Preparation method of larimichthys crocea polypeptide and application of larimichthys crocea polypeptide in anti-Alzheimer's disease functional food
Through ultrasound-assisted complex enzyme-enzymatic calcined croaker meat protein, a polypeptide with acetylcholinesterase inhibitory activity was prepared, which solved the shortcomings of the croaker polypeptide in the prior art in alleviating Alzheimer's disease, significantly improved the memory and motor ability of the Alzheimer's disease model, and provided a way to develop functional foods against Alzheimer's disease.
Patent Information
- Application Number
- CN202510348423.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-24
- Publication Date
- 2025-07-25
AI Technical Summary
The prior art fails to effectively utilize yellow croaker polypeptides to relieve and improve Alzheimer's disease, and lacks acetylcholinesterase inhibitors, resulting in a lack of drugs for the treatment of AD.
Using ultrasonic-assisted complex enzymatic enzyme lysis of yellow croaker meat protein, active polypeptides with acetylcholinesterase inhibitory activity were prepared, including CMSCGP, CPNEEKCCHN, CSNDSDC and GCGHECIAP, and obtained by ultrasonic-assisted hydrolysis and gel chromatography separation and purification.
The enzymatic lysis efficiency of the big yellow croaker meat protein was significantly improved, and a polypeptide with strong acetylcholinesterase inhibitory activity was prepared, which significantly improved the memory and motor ability of the Alzheimer's zebrafish model, and provided the development direction of functional foods against Alzheimer's disease.
Smart Images
Figure CN120366407A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly relates to a method for preparing large yellow croaker polypeptide and its application in functional foods for anti-Alzheimer's disease.
Background Art
[0002] Alzheimer's disease (AD) is the most common dementia among the aging population. Patients with AD have memory loss, confused thinking, inarticulate speech, and decreased judgment ability, and the symptoms become more and more serious over time, seriously affecting the physical health and quality of life of the elderly. More severely, there is currently no cure for AD, and it can only be resisted through prevention and alleviation. This is undoubtedly a heavy blow to families with AD patients and also causes a very serious economic burden on today's medical system. Therefore, it is urgent to strengthen the prevention and treatment of AD, slow down the occurrence and development of AD, and reduce the economic pressure on families and society.
[0003] Research believes that there are many hypotheses about the pathogenesis of AD, mainly including the cholinergic hypothesis, free radical damage theory, gene mutation theory, amyloid peptide hypothesis, etc. Clinically, the treatment of AD is mainly based on the cholinergic hypothesis. This theory believes that the lack of the neurotransmitter acetylcholine in the brains of AD patients is the main cause, and the degradation of acetylcholine by acetylcholinesterase (AChE) is the most direct cause of the lack of acetylcholine. Galantamine, an alkaloid compound derived from the Amaryllidaceae family, as a selective and reversible competitive AChE inhibitor, was first clinically approved in Australia due to its significant efficacy. Therefore, inhibitors can be developed or searched to inhibit the activity of acetylcholinesterase to slow down the occurrence and development of AD.
[0004] Currently, through the CNKI search of domestic and foreign patents, no invention application that uses the extracted large yellow croaker polypeptide to relieve and improve Alzheimer's disease has been retrieved.
Summary of the Invention
[0005] One of the technical problems to be solved by the present invention is to provide a method for efficiently hydrolyzing large yellow croaker fish meat into active polypeptides by ultrasonic-assisted complex enzymes, which uses ultrasonic-assisted complex enzymatic hydrolysis of large yellow croaker fish meat protein, significantly improves the enzymatic hydrolysis efficiency of large yellow croaker fish meat protein, and simultaneously separates and purifies to obtain large yellow croaker active polypeptides with strong acetylcholinesterase inhibitory activity, which can be used to prepare functional foods for anti-Alzheimer's disease, thereby relieving and improving Alzheimer's disease.
