Cyperus esculentus polypeptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of extraction and application of bioactive peptides, in particular to a cyperus esculentus polypeptide and a preparation method and application thereof.The cyperus esculentus polypeptide is obtained by taking cyperus esculentus meal as a raw material, extracting through an alkali-solution and acid-precipitation treatment and enzymolysis method and separating through an ultrafiltration membrane with the molecular weight cutoff of 3 kDa; the cyperus esculentus polypeptide contains peptide fragments as shown in SEQ ID NO. 1 to SEQ ID NO. 30. The cyperus esculentus polypeptide prepared by the preparation method disclosed by the invention is beneficial to improving a cognitive function, improving memory and learning ability, relieving oxidative stress of brain tissues, recovering a cholinergic system function and inhibiting neuroinflammation, and also can be used for remodeling an intestinal flora structure and increasing abundance of beneficial bacteria.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide extraction and application technology, specifically to a tiger nut polypeptide and its preparation method and application. Background Technology

[0002] With the accelerating aging of society and the fast pace of life, health problems related to cognitive impairment, including Alzheimer's disease (AD), are becoming increasingly prominent. The gut microbiota plays a crucial role in regulating central nervous system function, directly or indirectly influencing neuronal development, neural signal transmission, and behavior through gut-brain interactions.

[0003] Currently, the main medications used in clinical practice to treat cognitive impairment are cholinergic drugs and cholinesterase inhibitors. While these drugs can provide short-term symptom relief, they cannot reverse the disease progression. Therefore, exploring ways to improve cognitive function is crucial. Summary of the Invention

[0004] To develop pathways to improve cognitive dysfunction, this invention provides a tiger nut polypeptide, its preparation method, and its applications. The tiger nut polypeptide provided by this invention is beneficial for improving cognitive dysfunction, enhancing memory and learning abilities, alleviating oxidative stress in brain tissue, restoring cholinergic system function, and inhibiting neuroinflammation. It can also reshape the gut microbiota structure and increase the abundance of beneficial bacteria.

[0005] This invention provides the application of tiger nut peptides in the preparation of drugs to improve cognitive impairment. The tiger nut peptides are obtained by using tiger nut meal as raw material, extracting them through alkaline dissolution and acid precipitation treatment and enzymatic hydrolysis, and separating them using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The tiger nut polypeptide contains the peptides shown in SEQ ID NO.1 to SEQ ID NO.30.

[0006] The tiger nut polypeptide provided by this invention is beneficial for improving cognitive dysfunction, improving memory and learning ability, relieving oxidative stress in brain tissue, restoring cholinergic system function and inhibiting neuroinflammation, and can also reshape the intestinal flora structure and increase the abundance of beneficial bacteria.

[0007] Furthermore, the drug is used to prevent or treat Alzheimer's disease.

[0008] Furthermore, the drug is used to reduce inflammation and improve neuronal damage.

[0009] Furthermore, the drug is also used to help improve memory.

[0010] Furthermore, the drug is also used to increase the abundance of beneficial bacteria in the gut.

[0011] Furthermore, the enhancement of the abundance of beneficial bacteria in the gut is achieved by increasing the ratio of the abundance of Firmicutes and Bacteroidetes in the gut, and increasing the abundance of Bifidobacterium, Akkermania, and Dubois.

[0012] Furthermore, the drug uses tiger nut polypeptide as the sole active ingredient and pharmaceutically acceptable solvents as excipients.

[0013] Furthermore, the drug is a tiger nut polypeptide solution with a concentration of 65 mg / mL to 130 mg / mL prepared with physiological saline.

[0014] This invention provides a tiger nut polypeptide for improving cognitive impairment. The tiger nut polypeptide is obtained by extracting tiger nut meal as raw material through alkaline dissolution and acid precipitation treatment and enzymatic hydrolysis, and then separating it using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The tiger nut polypeptide contains the peptides shown in SEQ ID NO.1 to SEQ ID NO.30.

