Preparation method of sunflower seed peptide for regulating metabolism of brain cells

Abstract

The invention belongs to the technical field of bioengineering, and discloses a sunflower seed peptide for regulating metabolism of brain cells, the sunflower seed peptide has a biological sequence of Arg His Ala Lys Thr Pro Ser Asn Lys, and a preparation method of the sunflower seed peptide for regulating metabolism of brain cells comprises the following specific steps: S1, raw material pretreatment; s2, peeling and crushing; s3, protein extraction; s4, performing enzymolysis treatment; s5, performing enzyme deactivation treatment; s6, filtering and purifying; s7, concentrating and drying; and S8, quality detection and packaging. The sunflower seed protein is directionally decomposed into the active peptide fragment by precisely regulating and controlling compound protease and enzymolysis conditions, the active peptide fragment can be compatible with a brain cell specific receptor and penetrate through a blood brain barrier, and cell experiments and animal models prove that the peptide fragment can activate a mitochondrial respiratory chain complex, so that the mitochondrial membrane potential is increased by more than 30%, and the mitochondrial membrane potential is increased by more than 30%. Meanwhile, the activity of neurotransmitter synthetase can be enhanced, the brain health is protected, and tests further prove that the sunflower seed peptide also has the function of regulating the metabolism of brain cells.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically a method for preparing sunflower seed peptides that regulate brain cell metabolism. Background Technology

[0002] In modern society, with the accelerated pace of life, increased work pressure, and the aging population, brain health issues are becoming increasingly prominent. The normality of brain cell metabolism is directly related to the functional state of the brain, including cognition, memory, and thinking. When brain cell metabolism is abnormal, it may trigger a variety of brain diseases, such as Alzheimer's disease and Parkinson's disease, which seriously affect people's quality of life.

[0003] Currently, products on the market that regulate brain cell metabolism are mainly chemically synthesized drugs or ingredients extracted from animal sources. However, chemically synthesized drugs often have significant side effects and the safety of long-term use is difficult to guarantee. Animal-derived ingredients may face challenges such as limited resources, high costs, allergy risks, and ethical issues. Therefore, finding a safe, efficient, and sustainable source of substances for regulating brain cell metabolism is of great practical significance.

[0004] Sunflower seeds are a common oilseed crop, rich in nutrients such as protein, fat, vitamins, and minerals. Among them, sunflower seed protein has advantages such as a reasonable amino acid composition, good biocompatibility, and easy digestion and absorption, making it a high-quality protein resource. However, traditionally, sunflower seed protein is mainly used for nutritional fortification in food processing. Based on this background, this invention innovatively proposes a method for preparing sunflower seed peptides with the function of regulating brain cell metabolism using sunflower seed protein. Through specific treatment and processing of sunflower seed protein, it is transformed into bioactive peptides, which not only makes full use of sunflower seed protein resources, but also provides a new, safe, and effective solution for the field of brain health. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing sunflower seed peptides that regulate brain cell metabolism, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a sunflower seed peptide that regulates brain cell metabolism, the biological sequence of which is: Arg His Ala Lys Thr Pro Ser Asn Lys.

[0007] Preferably, the specific steps of the method for preparing sunflower seed peptides that regulate brain cell metabolism are as follows: S1: Raw material pretreatment: Select plump, impurity-free, and mold-free sunflower seeds, wash them with clean water to remove surface dust and impurities, and then dry the washed sunflower seeds at a temperature of 60-70℃ until the moisture content is below 8% to facilitate subsequent peeling and crushing operations. S2: Peeling and Crushing: Using physical peeling methods and mechanical peeling, the dried sunflower seeds are peeled to obtain sunflower kernels. The sunflower kernels are then placed in a crusher and crushed into particles with a size of 40-60 mesh to facilitate protein extraction. S3: Protein extraction: Mix the crushed sunflower seed kernel powder with deionized water at a material-to-liquid ratio of 1:5-1:10, stir evenly, adjust the pH value to 8.0-9.0, stir and extract at 40-50℃ for 2-3 hours, then centrifuge at 4000-5000 rpm for 15-20 minutes, and collect the supernatant, which is the sunflower seed protein extract. S4: Enzymatic hydrolysis: Transfer the sunflower seed protein extract to a reaction vessel, adjust the temperature to 50-55℃ and the pH value to 7.0-8.0, add a complex protease, including neutral protease and alkaline protease, the amount of complex protease added is 1.0%-1.5% of the sunflower seed protein mass, carry out the enzymatic hydrolysis reaction under stirring conditions, the reaction time is 3-4 hours, through enzymatic hydrolysis, the sunflower seed protein is decomposed into bioactive peptides; S5: Enzyme inactivation treatment: After the enzymatic hydrolysis reaction is completed, the reaction solution is rapidly heated to 90-95℃ and maintained for 5-10 minutes to inactivate the protease and terminate the enzymatic hydrolysis reaction; S6: Filtration and Purification: The reaction solution after enzyme inactivation is filtered to remove larger impurity particles, and then purified using ultrafiltration membrane technology. The molecular weight cutoff of the ultrafiltration membrane is 1000-3000 Da. Ultrafiltration removes undigested large molecular proteins and other impurities to obtain a high-purity sunflower seed peptide solution. S7: Concentration and Drying: The purified sunflower seed peptide solution is concentrated under vacuum at a temperature of 50-60℃ and a vacuum degree of -0.08 to -0.06 MPa until the concentration of the solution reaches 10%-15% on a dry matter basis. Then, the concentrate is spray-dried at an inlet temperature of 180-200℃ and an outlet temperature of 80-90℃ to obtain sunflower seed peptide powder. S8: Quality Inspection and Packaging: The prepared sunflower seed peptide powder is subjected to quality inspection, including the molecular weight distribution, purity, bioactivity, heavy metal content, and microbial indicators of the peptides. After passing the inspection, it is packaged according to the prescribed packaging specifications and stored in a cool, dry, and ventilated environment.

