Anti-fatigue polypeptide in yak blood as well as screening method and application thereof

Abstract

The invention belongs to the technical field of peptiomics, and particularly relates to an anti-fatigue polypeptide in yak blood and a screening method and application thereof, applied anti-fatigue products are specifically food and drugs, and the screening method of the anti-fatigue polypeptide comprises the following steps: carrying out sequence identification on an extracted wild blood yak blood polypeptide mixture; identifying polypeptide sequences with higher scores, and screening functional polypeptide sequences; the screened polypeptide sequence is synthesized into a target polypeptide sequence by adopting a biochemical method, the target polypeptide sequence is detected, the synthesized polypeptide is subjected to antioxidant detection, and the polypeptide KD-8 is subjected to anti-fatigue function verification, so that the polypeptide KD-8 provided by the invention has a remarkable anti-fatigue effect.

Description

Technical Field

[0001] The invention belongs to the technical field of peptideomics, and specifically relates to an anti-fatigue polypeptide in yak blood and a screening method and application thereof. Background Art

[0002] Peptides are compounds formed by α-amino acids linked together in peptide chains, and are also intermediate products of protein hydrolysis. All endogenous polypeptide components in organisms, including polypeptides produced by protein degradation or generated in non-coding regions, usually have a molecular weight of less than 5 kDa. Some polypeptides (active peptides) play an important role in the growth, development and metabolism of organisms, such as semaglutide for the treatment of type 2 diabetes and endorphins with analgesic effects. Some peptides also have a taste effect, such as umami peptides.

[0003] However, there has been no relevant progress in the anti-fatigue peptide KD-8 and its related applications. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a polypeptide extracted from yak blood, wherein the polypeptide is specifically polypeptide KD-8, and its amino acid sequence is shown in SEQ ID NO.1.

[0005] The present invention also provides an application of a polypeptide extracted from yak blood in the preparation of an anti-fatigue product, wherein the anti-fatigue product is specifically food and medicine.

[0006] The present invention also provides a method for screening anti-fatigue polypeptides in yak blood, comprising: S1, performing sequence identification on the extracted wild yak blood polypeptide mixture; S2, screening the polypeptide sequences with higher identification scores in step S1 for functional polypeptide sequences; S3. The polypeptide sequence screened in step S2 is synthesized into a target polypeptide sequence by a biochemical method and detected, wherein the target polypeptide sequence includes the polypeptide KD-8 according to claim 1, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] Further, 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; polypeptide LK-23, whose amino acid sequence is shown in SEQ ID NO.4.

[0008] Furthermore, the sequence identification in step S1 is achieved by reductive alkylation, desalting and liquid chromatography-tandem mass spectrometry on the polypeptide mixture.

[0009] Furthermore, the process of screening the functional polypeptide sequences in step S2 is to first perform significance scoring and sorting on the polypeptide sequences with higher identification scores in step S1; then select polypeptide sequences with higher significance scores to calculate their antioxidant amino acid ratios and sort them; and query the selected polypeptide sequences in the database for valid functional polypeptide sequences.

[0010] Furthermore, in step S3, the chemical synthesis adopts liquid phase peptide synthesis and solid phase peptide synthesis, and the synthesized polypeptides KD-8, PD-11, LG-24 and LK-23 are tested for antioxidant activity and ranked, and then the polypeptide sequences with stronger antioxidant activity are selected to verify the anti-fatigue function in mice.

[0011] Furthermore, the antioxidant activity was detected by DPPH antioxidant test, wherein the antioxidant effects were ranked from strong to weak as follows: polypeptide LK-23, polypeptide KD-8, polypeptide LG-24, polypeptide PD-11.

[0012] Compared with the prior art, the present invention has the following beneficial technical effects: The polypeptide KD-8 sequence provided by the present invention is derived from yak blood, has functional activities such as anti-oxidation and anti-fatigue, and has multiple potential application scenarios, including but not limited to applications in food, medicine or health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a comparison chart of mouse weight growth in the verification of the anti-fatigue function of the polypeptide mixture provided in Example 2 of the present invention, wherein (a) is a weight fluctuation trend chart; (b) is a weight growth rate change chart.