[0015] The present invention also provides a method for preparing the tiger nut polypeptide for improving cognitive impairment, comprising the following steps: Tiger nut meal was defatted with n-hexane, and the defatted tiger nut meal was subjected to alkali dissolution and acid precipitation treatment. The pH value of the acid precipitation step was adjusted to 4.2-4.5 to obtain tiger nut protein isolate. The alkaline dissolution and acid precipitation treatment steps include: mixing defatted tiger nuts with water, adjusting the pH to 8.0-9.0 with alkaline solution to dissolve them, centrifuging and taking the supernatant, then adjusting the pH to 4.2-4.5 with acid solution to precipitate the protein; Tiger bean protein isolate is first hydrolyzed with pepsin under acidic conditions, and then hydrolyzed with trypsin under neutral conditions to obtain the hydrolysate. The conditions for the first step of enzymatic hydrolysis are: pH 1.5–2.5, temperature 35℃–39℃, enzyme to substrate mass ratio of 1:40–60, and hydrolysis time of 60 min–120 min; the conditions for the second step of enzymatic hydrolysis are: pH 6.5–7.5, temperature 35℃–39℃, enzyme to substrate mass ratio of 1:40–60, and hydrolysis time of 150 min–210 min. The enzymatic hydrolysis product was centrifuged, and the supernatant was collected. Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was collected and dried to obtain the tiger nut polypeptide.

[0016] Furthermore, during the defatting process, the ratio of tiger nut meal to hexane liquid is 1:3 g to 4 mL.

[0017] Furthermore, the centrifugation conditions were: 4000 r / min for 15 minutes.

[0018] This invention provides an application of the aforementioned tiger nut polypeptide in the preparation of a health product for regulating intestinal microbiota, wherein the health product is used to increase the abundance of beneficial bacteria.

[0019] This invention provides an application of the aforementioned tiger nut polypeptide in the preparation of health products that help improve memory.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: The tiger nut polypeptides prepared by this invention are beneficial for improving cognitive function, improving memory and learning ability, relieving oxidative stress in brain tissue, restoring cholinergic system function and inhibiting neuroinflammation, and can also reshape the intestinal flora structure and increase the abundance of beneficial bacteria.

[0021] This invention utilizes readily available and inexpensive tiger nut meal (after cold pressing) as raw material, and prepares it through a specific stepwise enzymatic hydrolysis process, achieving high-value comprehensive utilization of the raw material. Network pharmacology analysis shows that the potential target of this peptide is significantly related to cognitive-related pathways such as the cAMP signaling pathway and neuroinflammation, suggesting its potential to improve cognitive function. Animal experiments have confirmed that this peptide can significantly improve spatial memory and learning ability in mice with cognitive impairment, effectively alleviate oxidative stress in brain tissue, restore cholinergic system function, and inhibit neuroinflammation. Further research has found that it can improve cognition through the gut-brain axis by reshaping the gut microbiota structure and increasing the abundance of beneficial bacteria, providing technical support and scientific basis for its application in related functional foods, health foods, and pharmaceuticals. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The amino acid composition and mass spectrum of the prepared tiger nut polypeptide; In the diagram, A represents the amino acid composition of tiger nut polypeptide; B is the mass spectrum of tiger nut peptides.

[0024] Figure 2 This is a diagram showing the association between potential targets of tiger barley peptides and cognitive-related pathways based on network pharmacology analysis. In the diagram, A represents the intersection target point; B represents the interaction diagram of the core target points; C represents the core target enrichment map; D is the core target-pathway interaction diagram; E represents GO functional enrichment analysis; F represents the KEGG pathway enrichment analysis.

[0025] Figure 3 To investigate the effects of tiger nut peptides on the behavioral phenotypes of cognitively impaired mice in the Barnes maze, new object recognition, and Y-maze. In the figure, A represents the typical trajectory of a cognitively impaired mouse in the Barnes maze experiment; B is to avoid the incubation period; C represents the exploration time; D represents the typical trajectory of a cognitively impaired mouse in recognizing new objects; E represents the new object recognition index; F represents the percentage of alternating movements in the Y maze.

[0026] Figure 4 The effect of tiger nut peptides on brain tissue morphology in mice with cognitive impairment.

[0027] Figure 5 The effects of tiger nut peptides on the expression of SOD, MDA, and GSH, indicators of oxidative stress, in the brain tissue of cognitively impaired mice.

[0028] Figure 6 The effects of tiger nut peptides on the expression of inflammatory factors TNF-α, IL-6 and iNOS genes in the brain tissue of cognitively impaired mice.