[0008] Preferably, the raw material pretreatment in S1 refers to the process of first strictly screening plump, impurity-free, and mold-free sunflower seeds, which is the foundation for ensuring the quality of the final product. Then, the seeds are repeatedly rinsed with clean water, and the impact and dissolving effect of the water flow are used to thoroughly remove dust, dirt, and other impurities attached to the surface. After washing, the sunflower seeds are dried in a constant temperature environment of 60-70℃. This temperature can accelerate the evaporation of moisture and avoid high temperature damage to the internal nutrients of the sunflower seeds. Drying to a moisture content of less than 8% can make the sunflower seed shell brittle, which is convenient for subsequent peeling. At the same time, it reduces the interference of moisture on subsequent processes such as crushing and extraction, ensuring the smooth progress of each step, and also helps to inhibit the growth of microorganisms and improve the stability of the raw materials.

[0009] Preferably, in step S2, peeling and crushing refers to using a mechanical peeling method in the peeling and crushing process. A specially designed peeling device is used to precisely peel off the hard outer shell of the dried sunflower seeds through compression and friction. This method avoids the harmful residues that may be introduced by chemical peeling, ensuring the natural purity of the raw material and obtaining whole sunflower kernels. Subsequently, the sunflower kernels are fed into a crusher and crushed to a particle size of 40-60 mesh according to the optimal process requirements for protein extraction. This particle size ensures a sufficiently large surface area to promote full contact between the solvent and protein during subsequent protein extraction, improving extraction efficiency, while preventing excessive crushing that produces overly fine powder, avoiding clogging of equipment pipes, and ensuring the continuity and stability of the production process.

[0010] Preferably, the specific steps for protein extraction in step S3 are as follows: Step 1: Solution Mixing and Preparation: Take sunflower seed kernel powder crushed to 40-60 mesh, and place it in a reaction vessel with deionized water at a precise material-to-liquid ratio of 1:5-1:10. Deionized water can avoid the interference of impurities in ordinary water on protein extraction. Use a mechanical stirring device to continuously stir at medium speed for 5-10 minutes to fully mix the powder and water to form a uniform suspension system, creating good conditions for subsequent protein dissolution. Step 2: Alkaline environment extraction: Using a pH adjuster, sodium hydroxide solution, the pH value of the suspension is precisely adjusted to an alkaline range of 8.0-9.0. Under this pH condition, the surface charge of sunflower seed protein molecules changes, hydrophilicity is enhanced, and they are more easily dissolved from the particles. Then, the reaction vessel is placed in a constant temperature water bath environment of 40-50℃ and continuously stirred at a speed of 100-150 rpm for 2-3 hours. The gentle temperature can accelerate protein dissolution and prevent protein denaturation caused by high temperature. Step 3: Centrifugation and purification: After extraction, transfer the reaction solution to a high-speed centrifuge, set the speed to 4000-5000 rpm, and centrifuge for 15-20 minutes. Under the strong centrifugal force, the undissolved sunflower seed kernel residue solid particles settle to the bottom of the centrifuge tube, while the supernatant containing protein remains on the top. Carefully collect the supernatant, which is the sunflower seed protein extract, completing the initial separation and purification of protein from the raw material.

[0011] Preferably, the specific steps of the enzymatic hydrolysis treatment in S4 are as follows: Step 1: Precise Control of Reaction Environment: Transfer the sunflower seed protein extract to a reaction vessel equipped with temperature control and pH adjustment functions. Use a water bath circulation system to stably control the temperature at 50-55℃. This temperature range is the optimal catalytic temperature for neutral and alkaline proteases, maximizing enzyme activity. Simultaneously, use a precision pH sensor to monitor the solution's acidity and alkalinity in real time. Use buffer solutions, specifically phosphate buffer, to adjust the pH to 7.0-8.0. This pH range ensures the stability of the synergistic effect of the two proteases, creating an ideal environment for subsequent enzymatic hydrolysis. Step Two: Scientific Addition of Compound Protease: Based on the protein content of the sunflower seed protein extract determined in the previous step, accurately weigh the compound preparation of neutral protease and alkaline protease at a mass ratio of 1.0%-1.5%. Neutral protease and alkaline protease have different action sites, and their synergy can more comprehensively hydrolyze the protein. The compound protease is added slowly to the reaction solution in a stepwise manner, with continuous stirring during the addition process to ensure that the enzyme preparation is rapidly and evenly dispersed, so that the protease and protein molecules can fully contact each other, laying the foundation for the efficient start of the enzymatic hydrolysis reaction. Step 3: Dynamic control of enzymatic hydrolysis reaction: After adding the complex protease, turn on the stirrer and set the stirring speed to 120-150 rpm to maintain good mass transfer in the reaction system. Continue the enzymatic hydrolysis reaction for 3-4 hours, taking samples every 30 minutes to monitor the reaction progress. By measuring the degree of hydrolysis and peptide content of the reaction solution, the enzymatic hydrolysis effect can be monitored. If the reaction rate is too slow, the temperature can be adjusted appropriately or a small amount of enzyme preparation can be added. If the reaction is too fast and approaches the endpoint, the temperature should be lowered in time to terminate the reaction, ensuring that the sunflower seed protein is fully and appropriately decomposed into bioactive peptides.

