Tetrapeptide KA4 with anti-fatigue function as well as preparation method and application thereof

Abstract

The invention discloses tetrapeptide KA4 with an anti-fatigue function as well as a preparation method and application of the tetrapeptide KA4, and belongs to the technical field of biology. The tetrapeptide KA4 is screened from pacific salmon protamine enzymatic hydrolysate, the amino acid sequence is KYPA, and the tetrapeptide KA4 has strong binding capacity with KEAP1, AMPK and LDH, can significantly reduce the serum lactic acid content of a fatigue model mouse and significantly increase the load swimming time of the fatigue model mouse, has an anti-fatigue function, and can be applied to development of functional food for relieving fatigue.

Description

Technical Field

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

[0002] Fatigue is a common health problem in modern society, manifested as persistent physical and mental burnout and energy metabolism imbalance, which seriously affects the quality of life of individuals. According to different occurrence mechanisms, fatigue can be divided into central fatigue (neurotransmitter disorder caused by the dysfunction of the hypothalamus-pituitary-adrenal axis) and peripheral fatigue (energy crisis caused by skeletal muscle mitochondrial dysfunction). Although proper rest can relieve fatigue, long-term accumulation of fatigue will lead to the imbalance of the oxidation-antioxidation system and exacerbate the decline of tissue and organ functions.

[0003] Current anti-fatigue research mainly focuses on three major pathways: (1) Activating the PGC-1α signaling axis to promote mitochondrial biogenesis; (2) Regulating the HO-1 / NQO1 pathway to enhance the antioxidant defense system; (3) Mediating the AMPK energy sensing network through changes in the AMP / ATP ratio.

[0004] 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 the change of the AMP / ATP ratio. Therefore, these three indicators can be used as important biological targets for predicting anti-fatigue function.

[0005] Chemically synthesized drugs are prone to side effects such as arrhythmia, and food-derived bioactive peptides show unique advantages in the development of anti-fatigue functional factors due to their multi-target regulation characteristics, high bioavailability and good metabolic adaptability.

[0006] Differentiated enzymatic hydrolysis of different proteins can generate active peptide libraries with different structural functions. For example, marine collagen is mainly hydrolyzed by 2709 alkaline protease to release short peptides rich in glycine, and whey protein is more likely to generate polypeptides rich in branched-chain amino acids after hydrolysis by a complex enzyme (trypsin and A.S1398 neutral protease). It is worth noting that bioactive peptides with the same function may exhibit completely different amino acid sequence characteristics and mechanisms of action. Studies have found that the wheat-derived pentapeptide PQLPQ and the cod-derived tripeptide GPH can both prolong the time from the start of mouse swimming to exhaustion. Two bioactive peptides with completely different amino acid sequence characteristics have the same function (anti-fatigue), but the former exerts its effect by enhancing glycogen reserve, while the latter mainly exerts its effect by improving the efficiency of the mitochondrial respiratory chain.

[0007] Recently, multiple studies have revealed new mechanisms of action of anti-fatigue peptides: (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 the protein hydrolysate of croceine croaker has anti-fatigue function, which can act by increasing the liver glycogen level, reducing the levels of blood urea nitrogen, lactic acid and malondialdehyde, and at the same time enhancing the activity of lactate dehydrogenase.

[0008] (2) The article "Experimental Study on Anti-Fatigue and Hypoxia Tolerance of Antarctic Krill Apoprotein Peptide" (Xu K, Liu Y, Wang YA, et al., Food Science, 2011, 32(11): 10 - 313) pointed out that high-dose Antarctic krill apoprotein peptide can significantly increase the weight-bearing swimming time of mice, the content of LDH in serum, the clearance rate of LA and the content of liver glycogen.

[0009] (3) In the field of plant proteins, the paper "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, J Sci Food Agric, 2022 May; 102(7): 2693-2703) pointed out that the hydrolysate of fermented soybean protein can increase the forced swimming time and significantly extend the anaerobic survival time. At the same time, it can reduce the concentrations of lactic acid and blood urea nitrogen. The paper "Bioactive Peptides from Marine Organisms" (Wang P, Zhang Y, Hu J, Tan BK, Protein Pept Lett, 2024; 31(8): 569-585) systematically summarized the bioactive peptides with anti-fatigue functions derived from marine organisms, and there are no sequences identical or similar to the structure of the present invention. In addition, if we want to modify based on the reported bioactive peptide sequences, since there are 20 common amino acids available for selection at each amino acid position, we will face the screening problem of exponential growth of 20ⁿ (n is the peptide chain length), which is not feasible.

