Preparation method of yak oligopeptide powder product with Alzheimer's disease intervention effect

Abstract

The invention provides a preparation method of a yak oligopeptide powder product with an Alzheimer's disease intervention effect, and belongs to the technical field of biology.The yak oligopeptide powder product contains peptide YP, and the peptide YP is composed of leucine-glutamic acid-tyrosine-glycine, the peptide YP has relatively strong binding affinity with acetylcholin esterase, the docking binding energy is-9.5 kcal / mol, the peptide YP and residues of Tyr124, Gly121, Phe338, Asp74, His447, Ser203 and Glu202 of the acetylcholin esterase form 9 hydrogen bonds, and two electrostatic interactions and three hydrophobic interactions exist, so that the function of regulating the enzymatic activity is achieved. And a more reliable natural choice can be provided for intervention of the Alzheimer's disease.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to the extraction and purification of bioactive peptide substances from yak peptide powder, and in particular to a preparation method of a yak oligopeptide powder product having the effect of intervening in Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a multiple, progressive neurodegenerative disease, the main pathological features of which are the deposition of amyloid protein (Aβ) outside brain cells to form plaques and the aggregation of Tau protein inside cells to form neurofibrillary tangles. At present, the US Food and Drug Administration (FDA) has approved a series of drugs for the treatment of Alzheimer's disease, among which the principle of using cholinesterase inhibitors (ChE1) is relatively extensive, including donepezil, galantamine and rivastigmine. Among them, donepezil can improve the integrity of neuronal cells by increasing the concentration of acetylcholinesterase (AChE) in the brain, and can also inhibit the neurodegeneration cascade by interfering with the exposure of the N-terminal part of APP to Aβ peptide. In addition, there are non-competitive NMDA receptor antagonists as drugs, such as memantine, which can restore normal glutamatergic neurotransmission. These drugs often target only a single mechanism and can only play a delaying role, and even have more serious side effects. Therefore, it is urgent to seek a multifunctional active substance to cope with the complexity and diversity of the pathogenesis of Alzheimer's disease.

[0003] Traditional bioactive peptide research usually focuses on demonstrating its biological activity by inhibiting specific enzyme activity or relying on protein-ligand interactions, which usually requires verification through wet chemical experiments, in vitro cell experiments, and animal experiments. However, these traditional methods are not only inefficient, but also costly, time-consuming, and dependent on expensive experimental equipment and techniques.

[0004] Food protein is a sustainable source for the preparation of bioactive peptides. Food-derived bioactive peptides have attracted much attention in the academic community in recent years. Among them, yak peptides show multiple biological activities, with significant advantages such as convenient material collection, good biocompatibility, and high safety. They have become an emerging research focus in the field of medicine and health. Yaks are a characteristic livestock species in plateau areas. They have firm meat, considerable protein content, and rich amino acid composition. They are a high-quality source of bioactive peptide extraction. Current studies have shown that yak peptides have multiple biological activities such as regulating neurotransmitter release, inhibiting neuroinflammation, anti-oxidative stress, and repairing nerve damage. However, existing research on yak peptides focuses on the overall activity of peptide mixtures, and research on the activity of specific peptides and their structures is still insufficient. There is still a lack of systematic research and verification on the specific role and mechanism of yak peptides in neurological diseases such as Alzheimer's disease (AD). In particular, there is no research specifically exploring the effects of specific peptide segments and their structures in yak peptides on improving cognitive function in patients with Alzheimer's disease. Therefore, it is urgent to develop a yak oligopeptide powder product to provide a natural, safe and effective treatment option for the treatment of AD, and it is expected to improve cognitive function and delay the progression of the disease. Summary of the invention

[0005] The purpose of the present invention is to address the above-mentioned problems in the existing technology and to propose a method for preparing a yak oligopeptide powder product with the efficacy of intervening in Alzheimer's disease.

[0006] The purpose of the present invention can be achieved by the following technical scheme: a method for preparing yak oligopeptide powder with the effect of intervening Alzheimer's disease, the method for preparing yak oligopeptide powder with the effect of intervening Alzheimer's disease specifically comprises the following steps: S1. Preparation of enzymatic hydrolysate: adding yak peptide powder to distilled water, stirring evenly to obtain an initial yak peptide solution, adjusting the pH value of the initial yak peptide solution under heating conditions, and performing ultrasonic dispersion treatment after adjusting the pH value to obtain a pre-enzymatic hydrolyzed yak peptide solution, and performing enzyme synergistic hydrolysis and enzyme inactivation on the pre-enzymatic hydrolyzed yak peptide solution using a composite enzyme to obtain an enzymatic hydrolyzate, wherein the weight ratio of yak peptide powder to distilled water is 1:15, and in the enzyme synergistic hydrolysis operation, the composite enzyme used includes trypsin and flavor protease, and the weight ratio of trypsin to flavor protease is 1:0.6; S2 filtration: The enzymatic hydrolysate is first centrifuged to remove solid residues, and then the supernatant is taken and ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of less than 2 kDa to obtain the target filtrate; S3 Gel chromatography separation: subjecting the target filtrate to gel chromatography separation to collect components with a molecular weight of less than 1 kDa to obtain a pre-purified yak peptide solution; S4 purification: using reverse phase high performance liquid chromatography technology, the pre-purified yak peptide solution was purified by gradient elution to obtain the initial yak peptide product; in the gradient elution, the mobile phases used included phase A and phase B, phase A was a 0.05% trifluoroacetic acid aqueous solution; phase B was a 0.05% trifluoroacetic acid acetonitrile aqueous solution, and the acetonitrile content in the trifluoroacetic acid acetonitrile aqueous solution was 75%.

[0007] S5 Drying: The initial yak peptide product is concentrated and dried to obtain yak oligopeptide powder. The yak oligopeptide powder product contains peptide YP. The composition of peptide YP is as follows: leucine-glutamic acid-tyrosine-glycine.

[0008] In step S1, the addition amount of the complex enzyme is 6000u / g; the complex enzyme hydrolysis conditions are: pH value is 7.3-8.0, hydrolysis temperature is 40-50°C, and hydrolysis time is 4-5 hours.

[0009] In step S1, the ultrasonic dispersion treatment parameters are as follows: temperature: 40-50°C; ultrasonic frequency: 40-60kHz, ultrasonic power: 200-300W, treatment time: 15-20 minutes.

[0010] In step S1, the sterilization operation parameters are as follows: temperature: 80-90°C; time: 10-15 minutes.

[0011] In step S2, the centrifugal separation operation parameters are as follows: rotation speed: 4000-6000r / min; centrifugal time: 10-15 minutes.

[0012] The gel chromatography separation operation in step S3 is designed as follows: the chromatographic column is a Sephadex G-25 chromatographic column, the detection wavelength of the ultraviolet detector is 220 nm, the equilibrium buffer is 0.01 M phosphate buffer, the pH value of 0.01 M phosphate buffer is 7.2; the injection concentration is 5 mg / mL.

[0013] In the purification operation of step S4, the design is as follows: the selected chromatographic column is Waters Symmetry C18, with a pore size of 100Å, a size of 25×150mm, and a filling particle size of 5µm; the detection wavelength is 215nm; and the column oven operating temperature is 32°C.

[0014] In step S4, the gradient elution was designed as follows: 0-45 minutes: the concentration of mobile phase A was uniformly reduced from 98% to 45%, 45-50 minutes: the concentration of mobile phase A was uniformly reduced from 45% to 0%, 50-55 minutes: the concentration of mobile phase A was 0%, and only mobile phase B was used.

[0015] In step S5, the drying operation is performed by freeze drying.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Use compound enzyme for enzymatic hydrolysis: Using compound enzyme (combination of trypsin and flavor protease) for synergistic hydrolysis not only improves the hydrolysis efficiency of peptide molecules, but also makes the molecular weight of oligopeptides moderate. The precise control of compound enzyme hydrolysis conditions (such as pH, temperature, and hydrolysis time) helps to ensure the quality and effect of peptides. Ultrasonic dispersion treatment can effectively improve the uniformity of the enzymatic solution and promote the action of enzymes through the action of ultrasonic waves, thereby improving the extraction effect of oligopeptides.

