Yak blood anti-fatigue polypeptide, screening method and application thereof

Abstract

The application belongs to the technical field of polypeptidomics, and particularly relates to a kind of anti-fatigue polypeptide in yak blood and its screening method and application, and the application of anti-fatigue product is specifically health care product, medicine, and the screening method of anti-fatigue polypeptide includes sequence identification of extracted wild yak blood polypeptide mixture;The polypeptide sequence with higher score identified is subjected to functional polypeptide sequence screening;The polypeptide sequence screened is subjected to target polypeptide sequence synthesis by using biochemical method and detection, and the synthesized polypeptide is subjected to antioxidant detection and anti-fatigue function verification of polypeptide KD-8, which shows that the polypeptide KD-8 provided by the application has significant anti-fatigue effect.

Description

Technical Field

[0001] This invention belongs to the field of peptide omics technology, specifically relating to an anti-fatigue peptide in yak blood, its screening method, and its application. Background Technology

[0002] Peptides are compounds formed by α-amino acids linked together in polypeptide chains, and they are also intermediate products of protein hydrolysis. All endogenous polypeptide components in organisms, including polypeptides produced by protein degradation or from non-coding regions, typically have a molecular weight below 5 kDa. Some polypeptides (bioactive peptides) play important roles in the growth, development, and metabolism of organisms, such as smegglutinin used to treat type 2 diabetes and endorphins with analgesic effects. Some peptides also have flavor-enhancing properties, such as umami peptides.

[0003] Currently, there has been no progress on the peptide KD-8, which has anti-fatigue effects, or its related applications. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a polypeptide extracted from yak blood, specifically polypeptide KD-8, whose amino acid sequence is shown in SEQ ID NO.1.

[0005] The present invention also provides the application of polypeptides extracted from yak blood in the preparation of anti-fatigue products, specifically health products and pharmaceuticals.

[0006] This invention also provides a method for screening anti-fatigue peptides in yak blood, comprising:

[0007] S1. Sequence identification of the extracted wild yak blood polypeptide mixture;

[0008] S2. Screen the polypeptide sequences with high identification scores in step S1 for functional polypeptide sequences.

[0009] S3. The peptide sequences screened in step S2 are used to synthesize target peptide sequences using biochemical methods and then detected. The target peptide sequence includes the peptide KD-8 described in claim 1, and its amino acid sequence is shown in SEQ ID NO.1.

[0010] Furthermore, in step S3, the target polypeptide sequence is specifically polypeptide KD-8, whose amino acid sequence is shown in SEQ ID NO.1; polypeptide PD-11, whose amino acid sequence is shown in SEQ ID NO.2; polypeptide LG-24, whose amino acid sequence is shown in SEQ ID NO.3; and polypeptide LK-23, whose amino acid sequence is shown in SEQ ID NO.4.

[0011] Furthermore, the sequence identification in step S1 is achieved by reducing alkylation, desalting, and liquid chromatography-tandem mass spectrometry of the polypeptide mixture.

[0012] Furthermore, the process of screening functional polypeptide sequences in step S2 is as follows: first, the polypeptide sequences with higher identification scores in step S1 are scored and ranked for significance; then, the polypeptide sequences with higher significance scores are selected, their antioxidant amino acid ratios are calculated and ranked; and the selected polypeptide sequences are queried in the database for valid functional polypeptide sequences.

[0013] Furthermore, in step S3, the chemical synthesis employs liquid-phase peptide synthesis and solid-phase peptide synthesis. The antioxidant activities of the synthesized peptides KD-8, PD-11, LG-24, and LK-23 are detected and sorted. Then, the peptide sequence with stronger antioxidant activity is selected for in vivo verification of anti-fatigue function in mice.

[0014] Furthermore, the antioxidant activity was detected by a DPPH antioxidant experiment, in which the antioxidant effects, from strongest to weakest, were: peptide LK-23, peptide KD-8, peptide LG-24, and peptide PD-11.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects:

[0016] The peptide KD-8 sequence provided by this invention is derived from yak blood and has antioxidant and anti-fatigue functional activities, with a variety of potential applications, including but not limited to pharmaceuticals or health products. Attached Figure Description

[0017] Figure 1 The graphs show the comparison of mouse weight gain during the verification of the anti-fatigue function of the polypeptide mixture provided in Example 2 of the present invention. (a) is a graph showing the trend of weight fluctuation; (b) is a graph showing the change in the rate of weight gain.