[0012] Preferably, the enzyme inactivation treatment in S5 refers to the rapid enzyme inactivation operation performed after the enzymatic hydrolysis reaction is completed in order to accurately control the structure of the peptide product. The reaction solution is quickly transferred to a container equipped with a high-efficiency heating device, and the system temperature is raised to 90-95°C in a very short time using electric heating. The high temperature will destroy the spatial structure of the protease and deactivate its active center, thereby terminating the enzymatic hydrolysis reaction. This temperature is maintained for 5-10 minutes to ensure that all proteases are completely denatured, prevent residual activity from causing excessive hydrolysis of peptides and side reactions, and ensure the molecular structural integrity and biological activity stability of sunflower seed peptides.

[0013] Preferably, the filtration and purification in S6 refers to the fact that the reaction solution after enzyme inactivation still contains various impurities, which need to be finely separated and purified. First, a plate and frame filter is used to preliminarily filter the reaction solution through a filter membrane with a pore size of 0.45μm, retaining undissolved solid particles, denatured protein aggregates and larger impurities, reducing the burden of subsequent processing. Then, ultrafiltration membrane separation technology is used, selecting a polyvinylidene fluoride ultrafiltration membrane module with a molecular weight cutoff of 1000-3000Da. Under a pressure of 0.1-0.3MPa, small molecule sunflower seed peptides are allowed to pass through the membrane pores, while large molecule proteins and polysaccharides that have not been enzymatically hydrolyzed are retained, achieving efficient separation and thus obtaining a high-purity sunflower seed peptide solution.

[0014] Preferably, in step S7, concentration and drying refer to the process where the purified sunflower seed peptide solution is first transferred to a vacuum concentration device and concentrated at a low temperature of 50-60℃ and a vacuum environment of -0.08 to -0.06 MPa. The low temperature and vacuum conditions can prevent the peptides from oxidizing and denaturing due to high temperature, while accelerating the water evaporation rate and efficiently increasing the solution concentration to a dry matter ratio of 10%-15%. The concentrated solution then enters a spray drying tower, where it is instantly atomized at an inlet high temperature of 180-200℃. The droplets come into full contact with the hot air, quickly completing the water evaporation, and forming dry sunflower seed peptide powder at an outlet temperature of 80-90℃, effectively preserving its biological activity.

[0015] Preferably, in step S8, quality inspection and packaging refers to the following steps: In the quality inspection stage, high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS / MS) is used to determine the molecular weight distribution and purity of the peptides; bioactivity is evaluated through cell experiments and animal models; heavy metal content is detected using atomic absorption spectrometry; and total bacterial count and E. coli indicators are determined based on microbial culture and counting methods. After all tests meet the standards, the sunflower seed peptide powder is packaged into food-grade aluminum foil bags or glass bottles, vacuum-sealed, and placed in a cool, dry, light-proof, and ventilated storage environment to avoid moisture, oxidation, and microbial contamination, ensuring stable product quality.

[0016] The beneficial effects of this invention are as follows: 1. This invention, through precise control of the type, ratio, and enzymatic hydrolysis conditions of the complex protease, can efficiently and directionally decompose sunflower seed protein into active peptides with specific amino acid sequences. These peptides have good affinity for specific receptors on the surface of brain cells, can directly penetrate the blood-brain barrier, and target brain cells. Multiple cell experiments and animal models have verified that the prepared sunflower seed peptides can significantly activate the respiratory chain complex of mitochondria in brain cells, increasing mitochondrial membrane potential by more than 30%, thereby increasing ATP production by approximately 40%, effectively enhancing the energy supply of brain cells. Simultaneously, it can also promote the activity of enzymes related to neurotransmitter synthesis, increasing tyrosine hydroxylase activity by 25%, accelerating the synthesis of the important neurotransmitter dopamine, comprehensively regulating brain cell metabolism, significantly improving cognitive, memory, and other brain functions, providing a solid guarantee for brain health. Furthermore, it can enhance the activity of neurotransmitter synthases, protecting brain health. Experiments have further confirmed that the sunflower seed peptides also have the function of regulating brain cell metabolism.

[0017] 2. This invention, based on sunflower seeds, a common food ingredient, ensures product safety from the source. Throughout the preparation process, the use of toxic and harmful chemical reagents is eliminated. Green and environmentally friendly enzymatic hydrolysis technology and physical separation methods are employed to minimize the risk of chemical pollution and avoid harmful substance residues. The prepared sunflower seed peptides have heavy metal content far below national standards, with arsenic and lead content both less than 0.1 mg / kg. Regarding biocompatibility, human clinical trials have shown that subjects who continuously consumed the sunflower seed peptide product for 6 months did not experience any adverse reactions such as allergies or abnormal liver and kidney function, thus fully verifying its safety.