[0017] 2. Accurate purification process: Multiple purification treatments using gel chromatography and reversed-phase high-performance liquid chromatography (HPLC) can effectively remove impurities and improve the purity of yak oligopeptides. The use of gel chromatography can initially separate peptides according to molecular weight, and further use HPLC for fine purification, effectively retaining the target components, removing unnecessary impurities and macromolecules, and improving purity and biological activity.

[0018] 3. Freeze drying: Freeze drying (lyophilization) can avoid the destruction of heat-sensitive substances, maintain the active ingredients of yak oligopeptides, and facilitate long-term storage and use of the product.

[0019] 4. Safety: The raw materials (yak peptide powder) and the entire process avoid the use of chemical solvents to ensure the naturalness and safety of the product.

[0020] In summary, the present invention provides a method for preparing yak oligopeptide powder with the effect of intervening in Alzheimer's disease. The present invention has an efficient enzymatic hydrolysis process, an accurate purification process, a freeze-drying technology to ensure biological activity, precise process control, and efficient extraction and production capabilities. These characteristics make the final product have significant advantages in quality, purity, biological activity and safety, and can provide a more reliable natural choice for the intervention of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a curve diagram of the escape latency change in the space positioning navigation experiment of the present invention. Figure 1 In the figure, Control, blank control group; AD, Alzheimer's disease model control group; Don, donepezil hydrochloride control group; YP-L: yak peptide low-dose group; YP-M: yak peptide medium-dose group; YP-H: yak peptide high-dose group. The escape latency change curves of mice in different groups are represented by different colors and shapes.

[0022] Figure 2 Schematic diagram of the effect of peptide YP of the present invention on the memory ability of Alzheimer's disease mice. Figure 2Control, blank control group; AD, Alzheimer's disease model control group; Don, donepezil hydrochloride control group; YP-L: yak peptide low-dose group; YP-M: yak peptide medium-dose group; YP-H: yak peptide high-dose group; (a) Number of crossing the platform, (b) target quadrant movement time, (c) swimming trajectories of mice in each group; Different letters represent significant differences among the groups (P < 0.05).

[0023] Figure 3 Schematic diagram of the effect of peptide YP on serum oxidative stress levels in AD mice. Figure 3 Control, blank control group; AD, Alzheimer's disease model control group; Don, donepezil hydrochloride control group; YP-L: yak peptide low-dose group; YP-M: yak peptide medium-dose group; YP-H: yak peptide high-dose group. MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; (a) serum MDA content, (b) serum SOD content, (c) serum GSH content; different letters represent significant differences among the groups (P < 0.05).

[0024] Figure 4 Schematic diagram of the effect of peptide YP on the deposition of Aβ1-42 amyloid protein in the hippocampus of AD mice. Figure 4 Control, blank control group; AD, Alzheimer's disease model control group; Don, donepezil hydrochloride control group; YP-L: yak peptide low-dose group; YP-M: yak peptide medium-dose group; YP-H: yak peptide high-dose group. (a) Aβ1-42 amyloid protein deposition in the hippocampus (scale = 100μm, magnification 200×), (b) Aβ1-4 protein content in the hippocampus; different letters represent significant differences among the groups (P < 0.05).

[0025] Figure 5 Schematic diagram of the effect of peptide YP on the level of neuroinflammation in the hippocampus of AD mice. Figure 5 Control, blank control group; AD, Alzheimer's disease model control group; Don, donepezil hydrochloride control group; YP-L: yak peptide low-dose group; YP-M: yak peptide medium-dose group; YP-H: yak peptide high-dose group; (a) microglial response in the hippocampus (scale = 100 μm, magnification 200×), (b) TNF-α content in the hippocampus; different letters represent significant differences among the groups (P < 0.05).

[0026] Figure 6 Schematic diagram of the effect of peptide YP on the content of short-chain fatty acids (SCFAs) in feces of AD mice. Figure 6Control, blank control group; AD, Alzheimer's disease model control group; Don, donepezil hydrochloride control group; YP-L: yak peptide low-dose group; YP-M: yak peptide medium-dose group; YP-H: yak peptide high-dose group; (a) Acetic acid content in feces, (b) Propionic acid content in feces, (c) Butyric acid content in feces; Different letters represent significant differences among the groups (P < 0.05).

[0027] Figure 7 Schematic diagram of the effect of peptide YP on the Alpha diversity of intestinal flora in AD mice. Figure 7 Control, blank control group; AD, Alzheimer's disease model control group; YP-M: medium-dose yak peptide group; (a) Shannon index, (b) Simpson index, (c) ACE index, (d) Chao1 index; different letters represent significant differences among the groups (P < 0.05).

[0028] Figure 8 is the OTUs intersection Venn diagram, Figure 8 Middle, Control, blank control group; AD, Alzheimer's disease model control group; YP-M: yak peptide medium dose group, different groups are represented by different colors.

[0029] Fig. 9 is the species abundance map at the level of intestinal flora. Fig. 9 Middle, Control, blank control group; AD, Alzheimer's disease model control group; YP-M: yak peptide medium dose group, different bacterial phyla are represented by different colors.

[0030] Figure 10 is a differential analysis diagram of the intestinal flora at the genus level. In Figure 10, Control, blank control group; AD, Alzheimer's disease model control group; YP-M: yak peptide medium dose group; (a) Circos diagram of intestinal flora distribution, (b) species abundance diagram of intestinal flora at the genus level, different genera are represented by different colors; (c) genus level clustering heat map, the color depth indicates the high and low species abundance.

[0031] Fig.11 This is the multivariate analysis diagram of serum metabolites. Fig.11 Middle, AD, Alzheimer's disease model control group; YP-M: yak peptide medium dose group; (a) orthogonal partial least squares discriminant analysis (OPLS-DA), (b) permutation test model validation of AD group and YP-M group.

[0032] Fig.12 is a schematic diagram of differential metabolites. Fig.12Middle, AD, Alzheimer's disease model control group; YP-M: yak peptide medium dose group. The X-axis represents the sample name, the Y-axis represents the metabolite name, and the different shades of color represent the relative expression of the metabolite in the group of samples. The redder the color, the higher the expression.

[0033] Fig.13 The schematic diagram of KEGG functional pathways of AD group and YP-M group. Fig.13 In the figure, the ordinate is the secondary classification of the KEGG metabolic pathway, and the abscissa is the number of metabolites under the pathway.

[0034] Fig.14 This is the KEGG pathway enrichment analysis diagram. Fig.14 In the figure, each column represents a KEGG Pathway pathway; the horizontal axis represents the relative importance of the metabolites in the pathway; the vertical axis represents the enrichment significance of the metabolites involved in the pathway -log10 (P value).

[0035] Figure 15 is a schematic diagram of the correlation between intestinal flora and serum metabolites; in Figure 15, the right side of the figure is the name of the intestinal microbiota, and the bottom is the name of the metabolite; the grid in the figure represents the association between two attributes (intestinal microbiota and associated features); (a) phylum level correlation, (b) genus level correlation. Red indicates positive correlation, and blue indicates negative correlation.

[0036] Fig.16 This is the mass spectrum of yak oligopeptide powder product.

[0037] Fig.17 Schematic diagram of the chemical structure of peptide YP.

[0038] Fig.18 This is the molecular structure model diagram of peptide YP - ball-and-stick model. DETAILED DESCRIPTION

[0039] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments. Specific embodiments

[0040] A method for preparing a yak oligopeptide powder product having the effect of intervening Alzheimer's disease, the method for preparing the yak oligopeptide powder product having the effect of intervening Alzheimer's disease specifically comprises the following steps: S1. Preparation of enzymatic hydrolysate: adding yak peptide powder to distilled water, stirring evenly to obtain an initial yak peptide solution, adjusting the pH value of the initial yak peptide solution under heating conditions, and performing ultrasonic dispersion treatment after adjusting the pH value to obtain a pre-enzymatic hydrolyzed yak peptide solution, and performing enzyme synergistic hydrolysis and enzyme inactivation on the pre-enzymatic hydrolyzed yak peptide solution using a composite enzyme to obtain an enzymatic hydrolyzate, wherein the weight ratio of yak peptide powder to distilled water is 1:15, and in the enzyme synergistic hydrolysis operation, the composite enzyme used includes trypsin and flavor protease, and the weight ratio of trypsin to flavor protease is 1:0.6; S2 filtration: The enzymatic hydrolysate is first centrifuged to remove solid residues, and then the supernatant is taken and ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of less than 2 kDa to obtain the target filtrate; S3 Gel chromatography separation: subjecting the target filtrate to gel chromatography separation to collect components with a molecular weight of less than 1 kDa to obtain a pre-purified yak peptide solution; S4 purification: using reverse phase high performance liquid chromatography technology, the pre-purified yak peptide solution was purified by gradient elution to obtain the initial yak peptide product; in the gradient elution, the mobile phases used included phase A and phase B, phase A was a 0.05% trifluoroacetic acid aqueous solution; phase B was a 0.05% trifluoroacetic acid acetonitrile aqueous solution, and the acetonitrile content in the trifluoroacetic acid acetonitrile aqueous solution was 75%.