[0014] Figure 2 This is a graph for evaluating the anti-fatigue index of mice in the anti-fatigue function verification of the polypeptide mixture provided in Example 2 of the present invention, wherein (a) represents the swimming exhaustion time; (b) represents the blood lactate content; (c) represents the serum urea nitrogen; (d) represents the liver glycogen content; (e) represents the muscle glycogen content; the symbol "*" represents a statistically significant difference compared with the blank control group (CK); "ns" represents no statistically significant difference compared with the blank control group (CK).

[0015] Figure 3 This is a diagram of the mass spectrometry detection results of the polypeptide KD-8 provided in Example 5 of the present invention.

[0016] Figure 4This is a graph showing the results of HPLC purity analysis of the polypeptide KD-8 provided in Example 5 of the present invention.

[0017] Figure 5 This is a comparison chart of the in vitro DPPH antioxidant effects of the four polypeptides (KD8, PD11, LG24, LK23) provided in Example 6 of the present invention.

[0018] Figure 6 This is a comparison chart of mouse weight growth in the verification of the anti-fatigue function of the KD-8 peptide provided in Example 6 of the present invention, wherein (a) is a weight fluctuation trend chart; (b) is a weight growth rate change chart.

[0019] Figure 7 These are graphs for evaluating the anti-fatigue index of mice in the anti-fatigue function verification of the KD-8 peptide provided in Example 6 of the present invention, wherein (a) represents the swimming exhaustion time; (b) represents the blood lactate content; (c) represents the serum urea nitrogen; (d) is the T test estimation graph in (a), (e) is the T test estimation graph in (b), and (f) is the T test estimation graph in (c); the symbol "*" indicates a statistically significant difference compared with the blank control group (CK). DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The experimental methods in the following examples are all conventional experimental methods unless otherwise specified. The pharmaceutical reagents used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified. In this example, wild yak blood was used as raw material, and a polypeptide mixture with anti-fatigue function was extracted with reference to the relevant processing technology of traditional Chinese medicine. The reference standards for the evaluation method of anti-fatigue efficacy at the mouse level in this example are: "Catalogue of Health Functions Allowed to Be Claimed by Health Foods: Non-Nutrient Supplements (2023 Edition)" and "Health Food Function Inspection and Evaluation Methods (2023 Edition)" and other supporting documents.

[0022] The following is explained in conjunction with specific embodiments: Example 1 Extraction of wild yak blood polypeptide mixture Preparation of freeze-dried wild yak red blood cell powder: freshly purified yak blood was centrifuged at 8000r for 10 min at 4°C, the upper plasma was discarded after centrifugation, the precipitate was added with 0.9% saline for resuspending, and all the precipitate was collected to obtain the yak red blood cell resuspension. The collected precipitate was placed in a -80°C refrigerator for 24 h, and then placed in a freeze dryer for freeze drying, freeze drying for 48 h to obtain yak red blood cell freeze-dried powder.

[0023] Preparation of wild yak red blood cell extract (main process of preparation): take an appropriate amount of yak red blood cell freeze-dried powder into a conical flask, autoclave at 121°C for 20 min, then add a certain proportion of pure water, autoclave again at 121°C for 20 min to obtain a sterilized yak red blood cell solid-liquid mixture; the solid-liquid mixture is first filtered through gauze to remove insoluble precipitates by rough separation, and the liquid part is collected. The liquid obtained by rough separation is centrifuged at 3000r for 15 min, the precipitate is discarded, and the upper clear liquid is collected for subsequent protein precipitation.

[0024] The crude mixed polypeptide (not desalted) was obtained from the extract by ammonium sulfate precipitation method: 50 ml of the extract was placed in a beaker and precooled at 4°C for 30 min. According to 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, and the solution was stirred while adding until the newly added solid ammonium sulfate could not be dissolved. The saturated ammonium sulfate solution was placed at 4°C to settle naturally for 30 min to completely precipitate the protein therein, and then centrifuged at 8000r15min, the supernatant was discarded, and the precipitate was resuspended with pure water 2-3 times. All the precipitates were collected, i.e., the crude mixed polypeptide (not desalted), for the next step of dialysis desalting.

