Preparation method and application of almond protein enzymatic hydrolysis peptide with improved memory function

Abstract

The application relates to the technical field of biotechnology, and particularly discloses a preparation method and application of almond protein enzymolysis peptides with improved memory function, and the technical key points are as follows: the inhibition rate of the almond protein enzymolysis peptides on acetylcholinesterase is 52.29+ / -1.91%. The preparation method comprises the following steps: extracting almond protein by an alkali extraction and acid precipitation method, carrying out enzymolysis on the almond protein by using trypsin and cellulase, and collecting a peptide component with a molecular weight less than 3 KDa by using ultrafiltration separation. The optimal preparation process conditions of the almond protein enzymolysis peptides are determined, and the peptide yield reaches 77.92+ / -0.92%. The almond protein enzymolysis peptides can significantly shorten the escape latency of memory disorder mice, increase the number of times of crossing the target platform, repair the CA1 area of hippocampal tissue, and increase the number of neuron cells; the almond protein enzymolysis peptides can significantly inhibit the acetylcholinesterase activity of brain tissue, improve the acetylcholine level, improve the SOD activity, and reduce the MDA level, so that the memory disorder is improved. The application fills the blank of the almond protein enzymolysis peptides in the field of improving memory function, and can be used for preparing medicines or functional foods for improving memory function.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for preparing and applying almond enzyme hydrolysate peptides with memory-improving functions. Background Technology

[0002] almonds ( Amygdalus communis Almonds (Prunus persica), also known as sweet almonds or sago palms, are the fruit of a tree belonging to the genus Prunus in the family Rosaceae. They are a specialty dried fruit resource of Xinjiang. Almonds are extremely nutritious, with their kernels rich in protein, unsaturated fatty acids, vitamins, and various minerals. The protein content can reach over 20%, making them a high-quality plant protein resource with broad prospects for development and utilization. Xinjiang is my country's main almond-producing area, with a long history of cultivation, abundant varieties, and considerable annual output. However, currently, almond processing and utilization mainly focus on direct consumption of the dried fruit, with relatively low levels of deep processing. The in-depth development and utilization of its protein resources are still insufficient, and high-value-added products are few, which to some extent restricts the further development of the almond industry.

[0003] Protein hydrolyzed peptides refer to a mixture of peptides produced by the hydrolysis of proteins under the action of proteases. They typically consist of 2-20 amino acid residues and have a molecular weight generally less than 10 kDa. In recent years, with the deepening research on bioactive peptides, more and more studies have shown that protein hydrolyzed peptides are not only a type of nutrient but also a class of bioactive substances with multiple physiological regulatory functions. Numerous studies have confirmed that protein hydrolyzed peptides derived from various plant proteins such as soybeans, corn, peanuts, and walnuts possess a variety of biological activities, including antioxidant, antihypertensive, hypolipidemic, immunomodulatory, and antibacterial effects. However, research on almond protein hydrolyzed peptides is currently limited both domestically and internationally, and the development and utilization of almond protein as a raw material for preparing protein hydrolyzed peptides is still in its early stages.

[0004] Memory impairment is a common neurological disorder, clinically manifested as decreased learning and memory abilities and cognitive decline. With the accelerating aging of the global population, the incidence of age-related memory impairment is increasing year by year, becoming a major public health problem seriously affecting the quality of life of the elderly. Furthermore, memory impairment can be caused by various factors such as Alzheimer's disease, cerebrovascular disease, brain injury, drug poisoning, and mental stress. In severe cases, it can develop into dementia, imposing a heavy economic and care burden on patients' families and society. Therefore, finding safe and effective products to improve memory function has significant social and economic value.

[0005] Currently, clinically used drugs for improving memory function mainly include acetylcholinesterase inhibitors (such as donepezil, rivastigmine, and galantamine) and N-methyl-D-aspartate (NMDA) receptor antagonists (such as memantine). Among them, acetylcholinesterase inhibitors are the most widely used class of drugs for improving memory function. Their mechanism of action is as follows: In the central nervous system, acetylcholine (ACh) is an important neurotransmitter that plays a crucial role in learning and memory. Acetylcholinesterase (AChE) is an enzyme that catalyzes the hydrolysis of acetylcholine into choline and acetic acid. Increased AChE activity leads to a decrease in acetylcholine levels in the synaptic cleft, thereby impairing cholinergic neurotransmission and causing learning and memory dysfunction. Acetylcholinesterase inhibitors reduce the hydrolysis of acetylcholine by inhibiting the activity of acetylcholinesterase, thereby increasing the level of acetylcholine in brain tissue, enhancing central cholinergic neuronal function, and improving memory and cognitive abilities. However, existing acetylcholinesterase inhibitors also have certain limitations in clinical application, such as gastrointestinal side effects, liver toxicity, and poor drug tolerance. The safety and compliance of long-term use need to be improved.

[0006] Furthermore, oxidative stress plays a crucial role in the development and progression of memory impairment. Studies have shown that under pathological conditions such as aging, ischemia, hypoxia, and neurodegenerative diseases, the production and clearance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the body become unbalanced. Excessive free radicals attack the unsaturated fatty acids in nerve cell membranes, triggering lipid peroxidation, which damages the structure and function of nerve cells, thereby leading to a decline in learning and memory abilities. Therefore, supplementing the body with exogenous active substances capable of scavenging free radicals, increasing the activity of antioxidant enzymes (such as superoxide dismutase SOD), and reducing the production of lipid peroxidation products (such as malondialdehyde MDA) is also an important strategy for improving memory impairment.

