Hexapeptide VA6 with anti-fatigue function and its preparation method and application

Abstract

The present invention discloses a hexapeptide VA6 with anti-fatigue function, as well as its preparation method and application, belonging to the field of biotechnology. The hexapeptide VA6, screened from Pacific salmon sperm protein hydrolysate, has an amino acid sequence of VGVVVA and strong binding ability to AMPK and LDH. It can significantly increase the liver glycogen content and significantly reduce the serum urea nitrogen content in fatigue model mice, alleviate the fatigue phenotype of fatigue model mice, and significantly increase the weighted swimming time of fatigue model mice. It has anti-fatigue function and can be used in the development of functional foods that relieve 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 hexapeptide VA6 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 phenomenon characterized by exhaustion and decreased energy levels, and is generally categorized as either central or peripheral. Central fatigue refers to an adverse mental state caused by chronic stress on the central nervous system or blocked signaling in peripheral motor neurons. In contrast, peripheral fatigue is a physiological condition resulting from decreased muscle function due to overtraining. Rest can typically help alleviate normal fatigue symptoms. However, inadequate fatigue relief can impair the immune system and contribute to the onset of various diseases, such as cancer, anemia, karoshi (death from overwork), and chronic fatigue syndrome, negatively impacting human health.

[0003] Existing research on anti-fatigue active substances has been carried out from three main mechanisms of action:

[0004] (1) Participate in metabolism to reduce blood lactate and blood urea nitrogen levels;

[0005] (2) Enhance glucose homeostasis to increase glycogenolysis and energy supply;

[0006] (3) Alleviate the harm caused by excessive reactive oxygen species levels by restoring oxidative stress-induced damage.

[0007] Among them, adenosine monophosphate-activated protein kinase (AMPK) is a sensor of cellular energy status, and lactate dehydrogenase (LDH) is a key enzyme in blood lactate metabolism. Currently, clinical options for treating fatigue are limited. Active peptides derived from the hydrolysis of food-derived proteins offer advantages such as high specificity, minimal toxicity and side effects, excellent efficacy, and a higher dose, making them a promising strategy for alleviating fatigue.

[0008] Protein raw materials from different sources, such as animals and plants, will produce different hydrolysates through different enzymatic hydrolysis processes. Enzymatic hydrolysates are a collection of active peptides with different structures, diverse sequences, and functional emphases. The amino acid composition and structure of active peptides with anti-fatigue function vary greatly, and there is no fixed or unified amino acid composition. The composition of food protein hydrolysis products is quite complex. Even if the same protein raw material is used, different product components will be produced due to different enzymatic hydrolysis processes. Therefore, the strategy of screening active peptides with anti-fatigue function from enzymatic hydrolysates obtained from different protein sources and different preparation processes is specific.

[0009] The article "Anti-fatigue effect of Lateolabrax japonicuspeptides in mice and the underlying action mechanism via in vitro and in 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 sea cucumber hydrolysate improved energy metabolism and reduced oxidative stress in a fatigue mouse model to exert anti-fatigue effects, but the identification of the active components and their sequences was not completed. Stichopus japonicusin an endurance swimming ratmodel”(Ye J, Shen C, Huang Y, Zhang X, Xiao M, J Sci Food Agric, 2017 Oct;97(13):4548-4556) reported that sea cucumber hydrolysate rich in glycine, glutamate and proline has anti-fatigue activity, which may play an anti-fatigue role by normalizing energy metabolism and reducing oxidative damage and inflammatory response. The article "Study on the Anti-fatigue Effect and Mechanism of Sea Cucumber Peptides" (Yu Yihao, Journal of Jiangnan University, 2021) reported that sea cucumber body wall hydrolysate plays an anti-fatigue role by promoting fat decomposition metabolism, 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 Tilapia Antioxidant Peptides and Their Anti-fatigue Efficacy" (Peng Shuo, Journal of Guangdong Ocean University, 2022) reported that tilapia enzymatic hydrolysates significantly prolonged exhaustive swimming time in mice and significantly reduced lactate, blood urea nitrogen, and malondialdehyde levels, further focusing on functional peptides with the sequences HHC and PCCVT. Meanwhile, 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, FoodBioscience, Volume 43, 2021, 101298, ISSN 2212-4292) systematically summarizes the preparation methods of enzymatic hydrolysates with anti-fatigue activity discovered in recent years and the structures of active peptides isolated from them.