[0006] The present invention realizes the above technical problem as follows:
[0007] A method for efficiently hydrolyzing large yellow croaker fish meat with ultrasonic-assisted composite enzymes to prepare bioactive peptides, comprising the following steps:
[0008] Step 1: Weigh large yellow croaker fish meat and grind it into minced meat. After fully mixing the fish meat with isopropanol, perform cyclic extraction at 60 ± 5 °C for 1 ± 0.5 h to remove fat and volatilize excess isopropanol;
[0009] Step 2: Heat with boiling water to inactivate endogenous proteases, adjust the pH to 7.0 ± 0.5, add 50 - 150 mg / g of compound protease, perform ultrasonic-assisted hydrolysis, and then place it in a water bath at 28 - 45 °C for constant-temperature enzymatic hydrolysis; the compound protease is at least one of acidic protease, neutral protease, compound protease, and trypsin;
[0010] Step 3: After the enzymatic hydrolysis ends and cools down, centrifuge to obtain the supernatant, which is the large yellow croaker protease hydrolysate;
[0011] Step 4: Fractional separation of large yellow croaker peptides: First, concentrate the large yellow croaker peptide mixture to remove small molecular compounds such as amino acids and inorganic salts, and obtain a small molecular peptide mixture with a molecular weight less than or equal to 3000 Da; then, perform gel chromatography separation on the small molecular peptide mixture, elute with distilled water, the detection wavelength is 220 nm, start collecting from 30 minutes, stop at 50 minutes, and concentrate the polypeptide solution collected during this period to obtain large yellow croaker bioactive peptides.
[0012] Further, in Step 1, the fish meat and isopropanol are fully mixed at a mass-to-volume ratio of 1:4.
[0013] Further, in Step 2, ultrasonic-assisted hydrolysis is performed three times with 150 V ultrasound, each time for 10 min; the addition amount of the compound protease is 100 mg / g, the water bath hydrolysis temperature is 30 °C, the hydrolysis time is 5 h, and the pH is 7.0.
[0014] Further, the compound protease is compounded by acidic protease, neutral protease, compound protease, and trypsin in a ratio of 1:2:3:2.
[0015] Further, Step 4 is specifically as follows: Fractional separation of large yellow croaker peptides: First, concentrate the large yellow croaker peptide mixture using an ultrafiltration tube with a molecular weight cut-off of 3 kDa to remove small molecular compounds such as amino acids and inorganic salts, and obtain a small molecular peptide mixture with a molecular weight less than or equal to 3000 Da; then, perform gel chromatography separation on the small molecular peptide mixture using Sephadex G-15 (1.6 x 100 cm), elute with distilled water, the detection wavelength is 220 nm, collect the components in the 30 - 50 minute time period, and obtain large yellow croaker bioactive peptides after rotary evaporation.
[0016] The second technical problem to be solved by the present invention is to provide an application of large yellow croaker polypeptide in the preparation of functional foods for anti-Alzheimer's disease. The large yellow croaker polypeptide has strong acetylcholinesterase inhibitory activity and can be used to relieve and improve Alzheimer's disease.
[0017] The present invention realizes the second above-mentioned technical problem as follows:
[0018] An application of large yellow croaker polypeptide in the preparation of functional foods for anti-Alzheimer's disease, wherein the large yellow croaker polypeptide is prepared by the method of preparing active polypeptide by ultrasonic-assisted complex enzyme hydrolysis of large yellow croaker fish meat, and has acetylcholinesterase inhibitory activity.
[0019] Furthermore, the large yellow croaker active polypeptide includes CMSCGP, CPNEEKCCHN, CSNDSDC, and GCGHECIAP, and the amino acid sequences are shown in SEQ ID No: 1-4.
[0020] The present invention has the following advantages:
[0021] 1. The present invention uses ultrasonic-assisted complex enzymatic hydrolysis of large yellow croaker fish meat protein, which significantly improves the enzymatic hydrolysis efficiency of large yellow croaker fish meat protein. Different molecular weight peptide components can be obtained through separation and purification, and large yellow croaker peptides can be efficiently prepared, providing a new route for the efficient preparation of molecular peptides.