[0007] In view of the shortcomings of the prior art, this application proposes an almond enzyme hydrolysate with memory-improving function, its preparation method and application, in order to solve the above problems. Summary of the Invention

[0008] The purpose of this invention is to solve the technical problems mentioned in the background section and provide a method for preparing and applying almond protein hydrolysate with memory-improving function. This invention extracts almond protein using an alkaline extraction and acid precipitation method, and optimizes the enzymatic hydrolysis process through single-factor and response surface methodology experiments, using the degree of hydrolysis and peptide yield as indicators, to determine the optimal preparation conditions for almond protein hydrolysate. Furthermore, the almond protein hydrolysate is separated and purified by ultrafiltration, and almond protein hydrolysate fractions with acetylcholinesterase inhibitory activity are screened using the acetylcholinesterase inhibition rate as an indicator, and their free radical scavenging ability is evaluated. Finally, the efficacy of almond protein hydrolysate in improving memory impairment is verified at the animal level by establishing a scopolamine-induced memory impairment mouse model. This invention fully utilizes Xinjiang's unique dried fruit resources, providing a new avenue for the deep processing and utilization of almond protein, and also providing new candidate substances for the research and development of drugs or functional foods that improve memory function.

[0009] The above-mentioned objective of the present invention is achieved as follows: One aspect of the present invention provides an almond protein hydrolysate with memory-improving function. The almond protein hydrolysate is obtained by enzymatic hydrolysis of almond protein with trypsin and cellulase, followed by centrifugation and fractionation using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa, 3 kDa, and 1 kDa. The molecular weight of the almond protein hydrolysate is less than 3 kDa.

[0010] Another aspect of the present invention provides a method for preparing almond enzyme hydrolysates with memory-improving function, comprising the following steps: S1. Extraction of almond protein: Defatted almond meal was dissolved in distilled water at a material-to-liquid ratio of 1:25 (g:mL), the pH was adjusted to 9.0 with 0.6 mol / L NaOH, sonicated for 15 min, water bath at 50 ℃ for 60 min, centrifuged at 6000 r / min for 20 min, the supernatant was collected, the pH was adjusted to 4.0 with 1 mol / L HCl, centrifuged at 6000 r / min for 20 min, the supernatant was discarded, the precipitate was adjusted to neutral pH, and freeze-dried to obtain almond protein; S2. Preparation of almond protein hydrolysate: The almond protein obtained in step S1 was prepared into a 5% almond protein solution. Trypsin was added at a [E] / [S] ratio of 10%, and cellulase was added at a trypsin:cellulase ratio of 4:1. After mixing, the solution was enzymatically hydrolyzed in a shaking water bath at 55 °C for 12 h, and then the enzyme was inactivated in a water bath at 95 °C for 15 min. After centrifugation at 8000 r / min for 20 min, the supernatant was collected and freeze-dried to obtain almond protein hydrolysate. S3. Ultrafiltration separation: The almond enzyme hydrolysate obtained in step S2 is centrifuged and fractionated using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 KDa, 3 KDa and 1 KDa. The ultrafiltration fraction with a molecular weight less than 3 KDa is collected, freeze-dried, and the almond enzyme hydrolysate with memory-improving function is obtained.

[0011] Furthermore, in step S1, the defatted almond meal is prepared by the following method: after the almonds are shelled, dried, and pulverized, they are defatted with petroleum ether for 6 hours, the petroleum ether is discarded, and the meal is passed through a 40-mesh sieve to obtain defatted almond meal.

[0012] Furthermore, in step S1, the almond protein extraction conditions are as follows: material-to-liquid ratio 1:25 (g:mL), pH 9.0, sonication for 15 min, water bath at 50 ℃ for 60 min, and centrifugation at 6000 r / min for 20 min.

[0013] Furthermore, in step S2, the enzymatic hydrolysis conditions are as follows: almond protein solution concentration 5%, [E] / [S] 10%, trypsin to cellulase addition ratio 4:1, and enzymatic hydrolysis at 55 °C for 12 h.

[0014] Furthermore, in step S3, the relative molecular weight cutoff of the ultrafiltration centrifuge tube is 10 kDa, 3 kDa, and 1 kDa.

[0015] This invention also provides the application of almond protein hydrolysate peptides in the preparation of drugs or functional foods that improve memory function.

[0016] This invention also provides the application of almond protease hydrolysate in the preparation of acetylcholinesterase inhibitors.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides, for the first time, almond protein hydrolysate peptides with memory-improving functions. No research reports on the memory-improving effects of almond protein hydrolysate peptides have been found in the prior art. This invention extracts almond protein using an alkaline extraction and acid precipitation method, and prepares almond protein hydrolysate peptides using an enzymatic hydrolysis method. Using the degree of hydrolysis and peptide yield as indicators, single-factor and response surface methodology experiments were conducted to determine the optimal preparation conditions for almond protein hydrolysate peptides: substrate concentration 4.8%, enzyme dosage 14%, hydrolysis time 8 h, and hydrolysis temperature 54℃. Under these conditions, the degree of hydrolysis of almond protein can reach 55.22 ± 0.56%, and the almond peptide yield can reach 77.92% ± 0.92%, providing technical support for the efficient utilization of almond protein.

[0018] 2. This invention fractionates almond protease-digested peptides using ultrafiltration and screens them for activity based on acetylcholinesterase inhibition rate. The results show that almond protease-digested peptide fractions with a molecular weight less than 3 kDa exhibit the highest acetylcholinesterase inhibitory activity, with an inhibition rate of 52.29 ± 1.91%, indicating the potential of this fraction to be developed as an acetylcholinesterase inhibitor. Since acetylcholinesterase inhibitors can improve memory function by increasing acetylcholine levels in brain tissue, the almond protease-digested peptide fractions screened in this invention are expected to become a novel candidate substance for improving memory function.

