Hexapeptide VA6 with anti-fatigue function as well as preparation method and application thereof

Abstract

The invention discloses a hexapeptide VA6 with an anti-fatigue function as well as a preparation method and application thereof, and belongs to the technical field of biology. The hexapeptide VA6 is screened from pacific salmon protamine enzymatic hydrolysate, the amino acid sequence is VGVVVA, the binding capacity with AMPK and LDH is high, the liver glycogen content of a fatigue model mouse can be remarkably increased, the serum urea nitrogen content of the fatigue model mouse can be remarkably reduced, the fatigue phenotype of the fatigue model mouse can be remarkably relieved, and the load swimming time of the fatigue model mouse can be remarkably prolonged; the food 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 hexapeptide VA6 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 phenomenon, characterized by exhaustion and decreased energy levels, and is usually divided into central or peripheral. Central fatigue refers to an adverse mental state caused by long-term stress on the central nervous system or blocked signal conduction of peripheral motor neurons. In contrast, peripheral fatigue is a physiological condition resulting from decreased muscle function due to overtraining. Usually, rest can help relieve normal fatigue symptoms. However, insufficient fatigue relief can damage the immune system and lead to the onset of various diseases, such as cancer, anemia, karoshi, and chronic fatigue syndrome, having a negative impact on human health.

[0003] The existing research on anti-fatigue active substances has been carried out from three main mechanisms of action: (1) Participating in metabolism to reduce the levels of blood lactic acid and blood urea nitrogen; (2) Enhancing glucose homeostasis to increase glycogenolysis and energy supply; (3) Alleviating the harm caused by excessive reactive oxygen species levels by restoring oxidative stress-induced damage.

[0004] Among them, AMP-activated protein kinase (AMPK) is a sensor of the cellular energy state, and lactate dehydrogenase (LDH) is a key enzyme for blood lactic acid metabolism. Currently, the clinical options for treating fatigue are limited. Active peptides derived from food protease hydrolysis have the advantages of high specificity, low toxicity and side effects, good curative effect, and larger available drug doses, and are a good strategy for relieving fatigue.

[0005] Protein raw materials from different sources such as animals and plants will obtain different enzyme hydrolysates in different enzymatic hydrolysis processes. The enzyme hydrolysate is a collection of active peptides with diverse structures, sequences, and functions. The amino acid compositions and structures of active peptides with anti-fatigue functions vary greatly, and there is no fixed or unified amino acid composition. The components of food protease hydrolysis products are quite complex, and even when using the same protein raw material, different product components will be produced due to different enzymatic hydrolysis processes. Therefore, the strategy of screening active peptides with anti-fatigue functions from enzyme hydrolysates obtained from different protein sources and different preparation processes has specificity.

[0006] The article "Anti-fatigue effect of Japanese seabass peptides in miceand the underlying action mechanism via in vitro andin vivo "Assays》 (Chen Li, Lichan Li, Jing Cheng, Xu Chen, Yi Yuan, Mohamed A. Farag, Baocai Xu, Xixi Cai, Shaoyun Wang, Food Bioscience, Volume 58, 2024, 103763, ISSN 2212-4292) reported that the enzymatically hydrolyzed solution of perch played an anti-fatigue role in improving energy metabolism and reducing oxidative stress in a fatigued mouse model, but did not further complete the identification of its active components and their sequences. The article "Anti-fatigue activity of sea cucumber peptides prepared from Japanese sea cucumber "in an endurance swimming rat model》 (Ye J, Shen C, Huang Y, Zhang X, Xiao M, J Sci Food Agric, 2017 Oct;97(13):4548-4556) reported that the enzymatically hydrolyzed solution of sea cucumber rich in glycine, glutamate and proline had anti-fatigue activity, and might play an anti-fatigue role by normalizing energy metabolism and reducing oxidative damage and inflammatory responses. The article "Study on the anti-fatigue effect and mechanism of sea cucumber peptides》 (Yu Yihao, Journal of Jiangnan University, 2021) reported that the enzymatically hydrolyzed solution of sea cucumber body wall played an anti-fatigue role by promoting fat catabolism, promoting gluconeogenesis and enhancing mitochondrial energy supply, and further focused on the functional peptides with the sequences of G(Hyp)LQADY and FD(Hyp)GA. The article "Preparation of antioxidant peptides from tilapia and their anti-fatigue efficacy》 (Peng Shuo, Journal of Guangdong Ocean University, 2022) reported that the enzymatically hydrolyzed solution of tilapia significantly prolonged the exhaustive swimming time of mice, significantly reduced the contents of lactic acid, blood urea nitrogen and malondialdehyde, and further focused on the functional peptides with the sequences of HHC and PCCVT. At the same time, the article "Natural bioactive peptides to beat exercise-induced fatigue: A review》 (Peixin Wang, Dehua Wang, Jiamiao Hu, Bee K. Tan, Yi Zhang, Shaoling Lin, Food Bioscience, Volume 43, 2021, 101298, ISSN 2212-4292) systematically summarized the preparation methods of enzymatically hydrolyzed solutions with anti-fatigue activity discovered in recent years and the structures of active peptides isolated from them.