[0022] 2. The key polypeptides obtained by separation and purification are CMSCGP, CPNEEKCCHN, CSNDSDC, and GCGHECIAP large yellow croaker active polypeptides, which show acetylcholinesterase inhibitory activity. That is, the four polypeptide molecules in the large yellow croaker active polypeptide can bind to acetylcholinesterase through hydrogen bonds and other interactions, thereby reducing the activity of acetylcholinesterase, providing a new direction for natural active substances that inhibit acetylcholine enzyme or reduce the activity of acetylcholine enzyme.
[0023] 3. Feeding the obtained large yellow croaker active polypeptide to a zebrafish model simulating Alzheimer's disease significantly improves the memory ability and motor ability of the model, can be used to prepare functional foods for anti-Alzheimer's disease, and further relieve and improve Alzheimer's disease.
Description of the Drawings
[0024] The following further describes the present invention with reference to the drawings in conjunction with the embodiments.
[0025] Figure 1 It is the optimization effect diagram of the enzymatic hydrolysis conditions of large yellow croaker fish meat in the embodiment of the present invention.
[0026] Figure 2 It is the high performance liquid chromatography analysis diagram of the enzymatically hydrolyzed polypeptide of large yellow croaker in the embodiment of the present invention.
[0027] Figure 3 This is a schematic diagram of the test results of the acetylcholinesterase inhibitory activity of the enzymatically hydrolyzed polypeptide component of large yellow croaker in the embodiments of the present invention.
[0028] Figure 4 This is an attached drawing showing the influence of large yellow croaker polypeptides at different concentrations on the motor ability of zebrafish models in the embodiments of the present invention.
[0029] Figure 5 This is an attached drawing showing the influence of large yellow croaker polypeptides at different concentrations on the behavior of Alzheimer's zebrafish models in the embodiments of the present invention.
[0030] Figure 6 This is an attached drawing showing the influence of large yellow croaker polypeptides at different concentrations on the behavior of zebrafish models in a maze in the embodiments of the present invention.
[0031] Figure 7 This is a schematic diagram of HE staining sections and TUNEL staining sections of the pathological changes in the brain tissues of zebrafish in different groups in the embodiments of the present invention.
[0032] Figure 8 This is a schematic diagram of the molecular docking of polypeptides with acetylcholinesterase in the embodiments of the present invention. Among them, A: Molecular docking of CMSCGP with acetylcholinesterase, B: Molecular docking of CSNDSDC with acetylcholinesterase, C: Molecular docking of CPNEEKCHN with acetylcholinesterase, D: Molecular docking of GCGHECIAP with acetylcholinesterase.
Detailed implementation manners
[0033] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "connection", and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.
[0034] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the attached Figure 1-8 drawings and specific implementation manners. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.
[0035] Embodiment
[0036] 1. Materials
[0037] The processing by-products of large yellow croaker were provided by Fujian Fuding Seagull Aquatic Food Co., Ltd. The head meat, adhering meat and minced meat remaining on the large yellow croaker frame were removed using a dissecting knife, scissors, etc.
[0038] Compound protease produced by Angel Yeast Co., Ltd.
[0039] 2. Pretreatment of large yellow croaker fish meat
[0040] Weigh 100 g of thawed large yellow croaker fish meat and put it into a blender to be minced into meat paste. After fully mixing the fish meat and isopropanol at a ratio of 1:4 (mass to volume), extract it at 60 °C for 1 h in a cycle to fully remove the fat contained in the large yellow croaker fish meat. Place the extracted fish meat at room temperature to volatilize the excess isopropanol.