[0041] S5 Drying: The initial yak peptide product is concentrated and dried to obtain yak oligopeptide powder.

[0042] In step S1, the added amount of the complex enzyme is 6000u / g; the enzymatic hydrolysis conditions of the complex enzyme are: pH value is 7.8, enzymatic hydrolysis temperature is 40°C, and enzymatic hydrolysis time is 5 hours.

[0043] The ultrasonic dispersion treatment parameters are as follows: temperature: 45°C; ultrasonic frequency: 60kHz, ultrasonic power: 300W, treatment time: 20 minutes.

[0044] In step S1, the sterilization operation parameters are as follows: temperature: 85°C; time: 15 minutes.

[0045] In step S2, the centrifugal separation operation parameters are as follows: rotation speed: 4000-6000 r / min; centrifugal time: 15 minutes.

[0046] The gel chromatography separation operation in step S3 is designed as follows: the chromatographic column is a Sephadex G-25 chromatographic column, the detection wavelength of the ultraviolet detector is 220 nm, the equilibrium buffer is 0.01 M phosphate buffer, the pH value of 0.01 M phosphate buffer is 7.2; the injection concentration is 5 mg / mL.

[0047] In the purification operation of step S4, the design is as follows: the selected chromatographic column is Waters Symmetry C18, with a pore size of 100Å, a size of 25×150mm, and a filling particle size of 5µm; the detection wavelength is 215nm; and the column oven operating temperature is 32°C.

[0048] In step S4, the gradient elution was designed as follows: 0-45 minutes: the concentration of mobile phase A was uniformly reduced from 98% to 45%, 45-50 minutes: the concentration of mobile phase A was uniformly reduced from 45% to 0%, 50-55 minutes: the concentration of mobile phase A was 0%, and only mobile phase B was used.

[0049] In step S5, the drying operation is performed by freeze drying.

[0050] The prepared yak oligopeptide powder product was subjected to mass spectrometry analysis, and the analysis results were as follows: Fig.16 As shown in Figure 2, the peptide YP content in this product is 48.18%. Fig.17 and Fig.18 As shown, peptide YP is composed of the following four amino acids: Leucine-Glutamic Acid-Tyrosine-Glycine, abbreviated as LEYG.

[0051] Peptide YP can play a multi-target role in improving cognitive impairment by improving amyloid protein deposition, oxidative stress damage, microglial inflammatory response, fecal short-chain fatty acid content, intestinal flora imbalance and metabolic disorders in the brain of AD mice.

[0052] Experimental methods: 72 SPF-grade 8-week-old female Kunming mice were selected, each weighing 20±2g, and were adaptively fed for one week before the experiment. After one week of adaptation, the mice were randomly divided into a blank control group (Control), an Alzheimer's disease model group (AD), a donepezil hydrochloride control group (Donepezil), and yak peptide low, medium, and high dose groups (YP-L, YP-M, and YP-H) (12 mice / group) according to their average body weight.

[0053] In this experiment, the Alzheimer's disease mouse model was established by intraperitoneal injection of 0.2mL120mg / kg D-galactose (D-gal) combined with gavage of 0.2mL20mg / kg aluminum chloride (AlCl3) every day. Starting from the second week, the blank control group mice were injected and gavaged with the same amount of normal saline, and the mice in the other groups were modeled according to the operation of intraperitoneal injection of D-gal (0.2mL120mg / kg) combined with gavage of AlCl3 (0.2mL20mg / kg); starting from the seventh week, on the basis of continuous modeling, the positive control group was gavaged with 0.2mL1mg / kg donepezil hydrochloride every day, the YP-L group was gavaged with 0.2mL100mg / kg yak peptide, the YP-M group was gavaged with 0.2mL200mg / kg yak peptide, and the YP-H group was gavaged with 0.2mL400mg / kg yak peptide. The intervention experiment lasted for five weeks, during which the mice were weighed weekly, and fecal specimens were collected aseptically and stored at -80℃. No adverse reactions were observed in all mice during the experiment.

[0054] The animal experiment groups are shown in the following table: Grouping Modeling and intervention methods Blank control group 0.2 mL normal saline Alzheimer's disease model group <![CDATA[Intraperitoneal injection of 120 mg / kg D-gal + intragastric administration of 20 mg / kg AlCl3]]> Donepezil hydrochloride control group <![CDATA[120mg / kg D-gal+20mg / kg AlCl3+1mg / kg Donepezil]]> Yak peptide low dose group <![CDATA[120mg / kg D-gal+20mg / kg AlCl3+100mg / kg YP]]> Yak peptide medium dose group <![CDATA[120mg / kg D-gal+20mg / kg AlCl3+200mg / kg YP]]> Yak peptide high dose group <![CDATA[120mg / kg D-gal+20mg / kg AlCl3+400mg / kg YP]]> Every other week, the feces of mice in each group were collected aseptically and stored in a -80℃ refrigerator. After the intervention, Morris water maze behavioral experiments were performed to evaluate the cognitive status of mice in each group. After the experiment, all mice were fasted but not watered for 12 hours. The mice were anesthetized before blood was collected from the eyeballs. The eyeballs were quickly removed when blood was collected, and the mice were killed by dislocation. After the whole blood of the mice was allowed to stand at room temperature for 20 minutes, it was centrifuged at 12000g for 10 minutes at 4℃ to obtain serum; the mice were dissected, tissue samples (whole brain, hippocampus, liver, spleen, kidney, colon, cecum) were taken, and the contents of the colon and cecum were collected. The tissues were washed with 0.9% saline, and the surface was dried with filter paper for weighing; part of the brain of each group was immersed in a centrifuge tube filled with 4% paraformaldehyde solution in advance, and the hippocampus of the remaining brains was dissected on ice and weighed. All serum, tissues, and contents were placed in liquid nitrogen and transferred to a -80℃ refrigerator for storage.

[0055] Water maze experiment: The Morris water maze experiment consists of two parts: a positioning navigation experiment and a space exploration experiment. The positioning navigation experiment can effectively evaluate the learning ability of mice by recording the escape latency. The experiment was carried out for 5 days; the space exploration experiment evaluated the memory function of mice by the activity time of mice in the quadrant where the original platform was located and the number of times they crossed the original underwater hidden platform. The experiment was carried out in a black circular pool (120 cm in diameter and 40 cm in height) filled with water. An opaque sunshade was installed around the pool. Four markers of different shapes were symmetrically pasted on the inner wall of the pool for mice to distinguish the direction. The pool was divided into four quadrants. A black hidden platform with a diameter of 8 cm was fixed in the center of the third quadrant. The water temperature was kept constant at 23±1℃ during the experiment. In the formal experiment, mice were placed in the water facing the inner wall of the pool in a clockwise order according to the four quadrants. The swimming trajectory of the mice and the time they found the underwater hidden platform and stayed there were recorded by the Morris software visual tracking system installed just above the pool.

[0056] After five weeks of yak peptide intervention, the Morris water maze navigation experiment was conducted. Every day, mice were placed in the water facing the inner wall of the pool in a clockwise order of the four quadrants. The swimming trajectory of the mice in the water was observed, and the time required for the mice to find the hidden platform in the third quadrant and stay on the platform was recorded. This time is called the escape latency. If the mouse still does not find the hidden platform within 60 seconds, the mouse is placed on the platform for 10 seconds, and the escape latency is recorded as 60 seconds. The entire experimental process was recorded in real time by the Morris software visual tracking system installed directly above the pool, and the escape latency of the mouse was used as an evaluation indicator of the mouse's learning ability.

