

[0052] (2) Mass spectrometry: Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific), positive ion detection mode, primary resolution 120,000, AGC setting 310, scan range 110–2000 m / z. MIPS mode for peptides, valence states 1–6, secondary resolution 17,500, separation window 1.6 m / z.

[0053] 3. Screening peak area ≥10 7 Active oligopeptides with amino acid number ≤ 6

[0054] From the above obtained peptide sequences, 11 peptide sequences with peak areas ≥10 7 The screening results of active oligopeptides with amino acid number ≤ 6 are shown in Table 1.

[0055] Table 1 Peak area ≥10 in Pacific salmon protamine peptides 7 Active oligopeptide sequence with amino acid number ≤ 6

[0056]

[0057] 4. Screening of active oligopeptides with strong binding ability to KEAP1, AMPK and LDH

[0058] Discovery Studio software was used to perform molecular docking of the 11 active oligopeptides in Table 1 with the crystal structure of E3 ubiquitin ligase adaptor protein (KEAP1) (PDB ID: 2FLU), the crystal structure of adenylate-activated protein kinase (AMPK) (PDB ID: 4ZHX), and the crystal structure of lactate dehydrogenase (LDH) (PDB ID: 7EPM). Before docking, the 2D structures of the active oligopeptides were converted into 3D structures by energy minimization, and active oligopeptides with strong binding ability to KEAP1, AMPK, and LDH were screened.

[0059] The docking results are expressed as docking scores (-CiE). The larger the -CiE value, the stronger the interaction between the active oligopeptide and KEAP1, AMPK and LDH, and the more likely it is to exhibit anti-fatigue function.

[0060] The molecular docking results of 11 active oligopeptides with KEAP1, AMPK and LDH are shown in Table 2, Table 3 and Table 4, respectively.

[0061] Table 2 Prediction results of the interaction between 11 active oligopeptides and KEAP1

[0062]

[0063] Table 3 Prediction results of the interaction between 11 active oligopeptides and AMPK

[0064]

[0065] Table 4 Prediction results of the interaction between 11 active oligopeptides and LDH

[0066]

[0067] As shown in Table 2, among the 11 active oligopeptides, the -CiE of DLERP, YVAAP, LGYAP, KYPA and KEAP1 all exceeded 60 kcal / mol.

[0068] As shown in Table 3, among the 11 active oligopeptides, the -CiE of DLERP, KYPA and AMPK all exceeded 90 kcal / mol.

[0069] It can be seen from Table 4 that among the 11 active oligopeptides, the -CiE of KYPA, LGEP and VGVVVA with LDH all exceeded 80 kcal / mol.

[0070] Based on Tables 2, 3, and 4, it was predicted that among the 11 active oligopeptides, only KYPA had strong interactions with KEAP1, AMPK, and LDH. Therefore, KYPA (denoted as tetrapeptide KA4) was selected for further molecular docking analysis.

[0071] After analysis, the binding mode of tetrapeptide KA4 and KEAP1 is as follows Figure 1 and Figure 2 As shown, the binding mode with AMPK is as follows Figure 3 and Figure 4 The binding mode with LDH is shown in Figure 5 and Figure 6 The molecular docking situation is as follows:

[0072] There are 9 HH bond interactions, 3 electrostatic interactions, and no C-H bond interactions between tetrapeptide KA4 and KEAP1. 17 amino acid residues are involved in the interaction between tetrapeptide KA4 and KEAP1.

[0073] There are 9 HH bond interactions, 3 C-H bond interactions, and 6 electrostatic interactions between the tetrapeptide KA4 and AMPK. There are 20 amino acid residues involved in the interaction between the tetrapeptide KA4 and AMPK.

[0074] There are five HH bond interactions, one CH bond interaction, and five electrostatic interactions between the tetrapeptide KA4 and LDH. There are 22 amino acid residues involved in the interaction between the tetrapeptide KA4 and LDH.

[0075] 5. Evaluation of the anti-fatigue function of tetrapeptide KA4

[0076] 1. Solid phase synthesis of tetrapeptide KA4

[0077] The tetrapeptide KA4 (amino acid sequence: KYPA) was synthesized on solid phase using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid phase support with a purity of >90%.

[0078] 2. Animal Experiment Process

[0079] Four- to six-week-old male ICR mice were randomly divided into three groups after one week of adaptive feeding: a control group, a positive drug group, and a tetrapeptide KA4 group. The control group received daily oral administration of normal saline (200 μL), the positive drug group received daily oral administration of American ginseng powder (0.1 g / kg), and the tetrapeptide KA4 group received daily oral administration of solid-phase synthesized tetrapeptide KA4 (10 mg / kg) for 30 days. Mice in each group had free access to water and food. Thirty minutes after the last administration of the test sample (normal saline, American ginseng powder, or tetrapeptide KA4), a 5% body weight lead sheet was placed at the base of the tail of each mouse. The mice were placed in a swimming tank (50 cm × 50 cm × 40 cm, 30 cm deep, 25°C ± 1.0°C) for a weighted swimming test. The time from the start of swimming to exhaustion (exhaustion was defined as the time the mouse took to sink into the water for more than 7 seconds) was recorded. The mice were then sacrificed at the end of the experiment.