[0041] 3. Screening of enzymatic hydrolysis conditions for large yellow croaker protease
[0042] To screen out the optimal enzymatic hydrolysis conditions and optimize the protein hydrolysis process of large yellow croaker, first, use 4 kinds of proteases and their compound proteases to carry out enzymatic hydrolysis reactions. The types of enzymes are trypsin, compound protease, acidic protease, and neutral protease; the compound protease is acidic protease: neutral protease: compound protease: trypsin = 1:2:3:2. Examine the polypeptide yields of each protease hydrolyzing large yellow croaker protein in the same time. Under the same conditions, the hydrolysis effect of the compound protease is much higher than that of other proteases. Use single-factor analysis to study the effects of different enzyme addition amounts, hydrolysis temperatures, hydrolysis times, and pH on the enzymatic hydrolysis of large yellow croaker fish meat. Finally, determine that the optimal enzyme addition amount, hydrolysis temperature, hydrolysis time, and pH are 100 mg / g, 30 °C, 5 h, and 7.0 respectively. For specific reference, see Figure 1 。
[0043] 4. Ultrasonic-assisted high-efficiency hydrolysis of large yellow croaker fish meat by compound enzymes
[0044] Heat the pretreated large yellow croaker fish meat in boiling water for 15 min to inactivate the endogenous protease. Adjust the pH to 7.0 using 0.2 M NaOH and 0.5 M HCl. Add 100 mg / g of compound protease, and perform ultrasonic treatment at 150 V three times, each time for 10 min. Then place it in a water bath at 30 °C for constant-temperature enzymatic hydrolysis for 5 h. After completion, inactivate the enzyme in boiling water for 10 min. After cooling, centrifuge at 12000 rpm for 2 min to take the supernatant. The supernatant is the enzymatic hydrolysate of large yellow croaker protease. Determine the polypeptide content by the Folin-Ciocalteu method, and the polypeptide content under the optimal combined conditions is 198 ± 15 mg / L. Folin-Ciocalteu method: Add 10% trifluoroacetic acid to the enzymatic hydrolysate of large yellow croaker to precipitate for 30 minutes, centrifuge at 8000 rpm for 10 minutes, take the supernatant, and use the Folin-Ciocalteu kit of Sangon Biotech (Shanghai) Co., Ltd. to determine the polypeptide content therein.
[0045] 5. Fractionation and separation of large yellow croaker polypeptides
[0046] First, an ultrafiltration tube with a molecular weight cut-off of 3 kDa was used to concentrate the mixture of large yellow croaker polypeptides, removing small molecule compounds such as amino acids and inorganic salts to obtain a mixture of small peptides with a molecular weight less than or equal to 3000 Da. Then, the mixture of small peptides was separated by gel chromatography using Sephadex G-15 (1.6 x 100 cm), eluted with distilled water, the detection wavelength was 220 nm, fractions were collected every 3 - 5 minutes, the same fractions were collected multiple times and mixed, and after concentration, peptide fractions 1 - 4 with different molecular weights were obtained according to the molecular weight. Among them, fraction 2 (the fraction collected in the time period of 30 - 50 minutes) had the strongest acetylcholinesterase inhibitory activity. At 50 mM, the enzyme activity inhibition rate was 42%. For specific reference, Figure 2 as shown in Figure 2 the test results of the acetylcholinesterase inhibitory activity of the enzymatically hydrolyzed large yellow croaker polypeptide components.