[0057] After the navigation experiment, the original underwater hidden platform was removed and a one-day Morris water maze space exploration experiment was conducted. The mice were placed in the water from the first quadrant farthest from the original platform, facing the inner wall of the pool. The Morris software visual tracking system installed just above the pool was used to track and record the swimming trajectory of the mice in real time within 60 seconds, and the number of crossing the original platform and the activity time in the third quadrant were used as important indicators to evaluate the spatial memory ability of the mice.

[0058] Determination of mouse serum biochemical indicators: Determination of serum MDA: The determination of mouse serum malondialdehyde MDA adopts the thiobarbituric acid (TBA) method. The specific operation method and calculation formula are strictly in accordance with the instructions of the kit. Determination of serum SOD: The determination of mouse serum superoxide dismutase SOD content adopts the WST-1 method. The specific operation method and calculation formula are strictly in accordance with the instructions of the kit. Determination of serum GSH-Px: The determination of mouse serum glutathione peroxidase GSH-Px content adopts the colorimetric method. The specific operation method and calculation formula are strictly in accordance with the instructions of the kit.

[0059] Determination of biochemical indices of mouse hippocampal homogenate: Under ice bath conditions, the hippocampal tissue mass (g) was mixed with phosphate buffered saline (PBS) (mL) at a ratio of 1:9, and placed on a high-speed tissue grinder for low-temperature grinding at 60 Hz for 60 s to obtain a 10% hippocampal tissue homogenate. The homogenate was centrifuged at 4 ° C and 6000 g for 20 min to collect a 10% hippocampal tissue homogenate. The protein content of 10% hippocampal homogenate was determined using the BCA protein quantitative detection kit. The specific operation method and calculation formula were strictly in accordance with the kit instructions.

[0060] Determination of Aβ1-42 amyloid protein content in hippocampus: The double antibody sandwich method was used to determine the content of Aβ1-42 amyloid protein in mouse hippocampus. The specific operation method and calculation formula were strictly carried out in accordance with the instructions of the kit.

[0061] Determination of TNF-α in hippocampus: The double antibody sandwich method was used to determine the content of TNF-α in mouse hippocampus. The specific operation method and calculation formula were strictly carried out according to the instructions of the kit.

[0062] Mouse hippocampal immunofluorescence staining analysis: After rapid eye blood sampling, mice were killed by cervical dislocation, the mice were dissected and the whole brain tissue was taken out, the brain tissue was soaked and cleaned with sterile saline, and the excess blood and water were absorbed with filter paper. A 10 ml centrifuge tube containing 4% paraformaldehyde fixative was prepared in advance to place the dissected brain tissue.

[0063] Slice preparation: Rinse the brain tissue fixed with 4% paraformaldehyde with running water overnight, and then dehydrate, embed and slice: Dehydration: 70% ethanol (2h) → 80% ethanol (2h) → 95% ethanol overnight → 100% ethanol I (1h) → 100% ethanol II (1h) → 100% ethanol III (1h) → xylene I (1h) → xylene II (1h). Embedding: The dehydrated brain tissue is immersed in wax, wax I (1h) → wax II (1h) → wax III (1h). After the wax immersion is completed, the brain tissue is placed in the middle of the bottom of the wax box, filled with wax and embedded, and left at room temperature for 24h. Sectioning: Place the embedded tissue in a 4°C refrigerator for 10 minutes, trim the shape of the wax slice, and use a tissue slicer to perform coronal continuous sectioning. Unfold the cut wax slice in a slide spreader filled with 45°C water, use a glass slide to scoop it out intact, and place it in a 40°C drying machine to dry it for later use.

[0064] Immunofluorescence double staining: The immunofluorescence double staining method of this experiment refers to the detection method of Ali, T, etc., and the operation is carried out according to the process of paraffin section dewaxing, dehydration → antigen repair → blocking → incubation of primary antibody and secondary antibody 1 → adding FITC reagent → removing the bound primary antibody and secondary antibody 1 → incubation of primary antibody and secondary antibody 2 → autofluorescence quenching → DAPI counterstaining → sealing. The primary antibody 1 and secondary antibody 1 used are: anti-amyloid Aβ1-42 rabbit polyclonal antibody (rabbit polyclonal antibody, cat. no. GB111197, 1:200) and HRP-enzyme-labeled goat anti-rabbit IgG (HRP-conjugated goat anti-rabbit IgG (cat. no. GB23303, 1:200)). The primary antibody 1 and secondary antibody 2 used were: anti-BAI mouse monoclonal antibody (mouse monoclonal antibody, cat. no. GB12105, 1:200) and HRP-labeled goat anti-mouse IgG (cat. no. GB23301, 1:200).

[0065] Microscopic examination and photography: Collect the prepared tissue sections, observe the fluorescent signal under a confocal laser scanning microscope, and retain the image.

[0066] Determination of short-chain fatty acid levels in mouse feces: Short-chain fatty acids (SCFAs) are a class of organic volatile fatty acids. Their chemical structures are composed of less than 7 carbon atoms. The main SCFAs are acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid. Studies have shown that short-chain fatty acids are an important metabolic signaling molecule that plays an important role in maintaining metabolic balance in the body, regulating intestinal microorganisms, and improving intestinal barrier function. As an important metabolite of intestinal flora, it can also work synergistically with multiple systems to exert a wide range of physiological effects. Currently, the commonly used determination methods for SCFAs are mainly high-performance liquid chromatography, gas chromatography, etc. In this experiment, gas chromatography was used to detect the content of acetic acid, propionic acid and butyric acid in mouse feces. The specific operations are as follows: Standard curve drawing: accurately measure 20 μL of acetic acid, propionic acid and butyric acid standards, use ether solution to prepare 2 μL / mL short-chain fatty acid standard solution, and then dilute with ether to 0.05 μL / mL, 0.1 μL / mL, 0.15 μL / mL to 0.5 μL / mL series of standard concentrations, filter with organic pinhole microporous filter membrane and perform gas chromatography detection. The test results are plotted with standard concentration as the horizontal axis and peak area as the vertical axis. The obtained standard curve equation is as follows: acetic acid: y=312.12x-7.7815, R2=0.9947; propionic acid: y=597.19x-10.051, R2=0.9988; butyric acid: y=638.28x+6.6844, R2=0.9959.

[0067] Extraction of short-chain fatty acids from samples: Place the fecal samples frozen in a -80°C refrigerator in a freeze dryer and freeze-dry. After freeze-drying, accurately weigh 50 mg of the sample, slowly add 500 μL of saturated sodium chloride solution, use a vortex oscillator to oscillate for 1 minute to fully mix, and let stand at room temperature for 30 minutes. Then slowly add 40 μL of sulfuric acid solution for acidification reaction, and vortex again for 30 seconds. Add 800 μL of ether, vortex for 30 seconds, place in a centrifuge, centrifuge at 4°C, 12000g for 15 minutes, and take the supernatant. Add 0.25 g of anhydrous sodium sulfate to the supernatant, let stand at room temperature for 10 minutes, and perform a second centrifugation. The centrifugation conditions are 4°C, 12000g, 15 minutes, and finally aspirate the supernatant into a gas phase vial, filter with an organic pinhole microporous filter membrane and set aside.

[0068] Gas chromatography analysis conditions: chromatographic column DB-WAX capillary column (30m×250mm×0.25μm); injection port temperature 220℃, detector temperature 230℃, injection volume 1.0μL, split ratio 10:1, carrier gas is high-purity nitrogen, flow rate 1mL / min, hydrogen flow rate 400mL / min, tail gas flow rate 40mL / min. Heating program: initial temperature 110℃ maintained for 2.5min, heating to 150℃ at 8℃ / min and maintained for 8min, total operation 15.5min.

[0069] Analysis of the structure of the colonic microbial community in mice: 0.5 g of colonic contents were weighed, and the genomic DNA of intestinal microorganisms in the contents was extracted using the HiPure fecal DNA kit. The quality of the extracted DNA was evaluated by microspectrophotometer and agarose gel electrophoresis. The 16SrRNA gene in the V3-V4 hypervariable region was amplified by bacteria using the CFX-linked PCR system and primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 806R (5'-GGACTACHVGGGTATCTAAT-3'). On this basis, a second round of amplification was performed, and the product was purified using the AMPureXPBeads kit and quantified and sequenced using the ABIStepOnePlus real-time PCR system. After filtering and splicing based on the original data, the effective PCR products were sequenced using the NovaSeqPE250 platform, and then OTUs (Operational Taxonomic Units) cluster analysis and species classification analysis were performed to obtain the abundance distribution of species information, and further explore the differential intestinal microorganisms between the samples of each group based on the database species information.