[0010] Salmon belongs to the order Salmoniformes, family Salmonidae, and genus Oncorhynchus in biological taxonomy. It is also called Pacific salmon in China, and its scientific name is Oncorhynchus keta , which is a migratory mid-upper layer fish. It is characterized by high protein, low fat, and rich in ω-3 polyunsaturated fatty acids. It is an important aquatic resource with both nutritional and ecological values. Protamine is a high-quality raw material for preparing antibacterial peptides, immunomodulatory peptides, and metabolic active peptides because it is rich in arginine and histidine. Summary of the Invention

[0011] The object of the present invention is to provide a small molecule peptide screened from the hydrolysate of Oncorhynchus gorbuscha protamine, which has anti-fatigue function and can be applied to functional foods for relieving fatigue.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions: A tetrapeptide KA4 with anti-fatigue function, the amino acid sequence of the tetrapeptide KA4 is KYPA, as shown in SEQ ID NO: 10 in the sequence listing, and it has anti-fatigue function.

[0013] Application of the aforementioned tetrapeptide KA4 with anti-fatigue function in functional foods for relieving fatigue.

[0014] Preparation method of the aforementioned tetrapeptide KA4 with anti-fatigue function, using solid-phase synthesis method, specifically: Using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier, solid-phase synthesis was carried out using the Fmoc solid-phase synthesis strategy.

[0015] The preparation method of the aforementioned tetrapeptide KA4 with anti-fatigue function adopts an enzymatic hydrolysis method. Specifically: (1) Take Pacific salmon sperm, remove impurities, add water, heat to 65 ± 2 °C, and maintain for 12 h; (2) Fish out the sperm and make a slurry, put it into a reaction kettle, add water, start the stirrer and heat up to 48 °C, add papain for enzymatic hydrolysis, continue to heat up to 57 °C, add nuclease, deaminase, alkaline protease and neutral protease for enzymatic hydrolysis for 3 h, then add flavor protease for enzymatic hydrolysis for 4 h, continue to heat up to 85 °C, and maintain for 30 min to obtain an enzymatic hydrolysate; (3) While cooling, add an aqueous solution of chitosan to the enzymatic hydrolysate. After flocculants appear, centrifuge to retain the supernatant and filter press 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 °C for 30 min, filter press again, desalt and concentrate the filtrate to obtain a concentrated solution; (5) Dry the above concentrated solution to obtain Pacific salmon sperm protein polypeptide, which contains tetrapeptide KA4.

[0016] The beneficial effects of the present invention are as follows: (1) The tetrapeptide KA4 screened from the enzymatic hydrolysate of Pacific salmon sperm in the present invention has strong binding abilities with KEAP1, AMPK and LDH, can significantly reduce the serum lactic acid content in fatigue model mice (P < 0.01), and significantly increase the load-bearing swimming time of fatigue model mice (P < 0.01). It has anti-fatigue function and can be applied to the development of functional foods for relieving fatigue; (2) The tetrapeptide KA4 provided by the present invention is screened from the enzymatic hydrolysate of Pacific salmon sperm, and has the advantages of low toxicity and side effects and high use safety compared with traditional drugs (such as traditional Chinese medicine Polygonum multiflorum); (3) Compared with the positive control American ginseng, the tetrapeptide KA4 provided by the present invention has the same effect in increasing the load-bearing swimming time and reducing the blood lactic acid content. Considering that the use dose of the positive control American ginseng is 100 mg / kg, while the use dose of tetrapeptide KA4 is only one-tenth of it (10 mg / kg), so tetrapeptide KA4 has made significant progress in anti-fatigue. Description of the Drawings

[0017] Figure 1 It is a 3D schematic diagram of the binding mode of tetrapeptide KA4 and KEAP1; Figure 2 It is a 2D schematic diagram of the binding mode of tetrapeptide KA4 and KEAP1; Figure 3 3D schematic diagram of the binding mode of tetrapeptide KA4 and AMPK; Figure 4 2D schematic diagram of the binding mode of tetrapeptide KA4 and AMPK; Figure 5 3D schematic diagram of the binding mode of tetrapeptide KA4 and LDH; Figure 6 2D schematic diagram of the binding mode of tetrapeptide KA4 and LDH; Figure 7 Effect diagram of tetrapeptide KA4 on the weight-bearing swimming time of fatigued model mice. ** indicates P < 0.01, and *** indicates P < 0.001; Figure 8 Effect diagram of tetrapeptide KA4 on the serum lactic acid content of fatigued model mice. ** indicates P < 0.01, and *** indicates P < 0.001; Figure 9 Effect diagram of tetrapeptide KA4 on the serum urea nitrogen content of fatigued model mice. *** indicates P < 0.001; Figure 10 Effect diagram of tetrapeptide KA4 on the liver glycogen content of fatigued model mice. *** indicates P < 0.001. Detailed implementation mode