[0025] The crude mixed polypeptide product was dialyzed for desalting and freeze-dried to obtain a mixed polypeptide freeze-dried powder: a dialysis bag with a molecular weight of 500-1000D was selected, and pure water was used as the dialysis fluid at 4°C. The ratio of sample volume to pure water was between 1:50-1:500, and the dialysis was performed for 24-48 hours. During the period, the conductivity (μs / cm) in the dialysis fluid was monitored until the conductivity in the dialysis fluid was close to that of pure water and remained unchanged, indicating that the dialysis was completed; after the dialysis was completed, all the solutions in the dialysis bag were collected, pre-frozen in a -80°C refrigerator for 24 hours, and then placed in a freeze dryer for freeze-drying. The freeze-drying was completed after about 48 hours, and all the freeze-dried powders were collected, which was the mixed polypeptide freeze-dried powder.

[0026] Example 2 Identification of the Anti-fatigue Function of Wild Yak Blood Peptide Mixture 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), blood 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), blood urea nitrogen (BUN), liver glycogen (LG), or muscle glycogen (MG) are positive, it can be determined that the test sample has the effect of relieving physical fatigue.

[0027] (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 test sample is administered for 7 days. One day after the last administration of the test sample, the mice with a 5% body weight lead sheet loaded at 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 immersed in the water for 10 s and still cannot surface, that is, the weight-bearing swimming time of the mouse.

[0028] The swimming time is measurement data, and analysis of variance is used. However, the homogeneity of variance test should be carried out first according to the procedure of analysis of variance. If the variances are homogeneous, calculate the F value. If the F value < F0.05, the conclusion is that there is no significant difference among the means of each group; if the F value ≥ F0.05 and P ≤ 0.05, pairwise comparison of the means between multiple experimental groups and one control group is used for statistics; appropriate variable transformation is carried out on non-normal or heterogeneous variance data. After meeting the requirements of normality or homogeneous variance, the transformed data is used for statistics; if the purpose of normality or homogeneous variance is still not achieved after variable transformation, the rank sum test is used for statistics. If the weight-bearing swimming time of the test sample group is significantly longer than that of the control group and the difference is significant, the positive result of this experiment can be determined.

[0029] (2)Measurement of three indicators: blood lactic acid, serum urea, and liver glycogen / muscle glycogen; The test was conducted using kits from Nanjing Jiancheng Bioengineering Institute 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, a homogeneity of variance test was first performed. If the variances were homogeneous, the F value was calculated. If the F value < F0.05, the conclusion was that there was no significant difference among the means of each group; if the F value ≥ F0.05 and P ≤ 0.05, pairwise comparisons of the means between multiple experimental groups and a control group were used for statistics; For non-normal or heterogeneous variance data, appropriate variable transformations were performed. After meeting the requirements of normality or homogeneous variance, the transformed data were used for statistics; If the purpose of normality or homogeneous variance was still not achieved after variable transformation, the rank sum test was used for statistics. If the blood lactic acid in the test sample group was lower than that in the control group and the difference was significant, the experimental result could be判定 as positive; if the serum urea in the test sample group was lower than that in the control group and the difference was significant, the experimental result could be判定 as positive; if the liver glycogen content in the test sample group was significantly higher than that in the control group and the difference was significant, the experimental result could be判定 as positive.

[0030] 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判定 that the test sample has the effect of relieving physical fatigue.

[0031] The specific experimental process was as follows: 40 SPF-grade Kunming mice purchased from Xi'an Keao Co., Ltd. were used in the experiment. During the entire 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 ordinary SPF-grade mouse feed, and the mouse bedding was changed regularly. No other treatments were given, allowing the mice to gradually adapt to the breeding environment. After the pre-feeding ended, the formal experiment was carried out for a period of 7 days. Each group of mice was given gavage at a fixed time, place, and dose every day. Among them, the control group was given distilled water by gavage; Experimental group 1 was given the whole extract of wild yak red blood cells (suspension) by gavage; Experimental group 2 was given the supernatant after centrifuging the wild yak red blood cell extract at 3000 r for 15 min by gavage; Experimental group 3 was given the precipitate after centrifuging the wild yak red blood cell extract at 3000 r for 15 min (after grinding, made up to the corresponding volume with distilled water and then given by gavage) by gavage; Experimental group 4 was given the polypeptide mixture extracted from the supernatant of the wild yak red blood cell extract by gavage. The simple grouping situation is shown in Table 1: Table 1 Grouping of experimental animals (crude screening components of the extract)

[0032] Data analysis was performed using GraphPad Prism, and one-way analysis of variance (ANOVA) or two-way analysis of variance was used to perform statistics on the significant differences between two or more groups of data caused by a single factor or two factors. When there was a significant difference between the data, the present embodiment further performed a Tukey test to determine the specific circumstances of the difference. The significance level was set to P<0.05, which means that there was a statistically significant difference between the two groups of data.