[0018] Figure 2 The graphs shown in Example 2 of this invention are evaluation charts of anti-fatigue indicators in mice during the verification of the anti-fatigue function of the polypeptide mixture. (a) The graph represents the time to exhaustion during swimming; (b) The graph represents the blood lactate content; (c) The graph represents the serum urea nitrogen; (d) The graph represents the liver glycogen content; (e) The graph represents the muscle glycogen content; the symbol " "ns" indicates a statistically significant difference compared to the blank control group (CK); "ns" indicates no statistically significant difference compared to the blank control group (CK).

[0019] Figure 3 This is a mass spectrometry detection result of the peptide KD-8 provided in Example 5 of the present invention.

[0020] Figure 4 The figure shows the HPLC purity analysis results of the polypeptide KD-8 provided in Example 5 of this invention.

[0021] Figure 5 Comparison of the in vitro DPPH antioxidant effects of four polypeptides (KD8, PD11, LG24, and LK23) provided in Example 6 of this invention.

[0022] Figure 6 The graphs show the comparison of mouse weight gain during the verification of the anti-fatigue function of KD-8 peptide provided in Example 6 of the present invention. (a) is a graph showing the trend of weight fluctuation; (b) is a graph showing the change in the rate of weight gain.

[0023] Figure 7 The graphs shown in Example 6 of this invention are evaluation charts of anti-fatigue indicators in mice during the verification of the anti-fatigue function of KD-8 peptide. In Figure (a), the time to exhaustion during swimming is shown; in Figure (b), the blood lactate content is shown; in Figure (c), the serum urea nitrogen content is shown; in Figure (d), the T-test estimation is shown in Figures (a), (e), (b), and (f), and (c), the T-test estimation is shown; the symbol " "" indicates a statistically significant difference compared to the blank control group (CK). Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional experimental methods. Unless otherwise specified, all reagents used in the following examples were purchased from conventional biochemical reagent companies. This example uses wild yak blood as raw material and, referring to relevant traditional Chinese medicine processing techniques, extracted a polypeptide mixture with anti-fatigue functions. The evaluation method for anti-fatigue efficacy at the mouse level in this example references the following standards: "Catalogue of Permitted Health Function Claims for Non-Nutritional Supplements (2023 Edition)" and "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)," etc.

[0026] The following is an explanation with reference to specific embodiments:

[0027] Example 1: Extraction of wild yak blood polypeptide mixture

[0028] Preparation of lyophilized wild yak erythrocyte powder: Fresh, purified yak blood was centrifuged at 8000 rpm for 10 minutes at 4°C. After centrifugation, the supernatant plasma was discarded, and the precipitate was resuspended in 0.9% physiological saline. All precipitates were collected to obtain the yak erythrocyte resuspension. The collected precipitate was frozen at -80°C for 24 hours, and then freeze-dried in a freeze dryer for 48 hours to obtain lyophilized yak erythrocyte powder.

[0029] Preparation of wild yak erythrocyte extract (main processing steps): Take an appropriate amount of lyophilized yak erythrocyte powder into an Erlenmeyer flask and autoclave at 121℃ for 20 min. Then add a certain proportion of pure water and autoclave again at 121℃ for 20 min to obtain a solid-liquid mixture of sterilized yak erythrocytes. Filter the solid-liquid mixture through gauze to remove insoluble precipitates through coarse separation. Collect the liquid portion and centrifuge the coarsely separated liquid at 3000 rpm for 15 min. Discard the precipitate and collect the clear supernatant for subsequent protein precipitation.

[0030] The crude mixed polypeptides (unsalted) were obtained from the extract using ammonium sulfate precipitation: 50 ml of extract was placed in a beaker and pre-cooled at 4°C for 30 min. Based on the saturation of the ammonium sulfate solution at 4°C, an appropriate amount of solid ammonium sulfate was added to the 50 ml extract to prepare a saturated ammonium sulfate solution. The solution was stirred while adding the solid ammonium sulfate until it could no longer dissolve. The saturated ammonium sulfate solution was allowed to settle naturally at 4°C for 30 min to completely precipitate the proteins. The solution was then centrifuged at 8000 rpm for 15 min. The supernatant was discarded, and the precipitate was resuspended 2-3 times with pure water. All the precipitate was collected, which is the crude mixed polypeptide (unsalted), for the next step of dialysis desalting.