[0029] Figure 7 The effects of tiger nut peptides on the expression of ACh, ChAT and AChE in the brain tissue of cognitively impaired mice.

[0030] Figure 8 The effects of tiger nut peptides on the expression of neurotrophic factors PSD-95, BDNF and NGF genes in the brain tissue of cognitively impaired mice.

[0031] Figure 9 The effects of tiger nut peptides on the structure and composition of gut microbiota in cognitively impaired mice; In the diagram, A is the Venn diagram of the OTU; B is the LEfSe clustering tree; C is the percentage of abundance of each community in the gut microbiota at the phylum level; D is the ratio (F / B) between Firmicutes and Bacteroidetes. E represents the percentage of abundance of each community of the gut microbiota at the genus level.

[0032] In the above figures, CON represents the control group, SCOP represents the scopolamine model group, CEP-L represents the low-dose tiger nut peptide group (600 mg / kg / day), CEP-H represents the high-dose tiger nut peptide group (1200 mg / kg / day), and DON represents the donepezil group. Detailed Implementation

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0034] Example 1: A tiger nut polypeptide and its preparation method.

[0035] Tiger nut meal was pulverized, passed through a 100-mesh sieve, and defatted with hexane at a ratio of 1 g:3 mL (fungus meal:solvent) in a stirrer for 0.5 h. The mixture was then filtered, and the filtrate was discarded. This defatting process was repeated four times until the hexane liquid became clear. The resulting defatted tiger nut meal was dried to completely remove residual hexane, yielding defatted tiger nut powder. The defatted tiger nut powder was added to pure water at a ratio of 1:15, and the pH was adjusted to 8.5 with 1 mol / L sodium hydroxide solution. The mixture was stirred at 25°C for 1.5 h, and then centrifuged at 4°C and 6000 r / min for 20 min. The pH of the supernatant was then adjusted to 4.5 with 1 mol / L hydrochloric acid, and incubated at 25°C for 1 h. The supernatant was then discarded, and the remaining mixture was centrifuged at 4°C and 6000 r / min for 20 min. The precipitate was washed three times with deionized water and centrifuged at 6000 r / min at 4℃. The precipitate was collected and freeze-dried using a vacuum freeze dryer (freezing at -80℃ for 48 h) to obtain tiger nut protein. Tiger nut protein was dissolved in distilled water at a mass-to-volume ratio of 1:20. The pH was adjusted to 2 in a 37℃ constant temperature water bath. Pepsin was added at an enzyme-to-substrate mass ratio of 1:50, and digestion was maintained at constant temperature and pH for 90 min. The enzyme was then inactivated by boiling in a water bath for 10 min. The pH was then adjusted to 7, and trypsin was added at an enzyme-to-substrate mass ratio of 1:50. Digestion was continued for 180 min, followed by a boiling in a water bath for 10 min to terminate the reaction. The mixture was then centrifuged at 4000 r / min for 15 min, and the supernatant was collected. Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. After concentration by rotary evaporation, the mixture was freeze-dried using a vacuum freeze dryer (freezing at -80℃ for 24 h) to obtain tiger nut peptides.

[0036] Example 2: Network Pharmacology Target Prediction The tiger nut peptides prepared in Example 1 were analyzed for peptide composition using LC-MS / MS mass spectrometry. The results are as follows: Figure 1 As shown, the prepared tiger nut polypeptide contains 19 amino acids, including 7 essential amino acids, accounting for 48.28% of the total. Among them, leucine (Leu), glycine (Gly), valine (Val), and alanine (Ala) are the most abundant, accounting for 15.71%, 10%, 9.99%, and 8.47%, respectively.

[0037] The top 30 peptides by peak area were selected, and their sequences, peak areas, sequencing scores, and molecular weights are shown in Table 1.

[0038] Table 1. Sequences, peak areas, sequencing scores, and molecular weights of the top 30 most abundant peptides in tiger nut polypeptides. The GeneCards database, Gene-NCBI database, and Swiss Target Prediction database were used to identify cognitive impairment-related targets in tiger nut peptides.