[0047] 6. Mass spectrometry identification of the large yellow croaker polypeptide sequence
[0048] The sample of Component 2 was freeze-dried, an appropriate amount of 0.1% TFA was added, and the mixture was homogenized. After centrifugation at 20,000 g for 5 min, the supernatant was taken and transferred to a 10 KD ultrafiltration centrifugal tube, followed by centrifugation at 12,000 g for 15 min. 200 μL of 0.1% TFA was added and centrifuged at 12,000 g for 15 min, and this was repeated twice. The filtrate was collected, desalted using a C18 StageTip, and vacuum-dried. After drying, the peptide segments were redissolved with 0.1% FA, and the peptide segment concentration was measured for LC-MS analysis. An appropriate amount of the peptide segments from the sample was used for chromatographic separation using a nano-flow rate Easy nLC 1200 chromatographic system (Thermo Scientific). Buffer: Solution A was an aqueous solution of 0.1% formic acid, and Solution B was a mixed solution of 0.1% formic acid, acetonitrile, and water (where acetonitrile was 80%). The chromatographic column was equilibrated with 100% Solution A. After the sample was injected into the Trap Column (100 μm * 20 mm, 5 μm, C18, Dr. Maisch GmbH), gradient separation was performed using a chromatographic analysis column (75 μm * 150 mm, 3 μm, C18, Dr. Maisch GmbH) at a flow rate of 300 nl / min. The liquid phase separation gradient was as follows: from 0 min to 2 min, the linear gradient of Solution B was from 2% to 5%; from 2 min to 44 min, the linear gradient of Solution B was from 5% to 28%; from 44 min to 51 min, the linear gradient of Solution B was from 28% to 40%; from 51 min to 53 min, the linear gradient of Solution B was from 40% to 100%; from 53 min to 60 min, Solution B was maintained at 100%. After the peptide segments were separated, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive HF mass spectrometer (Thermo Scientific). The analysis duration was 60 min, the detection mode: positive ion, the precursor ion scan range: 400 - 1500 m / z, the first-stage mass spectrometry resolution: 120,000 @ m / z 200, AGC target: 3e6, the first-stage Maximum IT: 30 ms. The second-stage mass spectrometry analysis of the peptide segments was acquired according to the following method: After each full scan, the second-stage mass spectrometry spectra (MS2scan) of the 20 most intense precursor ions were triggered for acquisition. The second-stage mass spectrometry resolution: 15,000 @ m / z 200, AGC target: 1e5, the second-stage Maximum IT: 35 ms, MS2Activation Type: HCD, Isolation window: 1.6 m / z, Normalized collision energy: 28. The high-performance liquid chromatogram is as Figure 3 shown.
[0049] The key polypeptides of the identified yellow croaker polypeptide component 2 are CMSCGP (amino acid sequence shown in SEQ ID No: 1), PNEEKCCHN (amino acid sequence shown in SEQ ID No: 2), CSNDSDC (amino acid sequence shown in SEQ ID No: 3), and GCGHECIAP (amino acid sequence shown in SEQ ID No: 4). The specific peptide sequence information is as follows in the table:
[0050] Peptide Identification Table of Yellow Croaker Polypeptide Component 2
[0051]
[0052]
[0053] 7. Establishment of Alzheimer's Disease Zebrafish Model and Evaluation of the Activity of Yellow Croaker Polypeptides (1) Model Establishment and Polypeptide / Drug Intervention
[0054] Select 4 hpf zebrafish embryos and culture them in a 6-well plate, with 20 zebrafish embryos in each well. Culture them in an incubator at 28 °C until 72 hpf. The zebrafish embryos are exposed to 140 μM aluminum chloride solution until 120 hpf (48 hpi) to establish an AD zebrafish model. After the model establishment, the normal group and the model group are given conventional fish-raising water, and the other groups are given donepezil hydrochloride, low-concentration active peptide, medium-concentration active peptide, and high-concentration active peptide respectively for 2 days of drug administration. The specific grouping is as follows:
[0055] 1) Normal group: No treatment is performed;
[0056] 2) Model group: Soak and administer 140 μM aluminum chloride hexahydrate for 2 days;
[0057] 3) Positive group: After soaking and administering 140 μM aluminum chloride hexahydrate for 2 days + soak and administer 8 μM donepezil hydrochloride for 2 days;
[0058] 4) Low-concentration group: After soaking and administering 140 μM aluminum chloride hexahydrate for 2 days + soak and administer 2 mg / L yellow croaker polypeptide component 2 for 2 days;
[0059] 5) Medium-concentration group: After soaking and administering 140 μM aluminum chloride hexahydrate for 2 days + soak and administer 10 mg / L yellow croaker polypeptide component 2 for 2 days;
[0060] 6) High-concentration group: After soaking and administering 140 μM aluminum chloride hexahydrate for 2 days + soak and administer 50 mg / L yellow croaker polypeptide component 2 for 2 days;
[0061] (2) Behavioral Trajectory Analysis
[0062] After 24 h of treatment with the administered (bioactive peptide), zebrafish in each group were placed in a 96-well plate, one fish per well, and the drug treatment was continued for 24 h, followed by incubation at 28.5 °C. A 5-min behavioral trajectory video was recorded using the DanioVision system to record the movement trajectory, swimming distance, and average speed.
