Preparation method of walnut blood fat reducing peptide

Abstract

The invention provides a preparation method of walnut blood fat reducing peptide. The preparation method comprises the following steps: (1) treating walnut protein by using alkaline protease to obtain an enzymolysis product; (2) the biological activity of an enzymolysis product is improved by ultrasonic-assisted sodium hydrogen sulfite treatment; (3) performing ultrafiltration treatment on the enzymolysis product to obtain walnut peptide with the molecular weight of 5kDa or below; (4) performing chromatographic separation and screening on the small molecule peptide by adopting Sephadex G-25 gel to obtain an active peptide fragment; (5) identifying by adopting LC-MS (liquid chromatography-mass spectrometry) to obtain a purified polypeptide amino acid sequence; (6) screening a peptide fragment with a blood fat reducing effect through computer-aided and analysis docking; and (7) carrying out artificial synthesis on the peptide fragments YFQ and FRPQ to obtain the walnut blood fat reducing peptide. The walnut blood fat reducing peptide prepared by the method has a good biological activity function, can effectively reduce the blood fat of a human body, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of food, and in particular to a preparation method of walnut blood lipid lowering peptide. Background Art

[0002] Walnut meal, the product of walnut kernel oil extraction, has a protein content of more than 30%, which is a high-quality protein source. The small molecule peptides obtained by enzymatic hydrolysis after extracting walnut protein from walnut meal have multiple biological activities, such as anti-oxidation, antihypertensive, anti-glycemic, antibacterial, etc., and compared with synthetic drugs, lipid-lowering peptides do not show any adverse effects. The use of enzymatic hydrolysis of walnut protein to prepare walnut lipid-lowering peptides can improve the utilization rate of walnut resources, reduce unnecessary waste, and increase the economic value of walnuts.

[0003] Food-derived lipid-lowering peptides can not only reduce blood lipid levels, but are also highly safe and have no toxic side effects. At present, protease hydrolysis is the main method for preparing lipid-lowering peptides, but the lipid-lowering activity of its hydrolysate is often not ideal. Studies have found that pretreatment of protein can improve the biological activity of its hydrolysate. Therefore, this study used ultrasound combined with sodium bisulfite to pretreat walnut protein, and then enzymatic hydrolysis to prepare walnut lipid-lowering peptides, in order to improve the lipid activity of walnut peptides. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one object of the present invention is to provide a preparation method of walnut lipid-lowering peptide.

[0005] Walnut meal obtained after walnut kernels are extracted for oil is rich in protein and amino acids that are important to the human body, so walnut meal can be developed. By processing walnut meal, the active polypeptide substances in walnut kernels can be obtained, which not only have lipid-lowering activity but also can further improve the absorption and utilization of human active peptides. It has guiding significance for the research and development and production of lipid-lowering functional products, and is also of great significance for the development of high value-added walnut products.

[0006] The inventors found during the research process that in the process of obtaining walnut polypeptides, walnut protein can be treated with enzymatic hydrolysis combined with ultrasound-assisted sodium bisulfite. The obtained polypeptide has the dual advantages of enzymatic hydrolysis and ultrasound-assisted sodium bisulfite treatment, with a small molecular weight, high biological activity, and controllable operation and simple process. For example, after the enzymatic hydrolysis product is obtained by adding alkaline protease to treat walnut protein, the enzymatic hydrolysis product is treated with ultrasound-assisted sodium bisulfite. Through enzymatic hydrolysis combined with ultrasound-assisted sodium bisulfite treatment, the operation process is simple, and the obtained polypeptide has a small molecular weight and high activity, which not only effectively improves the biological activity of the peptide but also improves the utilization rate of walnut meal. Furthermore, the active peptide obtained by virtual screening has good lipid-lowering activity. The obtained walnut lipid-lowering peptide can be used as a lipid-lowering functional product and polypeptide nutrient with high bioabsorption rate.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In the first aspect of the present invention, the present invention provides a walnut lipid-lowering preparation method, comprising: (1) treating walnut protein with alkaline protease to obtain an enzymatic hydrolysis product; (2) using ultrasound-assisted sodium bisulfite treatment to improve the biological activity of the enzymatic hydrolysis product; (3) ultrafiltration treatment of the enzymatic hydrolysis product to obtain walnut peptides with a molecular weight of less than 5 kDa; (4) using Sephadex G-25 gel to perform chromatography separation and screening of small molecule peptides to obtain active peptide segments; (5) using LC-MS identification to obtain the purified polypeptide amino acid sequence; (6) using computer-assisted and analytical docking to screen out peptide segments with lipid-lowering effects; (7) preparing the walnut lipid-lowering peptides by artificial synthesis of peptide segments YFQ and FRPQ. After walnut protein is hydrolyzed by combining ultrasound-assisted sodium bisulfite treatment, the obtained product has a high content of polypeptides with small molecular weight, and the polypeptides in the product have a good taste and flavor. Through virtual screening, the obtained walnut lipid-lowering peptide can be efficiently absorbed and utilized by the human body, not only has a good lipid-lowering effect, but also improves the utilization value of walnut meal, and has a good application prospect.

[0009] According to an embodiment of the present invention, the preparation method of the walnut lipid-lowering peptide described above may further include the following technical features:

[0010] In some embodiments of the present invention, alkaline protease is used in step (1). Alkaline protease hydrolysis has high efficiency and specificity, can effectively decompose proteins in a relatively short time, improve reaction efficiency, and has a selective cleavage effect on specific peptide bonds, thereby controlling the molecular weight and peptide chain length of the hydrolyzate.

[0011] In some embodiments of the present invention, the substrate concentration of the enzymolysis reaction is 3% to 7%, preferably 5%. The efficiency of the enzymolysis reaction is greatly affected by the concentration of the protein substrate. When the amount of enzyme added is small, the degree of hydrolysis will gradually increase with the increase of the substrate concentration. However, if the substrate concentration is too high, the diffusion of the protease is affected, which hinders the effective binding with the substrate, thereby slowing down the enzymolysis reaction.

[0012] In some embodiments of the present invention, the amount of enzyme added is 3% to 7%, preferably 5%. In the process of enzymolysis of walnut meal by alkaline protease, the appropriate amount of alkaline protease can quickly enzymolyze the walnut meal. Under this content condition, the rapid enzymolysis of walnut meal can be achieved without causing excessive enzymolysis and producing unnecessary small molecule peptides that are not beneficial to the human body.

[0013] In some embodiments of the present invention, the enzymatic hydrolysis pH is 7 to 11, preferably 10. This allows rapid enzymatic hydrolysis of walnut protein to obtain the target polypeptide.