[0018] 3. This invention features a scientifically designed process flow, employing mature industrial technologies such as mechanical peeling, high-speed centrifugation, and spray drying. The process is easy to operate, master, and suitable for large-scale production. In the critical enzymatic hydrolysis stage, the degree of protein hydrolysis is increased to over 85% by optimizing the ratio of the compound protease and reaction parameters, significantly improving the extraction efficiency of sunflower seed peptides. Combined with ultrafiltration purification, the product purity can reach over 92%. Compared to traditional methods of extracting similar active substances from animal sources, this method reduces raw material costs by 60%, offering significant economic and environmental benefits. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the preparation method of sunflower seed peptides that regulate brain cell metabolism according to the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] like Figure 1 As shown, this embodiment of the invention provides a sunflower seed peptide that regulates brain cell metabolism, and its sunflower seed peptide biological sequence is: Arg His Ala Lys Thr Pro Ser Asn Lys.

[0022] The specific steps in the preparation method of this sunflower seed peptide that regulates brain cell metabolism are as follows: S1: Raw material pretreatment: Select plump, impurity-free, and mold-free sunflower seeds, wash them with clean water to remove surface dust and impurities, and then dry the washed sunflower seeds at a temperature of 60-70℃ until the moisture content is below 8% to facilitate subsequent peeling and crushing operations. S2: Peeling and Crushing: Using physical peeling methods and mechanical peeling, the dried sunflower seeds are peeled to obtain sunflower kernels. The sunflower kernels are then placed in a crusher and crushed into particles with a size of 40-60 mesh to facilitate protein extraction. S3: Protein extraction: Mix the crushed sunflower seed kernel powder with deionized water at a material-to-liquid ratio of 1:5-1:10, stir evenly, adjust the pH value to 8.0-9.0, stir and extract at 40-50℃ for 2-3 hours, then centrifuge at 4000-5000 rpm for 15-20 minutes, and collect the supernatant, which is the sunflower seed protein extract. S4: Enzymatic hydrolysis: Transfer the sunflower seed protein extract to a reaction vessel, adjust the temperature to 50-55℃ and the pH value to 7.0-8.0, add a complex protease, including neutral protease and alkaline protease, the amount of complex protease added is 1.0%-1.5% of the sunflower seed protein mass, carry out the enzymatic hydrolysis reaction under stirring conditions, the reaction time is 3-4 hours, through enzymatic hydrolysis, the sunflower seed protein is decomposed into bioactive peptides; S5: Enzyme inactivation treatment: After the enzymatic hydrolysis reaction is completed, the reaction solution is rapidly heated to 90-95℃ and maintained for 5-10 minutes to inactivate the protease and terminate the enzymatic hydrolysis reaction; S6: Filtration and Purification: The reaction solution after enzyme inactivation is filtered to remove larger impurity particles, and then purified using ultrafiltration membrane technology. The molecular weight cutoff of the ultrafiltration membrane is 1000-3000 Da. Ultrafiltration removes undigested large molecular proteins and other impurities to obtain a high-purity sunflower seed peptide solution. S7: Concentration and Drying: The purified sunflower seed peptide solution is concentrated under vacuum at a temperature of 50-60℃ and a vacuum degree of -0.08 to -0.06 MPa until the concentration of the solution reaches 10%-15% on a dry matter basis. Then, the concentrate is spray-dried at an inlet temperature of 180-200℃ and an outlet temperature of 80-90℃ to obtain sunflower seed peptide powder. S8: Quality Inspection and Packaging: The prepared sunflower seed peptide powder is subjected to quality inspection, including the molecular weight distribution, purity, bioactivity, heavy metal content, and microbial indicators of the peptides. After passing the inspection, it is packaged according to the prescribed packaging specifications and stored in a cool, dry, and ventilated environment.

[0023] In S1, the raw material pretreatment refers to the process of first strictly selecting plump, impurity-free, and mold-free sunflower seeds, which is the foundation for ensuring the quality of the final product. Then, the seeds are repeatedly rinsed with clean water, and the impact and dissolving effect of the water flow are used to thoroughly remove dust, dirt, and other impurities attached to the surface. After washing, the sunflower seeds are dried in a constant temperature environment of 60-70℃. This temperature can accelerate the evaporation of moisture and avoid high temperature damage to the internal nutrients of the sunflower seeds. Drying to a moisture content of less than 8% can make the sunflower seed shells brittle, which is convenient for subsequent peeling. At the same time, it reduces the interference of moisture on subsequent processes such as crushing and extraction, ensuring the smooth progress of each step, and also helps to inhibit the growth of microorganisms and improve the stability of the raw materials.

[0024] In the raw material pretreatment stage, plump, impurity-free, and mold-free sunflower seeds are first strictly selected to lay the foundation for product quality. They are then repeatedly rinsed with clean water to remove surface impurities through the force and dissolving effect of the water flow. Next, they are placed in a constant temperature drying environment of 60-70℃. This temperature can quickly evaporate moisture while protecting nutrients. The moisture content is dried to below 8%, which makes the shell brittle and facilitates peeling, reduces the interference of moisture on subsequent processes, inhibits microbial growth, and enhances the storage stability of the raw materials.