[0070] Non-targeted metabolomics analysis of mouse serum: Non-targeted metabolomics study of mouse serum was performed using UPLC / Q-TOF-MS. 100 μL of serum was measured, added with 80% methanol solution and 0.1% formic acid solution, pre-cooled in a vortex, ultrasonically extracted on ice for 5 minutes, and allowed to stand at -20°C for 30 minutes. After standing, centrifuged at 15,000g and 4°C for 20 minutes. The supernatant was collected and diluted with LC-MS grade water to a concentration of 53% methanol solution, and centrifuged at 15,000g for 20 minutes at 4°C. The obtained supernatant was injected into the LC-MS / MS system for analysis.

[0071] UHPLC-MS / MS analysis was performed on a Vanquish UHPLC system and an Orbitrap Q Exactive TMYP-X mass spectrometer. The sample was injected into a Hypesil Gold column (100×2.1 mm, 1.9 μm) at a rate of 0.2 mL / min, and the gradient elution was 17 min. In the positive ion mode, 0.1% formic acid in water was used as the mobile phase A and methanol solution as the mobile phase B; in the negative ion mode, 5 mM ammonium acetate solution (pH 9.0) was used as the mobile phase A and methanol solution as the mobile phase B. The gradient elution program was set as follows: 2% B, 1.5 min; 2-100% B, 12.0 min; 100% B, 14.0 min; 100-2% B, 14.1 min; 2% B, 17 min. The sample mass spectrometry signal was collected using a QExactiveTMYP-X mass spectrometer with a spray capillary voltage of 3.2 kV, a capillary temperature of 320°C, a sheath gas flow rate of 40 arb, an auxiliary gas flow rate of 10 arb, and positive and negative ion scanning modes, respectively.

[0072] The obtained data matrix was imported into SIMCA-P+14.0 software to obtain the raw data of metabolomics, and ProgenesisQI software (Waters) was used to process all the raw data. The metabolite information was imported into EZinfo2.0 software for multivariate statistical analysis, and the orthogonal partial least squares method OPLS-DA (Orthogonal Partial Lease Squares Discrimination Analysis) was used to distinguish the overall differences in the metabolism of each group and identify the marker differential ions. Potential differential biomarkers were screened from the Human Metabolome Database (HMDB) to explore the biological significance of the changes in differential metabolites, and MetaboAnalyst4.0 was used for subsequent analysis.

[0073] Correlation analysis between intestinal flora and serum metabolomics of mice: To explore the relationship between intestinal flora and serum metabolomics differential metabolites in the Alzheimer's disease model group and the yak peptide intervention group, GraphPad Prism 7.0 software was used for Pearson correlation analysis. A heat map was used to represent the correlation between intestinal microorganisms and differential metabolites in each group of mice.

[0074] Statistical analysis: All experimental data were expressed as mean ± standard deviation, and SPSS11.0 software was used to perform parameter significance test analysis on the data using variance analysis and Duncan multivariate range test (95% confidence interval). When P < 0.05, different letters indicate that there is a significant difference between the data in the groups.

[0075] The experimental results show that the positioning and navigation experiment lasted for 5 days. The Morris visual tracking camera installed just above the pool recorded the movement trajectory of the mice. The Morris software system calculated the time it took for the mice to reach the underwater hidden platform, which is called the escape latency. The escape latency can be used as an important indicator to evaluate the learning ability of mice. Figure 1 As shown in the figure, on the second day of the positioning navigation experiment, the escape latency of AD mice increased significantly compared with the blank control group (P < 0.05), indicating that the learning ability of AD mice decreased. Compared with AD mice, the escape latency of mice in the YP-L, YP-M, and YP-H groups began to decrease from the second day, and there was no significant difference compared with the blank control group mice from the fourth day (P > 0.05), indicating that the intake of yak peptides can significantly reverse the trend of decreased learning ability of AD mice.

[0076] After the navigation experiment, a space exploration experiment was conducted. The original underwater hidden platform was removed, and the movement trajectory of the mouse was recorded by a Morris visual tracking camera installed just above the pool. The Morris software system calculated the mouse's swimming trajectory, the number of times it crossed the platform, and the time it stayed in the quadrant where the original platform was located. The results of the space exploration experiment are shown in Figure 2. Figure 2 As shown in the figure, compared with the blank control group, the number of times AD mice crossed the platform was significantly reduced (P < 0.05), and the movement time in the target quadrant was significantly shortened (P < 0.05), indicating that the memory function of AD mice decreased. After the intervention of yak peptide, the number of times AD mice crossed the platform and the activity time in the quadrant where the original platform was located were significantly increased (P < 0.05). The relevant performance can also be intuitively seen from the swimming trajectories of each group of mice: the swimming trajectory of AD mice was obviously discrete, while the swimming trajectories of mice in the YP-M and YP-H groups showed a tendency centered on the third quadrant, which was closer to the blank control group, indicating that yak peptide can improve the memory ability of AD mice. Among them, the medium-dose yak peptide group had no significant difference from the blank control group in the number of times crossing the platform and the time staying in the quadrant of the original platform (P > 0.05). The above results show that AD mice modeled with D-gal combined with AlCl3 showed obvious cognitive dysfunction, and the intake of yak peptide can improve the learning and memory ability of AD mice, among which YP at a dose of 200 mg / kg / d had the best effect on improving cognitive dysfunction in AD mice.

[0077] When the body is stimulated by harmful factors for a long time, an overload of free radicals will be produced in the body, causing an imbalance in the body's antioxidant system, leading to a series of physiological and pathological damage. This phenomenon is called oxidative stress. Oxidative stress damage is an important factor leading to Alzheimer's disease. Previous studies have shown that increased oxidative stress and activation of related pathways can promote the development of AD, and oxidative stress can cause damage to the presynaptic membrane of the brain, thereby affecting learning and memory abilities. Therefore, this experiment used a commercial enzyme kit method to detect antioxidant enzymes and lipid peroxidation products in the serum of each group of mice to explore the oxidative stress damage of Alzheimer's mice and the intervention effect of yak peptide on it. The results are as follows: Figure 3 As shown. MDA is an important product of lipid peroxidation reaction and can indirectly reflect the severity of oxidative stress damage in the body. Compared with the blank control group, the serum MDA level of AD mice was significantly increased (P < 0.05), indicating that the AD mice were more severely attacked by free radicals. Compared with AD mice, the MDA content of the low, medium and high dose groups of yak peptide was significantly reduced (P < 0.05), among which the MDA content of the YP-M group was not statistically different from that of the blank control group mice (P > 0.05), indicating that the intake of yak peptide can reduce the degree of oxidative stress damage to the body, thereby reducing the MDA level in AD mice. SOD and GSH, as antioxidant enzymes in the body, can reflect the body's ability to scavenge oxygen free radicals. By Figure 3-4 In (bc), we can see that compared with the blank control group, the levels of SOD and GSH in the serum of AD mice were significantly reduced (P < 0.05), indicating that the antioxidant capacity of AD mice was reduced. Compared with the AD group mice, the low, medium and high dose groups of yak peptide can significantly increase the activity of antioxidant enzymes in the serum of AD mice (P < 0.05), among which there is no statistical difference in the SOD content between the medium dose group of yak peptide and the blank control group mice (P > 0.05). The above results suggest that yak peptide can alleviate the serum oxidative stress damage of Alzheimer's mice to a certain extent.