[0014] In some embodiments of the present invention, the enzymatic hydrolysis time is 1 to 5 hours, preferably 3 hours. Under this time condition, enzymatic hydrolysis can achieve rapid enzymatic hydrolysis of walnut protein, quickly obtain polypeptides with good taste and flavor, and the obtained polypeptides are of appropriate size.

[0015] In some embodiments of the present invention, the enzymolysis temperature is 45-65° C., preferably 55° C. Under the conditions of temperature and time, enzymolysis can achieve rapid enzymolysis of walnut protein, quickly obtain polypeptides with good taste and flavor, and the obtained polypeptides are of appropriate size.

[0016] In some embodiments of the present invention, the ultrasound-assisted sodium bisulfite treatment in step (2) changes the secondary and tertiary structures of the walnut protein. This structural adjustment results in more exposure of the hydrophobic groups originally hidden inside the walnut polypeptide. At the same time, by destroying the disulfide bonds inside the walnut protein, this pretreatment also increases the content of free thiol groups in the polypeptide and the hydrophobicity of its surface. These changes work together to improve the lipid-lowering activity of the polypeptide.

[0017] In some embodiments of the present invention, the concentration of sodium bisulfite is 0.5-2.5 g / L, preferably 1.5 g / L, which can improve the hydrolysis degree and biological activity of the enzymatic hydrolysis product.

[0018] In some embodiments of the present invention, the ultrasonic power is 0-500 W, preferably 400 W. More hydrophobic groups can be exposed, thereby improving the hydrolysis degree and biological activity of the enzymatic hydrolysis product.

[0019] In some embodiments of the present invention, in step (3), the enzymatic hydrolysis product is separated and treated using an ultrafiltration membrane with a molecular weight of 5 kDa. The separation and treatment using an ultrafiltration membrane has mild conditions and will not damage the walnut polypeptide.

[0020] In some embodiments of the present invention, in step (4), walnut peptides below 5 kDa are separated by chromatography using Sephadex G-25 gel. Separation by chromatography using Sephadex G-25 gel is beneficial for screening active peptide segments.

[0021] In some embodiments of the present invention, the peptides screened are identified by LC-MS in step (5). The identification of the peptides screened by LC-MS is helpful to clarify the structure of the active peptides.

[0022] In some embodiments of the present invention, molecular docking virtual screening is used in step (6). Active peptides can be quickly screened out through virtual screening.

[0023] In some embodiments of the present invention, the active peptide is artificially synthesized in step (7). Artificial synthesis is beneficial for preparing a large amount of active peptides.

[0024] In some embodiments of the present invention, before step (1), the method further comprises:

[0025] The walnut meal is defatted by an organic solvent, the obtained defatted walnut meal is sieved, and then walnut protein is extracted by alkali dissolution and acid precipitation. Excess oil can be removed by defatting. Then, the defatted walnut meal is sieved, for example, by a 40-mesh sieve, so that the defatted walnut meal is homogenized, the surface area for subsequent enzymatic hydrolysis and ultrasound-assisted sodium bisulfite treatment is increased, and highly active small molecule peptides are quickly obtained.

[0026] In some embodiments of the present invention, before step (2), the method further comprises: placing the walnut protein solution in a water bath at 90° C. for 15 minutes, cooling it to room temperature, and adjusting the protein solution to an optimal pH using a NaOH solution. Enzymatic hydrolysis under such conditions can retain the maximum polypeptide activity while ultrasonically assisting sodium bisulfite treatment and inactivating the enzymatic hydrolysis reaction, thereby preventing excessive enzymatic hydrolysis and obtaining a walnut polypeptide with a certain activity.

[0027] In a second aspect of the present invention, the present invention provides a method for preparing a walnut lipid-lowering peptide, comprising:

[0028] 1) treating walnut protein with alkaline protease to obtain an enzymatic hydrolysis product; 2) using ultrasound-assisted sodium bisulfite treatment to improve the biological activity of the enzymatic hydrolysis product; 3) performing ultrafiltration treatment on the enzymatic hydrolysis product to obtain walnut peptides with a molecular weight below 5 kDa; 4) using Sephadex G-25 gel to perform chromatography separation and screening of small molecule peptides to obtain active peptide segments; 5) using LC-MS to identify the amino acid sequence of the purified polypeptide; 6) using computer-assisted and analytical docking to screen out peptide segments with lipid-lowering effects; 7) preparing the walnut lipid-lowering peptides by artificial synthesis of peptide segments YFQ and FRPQ.

[0029] In the third aspect of the present invention, the present invention provides a walnut lipid-lowering peptide, which is prepared by the preparation method described in any embodiment of the first aspect of the present invention or the preparation method described in the second aspect of the present invention.

[0030] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0032] Figure 1 It is a schematic diagram of the process for preparing walnut lipid-lowering peptide according to one embodiment of the present invention;

[0033] Figure 2 A graph showing the effect of substrate concentration on the hydrolysis degree of walnut protein hydrolysate and the inhibition rate of cholesterol micelle solubility according to an embodiment of the present invention;

[0034] Figure 3 is a graph showing the effect of the amount of enzyme added on the hydrolysis degree of walnut protein hydrolysate and the inhibition rate of cholesterol micelle solubility according to one embodiment of the present invention;

[0035] Figure 4 is a graph showing the effect of enzymatic hydrolysis pH on the hydrolysis degree of walnut protein hydrolysis products and the inhibition rate of cholesterol micelle solubility according to one embodiment of the present invention;

[0036] Figure 5 is a graph showing the effect of enzymatic hydrolysis time on the hydrolysis degree of walnut protein hydrolysate and the inhibition rate of cholesterol micelle solubility according to an embodiment of the present invention;

[0037] Figure 6 is a graph showing the effect of enzymatic hydrolysis temperature on the hydrolysis degree of walnut protein hydrolysate and the inhibition rate of cholesterol micelle solubility according to one embodiment of the present invention;

[0038] Figure 7is a response surface diagram of the inhibition rate of polypeptide cholesterol micelle solubility due to the interaction of various factors according to one embodiment of the present invention;

[0039] Figure 8 is a graph showing the effect of sodium bisulfite concentration on cholesterol micelle solubility inhibition rate and polypeptide content according to an embodiment of the present invention;

[0040] Fig. 9 is a graph showing the effect of ultrasound power on the solubility inhibition rate of cholesterol micelles and the polypeptide content according to one embodiment of the present invention;

[0041] Fig.10 is a graph showing the effect of ultrasound time on the solubility inhibition rate of cholesterol micelles and the polypeptide content according to an embodiment of the present invention;

[0042] Fig.11 is a graph of bile salt adsorption rate of walnut polypeptide according to one embodiment of the present invention;

[0043] Fig.12 is a chromatogram of Sephadex G-25 chromatography separation and purification of active peptides according to one embodiment of the present invention;