[0063] Specifically refer to Figure 4 , and it can be seen from the figure that in the model group, treatment with aluminum chloride led to a significant decrease in the locomotor ability of zebrafish, and the tip contacted the back and tail of the larval large yellow croaker twice. The addition of large yellow croaker polypeptides at different concentrations (2, 10, 50 mg / L) could improve the locomotor ability of the zebrafish model, and the treatment effect of the low-concentration polypeptide was comparable to that of the positive drug. That is, the large yellow croaker polypeptide improved the locomotor ability of the zebrafish model.
[0064] (3) Light-dark response test
[0065] After 24 h of treatment with the administered (bioactive peptide), zebrafish in each group were placed in a 96-well plate, one fish per well, and the drug treatment was continued for 24 h, followed by incubation at 28.5 °C. The DanioVision system was used to track the behavioral trajectory for 60 min, which was carried out in two alternating cycles of light and dark, with each stimulation lasting for 10 minutes, starting from a light cycle, and the movement trajectory, distance, and average speed were recorded.
[0066] Specifically refer to Figure 5 , which is the attached figure showing the effects of different concentrations of large yellow croaker polypeptides on the behavior of the Alzheimer's zebrafish model. The figure shows the changes in the total distance and speed of zebrafish in the healthy group, model group, positive group (using donepezil hydrochloride), low-concentration group, medium-concentration group, and high-concentration group at different time periods (0 - 10, 10 - 20, 20 - 30, 30 - 40, 40 - 50, 50 - 60 minutes). In the model group, treatment with aluminum chloride led to a decrease in the light and dark stress response ability of zebrafish; the addition of large yellow croaker polypeptides at different concentrations (2, 10, 50 mg / L) could improve the light response ability of the zebrafish model, and the high-concentration polypeptide treatment basically restored the light and dark memory ability of the AD zebrafish model to the healthy state, with a better effect than the positive drug.
[0067] (4) Evaluation of the improvement effect of large yellow croaker polypeptide on the memory ability of the zebrafish model by the T-maze experiment
[0068] To analyze the improvement effect of the learning and memory ability of AD zebrafish, a T-maze behavioral experiment was used to record the behavioral trajectory of zebrafish. The effect of the bioactive peptide on the learning and memory ability of the AD zebrafish model was evaluated by recording and comparing the latency of each group of zebrafish entering the EC area.
[0069] 1) The testing device is a T-shaped maze made of opaque resin material. White stickers are pasted on the inner walls of the aisle and the left and right arms of equal length. The right arm leads to the enriched chamber (EC). The inner wall of the EC area is made of black resin, and the water depth is 5 cm deeper than other parts of the maze. The bottom is covered with fine sand and gravel, and artificial green plants are planted in the middle.
[0070] 2) At the start of the test, a fish is placed at the starting point of the aisle. The test lasts for 6 minutes. Record the latency time of the fish from the starting point to entering the EC area (it is considered to have truly entered the EC area only when the fish completely enters and continuously stays for more than 20 seconds), and the total stay time in the EC. After each fish is tested, it is placed in a numbered independent container. After all the tests, the 24-hour, 48-hour, 72-hour, and 96-hour tests are carried out in sequence.
[0071] 3) In this experiment, the T-maze device is used to test its spatial learning and memory function, and a region that conforms to the natural preference of zebrafish, namely the EC area, is artificially set. This area has a deeper water depth than other positions of the T-maze and is rich in aquatic plants. This experiment aims to verify whether normal zebrafish can significantly form a preference for the EC area and memory of spatial cues after multiple trainings.