[0078] One of the characteristic clinical manifestations of Alzheimer's disease patients is the appearance of extracellular β-amyloid protein deposition in the brain, which exerts neurotoxicity through specific receptors in the destructive cycle of AD, and Aβ oligomers (Aβ1-42) composed of 42 amino acids are the main toxic components of senile plaques. Aβ1-42 can be degraded by cells to form extracellular aggregates, leading to the formation of amyloid plaque deposition and neurofibrillary tangles in the brain of AD patients, thereby inducing neuronal dysfunction in the brain. In this experiment, the effect of yak peptide on Aβ1-42 protein deposition in the hippocampus of AD mice was explored by immunostaining Aβ1-42 in hippocampal tissue and detecting the content of Aβ1-42 protein in mouse hippocampal tissue. The results are as follows: Figure 4As shown. Compared with the blank control group, the Aβ1-42 content in the hippocampus of AD mice increased significantly, and the Aβ1-42 content decreased to a certain extent after yak peptide intervention. ELISA kit quantitative analysis showed that compared with the blank control group, the Aβ1-42 content in the hippocampus of AD mice increased significantly (P < 0.05), while its content decreased significantly after YP intervention. There was no significant difference in the Aβ1-42 content between the medium-dose and high-dose yak peptide groups and the blank control group (P > 0.05), indicating that the intake of yak peptide can significantly reduce the deposition of Aβ1-42 amyloid protein in the hippocampus of AD mice, thereby alleviating the deposition of toxic proteins in the brain.

[0079] Studies have shown that neuroinflammation is an important factor in causing hippocampal damage in patients with Alzheimer's disease. Microglia, as important innate immune cells in the central nervous system, can promote brain homeostasis and resist neurological diseases in a healthy state. However, when attacked by too many inflammatory cytokines, microglia will be abnormally activated, triggering a new pro-inflammatory cascade reaction, secreting pro-inflammatory factors such as tumor necrosis factor (TNF-α), causing neurotoxicity and synaptic loss, forming a vicious cycle. In this experiment, microglia were immunostained with the microglia marker IBA1+, and the ELISA kit was used to detect the content of TNF-α in the mouse hippocampal tissue to explore the effect of yak peptide on neuroinflammation in the hippocampus of AD mice. The results are as follows: Figure 5 As shown. The immunofluorescence images show that the expression of IBA1+ markers in the hippocampus of AD mice was significantly upregulated compared with the blank control group, and the number of somatic cells increased significantly, showing a unique abnormal activation response. Compared with the AD group mice, the intake of medium and high doses of yak peptide significantly inhibited the abnormal activation state of microglia. Compared with the blank control group, the level of TNF-α in the hippocampus of AD mice was significantly increased (P < 0.05), while compared with the AD group mice, the TNF-α levels in the low, medium and high dose groups of yak peptide were significantly reduced (P < 0.05), among which there was no significant difference in the TNF-α levels between the low and medium doses of yak peptide intervention groups and the blank control group (P > 0.05). The above results indicate that yak peptide can alleviate brain neuroinflammation in AD mice by inhibiting the abnormal activation of microglia and reducing the production of proinflammatory factors.

[0080] As intestinal microbial metabolites, short-chain fatty acids play an important role in the communication of the gut-brain axis. Among them, acetate, propionate and butyrate are important neurotransmitters that can cross the blood-brain barrier (BBB) ​​to affect brain-related functions and participate in the biosynthesis changes of neurotransmitters, neurotrophic factors, etc. Among them, butyrate can enhance the expression of tight junction proteins in mice and increase the integrity of the blood-brain barrier. It can also regulate cell autophagy through the P13K / Akt / mTOR signaling pathway and increase the RNA expression of α-synuclein. In order to clarify the effect of yak peptide on intestinal microbiota metabolites, this experiment detected the content of major SCFAs in mouse feces. The results are as follows. Figure 6 As shown in the figure, compared with the blank control group, the contents of acetic acid, propionic acid, and butyric acid in the feces of AD mice were significantly decreased (P < 0.05); compared with the AD group, the content of propionic acid in the feces of the YP low-dose group mice was significantly increased (P < 0.05), and there was no significant difference with the blank control group (P > 0.05); the contents of acetic acid and butyric acid in the feces of the YP medium-dose group mice were significantly increased (P < 0.05). The above results show that low-dose and medium-dose yak peptides can reverse the trend of the decrease in the content of SCFAs in the feces of AD mice.

[0081] At present, there is a consensus on the role of intestinal microorganisms and their metabolites in the development of AD. Intestinal flora imbalance can lead to the production of harmful substances that are not conducive to the health of the body, and induce neuroinflammation in the brain through the brain-gut axis, affecting the central nervous system and greatly increasing the incidence of neurodegenerative diseases. Recent studies have shown that intestinal microbial imbalance occurs even before neuroinflammation caused by Aβ amyloid deposition and Tau protein hyperphosphorylation. In previous studies in this experiment, the medium dose of yak peptide performed well in improving cognitive impairment, oxidative stress, and neuroinflammation. Therefore, the medium dose group of yak peptide was selected to continue to explore the possibility of yak peptide regulating intestinal flora imbalance in AD mice, and the effect of 200 mg / kg / day of yak peptide on the colonic microbiota of AD mice was evaluated by 16SrRNA pyrophosphate high-throughput sequencing technology.

[0082] Alpha diversity analysis was performed based on a sequence threshold of more than 97% identity, such as Figure 7As shown in the results, compared with the blank control group, the Simpson index, ACE index and Chao1 index of AD mice decreased (P < 0.05), and the Shannon index increased significantly (P < 0.05), indicating that the richness and diversity of intestinal flora in AD mice decreased. Compared with the AD group, YP-M significantly reduced the Shannon index of AD mice and increased the Simpson, ACE and Chao1 indexes of AD mice (P < 0.05). In addition, there was no significant difference between the Shannon, Simpson and Chao1 indexes of the medium-dose yak peptide group and the blank control group (P > 0.05), indicating that yak peptide can effectively reverse the reduction in the richness and diversity of intestinal flora in AD mice.

[0083] Under the screening condition of sequence threshold of more than 97% consistency, the intestinal flora was subjected to OTUs (Operational Taxonomic Units) cluster analysis based on Effective Tags. The OTUs of each group were calculated according to the cluster analysis results, and the averaged OTUs number was represented by Veen diagram, as shown in the figure below: Figure 8 As shown. The Veen diagram shows that there are 479 core OTUs in each group, of which the blank control group has 103 unique OTUs, the AD group has 153 unique OTUs, and the YP-M group has 132 unique OTUs. In addition, the blank control group and the YP-M group share 564 OTUs, and the AD group and the YP-M group share 535 OTUs, indicating that the intestinal flora composition of AD mice after taking yak peptides has more shared OTUs with the blank control group.

[0084] In recent years, with the deepening of research on the brain-gut axis, it has been gradually clarified that the intestinal flora can be connected to the brain by regulating the immune system, participating in tryptophan metabolism, affecting the enteric nervous system and vagus nerve, etc. The above results also show that yak peptide can effectively change the intestinal flora of AD mice. In order to further explore the specific changes in the intestinal microbial community of each group of mice, the species abundance of the intestinal flora was measured and analyzed at the phylum level and genus level.

[0085] The abundance of intestinal flora species at the phylum level Fig. 9As shown in the figure, the main dominant bacteria in the colon contents of mice are Firmicutes, Bacteroidetes and Proteobacteria. It can be seen from the figure that the relative abundance of Firmicutes in AD mice is significantly lower than that in the blank control group, and the relative abundance of Proteobacteria is significantly higher than that in the blank control group. This trend is consistent with the results of a previous study that found that the abundance of Firmicutes decreased and the abundance of Proteobacteria increased in AD patients. In addition, studies have reported that Firmicutes are negatively correlated with amyloid deposition in the brain, and Proteobacteria can destroy the ecological balance of the intestine, lead to immune disorders, and promote the production of proinflammatory cytokines. In this experiment, medium-dose yak peptide intervention can effectively reverse the changes in the intestinal flora of AD mice at the phylum level, indicating that it may alleviate symptoms such as inflammation and cognitive impairment by improving the intestinal flora imbalance of AD mice.