[0010] 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.

[0011] 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

[0012] 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.

[0013] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0014] 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.

[0015] The application of the aforementioned hexapeptide VA6 with anti-fatigue function in fatigue-relieving functional foods.

[0016] The preparation method of the aforementioned hexapeptide VA6 with anti-fatigue function adopts a solid phase synthesis method, specifically:

[0017] 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.

[0018] The preparation method of the aforementioned hexapeptide VA6 with anti-fatigue function adopts an enzymatic hydrolysis method, specifically:

[0019] (1) Take Pacific salmon sperm, remove impurities, add water, and heat to 65±2℃ for 12 hours;

[0020] (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;

[0021] (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;

[0022] (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;

[0023] (5) The concentrated liquid is dried to obtain Pacific salmon protamine polypeptide, which contains a large amount of hexapeptide VA6.

[0024] The present invention is beneficial in that:

[0025] (1) The hexapeptide VA6 obtained from the proteolytic solution of Pacific salmon sperm has a strong ability to bind to AMPK and LDH, and can significantly increase the liver glycogen content of fatigue model mice (P<0.05), significantly reduce the serum urea nitrogen content of fatigue model mice (P<0.01), alleviate the fatigue phenotype of fatigue model mice, and significantly increase the weighted swimming time of fatigue model mice (P<0.001). It has anti-fatigue function and can be used to develop functional foods that relieve fatigue;

[0026] (2) The hexapeptide VA6 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;

[0027] (3) The hexapeptide VA6 provided by the present invention has comparable effects on increasing weighted swimming time and reducing blood urea nitrogen 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 hexapeptide VA6 is only one tenth of that (10 mg / kg), hexapeptide VA6 has a significant improvement in anti-fatigue. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 2D schematic diagram of the binding mode of hexapeptide VA6 and AMPK;

[0029] Figure 2 2D schematic diagram of the binding mode of hexapeptide VA6 and LDH;

[0030] Figure 3 This is the effect of hexapeptide VA6 on the weighted swimming time of fatigue model mice. *** indicates P < 0.001;

[0031] Figure 4 This is the effect of hexapeptide VA6 on serum lactate content in fatigue model mice, *** indicates P < 0.001;

[0032] Figure 5 This is the effect of hexapeptide VA6 on the serum urea nitrogen content in fatigue model mice, ** indicates P < 0.01, *** indicates P < 0.001;

[0033] Figure 6 This is a graph showing the effect of hexapeptide VA6 on liver glycogen content in fatigue model mice. * indicates P < 0.05, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] 1. Preparation of Pacific salmon protamine polypeptide

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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).

[0041] 2. Obtaining the Sequence of the Pacific Salmon Protamine Polypeptide

[0042] The Pacific salmon protamine polypeptides obtained above were detected by LC-MS / MS, and the detection results were analyzed by mass spectrometry analysis software to obtain several polypeptide sequences.

[0043] LC-MS / MS detection conditions are:

[0044] (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;

[0045] (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.

[0046] 3. Screening peak area ≥10 7 Active oligopeptides with amino acid number ≤ 6

[0047] 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.

[0048] Table 1 Peak area ≥10 in Pacific salmon protamine peptides 7 Active oligopeptide sequence with amino acid number ≤ 6

[0049]

[0050] 4. Screening of active oligopeptides with strong binding ability to AMPK and LDH

[0051] The 11 active oligopeptides in Table 1 were molecularly docked with the AMPK crystal structure (PDB ID: 4ZHX) and the LDH crystal structure (PDB ID: 7EPM) using Discovery Studio software. 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 AMPK and LDH were screened.

[0052] The docking results are expressed as docking scores (-CiE). The larger the -CiE value, the stronger the interaction between the active oligopeptide and AMPK and LDH, and the more likely it is to exhibit anti-fatigue function.

[0053] The molecular docking results of 11 active oligopeptides with AMPK and LDH are shown in Tables 2 and 3, respectively.