[0044] Fig.13 is a graph showing the pancreatic lipase inhibition rate of different components of active peptides in Sephadex G-25 chromatography according to one embodiment of the present invention;

[0045] Fig.14 is a primary mass spectrum of an active peptide segment and a distribution diagram of the number of peptides and peak areas according to an embodiment of the present invention;

[0046] Fig.15 Schematic diagram of the chemical structure of lipid-lowering peptides YFQ and FRPQ according to one embodiment of the present invention;

[0047] Fig.16 is a secondary mass spectrum of lipid-lowering peptides YFQ and FRPQ according to an embodiment of the present invention;

[0048] Fig.17 It is a schematic diagram of molecular docking of lipid-lowering active peptide segments YFQ and FRPQ according to one embodiment of the present invention. DETAILED DESCRIPTION

[0049] Embodiments of the present invention are described in detail below with reference to the accompanying drawings. It should be noted that the described embodiments are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0050] The invention provides a preparation method of walnut hypolipidemic peptide calcium, comprising: (1) treating walnut protein with alkaline protease to obtain an enzymatic hydrolysis product; (2) using ultrasound-assisted sodium bisulfite treatment to improve the biological activity of the enzymatic hydrolysis product; (3) ultrafiltration treatment of the enzymatic hydrolysis product to obtain walnut peptide with a molecular weight below 5 kDa; (4) using Sephadex G-25 gel to perform chromatography separation and screening of small molecule peptides to obtain active peptide segments; (5) using LC-MS identification to obtain the purified polypeptide amino acid sequence; (6) using computer-assisted and analytical docking to screen out peptide segments with hypolipidemic effects; (7) preparing the walnut hypolipidemic peptide by artificial synthesis of peptide segments YFQ and FRPQ. The preparation method can improve the utilization rate of walnut meal and produce a hypolipidemic peptide with high biological activity.

[0051] It should be noted that the raw material used in the preparation method of walnut lipid-lowering peptide provided by the present invention is walnut meal after oil extraction from walnut kernels. The walnut meal can be purchased directly, or it can be obtained by extracting oil from walnut kernels through conventional techniques in the art. Although the variety, quality and oil extraction conditions of walnuts will have some influence on the quality and lipid-lowering activity of the final product, walnut lipid-lowering peptides with lipid-lowering activity and high bioavailability can be obtained through the method provided by the present invention. In at least some embodiments of the present invention, in order to reduce the influence of oil in walnut meal, the walnut meal used can be defatted with an organic solvent, and the protein content in the walnut meal after defatting with an organic solvent is about 60% to 65%, for example, the protein content is about 63%. Thus, the walnut meal with a high protein content can be subjected to ultrasound-assisted sodium bisulfite treatment combined with enzymatic hydrolysis to obtain a large amount of target walnut peptides.

[0052] During the degreasing process of walnut meal, some commonly used organic solvents can be used for degreasing. For example, petroleum ether can be used to degrease the walnut meal to remove the remaining oil in the walnut meal.

[0053] In the experiment or production process, the walnut meal obtained after oil extraction or the defatted walnut meal can be mixed with water to obtain a walnut meal mixture, and then the protease is used for enzymolysis reaction. Of course, in order to increase the reaction area of ​​ultrasound-assisted sodium bisulfite treatment and enzymolysis reaction, the walnut meal after oil extraction or the defatted walnut meal can be sieved, for example, through a 40-mesh sieve. In some other embodiments of the present invention, when the protease is used for enzymolysis reaction, an alkaline protease with good enzymolysis effect of walnut protein, easy to obtain and low price can be selected as a hydrolase. The enzymolysis conditions can be that the substrate concentration of the enzymolysis reaction is 3% to 7%, preferably 5%; the enzyme addition amount is 3% to 7%, preferably 5%; the enzymolysis pH is 7 to 11, preferably 10; the enzymolysis reaction is at 55°C, and the enzymolysis treatment is 3h. After the enzymolysis and fermentation are completed, the enzymolysis and fermentation system is treated with a boiling water bath to kill the enzyme to prevent excessive enzymolysis, so that the system contains walnut polypeptides with certain biological activity for subsequent separation treatment.

[0054] In some other embodiments of the present invention, when the enzymatic hydrolyzate is treated with ultrasound-assisted sodium bisulfite, the biological activity of walnut polypeptide in reducing blood lipids can be more effectively improved. The treatment conditions can be: sodium bisulfite concentration 0.5-2.5g / L, preferably 1.5g / L; ultrasonic power 100-500W, preferably 400W; ultrasonic time 5-25min, preferably 15min.

[0055] In some other embodiments of the present invention, the enzymatic hydrolyzate is ultrafiltered through a 5kDa ultrafiltration membrane to obtain a walnut peptide liquid with a molecular weight less than 5kDa. The prepared walnut peptide liquid can be directly used for subsequent separation and purification. The walnut peptide liquid can also be freeze-dried to obtain walnut peptide powder, which can be stably stored in this way and is not easy to decompose. In some other embodiments of the present invention, the prepared walnut peptide powder can be added with water to prepare a walnut peptide solution of a certain concentration, and then separated by Sephadex G-25 gel chromatography. In at least some embodiments of the present invention, the peptide segments screened are identified by LC-MS to further screen active peptides. In some other embodiments of the present invention, molecular docking virtual screening is used to screen active peptides. In some other embodiments of the present invention, the walnut hypolipidemic peptide is prepared by artificial synthesis. Through this preparation method, while improving the utilization rate of walnut meal, a hypolipidemic active peptide with high bioabsorption rate can also be produced.

[0056] The IC value of the walnut lipid-lowering peptide obtained by the method provided by the present invention for inhibiting pancreatic lipase was determined. 50The values ​​were 506.33±25.85 and 682.07±56.12 μg / mL respectively. The prepared walnut hypolipidemic peptide calcium can be directly packaged as a ready-to-eat food, or can be mixed or processed with other edible substances and used as food.