[0031] The crude mixed peptides were desalted by dialysis and lyophilized to obtain lyophilized mixed peptide powder: Dialysis bags with a molecular weight of 500-1000D were selected. At 4℃, pure water was used as the dialysate, with the sample volume to pure water ratio between 1:50 and 1:500. Dialysis was performed for 24-48 hours, during which the conductivity (μs / cm) in the dialysate was monitored until the conductivity in the dialysate was close to that of pure water and remained constant, indicating that dialysis was complete. After dialysis, all solutions in the dialysis bag were collected, pre-frozen in a -80℃ freezer for 24 hours, and then lyophilized in a freeze dryer. Lyophilization was completed after about 48 hours, and all lyophilized powder was collected, which is the lyophilized mixed peptide powder.

[0032] Example 2: Identification of the anti-fatigue function of wild yak blood polypeptide mixture

[0033] It is carried out according to the method for evaluating physical fatigue relief in the "Methods for Function Testing and Evaluation of Health Foods (2022 Edition)". The improvement of exercise endurance is the most direct manifestation of enhanced anti-fatigue ability, and the length of swimming time can reflect the degree of exercise fatigue in animals. The experimental items for relieving physical fatigue include the weight-bearing swimming test for mice, blood lactic acid (LD), serum urea nitrogen (BUN), liver glycogen (LG), or muscle glycogen (MG). On the premise that the result of the weight-bearing swimming test is positive, if any two of the three biochemical indexes of blood lactic acid (LD), serum urea nitrogen (BUN), liver glycogen (LG), or muscle glycogen (MG) are positive, it can be determined that the tested sample has the effect of relieving physical fatigue.

[0034] (1) Weight-bearing swimming test for mice; The experimental animals are pure-line Kunming white mice, 4 - 5 weeks old. Generally, three dose groups and one negative control group are set up in the experiment. One of the dose groups is 10 times the human recommended dose, and two other dose groups are set up. A positive control group is set up if necessary. The tested sample is given for 7 days. One day after the last administration of the tested sample, mice with a 5% body weight lead sheet attached to the tail root are placed in a swimming bucket to swim. The water depth is not less than 30 cm, and the water temperature is 25°C ± 1.0°C. Record the time from the start of swimming until the mouse's head is submerged below the water surface for 10 s and still cannot surface, that is, the weight-bearing swimming time of the mouse.

[0035] The swimming time is measurement data, and analysis of variance is used. However, according to the procedure of analysis of variance, a homogeneity of variance test should be carried out first. If the variances are homogeneous, calculate the F value. If the F value < F0.05, the conclusion is that there is no significant difference between the means of each group; if the F value ≥ F0.05, P ≤ 0.05, statistical analysis is carried out using the pairwise comparison method between the means of multiple experimental groups and one control group; for non-normal or non-homogeneous variance data, appropriate variable transformation is carried out. After meeting the requirements of normality or homogeneity of variance, the transformed data is used for statistics; if the purpose of normality or homogeneity of variance is still not achieved after variable transformation, the rank sum test is used for statistics. If the weight-bearing swimming time of the tested sample group is significantly longer than that of the control group and the difference is significant, it can be determined that the experimental result is positive.

[0036] (2)Measurement of three indicators: blood lactic acid, serum urea, and liver glycogen / muscle glycogen; Using the kits from Nanjing Jiancheng Bioengineering Institute, the tests were conducted according to the methods required in the instructions. The data of blood lactic acid, urea, and liver glycogen are measurement data, and analysis of variance was used. However, according to the procedure of analysis of variance, homogeneity of variance test should be carried out first. If the variances are homogeneous, calculate the F value. If the F value < F0.05, the conclusion is that there is no significant difference between the means of each group; if the F value ≥ F0.05 and P ≤ 0.05, pairwise comparison method for the means between multiple experimental groups and a control group was used for statistics; for non-normal or non-homogeneous variance data, appropriate variable transformation was carried out. After meeting the requirements of normality or homogeneity of variance, the transformed data was used for statistics; if the purpose of normality or homogeneity of variance was still not achieved after variable transformation, rank sum test was used for statistics. If the blood lactic acid in the test sample group is lower than that in the control group and the difference is significant, the experimental result can be judged as positive; if the serum urea in the test sample group is lower than that in the control group and the difference is significant, the experimental result can be judged as positive; if the liver glycogen content in the test sample group is significantly higher than that in the control group and the difference is significant, the experimental result can be judged as positive.

[0037] If the result of the load-bearing swimming experiment is positive and any two of the three biochemical indicators of blood lactic acid, serum urea, and liver glycogen / muscle glycogen are positive, it can be determined that the test sample has the effect of relieving physical fatigue.