[0039] The results are as follows Figure 2 As shown, based on the prediction results, there are 538 predicted targets for tiger nut peptides and 4387 predicted targets related to cognitive impairment. Figure 2 The intersection of the target A with the predicted target of tiger barley peptide was matched, resulting in 113 potential intersection targets. Protein-protein interaction (PPI) network analysis was performed using the String database, and the results were visualized using Cytoscape software. Figure 2 B~ Figure 2 (D), and found that the core targets involved IL-6, AKT1, TNF, CASP3, etc. GO functional enrichment analysis showed that ( Figure 2 These targets (E) are significantly enriched in biological processes such as neuronal signal transduction and synaptic regulation. KEGG pathway enrichment analysis showed that (E) Figure 2 The predicted targets of tiger barley peptides (F) were significantly enriched in the cAMP signaling pathway, neuroactive ligand-receptor interactions, and the TNF signaling pathway. These results suggest that tiger barley peptides may improve cognitive function through multiple pathways, including regulating neurotransmitters and inflammatory responses.

[0040] Example 3: The effect of tiger nut peptides on improving learning and memory abilities in cognitively impaired mice The experimental animals were eight-week-old male C57BL / 6J mice weighing 20g–25g, purchased from Shaanxi Nuoyou Biotechnology Co., Ltd., and housed in the animal facility of Northwest A&F University under a 25±2℃, 12h light / dark cycle environment, with free access to water and food. After 7 days of acclimatization, the mice were randomly divided into five experimental groups, with eight mice in each group:

[0041] (1) Control group (CON): 0.2 mL of normal saline was administered by gavage daily; (2) Scopolamine model group (SCOP): 0.2 mL of physiological saline was administered by gavage daily; (3) Low-dose tiger nut polypeptide group (600 mg / kg / day by gavage, CEP-L): 0.2 mL of tiger nut polypeptide solution with a concentration of 65 mg / mL prepared by gavage was administered daily; (4) High-dose tiger nut polypeptide group (1200 mg / kg / day by gavage, CEP-H): 0.2 mL of tiger nut polypeptide solution with a concentration of 130 mg / mL prepared by gavage was administered daily by gavage; (5) Dopenezil group (3 mg / kg / day by gavage, DON): 0.2 mL of donepenezil solution with a concentration of 0.33 mg / mL prepared by normal saline was administered by gavage daily.

[0042] Four weeks after intervention, except for the control group, the other four groups were intraperitoneally injected with scopolamine (1.5 mg / kg / day) to establish a mouse model of cognitive impairment. The control group was intraperitoneally injected with 0.9% saline. Behavioral assessments were performed 30 minutes later. After the behavioral assessments, fecal samples were collected under sterile conditions. The animals were euthanized under anesthesia, and all measures were taken to minimize suffering throughout the experiment. Tissue and serum samples were immediately frozen in liquid nitrogen for further examination.

[0043] To assess whether tiger nut peptides could improve spatial memory in mice, the Barnes maze test was conducted, and the results were as follows: Figure 3 A~ Figure 3 As shown in Figure C, after 5 consecutive days of learning and training, the escape latency of mice in the SCOP group significantly increased during the test period, while the exploration time significantly decreased. Supplementation with high and low doses of tiger nut peptides significantly shortened the escape latency and significantly increased the exploration time. The novel object recognition experiment assessed the short-term recognition memory ability of mice using a two-stage paradigm. The experimental results showed ( Figure 3 D and Figure 3 Compared to the CON group, SCOP significantly reduced the novel object recognition index in mice, while intervention with tiger nut peptides improved the novel object recognition index and alleviated short-term memory impairment. Additionally, the Y-maze has been used to assess animals' learning and reference memory abilities, such as... Figure 3 In the F group of mice, the percentage of alternation induced by SCOP was significantly reduced. Supplementation with tiger barley peptides significantly increased the reduction in the percentage of alternation induced by SCOP. These results indicate that tiger barley peptides can improve SCOP-induced cognitive dysfunction in mice.

[0044] Example 4: Effects of tiger nut peptides on brain tissue morphology in cognitively impaired mice The experimental animals were grouped and fed in the same manner as in Example 3. The morphological structure of neurons in the mouse hippocampus was observed by hematoxylin-eosin (H&E) staining and Nissl staining.