[0057] In at least some embodiments of the present invention, the present invention provides a method for preparing walnut lipid-lowering peptide, the method comprising: 1) soaking walnut meal after hydraulic oil pressing in petroleum ether to remove oil; 2) passing the defatted walnut meal through a 40-mesh sieve to obtain defatted walnut powder; 3) preparing the defatted walnut powder into a solution, adding 1 mol / L sodium hydroxide to adjust the pH of the solution to 11.0, and stirring for 180 minutes at room temperature. Then, the precipitate is removed by centrifugation at 8000r / min for 15 minutes, and finally, the pH of the supernatant is adjusted to 4.5. Under the same conditions, the walnut protein precipitate is collected by centrifugation, thoroughly washed with deionized water to remove impurities, and freeze-dried to obtain walnut protein; 4) the walnut protein is treated with alkaline protease to obtain an enzymatic hydrolysis product; 5) the biological activity of the enzymatic hydrolysis product is improved by ultrasound-assisted sodium bisulfite treatment; 6) the enzymatic hydrolysis product is ultrafiltered to obtain a walnut peptide with a molecular weight of less than 5kDa; 7) the small molecule peptide is chromatographically separated and screened using Sephadex G-25 gel to obtain an active peptide segment; 8) the amino acid sequence of the purified polypeptide is obtained by LC-MS identification; 9) the peptide segment with lipid-lowering effect is screened out by computer-assisted and analytical docking; 10) the peptide segments YFQ and FRPQ are prepared by artificial synthesis to obtain the walnut lipid-lowering peptide

[0058] The scheme of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following embodiments are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in the art or in accordance with the product instructions are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be purchased commercially. The alkaline protease used was purchased from Beijing Solebow Technology Co., Ltd., and the item number is B8360.

[0059] The instruments and manufacturers used in the examples are as follows:

[0060] pH meter, Mettler-Toledo Instruments,

[0061] BioSafer Ultrasonic Cell Disruptor, Saifei China Co., Ltd.,

[0062] DK-98-II Electric Constant Temperature Water Bath, Tianjin Test Instrument Co., Ltd.,

[0063] SHA-BA water bath constant temperature oscillator, Jintan Ronghua Instrument Manufacturing Co., Ltd.,

[0064] HC-2518R high-speed refrigerated centrifuge, Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.,

[0065] Sartorius Vivaflow tangential flow membrane package, Sartorius Group, Germany,

[0066] SCIENTZ-12N freeze dryer, Ningbo Xinzhi Biotechnology Co., Ltd.

[0067] Example 1

[0068] Reference Figure 1 As shown, Example 1 provides a method for preparing walnut lipid-lowering peptide, comprising the following steps:

[0069] (1) Walnut meal after oil extraction is selected as raw material, 5 times the volume of petroleum ether is added for defatting, and after evaporation, it is passed through a 40-mesh sieve to obtain defatted walnut powder.

[0070] (2) Weigh an appropriate amount of defatted walnut meal, add purified water at a ratio of 1:20 (w / v), stir magnetically for 5 minutes to fully mix to obtain a defatted walnut meal solution, and add 1 mol / L sodium hydroxide to adjust the solution pH to 11.0, and stir for 180 minutes at room temperature. Then centrifuge at 8000 r / min for 15 minutes to remove the precipitate, and finally adjust the pH of the supernatant to 4.5. Collect the walnut protein precipitate by centrifugation under the same conditions, wash thoroughly with deionized water to remove impurities, and freeze-dry the precipitate.

[0071] (3) Weigh 1 g of walnut protein into 20 mL of distilled water, stir and dissolve it with a magnetic stirrer for 15 min, then add 1.5 g / L sodium bisulfite and stir and dissolve it for another half an hour for subsequent enzymatic hydrolysis experiments.

[0072] (4) Weigh 1 g of protein and add deionized water to prepare walnut protein solutions of different concentrations. Place the solution in a water bath at 90°C for 15 min, cool to room temperature, adjust the pH of the protein solution to 11 using a NaOH solution, and perform enzymatic hydrolysis at 55°C. After the enzymatic hydrolysis, inactivate the enzyme at high temperature, centrifuge, collect the supernatant, filter through a 0.45 μm filter membrane, freeze-dry the filtrate, and store it at -20°C for later use.

[0073] (5) At the optimal sodium bisulfite concentration, the walnut protein solution was treated with an ultrasonic disruptor, and the ultrasonic treatment time and interval time were both set to 2s. The enzymatic hydrolysate was treated with 400W ultrasound for 15min, and the ultrasound working parameters were optimized based on the lipid-lowering activity and polypeptide content.

[0074] (6) The enzymatic hydrolyzate is ultrafiltered through an ultrafiltration membrane with a molecular weight of 5 kDa to obtain a walnut polypeptide liquid with a molecular weight less than 5 kDa, and then freeze-dried to obtain a walnut polypeptide powder.

[0075] (7) The Sephadex G-25 gel was placed in a boiling water bath for 2 hours to expand, and then stirred with the eluent to form a suspension and added to the column to avoid the generation of bubbles, and the eluent was balanced with a vacuum pump for 2 hours. A walnut polypeptide solution with a concentration of 50 mg / mL was prepared and loaded after passing through a 0.22 μm filter membrane. The single loading volume was 3 mL, and the flow rate was adjusted to 1 mL / min. Then, an automatic component collector was used to collect the eluted components. The absorbance value of the collected eluate was measured at 220 nm, and each tube was collected for 3 minutes. The eluates of different components were collected according to the peak width in the elution spectrum, and freeze-dried to obtain different peptide segments.

[0076] (8) The amino acid sequence of the purified polypeptide was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The following chromatographic conditions were used in the experiment: a C18 column (0.15×150 mm) was used; the mobile phase was a 0.1% formic acid aqueous solution (A) and an aqueous solution containing 0.1% formic acid and 84% acetonitrile (B), and the elution gradient was: 0-50 min, B gradually increased from 4% to 50%; 50-54 min, B increased from 50% to 100%; 54-60 min, B was maintained at 100%. After capillary high performance liquid chromatography separation, mass spectrometry analysis was performed using a QExactive mass spectrometer. The corresponding database was searched using MaxQuant1.5.5.1 software to determine the amino acid sequence of the polypeptide.

[0077] (9) Autodock vina 1.1.2 was used for molecular docking. The protein receptor selected for docking was pancreatic lipase (PDBID: 1LPB), whose three-dimensional structure was downloaded from the protein data bank (PDB). The three-dimensional structure of the identified peptide was drawn using ChemDrew software. The original ligand methoxyundecylphosphonic acid (MUP) of protein 1LPB was isolated. Next, the protein receptor was dehydrated and hydrogenated, and then the ligand polypeptide was hydrogenated and its rotation bonds were detected. After processing, both the receptor and the ligand were saved as pdbqt files for subsequent docking. After importing the receptor and ligand into the software, the parameters of the docking box were set. The one with the lowest binding energy was selected as the optimal conformation. Finally, the docking diagram was viewed and analyzed using Discovery Studio client 2019.

[0078] (10) Artificially synthesized peptides YFQ and FRPQ with lipid-lowering activity.