[0038] The specific experimental process is as follows: 40 SPF-grade Kunming mice purchased from Xi'an Keao Co., Ltd. were used in the experiment. During the whole experiment period, the room temperature in the animal house was maintained at 23 ± 2 °C, the relative humidity was 50 ± 5%, and a 12-hour light / dark cycle was followed. First, the 40 mice were randomly divided into 5 groups (n = 8), with 8 mice in each group. The mice were pre-fed for 7 days, during which they had free access to water and normal SPF-grade common mouse feed, and the mouse bedding was replaced regularly. No other treatment was done, so that the mice could gradually adapt to the feeding environment. After the pre-feeding, the formal experiment was carried out for 7 days. Each group of mice was intragastrically administered at a fixed time, place, and dose every day. Among them, the control group was intragastrically administered distilled water; the experimental group 1 was intragastrically administered the whole extract of wild yak red blood cells (suspension); the experimental group 2 was intragastrically administered the supernatant after centrifuging the wild yak red blood cell extract at 3000 r for 15 min; the experimental group 3 was intragastrically administered the precipitate after centrifuging the wild yak red blood cell extract at 3000 r for 15 min (after grinding, add distilled water to make up the volume to the corresponding volume and then intragastrically administer); the experimental group 4 was intragastrically administered the polypeptide mixture extracted from the supernatant of the wild yak red blood cell extract. The simple grouping is shown in Table 1:

[0039] Table 1 Grouping of experimental animals (crude screening components of extract)

[0040]

[0041] Data analysis was performed using GraphPad Prism, employing either one-way ANOVA or two-way ANOVA to statistically analyze the significant differences between two or more groups of data caused by a single factor or two factors. In cases where significant differences existed, this embodiment further performed a Tukey test to determine the specific nature of the difference. A significance level of P < 0.05 was set, signifying a statistically significant difference between the two groups of data.

[0042] The weight result is as follows Figure 1 As shown, the body weight of mice in both groups increased over time, with the later-treatment group showing a higher weight gain rate than the control group. Anti-fatigue indicators include... Figure 2 As shown, all three groups (YM, YM1, and YMP) met the anti-fatigue standard. Specifically, the YM group, with a positive result in the weight-bearing swimming test, showed positive results for blood lactate (LD) and serum urea (BUN); the YM1 group, with a positive result in the weight-bearing swimming test, showed positive results for blood lactate (LD), serum urea (BUN), and liver glycogen (LG) / muscle glycogen (MG); and the YMP group, with a positive result in the weight-bearing swimming test, showed positive results for blood lactate (LD) and liver glycogen (LG) / muscle glycogen (MG). Therefore, all three samples (YM, YM1, and YMP) have the effect of relieving physical fatigue.

[0043] Example 3: Amino acid sequence analysis and identification of the mixed polypeptide

[0044] (1) Dissolution and ultrafiltration of mixed polypeptide lyophilized powder

[0045] Take 2 mg of the mixed peptide lyophilized powder sample and dissolve it in 0.1 mL of pure water. After dissolving the sample, accurately transfer 100 μL of the sample into a 10 kDa ultrafiltration tube using a pipette. Centrifuge at 12000 rcf for 10 min at 4℃. Take the fraction less than 10 kDa.

[0046] (2) Reductive alkylation

[0047] The steps included: accurately pipetting 1 μL of 1M DTT solution into the sample to achieve a final DTT concentration of 10 mmol / L, and reducing it in a 56°C water bath for 1 h; accurately pipetting 2 μL of 1M IAM solution into the sample to achieve a final IAM concentration of 20 mmol / L, and reacting it in the dark at room temperature for 40 min; and accurately pipetting 1 μL of 1M DTT solution into the sample to achieve a final DTT concentration of 10 mmol / L to neutralize unreacted IAM.

[0048] Table 2 Details of reagents used in reduction alkylation

[0049]

[0050] (3) Desalination (Stage-Tip)

[0051] The sample was desalted using a C18 stage-tip and then vacuum dried at 45°C.

[0052] (4) Separation and analysis of mixed peptides by liquid chromatography-tandem mass spectrometry

[0053] The liquid chromatography-mass spectrometry system used was a Vanquish Neo / Orbitrap Exploris 480, manufactured by Thermo Fisher Scientific.