[0019] 3. This invention clarifies that almond protease hydrolysate fractions with a molecular weight less than 3 kDa possess excellent free radical scavenging capabilities. Experimental results show that the DPPH free radical scavenging rate of this ultrafiltration fraction reaches 87.39%, with an IC50 of 5.11 mg / mL; the hydroxyl free radical scavenging rate reaches 90.67%, with an IC50 of 4.34 mg / mL; the superoxide anion free radical inhibition rate reaches 78.18%, with an IC50 of 28.74 mg / mL; and the total antioxidant capacity is 1.23 mM. Furthermore, both the free radical scavenging ability and the total antioxidant capacity increase with increasing concentration. These results indicate that almond protease hydrolysate peptides can improve memory function both by inhibiting acetylcholinesterase activity and by scavenging excess free radicals and reducing oxidative stress damage, demonstrating a dual mechanism of action.

[0020] 4. This invention establishes a scopolamine-induced memory impairment mouse model and verifies the efficacy of almond protein hydrolysate peptides in improving memory impairment at the animal level. Morris water maze behavioral experiments showed that after 28 days of oral administration of almond protein hydrolysate peptides, the high-dose group mice exhibited simplified movement trajectories, the shortest movement paths, and were able to find platforms more quickly. The escape latency of mice in the medium-dose and high-dose groups was significantly reduced. p <0.001), decreasing by 38.36±19.30 s and 92.67±16.32 s respectively, and significantly increasing the number of times the target platform region was traversed ( p <0.05); pathological sections showed that almond protease hydrolysate had a certain repair effect on the damaged CA1 region of the mouse hippocampus and significantly increased the number of neurons; the acetylcholine level in the brain tissue of mice in the medium-dose group and the high-dose group increased significantly by 39.58% ( p <0.05) and 112.43% ( p <0.001), the content of acetylcholinesterase in the brain tissue of mice in the low-dose, medium-dose and high-dose groups was significantly reduced ( p<0.001), reducing acetylcholine levels by 66.49%, 70.93%, and 73.85% respectively, indicating that almond protein hydrolysate can effectively increase acetylcholine levels in the brain tissue of memory-impaired mice and inhibit acetylcholinesterase activity, thereby improving cholinergic nerve function. Simultaneously, almond protein hydrolysate significantly increased serum superoxide dismutase (SOD) levels in memory-impaired mice (increasing by 5.59%, 9.94%, and 10.03% in the low, medium, and high dose groups, respectively) and significantly decreased malondialdehyde (MDA) levels (reducing by 14.97%, 23.20%, and 30.84% ​​in the low, medium, and high dose groups, respectively), indicating that almond protein hydrolysate can enhance the activity of antioxidant enzymes in the body, reduce the generation of lipid peroxidation products, and improve memory impairment through the antioxidant stress pathway.

[0021] 5. This invention confirmed through HE staining of mouse liver and kidney that almond protein hydrolysate had no significant effect on the morphology of liver and kidney tissues in each group of mice, and no pathological changes such as necrosis, degeneration, hyperplasia and fibrosis were found, indicating that almond protein hydrolysate has no toxic effect on mice and has good biosafety, providing a safety guarantee for its application in the fields of pharmaceuticals or functional foods.

[0022] 6. This invention uses almonds, a specialty dried fruit from Xinjiang, as raw material to prepare protease-hydrolyzed peptides with memory-improving functions. This provides a new approach for the deep processing and utilization of almonds and the development of high-value-added products, which is conducive to promoting the upgrading and development of the Xinjiang almond industry and has good economic and social benefits. Attached Figure Description

[0023] Figure 1 This is a graph showing the acetylcholinesterase inhibition rate of the almond protein hydrolysate ultrafiltration fraction (100 mg / mL) in an embodiment of the present invention. Figure 2 These are the in vitro antioxidant capacity results of different concentrations of almond protein hydrolysate (UF3) in the embodiments of the present invention (b is the result of scavenging hydroxyl radicals; c is the result of inhibiting superoxide anion radicals; d is the result of total antioxidant capacity (ABTS method)). Figure 3 This is a graph showing the effect of almond protein hydrolysate (AP) on mouse body weight in an embodiment of the present invention; Figure 4 This is an HE staining image (n=3; 400×; A: liver; B: kidney) showing the effects of almond protein hydrolysate (AP) on the morphology of the liver and kidneys in mice with memory impairment, as described in this embodiment of the invention. Figure 5 This is a diagram showing the effect of almond protein hydrolysate (AP) on the localization and navigation trajectory of mice with memory impairment in an embodiment of the present invention; Figure 6This is a diagram showing the effect of almond protein hydrolysate (AP) on the spatial exploration trajectory of mice with memory impairment in an embodiment of the present invention; Figure 7 This is a graph showing the effect of almond peptide (AP) on the spatial exploration ability of mice with memory impairment in this invention (a is the escape latency result; b is the result of the number of times the target platform area was crossed). Figure 8 This is an HE staining image (n=3; 400×) showing the effect of almond peptide (AP) on the morphology of mouse hippocampus tissue in an embodiment of the present invention. Figure 9 This is a graph showing the effect of almond peptide (AP) on the cholinergic system in the brain tissue of mice with memory impairment in this invention (a is the acetylcholine result; b is the acetylcholinesterase result). Figure 10 The following is a graph showing the effect of almond protein hydrolysate (AP) on oxidative stress in the serum of mice with memory impairment in this invention (a is the SOD result graph; b is the MDA result graph). Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] This invention provides a method for preparing and applying almond protein hydrolysate with memory-improving functions, aiming to address the lack of research on almond protein hydrolysate with memory-improving effects and its preparation process in the existing technology, as well as the lack of reports on the application of almond protein hydrolysate in improving memory. This invention extracts almond protein using an alkaline extraction and acid precipitation method, and prepares almond peptides using enzymatic hydrolysis. Using the degree of hydrolysis and peptide yield as indicators, the optimal preparation conditions for almond peptides were determined through single-factor and response surface methodology experiments. Furthermore, the almond peptides were separated and purified by ultrafiltration, and almond peptide components with potential memory-improving effects were screened using the acetylcholinesterase inhibition rate as an indicator. It was also confirmed that the almond peptide components have good free radical scavenging ability. Simultaneously, a mouse model of memory impairment was established, and the almond peptide components were applied to the mice, demonstrating that almond peptides have an improving effect on memory impairment in mice.