[0007] The above reports do not mention active peptides with the same or similar structures as the active peptides of the present invention that have anti-fatigue functions. In addition, if modifications are to be made based on the reported active peptide sequences, the amount of data will increase exponentially, making it impractical, as there are 20 common amino acids to choose from at each amino acid position.

[0008] 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 Chum salmon 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

[0009] 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.

[0010] In order to achieve the above objectives, the present invention adopts the following technical solutions: A hexapeptide VA6 with anti-fatigue function, wherein the amino acid sequence of the hexapeptide VA6 is VGVVVA, as shown in SEQ ID NO: 3 in the sequence table, and has anti-fatigue function.

[0011] The application of the aforementioned hexapeptide VA6 with anti-fatigue function in fatigue-relieving functional foods.

[0012] The preparation method of the aforementioned hexapeptide VA6 with anti-fatigue function adopts a solid phase synthesis method, 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.

[0013] The preparation method of the aforementioned hexapeptide VA6 with anti-fatigue function adopts an enzymatic hydrolysis method, 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, and 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 protamine polypeptide from Pacific salmon, which contains a relatively large amount of hexapeptide VA6.

[0014] The beneficial effects of the present invention are as follows: (1) The hexapeptide VA6 screened from the enzymatic hydrolysate of Pacific salmon protamine in the present invention has a strong binding ability to AMPK and LDH, can significantly increase the liver glycogen content in fatigued model mice (P < 0.05), significantly reduce the serum urea nitrogen content in fatigued model mice (P < 0.01), relieve the fatigue phenotype of fatigued model mice, and significantly increase the load-bearing swimming time of fatigued model mice (P < 0.001). It has an anti-fatigue function and can be applied to the development of functional foods for relieving fatigue; (2) The hexapeptide VA6 provided by the present invention is screened from the enzymatic hydrolysate of Pacific salmon protamine, 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 hexapeptide VA6 provided by the present invention has the same effect in increasing the load-bearing swimming time and reducing the blood urea nitrogen content. Considering that the dosage of the positive control American ginseng is 100 mg / kg, while the dosage of the hexapeptide VA6 is only one-tenth of it (10 mg / kg), the hexapeptide VA6 has made significant progress in anti-fatigue. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a 2D schematic diagram of the binding mode of hexapeptide VA6 to AMPK; Figure 2 It is a 2D schematic diagram of the binding mode of hexapeptide VA6 to LDH; Figure 3 It is a diagram showing the effect of hexapeptide VA6 on the load-bearing swimming time of fatigued model mice, *** indicates P < 0.001; Figure 4 It is a diagram showing the effect of hexapeptide VA6 on the serum lactic acid content of fatigued model mice, *** indicates P < 0.001; Figure 5 It is a diagram showing the effect of hexapeptide VA6 on the serum urea nitrogen content of fatigued model mice, ** indicates P < 0.01, *** indicates P < 0.001; Figure 6 It is a diagram showing the effect of hexapeptide VA6 on the liver glycogen content of fatigued model mice, * indicates P < 0.05, *** indicates P < 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

[0017] I. Preparation of protamine polypeptide from Pacific salmon Take Pacific salmon milt, 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.

[0018] Fish out the milt and beat it into pulp twice with a bone mill, then put it 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 flavour 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 enzymolyzed solution.

[0019] While cooling, add an aqueous solution of deacetylated chitin with a concentration of 1 wt% (food grade, to promote the sedimentation of impurities) to the enzymolyzed solution. After floccules appear, centrifuge (5000 rpm, 3 min) and retain the supernatant, and perform pressure filtration with a plate and frame filter press at a pressure of 0.3 MPa to obtain a clear and transparent filtrate.