[0054] Liquid chromatography conditions: Analytical column: 75 μm·d. × 25 cm, NanoViper C18 1.9 μm, 100A; Mobile phase A: 0.1% FA; Mobile phase B: 0.1% FA, 80% ACN; Flow rate: 600 nL / min; Analysis time for each component: 35 min; Specific chromatographic conditions are shown in Table 3.

[0055] Table 3 Chromatographic conditions

[0056]

[0057] Mass spectrometry conditions: The full scan range of the mass spectrometer is 100-1500 m / z, the resolution of the first-stage mass spectrometer is set to 60000, AGC is set to Custom, and Maximum IT is set to Custom; the resolution of the second-stage mass spectrometer is set to Resolution: 15000, AGC is set to Custom, Maximum IT is set to Custom, Cycle time is 2 s, and peptide fragmentation collision energy is set to 32. Generate raw mass spectrometry detection data (.raw).

[0058] (5) Analysis of peptide mass spectrometry database

[0059] The raw mass spectrometry files were retrieved using software to search the target protein database. The raw data files acquired by mass spectrometry were searched by the software database to obtain the identification results. Table 4 shows the results of the 10 peptides with the highest scores.

[0060] Table 4. Information on identified peptides

[0061]

[0062] Example 4: Screening of functional peptide sequences after sequence identification of peptide sequences.

[0063] The first step involved sorting the 10 peptide sequences obtained from the above identification according to their significance score (-10lgP: protein confidence score, a higher value indicates that the protein contains more reliable peptides). The six peptide sequences with the highest significance scores were then analyzed. The six selected peptide sequences were arranged in descending order of confidence as follows: LSFPTTKTYFPHFDLSQGSAQVKG, LPGALSELSELSDLHAHKLRVD, LSFPTTKTYFPHFDLSQGSAQVK, KTYFPHFD, PTTKTYFPHFD, and KAAP.

[0064] The second step is to calculate the proportion of antioxidant amino acids. Existing technologies indicate that hydrophobic amino acids rich in antioxidants (Leu, Ala, Ile, Pro, Val, and Phe(F)) can effectively delay fatigue caused by free radicals. The proportions of antioxidant amino acids in the above six peptides are detailed in Table 5. The order of the proportions of antioxidant amino acids in the six peptides from largest to smallest is: KAAP, LSFPTTKTYFPHFDLSQGSAQVK, LSFPTTKTYFPHFDLSQGSAQVKG, KTYFPHFD, LPGALSELSDLSDLHAHKLRVD, PTTKTYFPHFD.

[0065] The proportion of antioxidant amino acids in peptides is calculated using the following formula (1):

[0066] (1)

[0067] Table 5. Proportion of Antioxidant Amino Acids

[0068]

[0069] The third step involved searching the AMP Database Search database. It was found that the peptide sequence KTYFPHFD is part of the sequence of the antimicrobial peptide AP01339, and that KTYFPHFD is highly likely to be the effective functional sequence.

[0070] Example 5: Synthesis of the target polypeptide sequence

[0071] Based on the analysis results of the aforementioned three steps and the comparison of six peptide sequences, peptides were synthesized using liquid-phase and solid-phase peptide synthesis processes. The products were purified using HPLC to obtain target peptide sequences with a purity ≥95%. Specifically, four sequences were synthesized: KD-8, PD-11, LG-24, and LK-23. All four peptide sequences had high significance scores, similar proportions of antioxidant amino acids, and all contained the KD-8 peptide sequence. The amino acid sequence of KD-8 is shown in SEQ ID NO.1; the amino acid sequence of peptide PD-11 is shown in SEQ ID NO.2; the amino acid sequence of peptide LG-24 is shown in SEQ ID NO.3; and the amino acid sequence of peptide LK-23 is shown in SEQ ID NO.4.

[0072] The KD-8 peptide sequence was analyzed by mass spectrometry and HPLC for purity. The mass spectrometry results are as follows: Figure 3 As shown, the HPLC purity analysis results are as follows: Figure 4 As shown.

[0073] Example 6 evaluates the in vitro antioxidant and in vivo anti-fatigue functions of the target peptides. First, the peptides with strong antioxidant activity are selected by in vitro antioxidant detection, and then their anti-fatigue function is verified in mice.