[0018] The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

[0019] I. Preparation of protamine polypeptide from Pacific salmon Take Pacific salmon sperm, remove impurities, add an appropriate amount of pure water, heat to 65 ± 2 °C, and maintain for 12 h (to fully swell the testis tissue for easy extraction and subsequent impurity removal), and turn it over every 4 h during this period.

[0020] Fish sperm was fished out and beaten into pulp twice with a bone mill, then put into a reaction kettle, add pure water according to the solid-liquid ratio of 1:5, start the stirrer (rotation speed 100 rpm) and heat up to 48 °C, add papain (enzyme-substrate ratio 1.0%) and enzymolyze for 2 h, continue to heat up to 57 °C, add nuclease (enzyme-substrate ratio 0.5%), deaminase (enzyme-substrate ratio 0.3%), alkaline protease (Alcalase® 2.4L, enzyme-substrate ratio 1.0%) and neutral protease (Neutrase® 0.8L, enzyme-substrate ratio 0.5%) and enzymolyze for 3 h, then add flavor protease (Flavourzyme® 500MG, enzyme-substrate ratio 1.0%) and enzymolyze for 4 h, continue to heat up to 85 °C, and maintain for 30 min (inactivate the enzyme) to obtain an enzymolysis solution.

[0021] While cooling, add an aqueous solution of chitosan (food grade, promoting sedimentation of impurities) with a concentration of 1 wt% to the enzymatic hydrolysate. After flocculants appear, centrifuge (5000 rpm, 3 min) to obtain the supernatant, and then filter press with a plate and frame filter press at a pressure of 0.3 MPa to obtain a clear and transparent filtrate.

[0022] Put the filtrate into a reaction kettle, adjust the pH value of the filtrate to 5.50 ± 1.50, then add 1% of the total liquid volume of coconut shell activated carbon, and decolorize and deodorize at 70 ± 2 °C for 30 min. Filter press again with a plate and frame filter press, desalt the obtained filtrate with a nanofiltration system, and transfer it to a double-effect concentrator for further concentration when no salt is detected in the discharge liquid to obtain a concentrated solution.

[0023] Spray dry the concentrated solution with a spray dryer, with a feeding pressure of 18.0 MPa, a flow rate of 300 L / h, an inlet air temperature controlled at 130 °C, and an outlet air temperature controlled at 95 °C to obtain protamine polypeptide from Pacific salmon (beige powder).

[0024] II. Obtaining the sequence of the above-mentioned protamine polypeptide from Pacific salmon Detect the protamine polypeptide from Pacific salmon obtained above by LC-MS / MS, and analyze the detection results with mass spectrometry analysis software to obtain several polypeptide sequences.

[0025] The LC-MS / MS detection conditions are as follows: (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 decreases uniformly from 95% to 65%; 55 - 63 min, phase A decreases uniformly from 65% to 50%; 63 - 64 min, phase A decreases uniformly from 50% to 0; 64 - 70 min, maintain 0% phase A; (2) In the mass spectrometry method: Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific), positive ion detection mode, the first-order resolution is 120000, the AGC is set to 310, and the scanning range is 110 - 2000 m / z. The MIPS mode is peptide, select valence states 1 - 6, the second-order resolution is 17500, and the separation window is 1.6 m / z.

[0026] III. Screening for bioactive oligopeptides with a peak area ≥ 10 7 and the number of amino acids ≤ 6 From the several polypeptide sequences obtained above, 11 bioactive oligopeptides with a peak area ≥ 10 7And active oligopeptides with the number of amino acids ≤ 6, and the screening results are shown in Table 1 specifically.

[0027] Table 1 Active oligopeptides with peak area ≥ 10 in protamine polypeptide of Pacific salmon 7 And the sequences of active oligopeptides with the number of amino acids ≤ 6

[0028] IV. Screening active oligopeptides with strong binding ability to KEAP1, AMPK and LDH Using Discovery Studio software, the 11 active oligopeptides in Table 1 were respectively docked with the crystal structures of E3 ubiquitin ligase adaptor protein (KEAP1) (PDB ID: 2FLU), adenosine monophosphate-activated protein kinase (AMPK) (PDB ID: 4ZHX) and lactate dehydrogenase (LDH) (PDB ID: 7EPM). Before docking, the 2D structures of the active oligopeptides were converted into 3D structures through energy minimization, and active oligopeptides with strong binding ability to KEAP1, AMPK and LDH were screened.