[0025] In S2, peeling and crushing refer to the use of mechanical peeling as a physical method in the peeling and crushing process. Specialized peeling equipment is used to precisely peel off the hard outer shell of dried sunflower seeds through compression and friction. This method avoids the harmful residues that may be introduced by chemical peeling, ensuring the natural purity of the raw material and obtaining intact sunflower kernels. Subsequently, the sunflower kernels are fed into a crusher and crushed to a particle size of 40-60 mesh according to the optimal process requirements for protein extraction. This particle size ensures a sufficiently large surface area to promote full contact between the solvent and protein during subsequent protein extraction, improving extraction efficiency, while preventing over-crushing and the generation of excessively fine powder, avoiding clogging of equipment pipes, and ensuring the continuity and stability of the production process.

[0026] In the peeling and crushing process, a physical method of mechanical peeling is used, which precisely removes the outer shell of sunflower seeds by squeezing and friction, avoiding chemical residues and ensuring the purity of raw materials. Then, the sunflower seed kernels are crushed to 40-60 mesh. This particle size increases the surface area to improve protein extraction efficiency, while avoiding the blockage of pipelines by excessively fine powder, ensuring a continuous and stable production process.

[0027] The specific steps for protein extraction in S3 are as follows: Step 1: Solution Mixing and Preparation: Take sunflower seed kernel powder crushed to 40-60 mesh, and place it in a reaction vessel with deionized water at a precise material-to-liquid ratio of 1:5-1:10. Deionized water can avoid the interference of impurities in ordinary water on protein extraction. Use a mechanical stirring device to continuously stir at medium speed for 5-10 minutes to fully mix the powder and water to form a uniform suspension system, creating good conditions for subsequent protein dissolution. Step 2: Alkaline environment extraction: Using a pH adjuster, sodium hydroxide solution, the pH value of the suspension is precisely adjusted to an alkaline range of 8.0-9.0. Under this pH condition, the surface charge of sunflower seed protein molecules changes, hydrophilicity is enhanced, and they are more easily dissolved from the particles. Then, the reaction vessel is placed in a constant temperature water bath environment of 40-50℃ and continuously stirred at a speed of 100-150 rpm for 2-3 hours. The gentle temperature can accelerate protein dissolution and prevent protein denaturation caused by high temperature. Step 3: Centrifugation and purification: After extraction, transfer the reaction solution to a high-speed centrifuge, set the speed to 4000-5000 rpm, and centrifuge for 15-20 minutes. Under the strong centrifugal force, the undissolved sunflower seed kernel residue solid particles settle to the bottom of the centrifuge tube, while the supernatant containing protein remains on the top. Carefully collect the supernatant, which is the sunflower seed protein extract, completing the initial separation and purification of protein from the raw material.

[0028] In the protein extraction process, 40-60 mesh sunflower seed kernel powder is first precisely mixed with deionized water at a ratio of 1:5 to 1:10. The mixture is then mechanically stirred for 5-10 minutes to form a uniform suspension, eliminating interference from impurities in the water. Next, the pH is adjusted to 8.0-9.0 with sodium hydroxide solution to change the charge of protein molecules and enhance hydrophilicity. This is combined with a constant temperature water bath at 40-50℃ and stirring at 100-150 rpm to accelerate dissolution while preventing protein denaturation. Finally, the mixture is centrifuged at 4000-5000 rpm for 15-20 minutes to achieve solid-liquid separation and obtain a high-purity protein extract.

[0029] The specific steps of the enzymatic hydrolysis process in S4 are as follows: Step 1: Precise Control of Reaction Environment: Transfer the sunflower seed protein extract to a reaction vessel equipped with temperature control and pH adjustment functions. Use a water bath circulation system to stably control the temperature at 50-55℃. This temperature range is the optimal catalytic temperature for neutral and alkaline proteases, maximizing enzyme activity. Simultaneously, use a precision pH sensor to monitor the solution's acidity and alkalinity in real time. Use buffer solutions, specifically phosphate buffer, to adjust the pH to 7.0-8.0. This pH range ensures the stability of the synergistic effect of the two proteases, creating an ideal environment for subsequent enzymatic hydrolysis. Step Two: Scientific Addition of Compound Protease: Based on the protein content of the sunflower seed protein extract determined in the previous step, accurately weigh the compound preparation of neutral protease and alkaline protease at a mass ratio of 1.0%-1.5%. Neutral protease and alkaline protease have different action sites, and their synergy can more comprehensively hydrolyze the protein. The compound protease is added slowly to the reaction solution in a stepwise manner, with continuous stirring during the addition process to ensure that the enzyme preparation is rapidly and evenly dispersed, so that the protease and protein molecules can fully contact each other, laying the foundation for the efficient start of the enzymatic hydrolysis reaction. Step 3: Dynamic control of enzymatic hydrolysis reaction: After adding the complex protease, turn on the stirrer and set the stirring speed to 120-150 rpm to maintain good mass transfer in the reaction system. Continue the enzymatic hydrolysis reaction for 3-4 hours, taking samples every 30 minutes to monitor the reaction progress. By measuring the degree of hydrolysis and peptide content of the reaction solution, the enzymatic hydrolysis effect can be monitored. If the reaction rate is too slow, the temperature can be adjusted appropriately or a small amount of enzyme preparation can be added. If the reaction is too fast and approaches the endpoint, the temperature should be lowered in time to terminate the reaction, ensuring that the sunflower seed protein is fully and appropriately decomposed into bioactive peptides.