[0074] (1) In vitro antioxidant assay:

[0075] The antioxidant activity of four synthetic peptides (KD-8, PD-11, LG-24, and LK-23) was detected by DPPH free radical scavenging ability. The specific procedure was as follows: 4 mg of each of the four peptides (KD8, PD-11, LG-24, and LK-23) was accurately weighed and added to 1 ml of distilled water. The mixture was vortexed to prepare a 4 mg / ml peptide aqueous solution, which was the test sample. 8 mg of DPPH was accurately weighed and added to anhydrous ethanol, bringing the volume to 100 ml. The solution was stored in the dark and prepared fresh each time. 100 μL of the clear solution was mixed with 100 μL of DPPH solution. A mixture of 100 μL of ethanol and 100 μL of DPPH was used as a control. The mixture was incubated in a 96-well plate at room temperature for 30 min, and the absorbance at 517 nm was measured. The DPPH free radical scavenging rate was calculated using the following formula:

[0076] Clearance rate = [1 - (Ai - Aj) / Ac] × 100%;

[0077] In the formula: Ac is the absorbance of 100 μL anhydrous ethanol + 100 μL LPPH at 517 nm; Ai is the absorbance of 100 μL test sample + 100 μL LPPH at 517 nm; Aj is the absorbance of 100 μL test sample + 100 μL anhydrous ethanol at 517 nm.

[0078] The in vitro DPPH antioxidant results of four peptides (KD-8, PD-11, LG-24, LK-23) are as follows: Figure 5 As shown, when the peptide synthesis purity is ≥95% and the concentration is 4 mg / ml, KD-8 peptide and PD-11, LG-24 and LK-23 peptides containing the KD-8 peptide sequence all have in vitro DPPH antioxidant activity. Among them, KD-8 peptide has a more obvious advantage. The antioxidant effects from strong to weak are: LK-23, KD-8, LG-24 and PD-11.

[0079] (2) In vivo anti-fatigue experiment in mice: Since the KD-8 peptide has a strong antioxidant effect and is easy to synthesize, the in vivo anti-fatigue experiment verifies the anti-fatigue function of the KD-8 peptide.

[0080] Sixteen SPF-grade Kunming mice, purchased from Xi'an Keao Biotechnology Co., Ltd., were used in the experiment. Throughout the experiment, the room temperature was maintained at 23±2℃, the relative humidity at 50±5%, and a 12-hour light / dark cycle was followed.

[0081] First, 16 mice were randomly divided into two groups (n=8), with 8 mice in each group. The mice underwent a 7-day pre-feeding period, during which they had free access to water and food (standard SPF grade mouse feed), and their bedding was changed regularly. No other treatment was given to allow the mice to gradually acclimatize to the environment. After the pre-feeding period, the formal experiment was conducted for 7 days, with each group of mice being administered the solution via gavage at fixed times and in fixed quantities each day. The mouse experiment consisted of two groups: the control group was administered distilled water via gavage, while the experimental group was administered KD-8 peptide solution via gavage. A simplified grouping diagram is shown in Table 1.

[0082] Table 1 Grouping of experimental animals

[0083]

[0084] According to the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)," provided the weight-bearing swimming test result is positive, any two of the three biochemical indicators—blood lactate (LD), serum urea (BUN), and liver glycogen (LG) / muscle glycogen (MG)—can be considered positive to determine that the tested sample has the effect of relieving physical fatigue. Data analysis was performed using GraphPad Prism, employing one-way ANOVA or two-way ANOVA to statistically analyze the significant differences between two or more groups of data caused by a single factor or two factors. When significant differences exist between the data, this embodiment further performs a Tukey test to determine the specific nature of the difference. A significance level of P < 0.05 was set, meaning that there is a statistically significant difference between the two groups of data.

[0085] Weight results as follows Figure 6As shown, the body weight of both groups of mice increased over time, with the experimental group showing a higher rate of weight gain than the control group; the results of the anti-fatigue index analysis are as follows. Figure 7 As shown, the KD-8 peptide group showed positive results in the weighted swimming test, and also showed positive results in blood lactate (LD) and serum urea (BUN), meeting the anti-fatigue standard; therefore, KD-8 peptide has anti-fatigue effects.

[0086] In summary, the polypeptide provided in this embodiment consists of at least 8 amino acids linked by peptide bonds and contains at least a KD-8 sequence. It can be obtained by extraction from biological materials or by biological or chemical synthesis. It has antioxidant and anti-fatigue functional activities and therefore has a variety of potential applications, including but not limited to applications in pharmaceuticals or health products.

[0087] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0088] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of a polypeptide extracted from yak blood in the preparation of anti-fatigue products, characterized in that, The anti-fatigue product is specifically a health supplement and a medicine; the polypeptide is specifically polypeptide KD-8, whose amino acid sequence is shown in SEQ ID NO.1.

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