[0029] The docking results are represented by the docking score (-CiE). The larger the -CiE value, the stronger the interaction between the active oligopeptide and KEAP1, AMPK and LDH, and the most likely to exhibit anti-fatigue function.

[0030] The molecular docking results of the 11 active oligopeptides with KEAP1, AMPK and LDH are shown in Table 2, Table 3 and Table 4 respectively.

[0031] Table 2 Prediction results of the interaction between 11 active oligopeptides and KEAP1

[0032] Table 3 Prediction results of the interaction between 11 active oligopeptides and AMPK

[0033] Table 4 Prediction results of the interaction between 11 active oligopeptides and LDH

[0034] It can be seen from Table 2 that among the 11 active oligopeptides, the -CiE values of DLERP, YVAAP, LGYAP and KYPA with KEAP1 all exceed 60 kcal / mol.

[0035] It can be seen from Table 3 that among the 11 active oligopeptides, the -CiE values of DLERP and KYPA with AMPK all exceed 90 kcal / mol.

[0036] As can be seen from Table 4: Among the 11 active oligopeptides, the -CiE of KYPA, LGEP, and VGVVVA with LDH all exceed 80 kcal / mol.

[0037] Based on Tables 2, 3, and 4, it is predicted that among the 11 active oligopeptides, only KYPA has strong interactions with KEAP1, AMPK, and LDH simultaneously. Therefore, KYPA (denoted as tetrapeptide KA4) was selected for further molecular docking analysis.

[0038] After analysis, the binding modes of tetrapeptide KA4 with KEAP1 are as Figure 1 and Figure 2 shown, the binding modes with AMPK are as Figure 3 and Figure 4 shown, and the binding modes with LDH are as Figure 5 and Figure 6 shown. The specific molecular docking situations are as follows: There are 9 H-H bond interactions and 3 electrostatic interactions between tetrapeptide KA4 and KEAP1, no C-H bond interactions, and 17 amino acid residues participate in the interaction between tetrapeptide KA4 and KEAP1; There are 9 H-H bond interactions, 3 C-H bond interactions, and 6 electrostatic interactions between tetrapeptide KA4 and AMPK, and 20 amino acid residues participate in the interaction between tetrapeptide KA4 and AMPK; There are 5 H-H bond interactions, 1 C-H bond interaction, and 5 electrostatic interactions between tetrapeptide KA4 and LDH, and 22 amino acid residues participate in the interaction between tetrapeptide KA4 and LDH.

[0039] V. Evaluation of the anti-fatigue function of tetrapeptide KA4 1. Solid-phase synthesis of tetrapeptide KA4 Using the Fmoc solid-phase synthesis strategy, with Fmoc-protected amino acids as raw materials and polystyrene resin as the solid-phase carrier, tetrapeptide KA4 (amino acid sequence: KYPA) was solid-phase synthesized with a purity > 90%.

[0040] 2. Animal experiment procedure Male ICR mice aged 4 - 6 weeks were used. After 1 week of adaptive feeding, they were randomly divided into 3 groups, namely: control group, positive drug group, and tetrapeptide KA4 group. The control group was intragastrically administered normal saline (200 μL) daily, the positive drug group was intragastrically administered American ginseng powder (0.1 g / kg) daily, and the tetrapeptide KA4 group was intragastrically administered solid-phase synthesized tetrapeptide KA4 (10 mg / kg) daily for 30 days. Mice in each group had free access to water and food every day. 30 minutes after the last administration of the test samples (normal saline, American ginseng powder, or tetrapeptide KA4), a lead sheet weighing 5% of the body weight was loaded at the tail root of each group of mice, and the mice were placed in a swimming tank (size 50 cm × 50 cm × 40 cm, water depth 30 cm, water temperature 25°C ± 1.0°C) for a weight-bearing swimming experiment. The time from the start of swimming to exhaustion (when the mouse sank into the water for more than 7 s was defined as exhaustion), that is, the weight-bearing swimming time of the mouse, was recorded. After the experiment, the mice were sacrificed.