[0079] Example 2

[0080] Example 2 The conditions of enzymatic hydrolysis and ultrasound-assisted sodium bisulfite treatment were optimized to investigate the effects of various factors on the walnut multi-peptide content and hydrolysis degree and the inhibition rate of cholesterol micelle solubility. The following process was included:

[0081] Weigh 1g of ultrasonic-assisted sodium bisulfite-treated walnut protein and add deionized water to prepare walnut protein solutions of different concentrations (3%, 4%, 5%, 6% and 7%), heat in a 90℃ water bath for 15min, cool to room temperature, adjust the pH of the protein solution to 7, 8, 9, 10, 11 with NaOH solution, and perform enzymolysis (1, 2, 3, 4, 5h) at the set enzymolysis temperature (5, 50, 55, 60, 65℃), keeping the pH constant. After the enzymolysis, the enzyme was inactivated at high temperature and centrifuged, the supernatant was collected and filtered through a 0.45μm filter membrane, and the filtrate was freeze-dried and stored at -20℃ for later use. The effects of various factors on the degree of hydrolysis of the enzymolysis product and the inhibition rate of cholesterol micelle solubility were investigated.

[0082] Weigh 1g of walnut protein in 20mL of distilled water, stir and dissolve it with a magnetic stirrer for 15 minutes, then add different concentrations of sodium bisulfite (0.5, 1, 1.5, 2.0 and 2.5g / L) and stir and dissolve for another half an hour. The effect of sodium bisulfite concentration on the lipid-lowering activity and content of polypeptides was investigated.

[0083] Weigh 1g walnut protein in 20mL distilled water, stir and dissolve it with a magnetic stirrer for 15min, then add 1.5g / L sodium bisulfite of different concentrations, and treat the walnut protein solution with an ultrasonic disruptor, with the ultrasonic treatment time and interval time set to 2s. Prepare various walnut protein solutions when the ultrasonic time is 15min, the power is (0, 100, 200, 300, 400, 500W) and when the ultrasonic power is 400W, the time is (5, 10, 15, 20, 25min), and use them for subsequent enzymatic hydrolysis experiments to investigate the effects of ultrasonic indicators on the lipid-lowering activity and content of polypeptides.

[0084] Determination of walnut polypeptide content:

[0085] Prepare 10 mg / mL standard bovine serum protein solution as the standard solution, then dilute the standard solution into protein solutions of different concentrations and add them to test tubes respectively, add biuret reagent, shake well, let stand at room temperature for 30 minutes, and then measure OD 540 The concentration of protein was used as the x-axis of the graph, and the absorbance value was used as the y-axis to draw the concentration-absorbance curve of the standard protein. After that, the sample solution was used to determine the polypeptide content of the experimental group, and a blank control group was set up with distilled water instead of the sample solution for comparative analysis.

[0086] Establishment of in vitro lipid-lowering model:

[0087] Dissolve the cholesterol standard in glacial acetic acid to prepare a solution with a concentration of 0.01% for subsequent experiments. Next, dilute the 85% phosphoric acid solution to 80%, weigh 2.2315g of ammonium ferric sulfate and mix it with 50mL of 80% phosphoric acid solution to prepare a solution, and after the solution is cooled to room temperature, take out 20mL and transfer it to a 250mL volumetric flask, then use concentrated sulfuric acid to make the volume, prepare the iron vanadium color developer, and store it at room temperature for use (freshly prepared). Then refer to the steps listed in Table 1, add the required reagents as indicated and mix thoroughly, and then let it stand at room temperature for 30 minutes. Use a spectrophotometer to measure its OD 560 By combining the concentration of cholesterol standard solution and the corresponding absorbance value, a cholesterol standard curve was drawn.

[0088] Table 1 Preparation of cholesterol standard solution

[0089]

[0090] Determination of cholesterol micelle solubility inhibition rate (MCI):

[0091] After preparing micelle solutions of equal concentration, ultrasonic emulsification was performed on the simulated micelle solution using an ultrasonic disruptor (power 400W) for 20 minutes. Then, the peptide sample was weighed and added to the micelle solution (5 mg / mL). Ultrasonic emulsification was performed for three minutes, and then the mixture was shaken in a shaker at 37°C for one day. Finally, the mixture was centrifuged (16000 r / min, 20 minutes), the supernatant was collected, and the OD was measured. 560 nm value. Substitute the cholesterol standard curve to obtain the cholesterol micelle solubility (mmol / L) in the supernatant, and calculate MCI by the formula:

[0092]

[0093] Where, S: cholesterol micelle solubility of blank group.

[0094] S 1 : Cholesterol micellar solubility of the sample set.

[0095] Determination of bile salt binding rate of walnut polypeptide:

[0096] (1) Phosphate buffer solution (pH 6.3, 0.1 mol / L), three bile salt standard solutions: accurately weigh 0.012 g sodium deoxycholate, 0.053 g sodium taurocholate, and 0.060 g sodium cholate, and dissolve them to 50 mL. (2) Standard curve and binding rate determination method: take 0 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 2.5 mL of bile salt standard solution in a test tube, add phosphate buffer to 2.5 mL, then add 7.5 mL of 60% sulfuric acid, react at 70°C for 20 min, and finally measure OD 387 nm value. The standard curve of sodium deoxycholate is y=1.0091x+0.0068, R 2 =0.9962; the standard curve of sodium taurocholate is y=2.82262x-0.0152, R 2 =0.9943; the standard curve of sodium cholate is y=0.66881x+0.0008, R 2 =0.9995. Take 1mL of polypeptide solution (20mg / mL), add 1mL of 0.01mol / L HCl, mix well, and incubate at 37℃ for 1h. Then adjust the solution pH to 6.3, add 4ml of porcine pancreatic enzyme solution (10mg / mL), and continue to oscillate for 1h. Then add 4mL of bile salt solution to each sample, oscillate at 37℃ for 2h, add four times the volume of 95% ethanol (v / v), centrifuge for 15min (10000r / min), and measure the concentration of bile salt in the supernatant. Use β-sitosterol as a positive control. The formula for calculating the bile salt binding rate is as follows:

[0097]

[0098] Among them, C 0 : Initial bile salt concentration (mmol / L).

[0099] C 1 : Concentration of bile salt after conjugation (mmol / L).

[0100] The results are as follows:

[0101] Depend on Figure 2It can be seen that the hydrolysis degree and MCI of walnut protein hydrolysate change with the change of substrate concentration in the range of 3% to 7%. When the substrate concentration reaches 5%, the hydrolysis degree and MCI reach the highest value, which are 24.33±0.63% and 42.32±0.43% respectively. This shows that the influence of substrate concentration on the hydrolysis reaction of walnut protein presents a certain trend, and then with the increase of substrate concentration, the hydrolysis degree and MCI all show a downward trend, which may be due to the substrate inhibition phenomenon, the insufficient combination between enzyme and substrate, and the insufficient interaction, which leads to the reduction of peptide bond hydrolysis efficiency. In addition, it may also be due to the fact that there is too little effective water in the reaction system, and the concentration of polypeptide produced by hydrolysis is too large to affect the continuation of the reaction.