[0020] 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, decolorize and deodorize at 70 ± 2 °C for 30 min, and perform pressure filtration again with a plate and frame filter press. The obtained filtrate is desalted by a nanofiltration system, and when no salt is detected in the discharged liquid, it is transferred to a double-effect concentrator for further concentration to obtain a concentrated solution.

[0021] Perform spray drying on the concentrated solution with a spray dryer. The feeding pressure is 18.0 MPa, the flow rate is 300 L / h, the inlet air temperature is controlled at 130 °C, and the outlet air temperature is controlled at 95 °C to obtain protamine polypeptide from Pacific salmon (beige powder).

[0022] II. Obtaining the sequence of the above-mentioned protamine polypeptide from Pacific salmon Use LC-MS / MS to detect the protamine polypeptide from Pacific salmon obtained above, and analyze the detection results with mass spectrometry analysis software to obtain several polypeptide sequences.

[0023] The LC-MS / MS detection conditions are as follows: (1) In the liquid phase method: The chromatographic column was C18, 3 μm, 250 mm × 75 μm (Eksigent), phase A was water with 0.1% formic acid; phase B was acetonitrile with 0.1% formic acid, the flow rate was 300 nL / min, the injection volume was 1 μL, and the chromatographic gradient was 70 min. The specific elution gradient was as follows: from 0 - 55 min, phase A decreased uniformly from 95% to 65%; from 55 - 63 min, phase A decreased uniformly from 65% to 50%; from 63 - 64 min, phase A decreased uniformly from 50% to 0; from 64 - 70 min, 0% phase A was maintained. (2) In the mass spectrometry method: Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific), positive ion detection mode, the primary resolution was 120,000, the AGC was set to 310, and the scanning range was 110 - 2000 m / z. The MIPS mode was peptide, the selected valence states were 1 - 6, the secondary resolution was 17,500, and the separation window was 1.6 m / z.

[0024] III. Screening for bioactive oligopeptides with peak area ≥ 10 7 and amino acid number ≤ 6 From the several polypeptide sequences obtained above, 11 bioactive oligopeptides with peak area ≥ 10 7 and amino acid number ≤ 6 were finally screened out. The screening results are shown in Table 1.

[0025] Table 1 Bioactive oligopeptide sequences in protamine polypeptides of Pacific salmon with peak area ≥ 10 7 and amino acid number ≤ 6

[0026] IV. Screening for bioactive oligopeptides with strong binding ability to AMPK and LDH Using Discovery Studio software, the 11 bioactive oligopeptides in Table 1 were respectively subjected to molecular docking with the crystal structure of AMPK (PDB ID: 4ZHX) and the crystal structure of LDH (PDB ID: 7EPM). Before docking, the 2D structure of the bioactive oligopeptide was converted to a 3D structure by energy minimization, and bioactive oligopeptides with strong binding ability to AMPK and LDH were screened out.

[0027] The docking results are represented by the docking score (-CiE). The larger the -CiE value, the stronger the interaction between the bioactive oligopeptide and AMPK and LDH, and the most likely to exhibit anti - fatigue function.

[0028] The molecular docking results of the 11 bioactive oligopeptides with AMPK and LDH are shown in Table 2 and Table 3 respectively.

[0029] Table 2 Prediction results of the interaction between 11 bioactive oligopeptides and AMPK

[0030] Table 3 Prediction results of the interaction between 11 bioactive oligopeptides and LDH

[0031] As can be seen from Table 2: Among the 11 bioactive oligopeptides, the -CiE of DLERP, KYPA, VGVVVA, LGYAP, and YVAAP with AMPK all exceeded 80 kcal / mol.

[0032] As can be seen from Table 3: Among the 11 bioactive oligopeptides, the -CiE of KYPA, LGEP, and VGVVVA with LDH all exceeded 80 kcal / mol.

[0033] It is predicted that KYPA interacts with both AMPK and LDH, and VGVVVA also interacts with both AMPK and LDH.

[0034] As can be seen from Table 1: The peak area of VGVVVA (18.3×10 7 ) is greater than the peak area of KYPA (1.2×10 7 ). Therefore, VGVVVA (denoted as hexapeptide VA6) was selected for further molecular docking analysis.

[0035] After analysis, the binding mode of hexapeptide VA6 with AMPK is as Figure 1 shown, and the binding mode with LDH is as Figure 2 shown. The specific molecular docking situation is as follows: There are 11 H-H bond interactions, 2 C-H bond interactions, and 3 electrostatic interactions formed between hexapeptide VA6 and AMPK. 15 amino acid residues are involved in the interaction between hexapeptide VA6 and AMPK, and the van der Waals force between them is -16.09 kcal / mol; There are 9 H-H bond interactions, 3 C-H bond interactions, and 6 electrostatic interactions formed between hexapeptide VA6 and LDH. 23 amino acid residues are involved in the interaction between hexapeptide VA6 and LDH, and the van der Waals force between them is -5.81 kcal / mol.