[0026] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0027] Example 1: Preparation process of almond peptides, screening of acetylcholinesterase inhibitory activity and free radical scavenging ability 1. Experimental Procedure (1) Extraction of almond protein Almond protein extraction was performed according to the method described by Sun Yue'e et al.

[11] The method involved shelling, drying, and pulverizing almonds, followed by defatting with petroleum ether for 6 hours. The petroleum ether was discarded, and the almonds were passed through a 40-mesh sieve. The defatted almond meal was dissolved in distilled water at a ratio of 1:25 (g:mL). The pH was adjusted to 9.0 with 0.6 mol / L NaOH, followed by sonication for 15 min, water bath at 50 ℃ for 60 min, and centrifugation at 6000 r / min for 20 min. The supernatant was collected, and the pH was adjusted to 4.0 with 1 mol / L HCl. The mixture was centrifuged at 6000 r / min for 20 min, and the supernatant was discarded. The precipitate was adjusted to neutral pH and freeze-dried.

[0028] (2) Preparation of almond peptides A 5% almond protein solution was prepared by adding trypsin at an [E] / [S] ratio of 10% and cellulase at a trypsin:cellulase ratio of 4:1. After mixing, the solution was incubated in a shaking water bath at 55 ℃ for 12 h, followed by enzyme inactivation in a water bath at 95 ℃ for 15 min. The solution was then centrifuged at 8000 r / min for 20 min, and the supernatant was collected and freeze-dried.

[0029] (3) Determination of degree of hydrolysis (DH) The degree of hydrolysis (DH) was determined using the o-phthalaldehyde (OPA) method, referring to Nielsen et al.

[12] The determination method is as follows: Mix 3 mL of OPA reagent and 0.4 mL of the test solution, let stand at room temperature for 2 min, and measure the absorbance at 340 nm. The concentration of the serine standard solution is 0.9516 mmol / L. The calculation of DH is shown in formula (1): (1) Where: OD 样品 : Absorbance of almond protein hydrolysate tube; OD 空白 : Distilled water tube absorbance; OD 标准 : Absorbance of serine standard solution tube; V: Total volume, L; N: Dilution factor; X: Sample mass, g; P: Protein content in sample, % (2) In the formula: α and β are constants, α = 1.00; β = 0.40; (3) In the formula: h of almond protein tot Take 7.58 mmol / g

[13] .

[0030] (4) Determination of peptide yield The peptide yield was determined using the biuret method, referring to Ye Kai et al.

[14] The determination method was as follows. A standard curve regression equation was constructed with bovine serum albumin (BSA) concentration (mg / mL) as the x-axis (X) and absorbance as the y-axis (Y). , (R 2 = 0.9996). The almond protein hydrolysate was mixed with an equal volume of 15% (w / v) trichloroacetic acid (TCA), allowed to stand for 10 min, centrifuged at 10000 r / min for 10 min, and 1 mL of the supernatant was mixed with 4 mL of biuret reagent and reacted at room temperature for 30 min. The absorbance was measured at 540 nm. The peptide concentration (mg / mL) of the almond protein hydrolysate was obtained by referring to the bovine serum albumin (BSA) standard curve. The peptide yield was calculated as shown in formula (4): (4) (5) Single-factor and response surface optimization experiments Based on single-factor experiments, Design-Expert 13 software was used to optimize the enzymatic hydrolysis process conditions of almond protein using the Box-Behnken design principle, with substrate concentration (A), enzyme dosage (B), hydrolysis time (C), and hydrolysis temperature (D) as experimental factors and degree of hydrolysis (DH) as the response value.

[0031] (6) Determination of acetylcholinesterase inhibition rate The assay was performed according to the method described by ELLMAN et al. 50 μL of almond peptide solution was mixed with 30 μL of thioacetylcholine iodide (ATCI, 7.5 mmol / L), 125 μL of 5,5'-dithiobisnitrosobenzoic acid (DTNB, 3 mmol / L), and 40 μL of HEPES (pH 8.0, 50 mM, containing 0.1% BSA). The mixture was incubated at 37 ℃ for 20 min, followed by the addition of 30 μL of acetylcholinesterase (AChE, 0.055 U / mL). A tannin assay was used as a positive control. The assay was performed at 412 nm with 30 s intervals for 30 min. The inhibition rate of acetylcholinesterase activity was calculated using formula (5).

[0032] (5) In the formula: A 样品 The absorbance value after the sample is added and reacted; A 样品空白 The absorbance value after adding the sample but not AChE and reacting; A 对照 The absorbance value after the reaction without adding a sample; A 对照空白 The absorbance value refers to the absorbance value after reacting with almond peptides without the addition of AchE.