[0102] Depend on Figure 3 It can be seen that when 3% to 7% of protease is added, the MCI of the enzymatic hydrolysis product shows a trend of first increasing and then decreasing. When the amount of protease added is 5%, it reaches the highest value of 44.57±1.54%, which shows that the amount of protease added has a significant effect on MCI. The degree of hydrolysis first increases rapidly and then tends to be flat. It is possible that when the amount of enzyme added is low, the binding site of walnut protein and alkaline protease is not saturated, the protease can fully contact with the substrate, effectively improve the hydrolysis efficiency, and enhance the functional activity of some biologically active peptides after enzymatic hydrolysis. When the amount of protease added is too much, the substrate protein is limited, so too much protease will continue to hydrolyze part of the enzymatic hydrolysis product into inactive short peptides, which will cause the MCI of walnut protein hydrolysis product to decrease instead. Therefore, the enzyme addition amount of 5% is selected.

[0103] Depend on Figure 4 It can be seen that the MCI of walnut protein hydrolysate first increases and then decreases with the increase of enzymatic pH. When the enzymatic pH is 10, the MCI of walnut protein hydrolysate reaches the highest value, which is 26.87±0.69%; the degree of hydrolysis shows the same trend as MCI, and the highest degree of hydrolysis is 44.3±0.95% when the enzymatic pH is 10. After that, the degree of hydrolysis and MCI both show a downward trend as the pH increases. This may be because when the enzymatic pH is not in the optimal pH range of alkaline protease, the group at the active site of the enzyme molecule is in a dissociated state, affecting the stability of the enzyme, causing the enzyme activity to decrease or even inactivate the enzyme. Therefore, this experiment selected pH 10 for subsequent experiments.

[0104] Depend on Figure 5It can be seen that from 1 to 3 hours, the MCI and hydrolysis degree of the enzymatic hydrolysis products of walnut protein increased rapidly, the inhibition rate increased from 28.85±0.83% to 43.68±1.15%, and the hydrolysis degree increased from 16.67±0.10% to 23.62±0.38%. After 3 hours, the hydrolysis degree and the MCI of the enzymatic hydrolysis products increased slowly and tended to be stable, with no significant difference from 3 hours. This may be because most substrates and proteases have basically reacted at 3 hours. Considering the comprehensive experimental cost and time, the enzymatic hydrolysis time was selected as 3 hours.

[0105] Depend on Figure 6 It can be seen that as the temperature increases, the reaction rate of the protease and the substrate accelerates, a large amount of walnut bioactive peptides are generated, and the MCI and hydrolysis degree of the walnut protein hydrolysate gradually increase. When the enzymatic hydrolysis temperature reaches 55°C, the inhibition rate and hydrolysis degree reach a peak. However, as the temperature further increases, the MCI decreases. Studies have shown that higher enzymatic hydrolysis temperatures will change the spatial structure of the protease active site to a certain extent, causing the protease activity to decrease or inactivate, and the efficiency of the enzymatic hydrolysis reaction to decrease; and too high a temperature will cause the bioactive peptides in the enzymatic hydrolysis product to be destroyed, and the corresponding MCI will also begin to decrease. Finally, the optimal enzymatic hydrolysis temperature of alkaline protease was determined to be 55°C.

[0106] Based on the single factor experiment, the response surface experiment was conducted using the central composite design of Design-Expert 13.0 software. The factor levels and results of the Box-Behnken experimental design in Design-Expert are shown in Table 2, and the analysis results are shown in Tables 3 and 4.

[0107] Table 2 Box-Behnken test design factors and levels

[0108]

[0109] The response surface experiment was conducted using the central composite design of Design-Expert 13.0 software. The factor levels and results of the Box-Behnken experimental design in Design-Expert are shown in Table 3.

[0110] Table 3 Box-Benhken test design and results

[0111]

[0112]

[0113] Table 4 shows the results of the multiple regression fitting analysis of Design Expert 13.0. The fitting regression equation is Y = 49.26 + 0.1763A-0.19B-0.235C-0.5875AB-0.44AC-0.225BC-2.17A 2 -3.54B 2 -0.7902C 2 (R 2 =0.9972, P<0.0001).

[0114] Table 4. Variance analysis table of regression model

[0115]

[0116] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).

[0117] As shown in Table 4, the regression model established with MCI had a significant effect (P<0.0001; the lack of fit P value was 0.2863, which was greater than 0.05, and the effect was not significant; the adjusted regression coefficient R2 = 0.9972, indicating that the regression equation model had a high degree of fit and was successfully established; the order of significance of the impact on MCI was: enzyme addition amount (B) > enzymatic pH (C) > substrate concentration (A), and the F test of the first-order terms A, B, and C in the model was extremely significant; the interaction terms AB, AC, and BC were all extremely significant (P<0.01), and the quadratic term A 2 , B 2 , C 2 The results were extremely significant (P<0.01), indicating that the relationship between A, B, C and MCI cannot be expressed in a simple linear manner, and there is a strong interaction between A, B, and C.

[0118] The response surface 3D interaction plot shows the interaction between two factors, such as Figure 7 As shown. The ratio of enzyme addition to substrate concentration, the ratio of substrate concentration to enzymatic hydrolysis pH, and the ratio of enzymatic hydrolysis pH to enzyme addition have extremely strong interactions. Through the analysis of the regression equation, the optimal theoretical process parameters for the enzymatic hydrolysis reaction are substrate concentration of 5.04%, enzyme addition of 4.91%, and enzymatic hydrolysis pH of 9.95. Under these conditions, the predicted value of the MCI of the enzymatic hydrolysis product is 49.36%. Taking into account the actual experimental conditions, the process parameters were modified to substrate concentration: 5%, enzyme addition: 5%, and enzymatic hydrolysis pH: 10. After verification experiments, the MCI was 49.23%, and the relative error with the theoretical prediction value was 0.13%, proving that this regression model is effective.

[0119] Depend on Figure 8It can be seen that the MCI of walnut protein hydrolysate significantly increased after sodium bisulfite pretreatment (P<0.05). With the increase of sodium bisulfite concentration, its MCI and degree of hydrolysis gradually increased. When the sodium bisulfite concentration was 1.5g / L, the inhibition rate reached a peak of 56.02±0.33%, which was 13.79% higher than the inhibition rate of protease hydrolysate not pretreated with sodium bisulfite, and then continued to decrease. The change trend of polypeptide content was similar to that of MCI. It also increased first and then decreased with the increase of sodium bisulfite concentration. The possible reason is that too high salt concentration will change the structure of walnut protein to a certain extent, making it unable to effectively react with the binding site of protease. Therefore, the optimal sodium bisulfite concentration was screened to be 1.5g / L.