[0033] The weight results are as follows Figure 1 As shown in Figure 2, the weight of both groups of mice increased over time, and the weight growth rate of the late treatment group was higher than that of the control group. Figure 2 As shown in the figure, the three groups of YM, YM1 and YMP all reached the anti-fatigue standard. Among them, the YM group had positive blood lactate (LD) and serum urea (BUN) under the premise of positive weight-bearing swimming test results; the YM1 group had positive blood lactate (LD), serum urea (BUN) and liver glycogen (LG) / muscle glycogen (MG) under the premise of positive weight-bearing swimming test results; the YMP group had positive blood lactate (LD) and liver glycogen (LG) / muscle glycogen (MG) under the premise of positive weight-bearing swimming test results, so the three samples of YM, YM1 and YMP all have the effect of relieving physical fatigue.

[0034] Example 3 Amino acid sequence analysis and identification of mixed polypeptides (1) Dissolution and ultrafiltration of mixed peptide freeze-dried powder Take 2 mg of mixed peptide freeze-dried powder sample and add 0.1 mL of pure water to dissolve. After the sample is dissolved, use a pipette to accurately transfer 100 μL of sample to a 10kD ultrafiltration tube. Centrifuge at 4°C 12000 rcf for 10 min. Take the fraction less than 10 kDa.

[0035] (2) Reductive alkylation Including: accurately pipetting 1 μL of 1M DTT solution into the sample with a pipette to make the final concentration of DTT to 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 with a pipette to make the final concentration of IAM to 20 mmol / L, and reacting it at room temperature in the dark for 40 min; accurately pipetting 1 μL of 1M DTT solution into the sample with a pipette to make the final concentration of DTT to 10 mmol / L to neutralize the unreacted IAM.

[0036] Table 2 Details of reagents used for reductive alkylation

[0037] (3) Desalination (Stage-Tip) The samples were desalted using a C18 stage-tip and dried under vacuum at 45°C.

[0038] (4) Liquid chromatography-tandem mass spectrometry separation and analysis of mixed peptides A liquid chromatography-mass spectrometer was used, with the specification model being Vanquish Neo / Orbitrap Exploris 480, and the manufacturer being Thermo Fisher Scientific.

[0039] Liquid chromatography conditions: Analytical column: 75 μm i.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: Table 3 Chromatographic conditions

[0040] Mass spectrometry conditions: The full scan range of the mass spectrometry was 100-1500 m / z, the primary mass spectrometry resolution was set to 60000, AGC was Custom, MaximumIT: Custom; the secondary mass spectrometry resolution was set to Resolution: 15000, AGC was Custom, MaximumIT: Custom, Cycle time: 2s, the peptide fragmentation collision energy was set to 32, and the mass spectrometry detection raw data (.raw) was generated.

[0041] (5) Peptide mass spectrometry data library search and analysis The mass spectrometry raw file uses software to search the target protein database. The raw data raw file collected by mass spectrometry is searched by the software database to obtain the identification results. Table 4 shows the results of the 10 peptides with higher scores.

[0042] Table 4 Information of identified peptides

[0043] Example 4 Screening of functional polypeptide sequences for polypeptide sequences after sequence identification In the first step, the 10 peptide sequences obtained from the above identification were sorted according to the significance score (-10lgP: protein credibility score, the larger the value, the more credible peptides the protein contains). The six peptides with higher significance scores were analyzed. The sequences of the 6 peptides screened out were arranged from large to small according to credibility: LSFPTTKTYFPHFDLSQGSAQVKG, LPGALSELSDLHAHKLRVD, LSFPTTKTYFPHFDLSQGSAQVK, KTYFPHFD, PTTKTYFPHFD, KAAP.

[0044] The second step is to calculate the proportion of antioxidant amino acids. The prior art shows that hydrophobic amino acids rich in antioxidants (Leu, Ala, Ile, Pro, Val and Phe (F)) can effectively delay fatigue caused by free radicals. The proportion of antioxidant amino acids contained in the above 6 peptides is calculated in Table 5, where the proportion of antioxidant amino acids contained in the 6 peptides is in the following order from large to small: KAAP, LSFPTTKTYFPHFDLSQGSAQVK, LSFPTTKTYFPHFDLSQGSAQVKG, KTYFPHFD, LPGALSELSDLHAHKLRVD, PTTKTYFPHFD.