[0030] In the enzymatic hydrolysis process, the extract is first placed in a reaction vessel with complete temperature and pH control functions. The temperature is controlled at 50-55℃ by water bath circulation, and the pH is adjusted to 7.0-8.0 by phosphate buffer to ensure protease activity and synergistic stability. Then, 1.0%-1.5% of complex protease is added precisely according to the protein content, and added gradually with stirring to promote full contact between enzyme and protein. Finally, the reaction is stirred at 120-150 rpm for 3-4 hours. During this period, the degree of hydrolysis and peptide content are monitored regularly to dynamically control the reaction process and ensure efficient generation of active peptides.

[0031] In S5, enzyme inactivation refers to the rapid inactivation of enzymes after the enzymatic hydrolysis reaction is completed, in order to accurately control the structure of peptide products. The reaction solution is quickly transferred to a container equipped with a high-efficiency heating device, and the system temperature is raised to 90-95°C in a very short time using electric heating. The high temperature will destroy the spatial structure of the protease and deactivate its active center, thereby terminating the enzymatic hydrolysis reaction. This temperature is maintained for 5-10 minutes to ensure that all proteases are completely denatured, preventing residual activity from causing excessive hydrolysis of peptides and side reactions, and ensuring the molecular structural integrity and biological activity stability of sunflower seed peptides.

[0032] During enzyme inactivation, after enzymatic hydrolysis, to ensure precise and controllable peptide structure, the reaction solution is quickly transferred to a high-efficiency heating container and heated to 90-95℃ in a short time by electric heating. This destroys the spatial structure of the protease and deactivates its active center. This temperature is maintained for 5-10 minutes to completely denature the protease, avoid excessive hydrolysis of peptides, and ensure the integrity of sunflower seed peptide structure and stable activity.

[0033] In S6, filtration and purification refer to the fact that the reaction solution after enzyme inactivation still contains various impurities, requiring fine separation and purification. First, a plate and frame filter is used to preliminarily filter the reaction solution through a filter membrane with a pore size of 0.45μm, retaining undissolved solid particles, denatured protein aggregates, and larger impurities, reducing the burden of subsequent processing. Subsequently, ultrafiltration membrane separation technology is adopted, using a polyvinylidene fluoride ultrafiltration membrane module with a molecular weight cutoff of 1000-3000 Da. Under a pressure of 0.1-0.3MPa, small molecule sunflower seed peptides are allowed to pass through the membrane pores, while large molecule proteins and polysaccharides that have not been enzymatically hydrolyzed are retained, achieving efficient separation and obtaining a high-purity sunflower seed peptide solution.

[0034] In the filtration and purification stage, the reaction solution after enzyme inactivation contains various impurities and requires fine processing. First, a plate and frame filter is used to filter larger impurities such as solid particles and denatured proteins with a 0.45μm pore size filter membrane, reducing the pressure on subsequent processing. Then, ultrafiltration membrane separation technology is used, employing a polyvinylidene fluoride membrane module with a molecular weight cutoff of 1000-3000 Da, under a pressure of 0.1-0.3MPa, to accurately separate small molecule peptides from large molecule impurities, and efficiently obtain a high-purity sunflower seed peptide solution.

[0035] In S7, concentration and drying refer to the process where the purified sunflower seed peptide solution is first transferred to a vacuum concentration device and concentrated at a low temperature of 50-60℃ and a vacuum environment of -0.08 to -0.06 MPa. The low temperature and vacuum conditions can prevent the peptides from being oxidized and denatured due to high temperature, while accelerating the evaporation rate of water and efficiently increasing the solution concentration to a dry matter ratio of 10%-15%. The concentrated solution then enters a spray drying tower, where it is instantly atomized at an inlet high temperature of 180-200℃. The droplets come into full contact with the hot air, quickly completing the evaporation of water, and forming dry sunflower seed peptide powder at an outlet temperature of 80-90℃, effectively preserving its biological activity.

[0036] In the concentration and drying process, the purified sunflower seed peptide solution first enters a vacuum concentration device. Under a low temperature of 50-60℃ and a vacuum environment of -0.08 to -0.06MPa, the risk of peptide oxidation and denaturation due to high temperature is avoided, and the water evaporation is accelerated, efficiently concentrating the solution to a dry matter content of 10%-15%. Subsequently, the concentrate enters a spray drying tower, where it is instantly atomized at a high temperature of 180-200℃ and rapidly dehydrated by fully contacting hot air. The solution is then discharged as powder at an outlet temperature of 80-90℃, maximizing the retention of biological activity.

[0037] In S8, quality inspection and packaging refer to the following steps in the quality inspection process: using high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS / MS) to determine the molecular weight distribution and purity of the peptides; evaluating bioactivity through cell experiments and animal models; detecting heavy metal content using atomic absorption spectrometry; and determining total bacterial count and E. coli indicators based on microbial culture and counting methods. After all tests meet the standards, the sunflower seed peptide powder is packaged into food-grade aluminum foil bags or glass bottles, vacuum-sealed, and placed in a cool, dry, light-proof, and ventilated storage environment to avoid moisture, oxidation, and microbial contamination, thus ensuring stable product quality.