[0033] (7) Evaluation of the free radical scavenging ability of almond peptides The DPPH radical, hydroxyl radical, and superoxide anion radical scavenging abilities and total antioxidant capacity of almond peptides were determined using a kit.

[0034] 2. Experimental Results (1) Results of the optimization experiment on the preparation process of almond peptides Response surface methodology (RSM) analysis of almond protein hydrolysis predicted the optimal conditions for preparing almond peptides as follows: substrate concentration 4.76%, enzyme dosage 14%, hydrolysis time 8 h, and hydrolysis temperature 53.5 °C. Under these conditions, the theoretical degree of hydrolysis of almond protein is 55.01%. Considering practical ease of operation, the optimal conditions were verified at a substrate concentration of 4.8%, enzyme dosage 14%, hydrolysis time 8 h, and hydrolysis temperature 54 °C. The results showed that the degree of hydrolysis of almond protein was 55.22 ± 0.56%, close to the predicted value. Under these conditions, the yield of almond peptides was 77.92% ± 0.92%.

[0035] (2) Ultrafiltration separation of almond peptides and screening of almond peptide components with acetylcholinesterase inhibitory activity Almond protein hydrolysate was fractionated by centrifugation using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa, 3 kDa, and 1 kDa, yielding six almond peptide fractions: UF1 (Mw < 1 kDa), UF2 (1 kDa < Mw < 1 kDa), UF3 (Mw < 3 kDa), UF4 (3 kDa < Mw < 10 kDa), UF5 (Mw < 10 kDa), and UF6 (Mw > 10 kDa). The results of each fraction inhibiting acetylcholinesterase activity are shown below. Figure 1 As shown in the figure, the low molecular weight ultrafiltration almond peptide fraction exhibits higher acetylcholinesterase inhibitory activity, with the ultrafiltration almond peptide fraction UF3 (M...) showing the highest activity. w The inhibition rate of acetylcholinesterase was highest at <3 kDa, at 52.29 ± 1.91%.

[0036] (3) Free radical scavenging ability of almond peptide (UF3) The in vitro antioxidant activity results of the ultrafiltration almond peptide component UF3 are shown in Figure 2. Figure 2 It can be seen that the DPPH free radical scavenging rate of the ultrafiltration component UF3 reaches 87.39%, and the IC50 value is [missing information]. 50 The concentration was 5.11 mg / mL; the hydroxyl radical scavenging rate reached 90.67%, and the IC50 concentration was [missing value]. 50 The concentration was 4.34 mg / mL; the superoxide anion radical inhibition rate reached 78.18%, and the IC50 concentration was [missing value]. 50The concentration was 28.74 mg / mL; the total antioxidant capacity was 1.23 mM. The free radical scavenging capacity and total antioxidant capacity of the ultrafiltration fraction UF3 increased with increasing concentration. At low concentrations, the free radical scavenging capacity and total antioxidant capacity of ultrafiltration fraction UF3 were weaker than those of vitamin C; however, the antioxidant capacity of the ultrafiltration fraction increased with increasing concentration.

[0037] Example 2: The effect of almond peptides on improving memory impairment in mice 1. Experimental Procedure (1) Animal experimental procedures and gavage strategies SPF-grade C57 mice were divided into groups after 7 days of acclimatization: the normal control group (NC) was administered an equal volume of 0.9% saline by gavage daily; the model control group (MC) was administered an equal volume of 0.9% saline by gavage daily; the positive control group (PC) was administered 400 mg / kg piracetam by gavage daily; and the low-dose peptide group (AP-L), medium-dose peptide group (AP-M), and high-dose peptide group (AP-H) were administered 200 mg / kg, 333 mg / kg, and 666 mg / kg almond peptide by gavage daily, respectively. After 28 days of continuous gavage, the mouse water maze test was performed.

[0038] In the water maze experiment, the normal control group (NC) was administered an equal volume of 0.9% saline by gavage, the model control group (MC) was administered an equal volume of 0.9% saline by gavage, the positive control group (PC) was administered 400 mg / kg piracetam by gavage, and the low, medium, and high dose groups of almond peptide were administered 200 mg / kg, 333 mg / kg, and 666 mg / kg almond peptide by gavage, respectively. Sixty minutes after gavage each day, except for the normal control group (NC), the other groups of mice were intraperitoneally injected with 1.0 mg / kg scopolamine. The normal control group (NC) mice were intraperitoneally injected with an equal volume of 0.9% saline. Thirty minutes after the intraperitoneal injection, the water maze behavioral test was performed for 6 consecutive days. Mice were sacrificed after the water maze experiment.

[0039] (2) Morris water maze experiment ① Positioning and navigation experiments In the orientation and navigation experiment, the platform was placed in the first quadrant. Mice were placed in four different initial quadrants. The escape latency (time to find the platform) and movement trajectory of the mice were continuously recorded for 60 seconds using a video tracking system. The changes in the mice during the training process were observed. If a mouse could not find the platform within 60 seconds, it was guided to the platform, and its latency was recorded as 60 seconds. After arriving at the platform, the mouse stayed on the platform for 10 seconds before the next experiment was conducted. This training was repeated for 5 consecutive days.

[0040] ② Space exploration experiment Twenty-four hours after the end of the navigation experiment, the platform location was marked as the target platform area. The platform was removed, and the mice were placed in the water from four different starting points (different quadrants). The swimming path of the mice within 120 seconds was recorded, as well as the time when the mice first found the target platform area and the number of times they crossed the target platform area. If the mice could not find the platform within 120 seconds, their latency period was recorded as 120 seconds.