[0086] At the genus level, (a) in circos Figure 10 shows that the dominant genera in each group are Lactobacillus, Acinetobacter, Dubosiella, Bacteroides, and Akkermansia. The abundance of intestinal microbial species at the genus level is shown in (b) in Figure 10. Compared with the blank control group, the Lactobacillus and Dubosiella in AD mice were significantly reduced, and Acinetobacter was significantly increased; after YP intervention, the relative abundance of Dubosiella, Alloprevotella, Candidatus_Saccharimonas, and Bifidobacterium in the intestinal flora of AD mice was increased, and the relative abundance of Acinetobacter, Akkermansia, and Staphylococcus was reduced. Studies have shown that Dubosiella is a beneficial bacterium that can produce SCFAs, which is positively correlated with butyrate metabolism and negatively correlated with the expression of inflammatory factors such as (interleukin 1) IL-1, (interleukin 6) IL-6 and TNF-α. The continuous increase in the number of Acinetobacter will destroy the body's immune defense, trigger lipopolysaccharide (LPS) toll-like receptor 4 (TLR4) to induce neuroinflammation, and mediate a variety of diseases. It is worth noting that the relative abundance of Alloprevotella and Bifidobacterium in the YP-M group was higher than that in the blank control group. Bifidobacterium is a recognized probiotic. Previous studies have shown that supplementation with Bifidobacterium can alleviate learning and memory disorders in AD patients and improve the MMSE (Minimum Mental State Examination) scores of AD patients. Alloprevotella can alleviate cognitive impairment by regulating glucose homeostasis in AD mice and having a positive effect on neuronal cells. (c) in the genus-level clustering heat map 10 shows that YP-M reshaped the abnormal changes in the genus level of the intestinal flora of AD mice, making the intestinal flora composition structure of AD mice closer to that of the blank control group. The above results show that yak peptide intervention can effectively alleviate the imbalance of intestinal flora in AD mice by increasing the abundance of probiotics in the intestinal flora and reducing the abundance of pathogenic bacteria, thereby increasing short-chain fatty acids, reducing intestinal inflammation, improving the body's immune defense ability, and alleviating cognitive impairment.

[0087] Orthogonal partial least squares discriminant analysis is a supervised discriminant analysis statistical method. By establishing a relationship model between different sample categories and metabolite expression levels, the sample category can be predicted and the metabolomics profiles of the two groups can be better distinguished. The OPLS-DA scores of the YP-M and AD groups in this experiment are shown in the figure. Fig.11 As shown in (a), the metabolic profiles of the AD and YP-M groups are clearly separated, indicating that the grouping effect is significant, indicating that the metabolism of AD mice has changed significantly after yak peptide intervention. Based on the OPLS-DA analysis, the arrangement of the sample data was randomly disrupted, and 200 random permutation tests were performed. The statistical inference method of the permutation test was used to determine whether the model is accurate and reliable. The permutation test diagram is shown in (b) in Figure 11. The horizontal axis represents the permutation retention (the Y value corresponding to the horizontal axis is 1, which is the R2 and Q2 value of the original model), the vertical axis represents the R2 and Q2 permutation test values, and the two dotted lines represent the regression lines of R2 and Q2. The evaluation criterion of the permutation test is whether the intersection of the Q2 regression line and the Y axis is less than 0. In this experiment, the intercept of Q2 is -0.03, indicating that the model does not have overfitting and the metabolic data is reliable.

[0088] In order to explore the potential intervention mechanism of yak peptide on AD mice from a metabolic perspective, the metabolites that were significantly changed after yak peptide intake were detected. The P value in unidimensional statistics can intuitively express the significance of the difference in differential metabolites, and the variable projection importance (VIP, Variable Importance for the Projection) value can reflect the contribution of the metabolite expression pattern to the model classification. Generally, P < 0.05 and VIP > 1 are used as the criteria for screening differential metabolites. Fig.12As shown, the UPLC-Q-TOF / MS method was used to detect the changes in serum metabolites, and all metabolites in the AD group and YP-M group were identified according to KEGG and matched with the human metabolome database. Screening revealed that there were 23 potential marker differential metabolites in the AD group and the YP-M group. Compared with the AD group, the YP-M group had higher levels of metabolites such as docosapentaenoic acid (DPA), sphingolipid (SM), phosphatidylcholine (PC), phosphatidylethanolamine (LPE) and lysophospolipid (LPC), while lower levels of metabolites such as succinic acid, hexadecanedioic acid, tetradecanedioic acid, 2-butoxyacetic acid, L-ascorbic acid 2-sulfate and (R)-3-hydroxymyristic acid.

[0089] Previous studies have shown that DPA, as a member of the long-chain polyunsaturated fatty acid family, has a great alleviating effect on neurodegenerative diseases. It can not only reduce the mRNA expression of inflammatory factors and microglial markers, but also improve the cognitive damage of brain Aβ and tau aggregation to 3xTg-AD mice through the PS1 presenilin pathway; SM is the most abundant sphingolipid in the cell membrane and is a regulator of cell membrane homeostasis and inflammation; PC and PE can both play an important role in the recognition and transduction of cell membrane signals, among which PC can protect neurons from the toxic effects of Aβ protein, and its hydrolysis product LPC can also inhibit lipopolysaccharide-mediated inflammatory response and abnormal activation of microglia. The above results show that yak peptide intervention improves neuroinflammation mediated by Aβ and tau aggregation and abnormal microglial activation in the brain of AD mice by regulating the content of some lipid metabolites, thereby alleviating cognitive impairment. It is worth noting that yak peptide intervention also reduced the levels of some toxic metabolites in AD mice. Among them, excessive (R)-3-hydroxymyristic acid content can cause liver toxicity accumulation, induce oxidative stress, and trigger mitochondrial dysfunction; 2-butoxyacetic acid can cause cell damage; hexadecanedioic acid and tetradecanedioic acid can also cause adverse reactions such as mitochondrial swelling, respiratory irritation, lipid metabolism disorders, and oxidative stress. It can be seen that the intake of yak peptides can increase the content of beneficial metabolites and reduce the content of toxic metabolites in AD mice, thereby regulating serum metabolic disorders.

[0090] After determining the potential bio-metabolite markers in each group, metabolic pathways were constructed based on the differential metabolites. KEGG and MetaboAnalyst were used to construct the pathways of the effects of yak peptide intervention on the metabolic pathways of AD mice, such as Fig.13 As shown. In the metabolic functional pathways of AD mice and yak peptide intervention group mice, a total of 4 types of KEGG metabolic pathways are involved, namely Metabolism, Organismal Systems, Human Diseases and Environmental Information Processing. Among them, the secondary classification of metabolic pathways is amino acid metabolism, carbohydrate metabolism, cofactor and vitamin metabolism, energy metabolism and other amino acid metabolism. The KEGG functional pathways after yak peptide intervention were further enriched and analyzed, and the results are shown as follows Fig.14 As shown in the figure, in the YP-MVSAD metabolic cluster, a total of 30 metabolic pathways were enriched, including GABAergic synapse, butanoate metabolism, citric acid cycle, glucagon signaling pathway, oxidative phosphorylation, alanine, aspartate and glutamate metabolism, glyoxylate and dicarboxylate metabolism, etc.

[0091] In order to further explore how yak peptide improves cognitive function by affecting intestinal flora and serum metabolism, Pearson correlation analysis was used to illustrate the correlation between intestinal flora and differential metabolites at the phylum level and genus level, and a heat map was used to represent the changes in intestinal flora OTU abundance and differential metabolite composition. The comparison of microorganisms and metabolites used |Correlation|>0.8000. The results are shown in Figure 15.

[0092] At the phylum level, Firmicutes was significantly positively correlated with docosapentaenoic acid (DPA), and significantly negatively correlated with DL-lysine, succinic acid, N1-piperidinocarbonyl-4-methylbenzene-1-sulfonamide, and valine. Epsilonbacteraeota (Proteobacteria) was significantly positively correlated with 18-9-ynoic acid, phosphatidylcholine (20:3 / 20:4) PC (20:3 / 20:4), sphingolipid (SM (d18:1 / 18:0), PC (16:0 / 17:1), and significantly negatively correlated with L-ascorbic acid 2-sulfate. At the same time, Actinobacteria was significantly positively correlated with creatinine. In addition, Proteobacteria was significantly positively correlated with N-acetyl-DL-tryptophan. The toxic metabolite tetradecanedioic acid mentioned above was significantly negatively correlated with Dubosiella, and hexadecanedioic acid was significantly positively correlated with Proteobacteria and Acinetobacter, suggesting that yak peptides may reduce the abundance of pathogenic bacteria such as Proteobacteria in AD mice and reduce the production of harmful metabolites such as hexadecanedioic acid and tetradecanedioic acid in AD mice.