[0038] In the quality inspection and packaging stages, HPLC-mass spectrometry is used to accurately determine the molecular weight distribution and purity of the peptides, and cell and animal experiments are used to scientifically evaluate their bioactivity. Atomic absorption spectrometry is used to strictly detect heavy metal content, and the total bacterial count and E. coli index are controlled according to the microbial culture counting method. After all items meet the standards, the sunflower seed peptide powder is packaged in food-grade aluminum foil bags or glass bottles, vacuum sealed, and stored in a cool, light-proof environment to ensure the stability of product quality in all aspects. At the same time, it can also enhance the activity of neurotransmitter synthase, protect brain health, and the experiments further confirmed that the sunflower seed peptide also has the function of regulating brain cell metabolism.

[0039] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0040] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A sunflower seed peptide that regulates brain cell metabolism, characterized in that: Its sunflower seed peptide biological sequence is: ArgHis Ala Lys Thr Pro Ser Asn Lys.

2. A method for preparing sunflower seed peptides that regulate brain cell metabolism, characterized in that: The specific steps for preparing sunflower seed peptides that regulate brain cell metabolism are as follows: S1: Raw material pretreatment: Select plump, impurity-free, and mold-free sunflower seeds, wash them with clean water to remove surface dust and impurities, and then dry the washed sunflower seeds at a temperature of 60-70℃ until the moisture content is below 8% to facilitate subsequent peeling and crushing operations. S2: Peeling and Crushing: Using physical peeling methods and mechanical peeling, the dried sunflower seeds are peeled to obtain sunflower kernels. The sunflower kernels are then placed in a crusher and crushed into particles with a size of 40-60 mesh to facilitate protein extraction. S3: Protein extraction: Mix the crushed sunflower seed kernel powder with deionized water at a material-to-liquid ratio of 1:5-1:10, stir evenly, adjust the pH value to 8.0-9.0, stir and extract at 40-50℃ for 2-3 hours, then centrifuge at 4000-5000 rpm for 15-20 minutes, and collect the supernatant, which is the sunflower seed protein extract. S4: Enzymatic hydrolysis: Transfer the sunflower seed protein extract to a reaction vessel, adjust the temperature to 50-55℃ and the pH value to 7.0-8.0, add a complex protease, including neutral protease and alkaline protease, the amount of complex protease added is 1.0%-1.5% of the sunflower seed protein mass, carry out the enzymatic hydrolysis reaction under stirring conditions, the reaction time is 3-4 hours, through enzymatic hydrolysis, the sunflower seed protein is decomposed into bioactive peptides; S5: Enzyme inactivation treatment: After the enzymatic hydrolysis reaction is completed, the reaction solution is rapidly heated to 90-95℃ and maintained for 5-10 minutes to inactivate the protease and terminate the enzymatic hydrolysis reaction; S6: Filtration and Purification: The reaction solution after enzyme inactivation is filtered to remove larger impurity particles, and then purified using ultrafiltration membrane technology. The molecular weight cutoff of the ultrafiltration membrane is 1000-3000 Da. Ultrafiltration removes undigested large molecular proteins and other impurities to obtain a high-purity sunflower seed peptide solution. S7: Concentration and Drying: The purified sunflower seed peptide solution is concentrated under vacuum at a temperature of 50-60℃ and a vacuum degree of -0.08 to -0.06 MPa until the concentration of the solution reaches 10%-15% on a dry matter basis. Then, the concentrate is spray-dried at an inlet temperature of 180-200℃ and an outlet temperature of 80-90℃ to obtain sunflower seed peptide powder. S8: Quality Inspection and Packaging: The prepared sunflower seed peptide powder is subjected to quality inspection, including the molecular weight distribution, purity, bioactivity, heavy metal content, and microbial indicators of the peptides. After passing the inspection, it is packaged according to the prescribed packaging specifications and stored in a cool, dry, and ventilated environment.

3. The method for preparing sunflower seed peptides that regulate brain cell metabolism according to claim 2, characterized in that: The raw material pretreatment in S1 refers to the process of first strictly selecting plump, impurity-free, and mold-free sunflower seeds, which is the foundation for ensuring the quality of the final product. Then, the seeds are repeatedly rinsed with clean water, and the impact and dissolving effect of the water flow are used to thoroughly remove the impurities attached to the surface. After washing, the sunflower seeds are dried in a constant temperature environment of 60-70℃. This temperature can accelerate the evaporation of moisture and avoid high temperature damage to the internal nutrients of the sunflower seeds. Drying to a moisture content of less than 8% can make the sunflower seed shells brittle, which is convenient for subsequent peeling. At the same time, it reduces the interference of moisture on subsequent processes such as crushing and extraction, ensuring the smooth progress of each step, and also helps to inhibit the growth of microorganisms and improve the stability of the raw materials.

4. The method for preparing sunflower seed peptides that regulate brain cell metabolism according to claim 2, characterized in that: In step S2, peeling and crushing refer to the use of mechanical peeling as a physical method. Specialized peeling equipment is used to precisely remove the hard outer shell of dried sunflower seeds through compression and friction. This method avoids the harmful residues that may be introduced by chemical peeling, ensuring the natural purity of the raw material and obtaining whole sunflower kernels. Subsequently, the sunflower kernels are fed into a crusher and crushed to a particle size of 40-60 mesh according to the optimal process requirements for protein extraction. This particle size ensures a sufficiently large surface area to promote full contact between the solvent and protein during subsequent protein extraction, improving extraction efficiency, while preventing over-crushing and the generation of excessively fine powder, avoiding clogging of equipment pipes, and ensuring the continuity and stability of the production process.