[0120] Depend on Fig. 9 It can be seen that with the increase of ultrasonic power, ultrasound-coupled sodium bisulfite pretreatment caused the MCI of the hydrolysate to increase first and then decrease. At 400W, the lipid-lowering activity of the hydrolysate reached the highest value (65.13±2.45%), which was 32.30% higher than that of the control group. After that, the lipid-lowering activity gradually decreased. This ultrasonic pretreatment combined with sodium bisulfite may enhance the cholesterol-lowering activity of the hydrolysate by exposing more hydrophobic groups. These newly exposed hydrophobic groups may bind to bile acids more effectively, thereby promoting the increase in activity. With the increase of ultrasonic power, ultrasound-assisted sodium bisulfite pretreatment caused the polypeptide content of the hydrolysate to have the same trend as MCI. The polypeptide content of the hydrolysate increased significantly when the ultrasonic power was 100-300W / L, and then the peptide content did not increase significantly with the increase of ultrasonic power density (P>0.05). In summary, the optimal ultrasonic power was determined to be 400W.

[0121] Depend on Fig.10 It can be seen that with the increase of ultrasound time, ultrasound-assisted sodium bisulfite pretreatment caused the MCI of the hydrolysate to increase first and then decrease. When the ultrasound time was 15min, the MCI of the hydrolysate reached a maximum value of 62.93±1.04%, which was 27.83% higher than that of the untreated group. After that, with the increase of ultrasound time, the inhibition rate decreased. This may be due to the denaturation of walnut protein by long-term ultrasound, or it may be due to the thermal effect of ultrasound that caused the lipid-lowering activity of walnut protein hydrolysate to decrease. With the increase of ultrasound time, the polypeptide content of walnut protein hydrolysate increased. When the ultrasound time was 15min, the polypeptide content of the hydrolysate was the highest, which was 12.94±0.08mg / mL. This is because as the ultrasound time increases, the protein particles dissolved in the solution are likely to re-aggregate, and the content of protease that can be enzymatically hydrolyzed is certain, resulting in a decrease in the polypeptide extraction rate. Therefore, the ultrasound time was selected as 15min.

[0122] Depend on Fig.11It can be seen that the binding rates of 20 mg / mL walnut polypeptide (PSBP) solution prepared by ultrasound-assisted sodium bisulfite pretreatment and enzymatic hydrolysis to sodium deoxycholate, sodium cholate and sodium taurocholate were 59.62±0.94%, 52.25±2.40% and 44.25±1.22%, respectively, and the binding rates of 20 mg / mL β-sitosterol to bile salt were 75.58±1.40%, 85.15±1.40% and 57.92±2.41%, respectively. β-sitosterol has a higher bile salt binding rate.

[0123] Example 3

[0124] Example 2 Screening and identification of walnut lipid-lowering peptides, the process is as follows:

[0125] 1. Prepare walnut protein hydrolysate by mixing freeze-dried hydrolysate and purified water at a ratio of 1:20. Filter the supernatant of the hydrolysate through a 0.45 μm filter and use a 5 kDa ultrafiltration membrane to perform preliminary separation on the hydrolysate. The membrane surface flow rate is 2.0 mL / min. Finally, collect the polypeptides with a molecular weight less than <5 kDa and freeze-dry the samples for storage.

[0126] 2. Boil the Sephadex G-25 gel in a boiling water bath for 2 hours to expand it, then stir it with the eluent to form a suspension and add it to the column to avoid bubbles, and use a vacuum pump to balance with the eluent for 2 hours. Prepare a walnut polypeptide solution with a concentration of 0 mg / mL, and then load it after passing through a 0.22μm filter membrane. The single loading volume is 3mL, and the flow rate is adjusted to 1mL / min. Then use an automatic component collector to collect the eluted components. The absorbance value of the collected eluate is measured at 220nm, and each tube is collected for 3min. And collect the eluents of different components according to the peak width in the elution spectrum and freeze-dry.

[0127] like Fig.12 As shown in the figure, after the walnut lipid-lowering peptide solution was separated by Sephadex G-25 chromatography, a total of 4 peaks appeared. Gel filtration chromatography separates and purifies the mixed polypeptide components according to their molecular size. The smaller the molecular weight, the more difficult it is to elute. Therefore, the molecular weights of the four components are F1>F2>F3>F4, and the differences are significant (P<0.05).

[0128] Depend on Fig.13 It can be seen that the IC of F1, F2, F3 and F4 50 The values ​​were 2.04±0.06, 1.67±0.02, 1.76±0.06 and 2.35±0.09 mg / mL, respectively. Among them, the IC 50 The lowest, but no significant difference with F3. Studies have shown that the activity of a peptide is related to its molecular weight.

[0129] 3. PyRx virtual screening

[0130] The highly water-soluble peptides predicted in the previous step were drawn in 2D and 3D structures using ChemDraw software, and energy minimization was performed to optimize the structure. Finally, the optimized peptides were saved as ligand files. Next, the 3D structure file (PDB format) of pancreatic lipase (PDB: 1LPB) was downloaded by visiting the RCSB PDB website (https: / / www.rcsb.org), and the structure file was preprocessed using Autodock Tools 1.5.6 software to save it as a receptor file suitable for molecular docking (pdbqt format). Subsequently, PyRx software was used to perform molecular docking on all prepared ligands and receptors, and a large docking search range was set to obtain a series of possible binding modes. According to the calculation results of docking energy, the interaction strength between the peptide and the receptor was evaluated, and the first six peptides with the lowest docking energy were selected as candidates. In order to further screen out potential peptides, preliminary screening was performed using the allergenicity assessment tool provided by the AllerTOP website (http: / / www.ddg-pharmfac.net / AllerTOP / index.html). Based on the evaluation results, the best performing peptides were selected to prepare for subsequent semi-flexible docking experiments.

[0131] The 242 peptides with high solubility were virtually screened, and the top six peptides with the highest docking energy were obtained, with the results shown in Table 5. Docking energy ≤ -8.4 kcal mol -1 , indicating that the binding force between the ligand and the receptor is relatively strong, and QPF has the strongest binding force, with a docking energy of -9.2 kcal mol -1 . The 9 peptides were compared with NCBI protein sequences, and all peptide sequences belonged to the protein sequences of walnut (Juglansregia L.). The 9 peptides were compared with the BIOPEP-UWM peptide database, and three of them were involved in the research.