[0093] At the genus level, DPA was significantly positively correlated with Bifidobacterium and Aerococcus. Creatinine was significantly positively correlated with Aerococcus, Odoribacter, Coriobacteriaceae_UCG-002 and Faecalibaculum, and 2-[3-Methyl-2-(methylimino)-4-oxo-1,3-thiazolan-5-yl]acetic acid was significantly positively correlated with Bifidobacterium and Ruminococcaceae_UCG-014. Dubosiella was significantly positively correlated with octadec-9-ynoic acid and negatively correlated with tetradecanedioic acid. Acinetobacter and Staphylococcus were positively correlated with N-acetyl-DL-tryptophan, PC (20:3 / 22:6), and L-ascorbic acid 2-sulfate. (R)-3-hydroxymyristic acid was positively correlated with Lachnospiraceae_NK4A136_group. As mentioned earlier, DPA has the effects of reducing inflammation and slowing down the aggregation of Aβ and tau. The study found that DPA was significantly positively correlated with the probiotic Bifidobacterium, suggesting that yak peptide increases the abundance of Bifidobacterium in the intestinal flora, increases the content of DPA in mice, and alleviates AD-related malignant changes such as neuroinflammation and cognitive impairment.

[0094] Yak oligopeptide powder products can intervene in the mechanism of Alzheimer's disease, mainly through the interaction between the peptide YP it contains and acetylcholinesterase. Acetylcholinesterase plays a key role in the pathogenesis of Alzheimer's disease, so inhibiting the activity of this enzyme is an important strategy to improve symptoms. The following is a specific analysis: The relationship between acetylcholinesterase and Alzheimer's disease: Acetylcholinesterase is an enzyme that mainly breaks down the neurotransmitter acetylcholine. Acetylcholine is a vital chemical in neurotransmission, especially playing an important role in cognitive function and memory formation. In Alzheimer's patients, acetylcholine levels are usually significantly reduced, which is closely related to the obstruction of neural signal transmission and memory loss. By inhibiting the activity of acetylcholinesterase, the level of acetylcholine can be effectively increased, thereby improving the cognitive function of Alzheimer's patients.

[0095] Effect of peptide YP in yak oligopeptide products: Peptide YP can bind to acetylcholinesterase, and its docking binding energy is -9.5 kcal / mol, which means that it has a strong binding affinity with acetylcholinesterase. This affinity shows that peptide YP can effectively bind to acetylcholinesterase and inhibit its enzymatic activity, thereby reducing the degradation of acetylcholine. Peptide YP forms 9 hydrogen bonds with Tyr124, Gly121, Phe338, Asp74, His447, Ser203, and Glu202 residues of acetylcholinesterase. Hydrogen bonds are important mechanical forces in protein-protein interactions and can enhance the binding affinity between peptides and enzymes. Electrostatic and hydrophobic interactions: There are also 2 electrostatic interactions and 3 hydrophobic interactions between peptide YP and acetylcholinesterase, which contribute to the stable binding of peptides to the enzyme. The binding ability of these non-covalent interactions ensures that peptide YP can effectively interfere with the normal function of acetylcholinesterase.

[0096] The specific embodiments described herein are merely examples of the spirit of the present invention. A person skilled in the art of the present invention may make various modifications or supplements to the specific embodiments described or replace them in a similar manner, but will not deviate from the spirit of the present invention or exceed the defined scope. Although the present invention is described and described in detail in the drawings and the foregoing description, such descriptions and descriptions are considered to be illustrative or exemplary rather than restrictive. It should be understood that within the scope of the following claims, changes and modifications may be made by a person of ordinary skill. Specifically, the present invention encompasses additional embodiments having any combination of features from the above-mentioned different embodiments. With respect to the use of the expression "generally" or "substantially", this patent application should be understood to disclose that these features and values ​​are also fully satisfied, i.e., there is no aforementioned characterization as "generally" or "substantially".

Claims

1. A method for preparing a yak oligopeptide powder product having the effect of intervening in Alzheimer's disease, characterized in that: The preparation method of the yak oligopeptide powder product having the effect of intervening in Alzheimer's disease specifically comprises the following steps: S1. Preparation of enzymatic hydrolysate: adding yak peptide powder to distilled water, stirring evenly to obtain an initial yak peptide solution, adjusting the pH value of the initial yak peptide solution under heating conditions, and performing ultrasonic dispersion treatment after adjusting the pH value to obtain a pre-enzymatic hydrolyzed yak peptide solution, and using a composite enzyme to perform enzyme synergistic hydrolysis and enzyme inactivation on the pre-enzymatic hydrolyzed yak peptide solution to obtain an enzymatic hydrolyzate, wherein the weight ratio of yak peptide powder to distilled water is 1:15, and in the enzyme synergistic hydrolysis operation, the composite enzyme used includes trypsin and flavor protease, and the weight ratio of trypsin to flavor protease is 1:0.6; S2 filtration: The enzymatic hydrolysate is first centrifuged to remove solid residues, and then the supernatant is taken and ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of less than 2 kDa to obtain the target filtrate; S3 Gel chromatography separation: subjecting the target filtrate to gel chromatography separation to collect components with a molecular weight of less than 1 kDa to obtain a pre-purified yak peptide solution; S4 purification: using reverse phase high performance liquid chromatography technology, the pre-purified yak peptide solution was purified by gradient elution to obtain the initial yak peptide product; in the gradient elution, the mobile phases used included phase A and phase B, phase A was a 0.05% trifluoroacetic acid aqueous solution; phase B was a 0.05% trifluoroacetic acid acetonitrile aqueous solution, and the acetonitrile content in the trifluoroacetic acid acetonitrile aqueous solution was 75%; S5 Drying: The initial yak peptide product is concentrated and dried to obtain a yak oligopeptide powder product. The yak oligopeptide powder product contains peptide YP. The composition of peptide YP is as follows: leucine-glutamic acid-tyrosine-glycine.

2. The method for preparing the yak oligopeptide powder product having the effect of intervening Alzheimer's disease according to claim 1, characterized in that: In step S1, the addition amount of the complex enzyme is 6000u / g; the complex enzyme hydrolysis conditions are: pH value is 7.3-8.0, hydrolysis temperature is 40-50°C, and hydrolysis time is 4-5 hours.

3. The method for preparing the yak oligopeptide powder product with the efficacy of intervening in Alzheimer's disease as claimed in claim 1, wherein in step S1, the ultrasonic dispersion treatment parameters are as follows: temperature: 40-50°C; ultrasonic frequency: 40-60kHz, ultrasonic power: 200-300W, and treatment time: 15-20 minutes.

4. The method for preparing the yak oligopeptide powder product with the efficacy of intervening in Alzheimer's disease as claimed in claim 1, wherein in step S1, the sterilization operation parameters are as follows: temperature: 80-90°C; time: 10-15 minutes.

5. The method for preparing the yak oligopeptide powder product with the effect of intervening in Alzheimer's disease as claimed in claim 1, wherein in step S2, the centrifugal separation operation parameters are as follows: rotation speed: 4000-6000r / min; centrifugation time: 10-15 minutes.

6. The method for preparing the yak oligopeptide powder product having the effect of intervening Alzheimer's disease according to claim 1, characterized in that: The gel chromatography separation operation in step S3 is designed as follows: the chromatographic column is a Sephadex G-25 chromatographic column, the detection wavelength of the ultraviolet detector is 220 nm, the equilibrium buffer is 0.01 M phosphate buffer, the pH value of 0.01 M phosphate buffer is 7.2; the injection concentration is 5 mg / mL.

7. The method for preparing the yak oligopeptide powder product having the effect of intervening Alzheimer's disease according to claim 1, characterized in that: In the purification operation of step S4, the design is as follows: the selected chromatographic column is Waters Symmetry C18, with a pore size of 100Å, a size of 25×150mm, and a filling particle size of 5µm; the detection wavelength is 215nm; and the column oven operating temperature is 32°C.

8. The method for preparing the yak oligopeptide powder product having the effect of intervening Alzheimer's disease according to claim 1, characterized in that: In step S4, the gradient elution was designed as follows: 0-45 minutes: the concentration of mobile phase A was uniformly reduced from 98% to 45%, 45-50 minutes: the concentration of mobile phase A was uniformly reduced from 45% to 0%, 50-55 minutes: the concentration of mobile phase A was 0%, and only mobile phase B was used.

9. The method for preparing the yak oligopeptide powder product having the effect of intervening Alzheimer's disease according to claim 1, characterized in that: In step S5, the drying operation is performed by freeze drying.

Images