[0041] (3) Sample collection All mice were processed after the behavioral experiments. They were anesthetized and fixed in a prone position on the operating table. The following samples were collected: Serum samples: 0.5-1 ml of whole blood was collected from each mouse. After centrifugation, the supernatant serum was separated, aliquoted into cryovials, and stored at -80°C. Brain tissue: 6 animals were used per group. Complete brain tissue was taken from each group of 6 mice, dissected along the coronal plane (a cross-section of the hippocampus, including the CA1-CA4 region), and placed in 4% formaldehyde solution for storage at room temperature. The remaining brain tissue was stored at -80°C. The remaining 6 mice had their complete brain tissue divided into two parts along the sagittal plane and stored at -80°C. Liver tissue: Half of the liver tissue was placed in cryovials, flash-frozen in liquid nitrogen, and stored at -80°C. The other half was placed in 4% formaldehyde solution for storage at room temperature. Kidney tissue: Half of the kidney tissue was placed in cryovials, flash-frozen in liquid nitrogen, and stored at -80°C. The other half was placed in 4% formaldehyde solution for storage at room temperature.

[0042] (4) The kit was used to detect acetylcholine (ACH), acetylcholinesterase (AChE), SOD and MDA in mouse brain tissue.

[0043] (5) Observation of HE staining of mouse tissue Brain tissue was fixed in formaldehyde and then in 4% paraformaldehyde at 4°C for 48 h. The tissue was then dissected in the coronal plane from the optic chiasm to the posterior end, and embedded in paraffin to prepare sections for hippocampal observation. Sections were 4 μm thick, stained with hematoxylin and eosin (HE), and neurons in the CA1 region of the hippocampus were observed under a microscope for neuronal cell counting.

[0044] Kidney and liver tissues from mice fixed in formaldehyde were collected from each group, and routine dehydration, clearing, paraffin embedding, sectioning, and HE staining were performed. The pathological conditions of the kidneys and livers were observed and described.

[0045] 2. Results of almond peptide's effect on improving memory impairment in mice (1) Effect of almond peptide (AP) on mouse body weight The effects of almond peptide (AP) on mouse body weight are as follows: Figure 3 As shown in the figure, after 35 days of gavage, the growth trends of mice in each group were basically the same. These results indicate that almond peptide and the positive control drug piracetam had no significant effect on the body weight of mice during gavage, and there were no significant differences in the physiological state of the mice.

[0046] (2) Effects of almond peptide (AP) on the morphology of liver and kidney tissues in mice with memory impairment Figure 4 shows the HE staining results of the effects of almond peptide (AP) on the morphology of the liver and kidneys of mice with memory impairment. No significant morphological differences were found between the groups, indicating that no pathological changes such as necrosis, degeneration, hyperplasia and fibrosis were observed in the liver and kidneys of the mice, confirming that almond peptide (AP) has no toxic effect on mice.

[0047] (3) Effects of almond peptide (AP) on learning and memory abilities in mice during the Morris water maze test ① Positioning and navigation experiments In the first phase of the orientation and navigation experiment, mice underwent a 5-day orientation and navigation experiment. The time it took for the mice to find the platform (escape latency) in the presence of the platform reflected their spatial learning and memory abilities. The trajectory results of the mouse orientation and navigation experiment are as follows: Figure 5 As shown, the circular area in the first quadrant of the water maze represents the location of the platform. The red dot indicates the starting position of the mouse, and the blue dot indicates the position where the mouse stopped when it found the platform or reached the platform after 60 seconds. The figure shows that, compared to the normal control group (NC) mice, the model control group (MC) mice still could not find the platform after 5 days of orientation and navigation experiments. Compared to the model control group (MC) mice with memory impairment, the medium-dose (AP-M) and high-dose (AP-H) almond peptide groups were able to find the platform after 5 days of orientation and navigation experiments. The high-dose almond peptide group (AP-H) mice exhibited a simplified movement trajectory and the shortest path, enabling them to find the platform more quickly.

[0048] The escape latency in the mouse navigation experiment was measured, and the results are shown in Table 1.

[0049] Table 1. Effects of almond protein hydrolysate (AP) on the latency of orientation-navigation escape in mice with memory impairment. ; As can be seen from the above, compared with the normal control group (NC) mice, the escape latency (time to find the platform) of the model control group (MC) mice was significantly prolonged. p <0.001), prolonged by 47.16±1.78 s; compared with the model control group (MC) memory-impaired mice, the escape latency of mice in the low-dose (AP-L), medium-dose (AP-M), and high-dose (AP-H) almond peptide groups was significantly reduced ( p <0.001), which reduced the escape latency by 37.20±5.77 s, 49.94±3.56 s and 53.37±2.09 s respectively. Moreover, the escape latency of mice in the almond peptide test group showed a decreasing trend with the extension of the positioning and navigation experiment time.

[0050] ② Space exploration experiment In the second phase of the spatial exploration experiment, after the platform was removed, the time it took for the mouse to first reach the target platform area (escape latency) and the number of times it traversed the target platform area reflected the mouse's memory ability. The mouse's trajectory in searching for the target platform area is shown below. Figure 6 .Depend on Figure 6 It was found that, compared with the normal control group (NC) mice, the model control group (MC) mice did not enter the target platform area; compared with the model control group (MC) memory-impaired mice, the low-dose (AP-L), medium-dose (AP-M), and high-dose (AP-H) almond peptide groups were able to find the target platform area, among which the high-dose almond peptide group (AP-H) mice had their movement area more concentrated in the target platform quadrant.