[0072] For specific reference Figure 6 , which is the attached figure of the effects of different concentrations of large yellow croaker polypeptide on the behavior of zebrafish models in the maze; it can be seen from the figure that in the model group, aluminum chloride treatment led to a decrease in the position memory of zebrafish for food in the maze. Compared with the healthy group, it took 2-3 times longer to find food. It can be seen that adding different concentrations (2, 10, 50 mg / L) of large yellow croaker polypeptide can improve the memory ability of zebrafish models for food in the maze. Among them, the treatment with high-concentration polypeptide is equivalent to the positive drug, and the treatment with high-concentration polypeptide basically restores the light-dark memory ability of the AD zebrafish model to the healthy state. That is, large yellow croaker polypeptide significantly improves the behavior of zebrafish models in the maze and significantly improves the memory ability of zebrafish.
[0073] (6) HE staining
[0074] Zebrafish treated with different treatments are collected separately and fixed with 4% paraformaldehyde at 4 °C for 4 hours. After dehydration with a series of ethanol, 100% xylene is used for treatment, and paraffin embedding is carried out to make blocks. Sections of 5 μm are cut, and stained and photographed with hematoxylin and eosin.
[0075] (7) TUNEL staining
[0076] The paraffin sections were dewaxed in xylene for 10 minutes, then dehydrated with graded alcohols, TUNEL fluorescence staining solution was added, and they were kept in the dark at 37 °C for 1 hour, washed and sealed for photography. The red fluorescence images were merged with the normal light field images by ImageJ software, and the red-labeled cells were defined as TUNEL-positive cells.
[0077] See specifically Figure 7 , which is a schematic diagram of HE staining sections and TUNEL staining sections of zebrafish brain tissue pathological changes in different groups; in the figure, the HE staining results show that: in the model group, aluminum chloride treatment caused damage to the zebrafish brain tissue, and the low-concentration and medium-concentration polypeptide groups improved the damage to the zebrafish brain tissue caused by aluminum chloride treatment. The TUNEL staining results show that: in the model group, that is, aluminum chloride treatment led to the formation of more apoptotic bodies in the zebrafish brain tissue cells, while the low-concentration polypeptide treatment improved the formation of apoptotic bodies caused by aluminum chloride treatment. That is, the large yellow croaker polypeptide repaired the nerve cell damage caused by aluminum chloride in the model group.
[0078] 8. Molecular docking of polypeptide with acetylcholinesterase
[0079] According to the acetylcholine theory, acetylcholinesterase is closely related to the occurrence and development of Alzheimer's disease. Multiple acetylcholinesterase inhibitors have been developed as Alzheimer's disease therapeutic drugs or lead compounds.
[0080] The human acetylcholinesterase structure was downloaded from the online database (https: / / alphafold.ebi.ac.uk / entry / P06276). The structures of 4 polypeptide molecules were drawn using Pymol software. The molecular docking experiment of polypeptide and acetylcholinesterase was carried out using Discovery Studio software, and the energy minimization model was adopted. The coordinates of the active center of acetylcholinesterase were: x = -66.4431, y = -66.4431, z = -16.6789.
[0081] Specifically as Figure 8 shown in A-8D, the molecular docking experiment can find that acetylcholinesterase binds closely to the polypeptide, revealing its acetylcholinesterase inhibitory activity. The results show that 4 large yellow croaker polypeptides can bind to acetylcholinesterase, suggesting their acetylcholinesterase inhibitory activity.
[0082] In summary, ultrasonic-assisted complex enzymatic hydrolysis of large yellow croaker fish meat protein significantly improves the enzymatic hydrolysis efficiency of large yellow croaker fish meat protein. Different molecular weight peptide components can be obtained through separation and purification, and key polypeptides CMSCGP, CPNEEKCCHN, CSNDSDC, and GCGHECIAP can be efficiently prepared as active polypeptides of large yellow croaker. The peptide segments in the active components are analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS), and it is found through molecular docking that the active polypeptides can specifically bind to acetylcholinesterase and inhibit its activity. Moreover, in this invention, the zebrafish model treated with aluminum chloride is tested with the active components of large yellow croaker polypeptides. The experimental results show that after feeding with large yellow croaker polypeptides, the memory ability and motor ability of zebrafish with AD have been significantly improved. It can be seen that the large yellow croaker polypeptides prepared and separated in this invention have important application potential in the development of functional foods for anti-Alzheimer's disease.