[0045] like Figure 4 As shown, chromatin blurring and nuclear condensation were observed in the CA1, CA3, and DG regions of the brains of mice in the SCOP group, indicating more significant neuronal damage. The intervention of tiger barley peptides significantly improved the neuronal state of the mice and alleviated the neuronal damage caused by SCOP.

[0046] Example 5: Effects of tiger nut peptides on the expression of oxidative stress markers in the brain tissue of cognitively impaired mice The experimental animals were grouped and fed in the same manner as in Example 3. Mouse cerebral cortex tissue was homogenized, and the activities of superoxide dismutase (SOD), malondialdehyde (MDA), and reduced glutathione (GSH) were measured using appropriate biochemical reagent kits, strictly following the instructions.

[0047] The results are as follows Figure 5 Compared with the CON group, the SCOP group mice showed a significant decrease of 67% and 21% in GSH and SOD levels in the cerebral cortex, respectively, and an increase of 12% in MAD level. These changes were improved by the intervention of tiger nut peptides, indicating that the antioxidant capacity of tiger nut peptides can inhibit oxidative stress response in brain tissue.

[0048] Example 6: Effects of tiger nut peptides on the expression of inflammatory factors in the brain tissue of cognitively impaired mice The experimental animals were grouped and fed in the same manner as in Example 3. Total RNA was extracted from the mouse cerebral cortex using the TRIzol method, and cDNA was synthesized by reverse transcription. Real-time quantitative PCR (RT-qPCR) was then performed using SYBR Green fluorescent dye. Primer design is shown in Table 2. The expression levels of inflammatory factor mRNA were detected.

[0049] Table 2 RT-qPCR primers The results are as follows Figure 6Compared with the CON group, the SCOP group mice showed significantly increased levels of IL-6, TNFα, and iNOS. Tiger barley peptide intervention could significantly inhibit the increase in these inflammatory factors, indicating that tiger barley peptide intervention can effectively alleviate neuroinflammation in the brains of cognitively impaired mice.

[0050] Example 7: Effects of tiger nut peptides on the functional expression of the cholinergic system in the brain tissue of cognitively impaired mice The experimental animals were grouped and fed in the same manner as in Example 3. The content of acetylcholine (ACh) was detected using a biochemical kit, and the content of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) was quantified using an enzyme-linked immunosorbent assay (ELISA) kit.

[0051] like Figure 7 As shown, ACh and CHAT levels were significantly decreased and AChE levels were significantly increased in the cerebral cortex of mice in the SCOP group, indicating that SCOP causes cholinergic dysfunction in mice. Compared with the SCOP group, treatment with tiger nut peptides significantly increased ACh and CHAT levels and significantly decreased AChE levels, indicating that supplementation with tiger nut peptides can improve cholinergic dysfunction.

[0052] Example 8: Effects of tiger nut peptides on the expression of neurotrophic factors in the brain tissue of cognitively impaired mice The experimental animals were grouped and fed in the same manner as in Example 3. The mRNA expression levels of postsynaptic density protein 95 (PSD-95), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) in mouse hippocampal tissue were detected using RT-qPCR, following the same method as in Example 6.

[0053] The results are as follows Figure 8 SCOP treatment significantly reduced the mRNA levels of PSD-95, BDNF, and NGF in the brain, while tiger nut peptide treatment restored the expression of PSD-95, BDNF, and NGF. The results indicate that tiger nut peptide can upregulate the expression of neurotrophic factor genes and improve neuronal damage.

[0054] Example 9: Effects of tiger nut peptides on the structure and composition of gut microbiota in cognitively impaired mice The experimental animals were grouped and fed in the same manner as in Example 3. The composition and structural changes of the mouse gut microbiota were analyzed using 16S rRNA high-throughput sequencing technology.