[0132] 4. Autodock molecular docking

[0133] Autodock vina 1.1.2 was used for molecular docking. The protein receptor selected for docking was pancreatic lipase (PDBID: 1LPB), whose three-dimensional structure was downloaded from the protein data bank (PDB). The three-dimensional structure of the identified peptide was drawn using ChemDrew software. The original ligand methoxyundecylphosphonic acid (MUP) of protein 1LPB was isolated. Next, the protein receptor was dehydrated and hydrogenated, and then the ligand polypeptide was hydrogenated and its rotation bond was detected. After processing, both the receptor and the ligand were saved as pdbqt files for subsequent docking. After importing the receptor and ligand into the software, the parameters of the docking box were set. The one with the lowest binding energy was selected as the best conformation. Finally, the docking diagram was viewed and analyzed using Discovery Studio client 2019.

[0134] Depend on Fig.17 It can be seen that FRPQ forms hydrogen bonds with 1LPB at His263 and Arg256, forms van der Waals forces with Phe77, Ile209, Ala259, Ala260, His151, Gly76, Tyr267, Leu154Glu179, forms carbon-hydrogen bonds with Ser152 and Tyr114, forms π-cation electrostatic interactions with Asp79, and forms alkyl and π-alkyl interactions with Ile87, Leu264, Phe215, Pro180 and Ala178. The above results indicate that YFQ and FRPQ can interact with 1LPB through hydrogen bonds, π-π bonds, hydrophobic interactions and van der Waals forces. These interactions can lead to the formation of a complex between the polypeptide and the receptor protein or affect the conformation of the enzyme, thereby affecting the normal physiological function of the receptor protein and showing lipid-lowering activity.

[0135] Table 5 Comparison results of some walnut peptides with PL docking and BIOPEP-UWM peptide database

[0136]

[0137] The prediction of allergens of walnut peptides is shown in Table 6. The results show that more than 50% of the peptides are predicted to be allergenic. Peptides YFQ, FRPQ, FQL and WSG are not allergenic. Because FQL has been reported, the docking energy result of WSG is low. Finally, two peptides YFQ (Tyr-Pro-Gln) and FRPQ (Phe-Arg-Pro-Gln) were selected for artificial synthesis to verify their PL inhibitory activity. The chemical structures and secondary mass spectra of peptides YFQ and FRPQ are shown in Figure 6. Fig.15 Shown and Fig.16 shown.

[0138] Table 6 Prediction of allergens of walnut peptides

[0139]

[0140] 5. Verification of synthetic peptide activity

[0141] As shown in Table 7, peptides YFQ and FRPQ both had good PL inhibitory activity in vitro, IC 50 The values ​​were 506.33±25.85 and 682.07±56.12 μg / mL, respectively. The IC 50 Value: 17.24±0.13μg / mL.

[0142] Table 7 Half-inhibitory concentrations of peptides YFQ and FRPQ on PL

[0143]

[0144] It is not difficult to see from the above experiments that the present invention has screened out two polypeptides with low binding energy, no allergenicity and not yet studied by optimizing the preparation process of walnut lipid-lowering peptides. The prepared walnut lipid-lowering peptides can be compounded or processed with other edible substances to prepare lipid-lowering functional foods.

[0145] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0146] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing walnut lipid-lowering peptide, characterized in that: include: (1) treating walnut protein with alkaline protease to obtain an enzymatic hydrolysis product; (2) Ultrasound-assisted sodium bisulfite treatment was used to improve the biological activity of the enzymatic hydrolysis product; (3) ultrafiltration treatment of the enzymatic hydrolysis product to obtain walnut peptides with a molecular weight below 5 kDa; (4) using Sephadex G-25 gel to perform chromatography separation and screening of small molecule peptides to obtain active peptide fragments; (5) using LC-MS to identify the amino acid sequence of the purified polypeptide; (6) Screening out peptides with lipid-lowering effects through computer-assisted and analytical docking; (7) The peptide fragments YFQ and FRPQ are prepared by artificial synthesis to obtain the walnut lipid-lowering peptide.

2. The preparation method according to claim 1, characterized in that: In step (1), alkaline protease is used to perform the enzymatic hydrolysis; Optionally, the substrate concentration of the enzymatic hydrolysis reaction is 3% to 7%, preferably 5%; Optionally, the enzyme addition amount is 3% to 7%, preferably 5%; Optionally, the enzymatic hydrolysis pH is 7 to 11, preferably 10; Optionally, the enzymatic hydrolysis time is 1 to 5 hours, preferably 3 hours; Optionally, the enzymolysis temperature is 45-65°C, preferably 55°C.

3. The preparation method according to claim 1, characterized in that: In step (2), the enzymatic hydrolysis product is treated with sodium bisulfite assisted by ultrasound; Optionally, the sodium bisulfite concentration is 0.5 to 2.5 g / L, preferably 1.5 g / L; Optionally, the ultrasonic power is 100-500W, preferably 400W; Optionally, the ultrasonic time is 5 to 25 min, preferably 15 min.

4. The preparation method according to claim 1, characterized in that: In step (3), the enzymatic hydrolysis product is separated by using an ultrafiltration membrane with a molecular weight of 5 kDa, in step (4), the peptides with a molecular weight less than 5 kDa are chromatographed and separated by using Sephadex G-25 gel, in step (5), the peptide segments are identified by using LC-MS, in step (6), the active peptides are screened by molecular docking, and in step (7), the walnut lipid-lowering peptide is artificially synthesized.

5. The preparation method according to claim 1, characterized in that: Before step (1), the method further comprises: The walnut meal is defatted by using an organic solvent, the obtained defatted walnut meal is sieved, and then walnut protein is extracted.

6. The preparation method according to claim 1, characterized in that: Before step (1), the method further comprises: placing the walnut protein in a water bath at 90° C. for 15 minutes, cooling to room temperature, and adjusting the protein solution to an optimal pH using a NaOH solution.

7. The preparation method according to claim 1, characterized in that: Step (2) further comprises: inactivating the enzyme at high temperature, centrifuging, collecting the supernatant, filtering through a 0.45 μm filter membrane, and freeze-drying the filtrate to obtain the polypeptide.

8. A method for preparing walnut lipid-lowering peptide, characterized in that: include: 1) treating walnut protein with alkaline protease to obtain an enzymatic hydrolysis product; 2) Using ultrasound-assisted sodium bisulfite treatment to improve the biological activity of enzymatic hydrolysis products; 3) ultrafiltration treatment of the enzymatic hydrolysis product to obtain walnut peptides with a molecular weight below 5 kDa; 4) Using Sephadex G-25 gel to separate and screen the small molecule peptides to obtain active peptide fragments; 5) Using LC-MS to identify the amino acid sequence of the purified polypeptide; 6) Screening out peptides with lipid-lowering effects through computer-assisted docking analysis; 7) The peptide segments YFQ and FRPQ are artificially synthesized to obtain the walnut lipid-lowering peptide.

9. A walnut lipid-lowering peptide, characterized in that: The walnut hypolipidemic peptide calcium is prepared by the preparation method described in any one of claims 1 to 7 or the preparation method described in claim 8.

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