[0054] Table 2 Prediction results of the interaction between 11 active oligopeptides and AMPK

[0055]

[0056] Table 3 Prediction results of the interaction between 11 active oligopeptides and LDH

[0057]

[0058] As shown in Table 2, among the 11 active oligopeptides, the -CiE of DLERP, KYPA, VGVVVA, LGYAP and YVAAP with AMPK all exceeded 80 kcal / mol.

[0059] It can be seen from Table 3 that among the 11 active oligopeptides, the -CiE of KYPA, LGEP and VGVVVA with LDH all exceeded 80 kcal / mol.

[0060] It is predicted that KYPA interacts with both AMPK and LDH, and VGVVVA also interacts with both AMPK and LDH.

[0061] From Table 1, we can see that the peak area of ​​VGVVVA (18.3×10 7 ) is larger than the peak area of ​​KYPA (1.2×10 7 Therefore, VGVVVA (denoted as hexapeptide VA6) was selected for further molecular docking analysis.

[0062] After analysis, the binding mode of hexapeptide VA6 and AMPK is as follows Figure 1 The binding mode with LDH is shown in Figure 2 The molecular docking situation is as follows:

[0063] There are 11 HH bond interactions, 2 C-H bond interactions, and 3 electrostatic interactions between the hexapeptide VA6 and AMPK. Fifteen amino acid residues are involved in the interaction between the hexapeptide VA6 and AMPK, and the van der Waals force between the two is -16.09 kcal / mol.

[0064] There are 9 HH bond interactions, 3 CH bond interactions, and 6 electrostatic interactions between the hexapeptide VA6 and LDH. There are 23 amino acid residues involved in the interaction between the hexapeptide VA6 and LDH, and the van der Waals force between the two is -5.81 kcal / mol.

[0065] 5. Evaluation of the anti-fatigue function of hexapeptide VA6

[0066] 1. Solid phase synthesis of hexapeptide VA6

[0067] The hexapeptide VA6 (amino acid sequence: VGVVVA) was synthesized in the solid phase using the Fmoc solid phase synthesis strategy with Fmoc-protected amino acids as raw materials and polystyrene resin as the solid phase support. The purity was >90%.

[0068] 2. Animal Experiment Process

[0069] 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 hexapeptide VA6 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 hexapeptide VA6 group received daily oral administration of solid-phase synthesized hexapeptide VA6 (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 hexapeptide VA6), 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.

[0070] 3. Sample collection and processing

[0071] 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.

[0072] 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).

[0073] The statistical results of the weight-bearing swimming time of each group of mice are shown in Figure 3 .Depend on Figure 3 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 hexapeptide VA6 group (426.2±31.0s) was also significantly increased (p<0.001).

[0074] The results of serum lactate content in each group of mice are shown in Figure 4 .Depend on Figure 4It 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 hexapeptide VA6 group (8.19±0.31mM) was reduced, but the difference was not significant.

[0075] The results of serum urea nitrogen content in each group of mice are shown in Figure 5 .Depend on Figure 5 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 hexapeptide VA6 group (5.75±0.37mM) was also significantly reduced (p<0.01).

[0076] The results of liver glycogen content test of mice in each group are shown in Figure 6 .Depend on Figure 6 It can be seen that compared with the liver glycogen content of mice in the control group (16.4±1.02 mg / g), the liver glycogen content of mice in the positive drug group (24.8±1.3 mg / g) was significantly increased (p<0.001), and the liver glycogen content of mice in the hexapeptide VA6 group (19.5±1.14 mg / g) was also significantly increased (p<0.05).

[0077] In summary, the active oligopeptide VGVVVA (hexapeptide VA6) obtained by screening from the protease hydrolysate of Pacific salmon sperm can significantly reduce the serum urea nitrogen content of fatigue model mice, increase the liver glycogen content of fatigue model mice, significantly increase the weighted swimming time of fatigue model mice, and alleviate the fatigue phenotype of fatigue model mice. It has anti-fatigue function and can be used in functional foods for relieving fatigue.

[0078] 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 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 table, and has anti-fatigue function.

2. Use of the hexapeptide VA6 with anti-fatigue function according to claim 1 in the preparation of functional foods for relieving fatigue.

3. The method for preparing the hexapeptide VA6 with anti-fatigue function according to 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.

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