[0083] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we described are illustrative rather than used to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered by the scope protected by the claims of the present invention.
Claims
1. A method for efficiently hydrolyzing large yellow croaker fish meat with ultrasonic-assisted composite enzymes to prepare bioactive peptides, characterized in that, It includes the following steps: Step 1: Weigh yellow croaker meat, crush it into meat paste, fully mix the fish meat with isopropanol, then carry out cyclic extraction at 60±5°C for 1±0.5 h to remove fat and volatilize the excess isopropanol; Step 2: Inactivate endogenous protease by heating with boiling water, adjust the pH to 7.0±0.5, add 50 - 150 mg / g of compound protease, carry out ultrasonic-assisted hydrolysis, and then place it in a water bath at 28 - 45°C for constant-temperature enzymatic hydrolysis; the compound protease is at least one of acidic protease, neutral protease, compound protease and trypsin; Step 3: After the enzymatic hydrolysis is completed and cooled, centrifuge to obtain the supernatant, which is the yellow croaker protease hydrolysate; Step 4: Fractionation and separation of yellow croaker polypeptides: First, concentrate the yellow croaker polypeptide mixture to remove small molecule compounds such as amino acids and inorganic salts to obtain a small molecule peptide mixture with a molecular weight less than or equal to 3000 Da; then, carry out gel chromatography separation on the small molecule peptide mixture, elute with distilled water, the detection wavelength is 220 nm, start collecting from 30 minutes and stop at 50 minutes, and concentrate the polypeptide solution collected during this time period to obtain yellow croaker active polypeptide.
2. The method for efficiently hydrolyzing large yellow croaker fish meat by ultrasonic-assisted composite enzymes to prepare bioactive polypeptides according to claim 1, wherein: In Step 1, the fish meat and isopropanol are fully mixed at a mass-to-volume ratio of 1:
4.
3. A method for efficiently hydrolyzing large yellow croaker fish meat by ultrasonic-assisted composite enzymes to prepare bioactive polypeptides according to claim 1, characterized in that: In Step 2, the ultrasonic-assisted hydrolysis is carried out with 150 V ultrasound three times, each time for 10 min; the addition amount of the compound protease is 100 mg / g, the water bath hydrolysis temperature is 30°C, the hydrolysis time is 5 h, and the pH is 7.
0.
4. A method for efficiently hydrolyzing large yellow croaker fish meat by ultrasonic-assisted composite enzymes to prepare bioactive polypeptides according to claim 1, characterized in that: The compound protease is compounded by acidic protease, neutral protease, compound protease and trypsin in a ratio of 1:2:3:
2.
5. A method for efficiently hydrolyzing large yellow croaker fish meat with ultrasonic-assisted composite enzymes to prepare bioactive polypeptides according to claim 1, characterized in that: The specific content of Step 4 is as follows: Fractionation and separation of yellow croaker polypeptides: First, use an ultrafiltration tube with a molecular weight cut-off of 3 kDa to concentrate the yellow croaker polypeptide mixture to remove small molecule compounds such as amino acids and inorganic salts to obtain a small molecule peptide mixture with a molecular weight less than or equal to 3000 Da; then, carry out gel chromatography separation on the small molecule peptide mixture using Sephadex G-15(1.6x100 cm), elute with distilled water, the detection wavelength is 220 nm, collect the components in the time period of 30 - 50 minutes, and obtain yellow croaker active polypeptide after rotary evaporation.
6. Use of a large yellow croaker polypeptide in the preparation of a functional food for preventing Alzheimer's disease, characterized in that: The yellow croaker polypeptide is prepared into yellow croaker active polypeptide by the method described in any one of claims 1 - 5 and has acetylcholinesterase inhibitory activity.
7. Use of a large yellow croaker polypeptide according to claim 6 in the preparation of a functional food for preventing Alzheimer's disease, characterized in that: The yellow croaker active polypeptide includes CMSCGP, CPNEEKCCHN, CSNDSDC and GCGHECIAP, and the amino acid sequences are as shown in SEQ ID No: 1 - 4.