[0045] The ratio of antioxidant amino acids in the peptide segment is calculated as shown in the following formula (1): (1) Table 5 Antioxidant amino acid ratio

[0046] The third step is to search in the database AMP Database Search. It is found that the polypeptide sequence KTYFPHFD is part of the antimicrobial peptide AP01339 sequence, and the polypeptide sequence KTYFPHFD is very likely to be an effective functional sequence.

[0047] Example 5 Synthesis of target polypeptide sequence According to the analysis results of the above three steps combined with the comparison of 6 polypeptide sequences, the peptides were synthesized by liquid phase polypeptide synthesis and solid phase polypeptide synthesis processes, and the products were purified by HPLC to obtain the target polypeptide sequence with a purity of ≥95%. Specifically, four sequences were synthesized: KD-8, PD-11, LG-24 and LK-23. The significance scores of the four polypeptide sequences were all high, the ratio of antioxidant amino acids was similar, and they 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 polypeptide PD-11 is shown in SEQ ID NO.2; the amino acid sequence of polypeptide LG-24 is shown in SEQ ID NO.3; the amino acid sequence of polypeptide LK-23 is shown in SEQ ID NO.4.

[0048] The KD-8 peptide sequence was subjected to mass spectrometry detection and HPLC purity analysis. The mass spectrometry detection results were as follows: Figure 3 As shown, the HPLC purity analysis results are as follows Figure 4 shown.

[0049] Example 6 The in vitro antioxidant and in vivo anti-fatigue functions of the target polypeptides were evaluated. First, the polypeptides with strong antioxidant activity were selected through in vitro antioxidant detection, and then the anti-fatigue functions of the polypeptides were verified in vivo in mice.

[0050] (1) In vitro antioxidant test: The antioxidant activity of four synthetic peptides (KD-8, PD-11, LG-24, LK-23) was detected by scavenging DPPH free radicals. The specific process was as follows: 4 mg of four peptides (KD8, PD11, LG24, LK23) were accurately weighed, 1 ml of distilled water was added to each, and vortexed to mix, and a 4 mg / ml peptide aqueous solution was prepared, which was the sample to be tested; 8 mg DPPH was accurately weighed, anhydrous ethanol was added, and the volume was adjusted to 100 ml, and it was placed away from light and prepared before use. 100 μL of the clear solution was mixed with 100 μL of DPPH solution, and the control was a mixture of 100 μL of ethanol and 100 μL of DPPH. After incubation at room temperature for 30 minutes in a 96-well plate, the absorbance at 517 nm was detected, and the scavenging rate of DPPH free radicals was calculated according to the following formula: Clearance rate = [1-(Ai-Aj) / Ac] × 100%; Wherein: Ac is the absorbance of 100 μL anhydrous ethanol + 100 μL DPPPH at 517 nm; Ai is the absorbance of 100 μL test sample + 100 μL DPPPH at 517 nm; Aj is the absorbance of 100 μL test sample + 100 μL anhydrous ethanol at 517 nm.

[0051] The in vitro DPPH antioxidant results of four peptides (KD-8, PD-11, LG-24, LK-23) are as follows Figure 6 As shown, it shows that the purity of polypeptide synthesis is ≥95%. When the concentration is 4 mg / ml, KD-8 peptide and PD-11, LG-24, and LK-23 peptides containing KD-8 peptide sequence all have in vitro DPPH antioxidant activity, among which KD-8 peptide has a more obvious advantage. The antioxidant effects are ranked from strong to weak as follows: LK-23, KD-8, LG-24, and PD-11.

[0052] (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.

[0053] Sixteen SPF grade Kunming mice purchased from Xi'an Keoko Biotechnology Co., Ltd. were used in the experiment. Throughout the experiment, the room temperature of the animal room was maintained at 23 ± 2 °C, the relative humidity was 50 ± 5%, and a 12-h light / dark cycle was followed.

[0054] First, 16 mice were randomly divided into 2 groups (n=8), 8 mice in each group. The mice were pre-fed for 7 days, during which they were allowed to drink water freely, eat ordinary SPF-grade ordinary mouse feed freely, and change the mouse bedding regularly. In addition, no other treatment was done to allow the mice to gradually adapt to the feeding environment. After the pre-fed period, the formal experiment was carried out for 7 days, and each group of mice was gavaged at a fixed time and fixed point every day. The mouse experiment was divided into two groups, among which the control group was gavaged with distilled water; the experimental group was gavaged with KD-8 peptide solution; the brief grouping is shown in Table 1.