[0055] Figure 9The Venn diagram of group A shows that groups CON, SCOP, CEP-L, CEP-H, and DON contain 1153, 1663, 1178, 1109, and 1089 unique OTU operational taxonomic units (OTUs), respectively. Gut microbiota composition was assessed using linear discriminant analysis of effect size (LDFSe), analyzing the relative abundance and changes of dominant species at the phylum level. Figure 9 (B and C groups) revealed that Bacteroidota, Desulfobacterota, Firmicutes, and Actinobacteriota dominated the gut microbiota, indicating that tiger barley peptide intervention altered the composition of the mouse gut microbiota. Furthermore, compared to the CON group, SCOP significantly reduced the F / B ratio and the relative abundance of Actinobacteriota, while increasing the relative abundance of Desulfobacterota. The F / B ratio represents the ratio of Firmicutes to Bacteroidetes abundance; this condition was restored by tiger barley peptide intervention. Figure 9 The intervention of tiger nut peptides (D) increased the F / B ratio. At the genus level ( Figure 9 (E) Alistipe (Alternaria) Desulfovibrio (Desulfovibrio) is the dominant bacterial group in SCOP-treated mice, while Lachnospiraceae_NK4A136_group (Spirophyceae_NK4A136_group) Bifidobacterium (Bifidobacterium) Akkermansia (Ackermania) and Dubosiella (Duboscirella) was the dominant gut microbiota in mice fed with tiger nut peptides. In summary, tiger nut peptide supplementation can improve gut microbiota and help protect mice from SCOP-induced cognitive impairment.

[0056] The above results indicate that the tiger nut polypeptide described in this invention has the function of improving cognitive impairment and regulating gut microbiota.

[0057] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0058] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of tiger nut peptides in the preparation of drugs to improve cognitive impairment, characterized in that, The tiger nut polypeptide is obtained by extracting tiger nut meal through alkaline dissolution and acid precipitation and enzymatic hydrolysis, and then separating it using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The tiger nut polypeptide contains the peptides shown in SEQ ID NO.1 to SEQ ID NO.

30.

2. The application according to claim 1, characterized in that, The drug is used to prevent or treat Alzheimer's disease.

3. The application according to claim 2, characterized in that, The drug is used to reduce inflammation and improve neuronal damage.

4. The application according to claim 2, characterized in that, The drug is also used to help improve memory.

5. The application according to claim 1, characterized in that, The drug is also used to increase the abundance of beneficial bacteria in the gut.

6. The application according to claim 5, characterized in that, The enhancement of beneficial intestinal bacteria abundance is achieved by increasing the ratio of Firmicutes to Bacteroidetes abundance in the gut, and by increasing the abundance of Bifidobacterium, Akkermania, and Dubois.

7. The application according to claim 1, characterized in that, The drug uses tiger nut polypeptide as the sole active ingredient and pharmaceutically acceptable solvents as excipients.

8. The application according to claim 7, characterized in that, The drug is a tiger nut polypeptide solution with a concentration of 65 mg / mL to 130 mg / mL prepared with physiological saline.

9. A tiger nut polypeptide for improving cognitive impairment, characterized in that, The tiger nut polypeptide is obtained by extracting tiger nut meal through alkaline dissolution and acid precipitation and enzymatic hydrolysis, and then separating it using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The tiger nut polypeptide contains the peptides shown in SEQ ID NO.1 to SEQ ID NO.

30.

10. The method for preparing the tiger nut polypeptide for improving cognitive impairment according to claim 9, characterized in that, Includes the following steps: Tiger nut meal was defatted with n-hexane, and the defatted tiger nut meal was subjected to alkali dissolution and acid precipitation treatment. The pH value of the acid precipitation step was adjusted to 4.2-4.5 to obtain tiger nut protein isolate. The alkaline dissolution and acid precipitation treatment steps include: mixing defatted tiger nuts with water, adjusting the pH to 8.0-9.0 with alkaline solution to dissolve them, centrifuging and taking the supernatant, then adjusting the pH to 4.2-4.5 with acid solution to precipitate the protein; Tiger bean protein isolate is first hydrolyzed with pepsin under acidic conditions, and then hydrolyzed with trypsin under neutral conditions to obtain the hydrolysate. The conditions for the first step of enzymatic hydrolysis are: pH 1.5–2.5, temperature 35℃–39℃, enzyme to substrate mass ratio of 1:40–60, and hydrolysis time of 60 min–120 min; the conditions for the second step of enzymatic hydrolysis are: pH 6.5–7.5, temperature 35℃–39℃, enzyme to substrate mass ratio of 1:40–60, and hydrolysis time of 150 min–210 min. The enzymatic hydrolysis product was centrifuged, and the supernatant was collected. Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The permeate was collected and dried to obtain the tiger nut polypeptide.

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