[0051] The escape latency and the number of times mice crossed the target platform area were measured in the space exploration experiment. The results are as follows: Figure 7 As shown. By Figure 7 As shown in a, compared with the normal control group (NC) mice, the escape latency (time to first reach the target platform area) of the model control group (MC) mice was significantly increased by 99.75 ± 21.74 s. p <0.001); Compared with the model control group (MC) of memory-impaired mice, the escape latency of mice in the medium-dose almond peptide group (AP-M) and the high-dose almond peptide group (AP-H) was significantly reduced ( p <0.001), which reduced the time by 38.36±19.30 s and 92.67±16.32 s, respectively.

[0052] Depend on Figure 7 As shown in b, compared with the normal control group (NC) mice, the model control group (MC) mice traversed the target platform region significantly less often. p<0.001); Compared with the model control group (MC) memory-impaired mice, the number of times mice in the medium-dose almond peptide group (AP-M) and the high-dose almond peptide group (AP-H) crossed the target platform region was significantly increased. p <0.05).

[0053] (4) Effects of almond peptide (AP) on the morphology of mouse brain tissue Mouse hippocampal tissue was stained with hematoxylin and eosin (HE) and observed under a 400x microscope. The results are as follows: Figure 8 As shown in the figure, the hippocampal neurons in the normal control group (NC) mice are intact and neatly and clearly arranged in the CA1 region. Compared with the normal control group (NC) mice, the hippocampal neurons in the model control group (MC) mice show edema, disordered arrangement, and a significant reduction in the number of neurons in the CA1 region of the hippocampus. p <0.001 (as shown in Table 2 below).

[0054] Table 2. Effects of almond peptide (AP) on the number of neurons in the CA1 region of the mouse hippocampus. ; Compared with the MC group of mice with memory impairment, the low-dose (AP-L), medium-dose (AP-M), and high-dose (AP-H) almond peptide groups showed varying degrees of improvement in the hippocampus, with reduced neuronal edema, orderly arrangement, and a significant increase in the number of neurons in the CA1 region. p <0.01). This indicates that almond peptide (AP) improves memory impairment in mice by increasing the number of neurons in the hippocampus and improving the neuronal state of the hippocampus.

[0055] (5) Regulation of the cholinergic system in the brain tissue of mice with memory impairment by almond peptide (AP). The results of the analysis of the effects of almond peptide (AP) on acetylcholine and acetylcholinesterase in the brain tissue of scopolamine-induced memory impairment mice are as follows: Figure 9 As shown. By Figure 9 It was found that, compared with the normal control group (NC) mice, after intraperitoneal injection of scopolamine to induce memory impairment in mice, the acetylcholine (ACH) content in the brain tissue of the model control group (MC) mice was significantly reduced by 58.29%. p <0.001); After oral administration of almond peptide (AP) for 28 days, compared with the model control group (MC) mice, the levels of acetylcholine (ACH) in the brain tissue of mice in the medium-dose group (AP-M) and the high-dose group (AP-H) of almond peptide were significantly increased, increasing by 39.58% ( ). p <0.05) and 112.43% ( p<0.001), and the content of acetylcholinesterase (AChE) in mouse brain tissue was significantly reduced ( p <0.001), which reduced memory impairment by 66.49%, 70.93% and 73.85% respectively, thereby improving memory impairment in mice.

[0056] (6) Effects of almond peptide (AP) on oxidative stress in mice with memory impairment The results of the analysis of the effects of almond peptide (AP) on the antioxidant defense in the serum of scopolamine-induced memory impairment mice are as follows: Figure 10 As shown. Figure 10 As can be seen from a, compared with the normal control group (NC) mice, after scopolamine-induced memory impairment in mice, the serum superoxide dismutase (SOD) content in the model control group (MC) mice was significantly reduced by 8.35%. p <0.001); After 28 days of intervention, compared with the model control group (MC) mice, the serum superoxide dismutase (SOD) levels in mice in the low-dose (AP-L), medium-dose (AP-M), and high-dose (AP-H) almond peptide groups were significantly increased, increasing by 5.59% ( ). p <0.01), 9.94% p <0.001) and 10.03% ( p <0.001).

[0057] Depend on Figure 10 As shown in b, compared with normal control (NC) mice, the serum malondialdehyde (MDA) content in the model control (MC) memory-impaired mice was significantly increased by 37.17%. p <0.001), after oral administration of almond peptide (AP) for 28 days, compared with the model control group (MC) mice, it significantly reduced the malondialdehyde (MDA) content in the brain tissue of mice in the low-dose group (AP-L), medium-dose group (AP-M), and high-dose group (AP-H) of almond peptide, by 14.97% ( ). p <0.05), 23.20% p <0.01) and 30.84% ​​( p <0.001).

[0058] The above results indicate that almond peptide (AP) can increase the activity of antioxidant enzymes in mice and reduce the content of malondialdehyde (MDA) in the body, thereby improving the learning and memory abilities of scopolamine-induced memory-impaired mice.