[0036] V. Evaluation of the anti-fatigue function of hexapeptide VA6 1. Solid-phase synthesis of hexapeptide VA6 Using the Fmoc solid-phase synthesis strategy, with Fmoc-protected amino acids as raw materials and polystyrene resin as the solid-phase carrier, hexapeptide VA6 (amino acid sequence: VGVVVA) was solid-phase synthesized with a purity >90%.

[0037] 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: control group, positive drug group, and hexapeptide VA6 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 hexapeptide VA6 group was intragastrically administered solid-phase synthesized hexapeptide VA6 (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 hexapeptide VA6), a lead sheet weighing 5% of the body weight was loaded at the root of the tail of each group of mice. 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 the 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.

[0038] 3. Sample Collection and Processing Serum: All the blood of the mice was collected by orbital blood sampling, placed in a 4°C refrigerator for 3 h, then centrifuged at 2000 rpm for 15 min. 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.

[0039] Liver: After sacrificing the mice, the liver was taken, rinsed with normal saline, then blotted dry with filter paper. Exactly 100 mg of the liver was weighed accurately for the determination of glycogen content in the liver (liver / muscle glycogen assay kit).

[0040] The statistical results of the weight-bearing swimming time of each group of mice are shown in Figure 3 . It can be seen from Figure 3 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 hexapeptide VA6 group mice (426.2 ± 31.0 s) was also significantly increased (p < 0.001).

[0041] The detection results of the serum lactic acid content of each group of mice are shown in Figure 4 . It can be seen from Figure 4 that: compared with the serum lactic acid content of the control group mice (8.79 ± 0.33 mM), the serum lactic acid content of the positive drug group mice (6.69 ± 0.41 mM) was significantly decreased (p < 0.001), and the serum lactic acid content of the hexapeptide VA6 group mice (8.19 ± 0.31 mM) decreased, but the difference was not significant.

[0042] The detection results of the serum urea nitrogen content of each group of mice are shown in Figure 5 . It can be seen from Figure 5It was found that: compared with the serum urea nitrogen content of the control group mice (8.35 ± 0.49 mM), the serum urea nitrogen content of the positive drug group mice (5.2 ± 0.38 mM) was significantly decreased (p < 0.001), and the serum urea nitrogen content of the hexapeptide VA6 group mice (5.75 ± 0.37 mM) was also significantly decreased (p < 0.01).

[0043] The detection results of the liver glycogen content of each group of mice are shown in Figure 6 . It was found from Figure 6 that: compared with the liver glycogen content of the control group mice (16.4 ± 1.02 mg / g), the liver glycogen content of the positive drug group mice (24.8 ± 1.3 mg / g) was significantly increased (p < 0.001), and the liver glycogen content of the hexapeptide VA6 group mice (19.5 ± 1.14 mg / g) was also significantly increased (p < 0.05).

[0044] In summary, the bioactive oligopeptide VGVVVA (hexapeptide VA6) screened from the enzymatic hydrolysate of Pacific salmon protamine can significantly reduce the serum urea nitrogen content of fatigue model mice, increase the liver glycogen content of fatigue model mice, significantly increase the weight-bearing swimming time of fatigue model mice, relieve the fatigue phenotype of fatigue model mice, has an anti-fatigue function, and can be applied to functional foods for relieving fatigue.

[0045] It should be noted that the above examples are merely illustrations for clearly explaining the present invention and are not 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 based on the above description. It is impossible to enumerate 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 hexapeptide VA6 with anti-fatigue function, characterized in that, The amino acid sequence of the hexapeptide VA6 is VGVVVA, as shown in SEQ ID NO: 3 in the sequence listing, and it has the function of anti-fatigue.

2. Use of the hexapeptide VA6 with anti-fatigue function described in claim 1 in a functional food for relieving fatigue.

3. The preparation method of the hexapeptide VA6 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 hexapeptide VA6 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 pulp, put it into a reaction kettle, add water, start the stirrer and raise the temperature to 48 °C, add papain for enzymatic hydrolysis for 2 h, continue to raise the temperature 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 raise the temperature 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 a relatively large amount of hexapeptide VA6.

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