[0041] 3. Sample Collection and Processing Serum: All the blood of the mice was collected by eye socket blood sampling, placed in a 4°C refrigerator for 3 h, then centrifuged at 2000 rpm for 15 min, and the upper-layer serum was aspirated with a pipette, aliquoted, and stored in an -80°C refrigerator for the determination of urea nitrogen and lactic acid contents in the serum.

[0042] Liver: After sacrificing the mice, the liver was taken, rinsed with normal saline, then blotted dry with filter paper, and 100 mg of the liver was accurately weighed for the determination of glycogen content in the liver (liver / muscle glycogen assay kit).

[0043] The statistical results of the weight-bearing swimming time of each group of mice are shown in Figure 7 . It can be seen from Figure 7 that: compared with the weight-bearing swimming time of the control group mice (308.2 ± 21.1 s), the weight-bearing swimming time of the positive drug group mice (452.1 ± 26.4 s) was significantly increased (p < 0.001), and the weight-bearing swimming time of the tetrapeptide KA4 group mice (400.4 ± 21.1 s) was also significantly increased (p < 0.01).

[0044] The detection results of the lactic acid content in the serum of each group of mice are shown in Figure 8 . It can be seen from Figure 8 that: compared with the lactic acid content in the serum of the control group mice (8.79 ± 0.33 mM), the lactic acid content in the serum of the positive drug group mice (6.69 ± 0.41 mM) was significantly decreased (p < 0.001), and the lactic acid content in the serum of the tetrapeptide KA4 group mice (7.14 ± 0.39 mM) was also significantly decreased (p < 0.01).

[0045] The detection results of the urea nitrogen content in the serum of each group of mice are shown in Figure 9 . It can be seen from Figure 9It can be seen that: compared with the serum urea nitrogen content of the mice in the control group (8.35 ± 0.49 mM), the serum urea nitrogen content of the mice in the positive drug group (5.2 ± 0.38 mM) was significantly decreased (p < 0.001), and there was no significant change in the serum urea nitrogen content of the mice in the tetrapeptide KA4 group (7.69 ± 0.34 mM) (p > 0.05).

[0046] The detection results of the liver glycogen content of the mice in each group are shown in Figure 10 . From Figure 10 It can be seen that: compared with the liver glycogen content of the mice in the control group (16.4 ± 1.02 mg / g), the liver glycogen content of the mice in the positive drug group (24.8 ± 1.3 mg / g) was significantly increased (p < 0.001), and there was no significant change in the liver glycogen content of the mice in the tetrapeptide KA4 group (17.94 ± 0.78 mM) (p > 0.05).

[0047] In summary, the active oligopeptide KYPA (tetrapeptide KA4) screened from the enzymatic hydrolysate of Pacific salmon protamine by the present invention has strong interactions with KEAP1, AMPK and LDH at the same time. Through animal experiments, it was found that this tetrapeptide KA4 can significantly reduce the serum lactic acid content of the fatigue model mice (p < 0.01), significantly increase the weight-bearing swimming time of the fatigue model mice (p < 0.01), has the function of anti-fatigue, and can be applied to functional foods for relieving fatigue.

[0048] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solution of the present invention still fall within the protection scope 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 listing, and it has the function of anti-fatigue.

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

3. The preparation method of the tetrapeptide KA4 with anti-fatigue function according to claim 1, characterized in that, Using the solid-phase synthesis method, specifically: Using Fmoc-protected amino acids as raw materials and polystyrene resin as the solid-phase carrier, solid-phase synthesis is carried out using the Fmoc solid-phase synthesis strategy.

4. The preparation method of the tetrapeptide KA4 with anti-fatigue function according to claim 1, characterized in that, Using the enzymatic hydrolysis method, specifically: (1) Take Pacific salmon sperm, remove impurities, add water, heat to 65 ± 2 °C, and maintain for 12 h; (2) Fish out the sperm and make a slurry, put it into a reaction kettle, add water, start the stirrer and heat up to 48 °C, add papain for enzymatic hydrolysis for 2 h, continue to heat up to 57 °C, add nuclease, deaminase, alkaline protease and neutral protease for enzymatic hydrolysis for 3 h, then add flavor protease for enzymatic hydrolysis for 4 h, continue to heat up to 85 °C, and maintain for 30 min to obtain an enzymatic hydrolysate; (3) While cooling, add an aqueous solution of chitosan to the enzymatic hydrolysate. After flocculants appear, centrifuge to retain the supernatant and filter press 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 °C for 30 min, filter press again, desalt and concentrate the filtrate to obtain a concentrated solution; (5) Dry the above concentrated solution to obtain Pacific salmon protamine polypeptide, which contains the tetrapeptide KA4.

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