[0059] In summary, through the above embodiments of the present invention, almond protein was extracted by alkaline extraction and acid precipitation. Using the degree of hydrolysis and peptide yield as indicators, the optimal preparation conditions for almond protein hydrolysis were determined through single-factor and response surface methodology experiments (substrate concentration 4.8%, enzyme dosage 14%, hydrolysis time 8 h, hydrolysis temperature 54 °C; under these conditions, the degree of hydrolysis reached 55.22 ± 0.56%, and the peptide yield reached 77.92% ± 0.92%). Furthermore, six fractions with different molecular weights were obtained through ultrafiltration fractionation. Using acetylcholinesterase inhibition rate as an activity tracking indicator, the almond protein hydrolysis peptide fraction (UF3) with a molecular weight less than 3 kDa was screened for the first time and found to have the highest acetylcholine content. The esterase inhibitory activity was 52.29±1.91%, and this component also exhibited excellent free radical scavenging ability (DPPH free radical scavenging rate 87.39%, hydroxyl free radical scavenging rate 90.67%, superoxide anion free radical inhibition rate 78.18%, and total antioxidant capacity 1.23 mM). Based on this, this invention established, for the first time, a scopolamine-induced memory impairment mouse model to verify the memory-improving effect of almond protease hydrolysate peptides in vivo. Experimental results showed that almond protease hydrolysate peptides could simplify the mouse's movement trajectory, shorten the movement path, enable it to find platforms more quickly, and significantly reduce the escape latency of mice (reduction of 38.36±19.30% in the medium-dose group). The high-dose group showed a reduction of 92.67 ± 16.32 s, significantly increased the number of times the target platform region was crossed, had a certain repair effect on the damaged CA1 region of the mouse hippocampus, and significantly increased the number of neurons. It could significantly increase the acetylcholine level in the brain tissue of mice with memory impairment (39.58% increase in the medium-dose group and 112.43% increase in the high-dose group), significantly reduce the acetylcholinesterase content (66.49% decrease in the low-dose group, 70.93% decrease in the medium-dose group, and 73.85% decrease in the high-dose group), significantly increase the serum superoxide dismutase activity (5.59%, 9.94%, and 10.03% increase in the low-, medium-, and high-dose groups, respectively), and significantly reduce the malondialdehyde content (14.97%, 23.20%, and 30.84% ​​decrease in the low-, medium-, and high-dose groups, respectively). Furthermore, the almond protease hydrolysate was confirmed to have no toxic effect by HE staining of the liver and kidneys. The above results indicate that the almond protease hydrolysate prepared in this invention can improve memory through a dual mechanism of inhibiting acetylcholinesterase activity, increasing acetylcholine levels in brain tissue, and enhancing the body's antioxidant capacity. This invention is the first to establish a link between almond protease hydrolysate and memory improvement, filling a gap in the field of almond protease hydrolysate for memory enhancement, and providing a novel technical solution and material basis for the deep processing and utilization of almond protein and the development of products that improve memory function.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An almond enzyme hydrolysate peptide with memory-improving function, characterized in that, The almond protein hydrolysate is obtained by enzymatic hydrolysis of almond protein with trypsin and cellulase, followed by centrifugation and fractionation using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa, 3 kDa, and 1 kDa. The molecular weight of the almond protein hydrolysate is less than 3 kDa.

2. A method for preparing almond protein hydrolysate peptides with memory-improving function, characterized in that, Includes the following steps: S1. Extraction of almond protein: Defatted almond meal was dissolved in distilled water at a material-to-liquid ratio of 1:25 (g:mL), the pH was adjusted to 9.0 with 0.6mol / L NaOH, sonicated for 15 min, heated in a 50 ℃ water bath for 60 min, centrifuged at 6000 r / min for 20 min, the supernatant was collected, the pH was adjusted to 4.0 with 1 mol / L HCl, centrifuged at 6000 r / min for 20 min, the supernatant was discarded, the precipitate was adjusted to neutral pH, and freeze-dried to obtain almond protein; S2. Preparation of almond protein hydrolysate: The almond protein obtained in step S1 was prepared into a 5% almond protein solution. Trypsin was added at a [E] / [S] ratio of 10%, and cellulase was added at a trypsin:cellulase ratio of 4:

1. After mixing, the mixture was enzymatically hydrolyzed in a shaking water bath at 55°C for 12 h, and then the enzyme was inactivated in a water bath at 95°C for 15 min. After centrifugation at 8000 r / min for 20 min, the supernatant was collected and freeze-dried to obtain almond protein hydrolysate. S3. Ultrafiltration separation: The almond enzyme hydrolysate obtained in step S2 is centrifuged and fractionated using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa, 3 kDa and 1 kDa. The ultrafiltration fraction with a molecular weight less than 3 kDa is collected, freeze-dried, and the almond enzyme hydrolysate with memory-improving function is obtained.

3. The preparation method according to claim 2, characterized in that, In step S1, the defatted almond meal is prepared by the following method: after the almonds are shelled, dried, and crushed, they are defatted with petroleum ether for 6 hours, the petroleum ether is discarded, and the meal is passed through a 40-mesh sieve to obtain defatted almond meal.

4. The preparation method according to claim 2, characterized in that, In step S1, the almond protein extraction conditions are as follows: material-to-liquid ratio 1:25 (g:mL), pH 9.0, sonication for 15 min, water bath at 50 ℃ for 60 min, and centrifugation at 6000 r / min for 20 min.

5. The preparation method according to claim 2, characterized in that, In step S2, the enzymatic hydrolysis conditions are as follows: almond protein solution concentration 5%, [E] / [S] 10%, trypsin to cellulase addition ratio 4:1, and enzymatic hydrolysis at 55 °C for 12 h.

6. The preparation method according to claim 2, characterized in that, In step S3, the relative molecular weight cutoff of the ultrafiltration centrifuge tube is 10 kDa, 3 kDa, and 1 kDa.

7. The use of the almond protease hydrolysate according to claim 1 or the almond protease hydrolysate prepared by any one of claims 2-6 in the preparation of drugs or functional foods for improving memory function.

8. The use of the almond protease hydrolysate according to claim 1 or the almond protease hydrolysate prepared by any one of claims 2-6 in the preparation of acetylcholinesterase inhibitors.

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