[0080] 3. Sample collection and processing

[0081] Serum: Collect all blood from mice by eyeball bleeding, place in a 4°C refrigerator for 3 hours, and then centrifuge at 2000 rpm for 15 minutes. Aspirate the upper serum with a pipette, aliquot, and store in a -80°C refrigerator for the determination of urea nitrogen and lactic acid content in serum.

[0082] Liver: After killing the mice, remove the liver, rinse with physiological saline, and then dry with filter paper. Accurately weigh 100 mg of the liver for the determination of liver glycogen content (liver / muscle glycogen determination kit).

[0083] The statistical results of the weight-bearing swimming time of each group of mice are shown in Figure 7 .Depend on Figure 7 It can be seen that compared with the weighted swimming time of mice in the control group (308.2±21.1s), the weighted swimming time of mice in the positive drug group (452.1±26.4s) was significantly increased (p<0.001), and the weighted swimming time of mice in the tetrapeptide KA4 group (400.4±21.1s) was also significantly increased (p<0.01).

[0084] The results of serum lactate content in each group of mice are shown in Figure 8 .Depend on Figure 8 It can be seen that compared with the serum lactic acid content of mice in the control group (8.79±0.33mM), the serum lactic acid content of mice in the positive drug group (6.69±0.41mM) was significantly reduced (p<0.001), and the serum lactic acid content of mice in the tetrapeptide KA4 group (7.14±0.39mM) was also significantly reduced (p<0.01).

[0085] The results of serum urea nitrogen content in each group of mice are shown in Figure 9 .Depend on Figure 9 It can be seen that compared with the serum urea nitrogen content of mice in the control group (8.35±0.49mM), the serum urea nitrogen content of mice in the positive drug group (5.2±0.38mM) was significantly reduced (p<0.001), and the serum urea nitrogen content of mice in the tetrapeptide KA4 group (7.69±0.34mM) did not change significantly (p>0.05).

[0086] The results of liver glycogen content test of mice in each group are shown in Figure 10 .Depend on Figure 10 It can be seen that compared with the liver glycogen content of mice in the control group (16.4±1.02mg / g), the liver glycogen content of mice in the positive drug group (24.8±1.3mg / g) increased significantly (p<0.001), and the liver glycogen content of mice in the tetrapeptide KA4 group (17.94±0.78mM) did not change significantly (p>0.05).

[0087] In summary, the active oligopeptide KYPA (tetrapeptide KA4) obtained by screening from Pacific salmon sperm protein hydrolysate in the present invention has a strong interaction with KEAP1, AMPK and LDH. Animal experiments have found that the tetrapeptide KA4 can significantly reduce the serum lactate content of fatigue model mice (p<0.01) and significantly increase the weighted swimming time of fatigue model mice (p<0.01). It has anti-fatigue function and can be used in functional foods that relieve fatigue.

[0088] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A tetrapeptide KA4 with anti-fatigue function, characterized in that: The amino acid sequence of the tetrapeptide KA4 is KYPA, as shown in SEQ ID NO: 10 in the sequence table, and has anti-fatigue function.

2. Use of the tetrapeptide KA4 with anti-fatigue function according to claim 1 in the preparation of functional foods for relieving fatigue.

3. A method for preparing the tetrapeptide KA4 having anti-fatigue function as claimed in claim 1, characterized in that: The solid phase synthesis method is used, specifically: The solid phase synthesis was carried out using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid phase carrier using the Fmoc solid phase synthesis strategy.

4. A method for preparing the tetrapeptide KA4 having anti-fatigue function as claimed in claim 1, characterized in that: Enzymatic hydrolysis method is used, specifically: (1) Take Pacific salmon sperm, remove impurities, add water, and heat to 65±2℃ for 12 hours; (2) Remove the fish essence and beat it into a pulp, put it into a reactor, add water, start the stirrer and heat it to 48°C, add papain for enzymatic hydrolysis for 2 hours, continue to heat it to 57°C, add nuclease, deaminase, alkaline protease and neutral protease for enzymatic hydrolysis for 3 hours, then add flavor protease for enzymatic hydrolysis for 4 hours, continue to heat it to 85°C and maintain it for 30 minutes to obtain the enzymatic hydrolyzate; (3) Adding the chitosan aqueous solution to the enzymatic hydrolysate while cooling, centrifuging after floccules appear, retaining the supernatant and filtering to obtain a clear and transparent filtrate; (4) Adjust the pH value of the filtrate to 5.50±1.50, then add coconut shell activated carbon, decolorize and deodorize at 70±2℃ for 30min, filter again, desalinate and concentrate the filtrate to obtain a concentrate; (5) The concentrated liquid is dried to obtain Pacific salmon protamine polypeptide, which contains tetrapeptide KA4.

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