[0055] Table 1 Experimental animal groups

[0056] According to the "Health Food Function Test and Evaluation Method (2023 Edition)", under the premise of positive results of the weighted swimming test, if any two of the three biochemical indicators of blood lactate (LD), serum urea (BUN), and liver glycogen (LG) / muscle glycogen (MG) are positive, it can be determined that the test sample has the effect of relieving physical fatigue. GraphPad Prism was used for data analysis, and one-way analysis of variance (ANOVA) or two-way analysis of variance was used to statistically analyze the significant differences between two or more groups of data caused by a single factor or two factors. When there is a significant difference between the data, this embodiment further performs a Tukey test to determine the specific circumstances of the difference. Setting the significance level to P<0.05 means that there is a statistically significant difference between the two groups of data.

[0057] Weight results are as follows Figure 6 As shown in the figure, the weight of both groups of mice increased over time, and the weight growth rate of the experimental group was higher than that of the control group. The results of the anti-fatigue index analysis are shown in the figure. Figure 7As shown, under the premise of positive results of the weighted swimming test, the blood lactate (LD) and serum urea (BUN) of the KD-8 peptide group were positive, reaching the anti-fatigue standard; therefore, the KD-8 peptide has an anti-fatigue effect.

[0058] In summary, the polypeptide provided in this embodiment is composed of at least 8 amino acids connected by peptide bonds and contains at least a KD-8 sequence. It can be obtained by extraction from biological materials, biological or chemical synthesis, etc., and has functional activities such as antioxidant and anti-fatigue. Therefore, it has a variety of potential application scenarios, including but not limited to applications in food, medicine or health products.

[0059] It should be noted that, in this article, terms such as "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0060] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A polypeptide extracted from yak blood, characterized in that: The polypeptide is specifically polypeptide KD-8, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The use of a polypeptide extracted from yak blood in the preparation of an anti-fatigue product according to claim 1, characterized in that: The anti-fatigue products are specifically foods and medicines.

3. A method for screening anti-fatigue polypeptides in yak blood, characterized in that: The screening method comprises: S1, performing sequence identification on the extracted wild yak blood polypeptide mixture; S2, screening the polypeptide sequences with higher identification scores in step S1 for functional polypeptide sequences; S3. The polypeptide sequence screened in step S2 is synthesized into a target polypeptide sequence by a biochemical method and detected, wherein the target polypeptide sequence comprises the polypeptide KD-8 according to claim 1, and its amino acid sequence is shown in SEQ ID NO.

1.

4. The method for screening anti-fatigue polypeptides in yak blood according to claim 3, characterized in that: 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; polypeptide LK-23, whose amino acid sequence is shown in SEQ ID NO.

4.

5. The method for screening anti-fatigue polypeptides in yak blood according to claim 3, characterized in that: The sequence identification in step S1 is achieved by performing reduction alkylation, desalting and liquid chromatography-tandem mass spectrometry on the polypeptide mixture.

6. The method for screening anti-fatigue polypeptides in yak blood according to claim 3, characterized in that: The process of screening the functional polypeptide sequences in step S2 is to first perform significance scoring and sorting on the polypeptide sequences with higher identification scores in step S1; then select polypeptide sequences with higher significance scores, calculate their antioxidant amino acid ratios and sort them; and query the selected polypeptide sequences in the database for effective functional polypeptide sequences.

7. The method for screening anti-fatigue polypeptides in yak blood according to claim 3, characterized in that: In step S3, the biochemical method adopts liquid phase peptide synthesis and solid phase peptide synthesis, and the synthesized polypeptides KD-8, PD-11, LG-24 and LK-23 are tested for antioxidant activity and ranked, and then the polypeptide sequences with strong antioxidant activity are selected to verify the anti-fatigue function in mice.

8. The method for screening anti-fatigue polypeptides in yak blood according to claim 7, characterized in that: The antioxidant activity was detected by DPPH antioxidant test, wherein the antioxidant effects were ranked from strong to weak as follows: polypeptide LK-23, polypeptide KD-8, polypeptide LG-24, polypeptide PD-11.

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