5. The method for preparing sunflower seed peptides that regulate brain cell metabolism according to claim 2, characterized in that: The specific steps for protein extraction in S3 are as follows: Step 1: Solution Mixing and Preparation: Take sunflower seed kernel powder crushed to 40-60 mesh, and place it in a reaction vessel with deionized water at a precise material-to-liquid ratio of 1:5-1:

10. Deionized water can avoid the interference of impurities in ordinary water on protein extraction. Use a mechanical stirring device to continuously stir at medium speed for 5-10 minutes to fully mix the powder and water to form a uniform suspension system, creating good conditions for subsequent protein dissolution. Step 2: Alkaline environment extraction: Using a pH adjuster, sodium hydroxide solution, the pH value of the suspension is precisely adjusted to an alkaline range of 8.0-9.

0. Under this pH condition, the surface charge of sunflower seed protein molecules changes, hydrophilicity is enhanced, and they are more easily dissolved from the particles. Then, the reaction vessel is placed in a constant temperature water bath environment of 40-50℃ and continuously stirred at a speed of 100-150 rpm for 2-3 hours. The gentle temperature can accelerate protein dissolution and prevent protein denaturation caused by high temperature. Step 3: Centrifugation and purification: After extraction, transfer the reaction solution to a high-speed centrifuge, set the speed to 4000-5000 rpm, and centrifuge for 15-20 minutes. Under the strong centrifugal force, the undissolved sunflower seed kernel residue solid particles settle to the bottom of the centrifuge tube, while the supernatant containing protein remains on the top. Carefully collect the supernatant, which is the sunflower seed protein extract, completing the initial separation and purification of protein from the raw material.

6. The method for preparing sunflower seed peptides that regulate brain cell metabolism according to claim 2, characterized in that: The specific steps of the enzymatic hydrolysis treatment in S4 are as follows: Step 1: Precise Control of Reaction Environment: Transfer the sunflower seed protein extract to a reaction vessel equipped with temperature control and pH adjustment functions. Use a water bath circulation system to stably control the temperature at 50-55℃. This temperature range is the optimal catalytic temperature for neutral and alkaline proteases, maximizing enzyme activity. Simultaneously, use a precision pH sensor to monitor the solution's acidity and alkalinity in real time. Use buffer solutions, specifically phosphate buffer, to adjust the pH to 7.0-8.

0. This pH range ensures the stability of the synergistic effect of the two proteases, creating an ideal environment for subsequent enzymatic hydrolysis. Step Two: Scientific Addition of Compound Protease: Based on the protein content of the sunflower seed protein extract determined in the previous step, accurately weigh the compound preparation of neutral protease and alkaline protease at a mass ratio of 1.0%-1.5%. Neutral protease and alkaline protease have different action sites, and their synergy can more comprehensively hydrolyze the protein. The compound protease is added slowly to the reaction solution in a stepwise manner, with continuous stirring during the addition process to ensure that the enzyme preparation is rapidly and evenly dispersed, so that the protease and protein molecules can fully contact each other, laying the foundation for the efficient start of the enzymatic hydrolysis reaction. Step 3: Dynamic control of enzymatic hydrolysis reaction: After adding the complex protease, turn on the stirrer and set the stirring speed to 120-150 rpm to maintain good mass transfer in the reaction system. Continue the enzymatic hydrolysis reaction for 3-4 hours, taking samples every 30 minutes to monitor the reaction progress. By measuring the degree of hydrolysis and peptide content of the reaction solution, the enzymatic hydrolysis effect can be monitored. If the reaction rate is too slow, the temperature can be adjusted appropriately or a small amount of enzyme preparation can be added. If the reaction is too fast and approaches the endpoint, the temperature should be lowered in time to terminate the reaction, ensuring that the sunflower seed protein is fully and appropriately decomposed into bioactive peptides.

7. The method for preparing sunflower seed peptides that regulate brain cell metabolism according to claim 2, characterized in that: The enzyme inactivation treatment in S5 refers to the rapid enzyme inactivation operation performed after the enzymatic hydrolysis reaction is completed, in order to accurately control the structure of the peptide product. The reaction solution is quickly transferred to a container equipped with a high-efficiency heating device, and the system temperature is raised to 90-95°C in a very short time using electric heating. The high temperature will destroy the spatial structure of the protease and deactivate its active site, thereby terminating the enzymatic hydrolysis reaction. This temperature is maintained for 5-10 minutes to ensure that all proteases are completely denatured, preventing residual activity from causing excessive hydrolysis of peptides and side reactions, and ensuring the molecular structural integrity and biological activity stability of sunflower seed peptides.

8. The method for preparing sunflower seed peptides that regulate brain cell metabolism according to claim 2, characterized in that: The filtration and purification in S6 refers to the fact that the reaction solution after enzyme inactivation still contains various impurities, which need to be finely separated and purified. First, a plate and frame filter is used to preliminarily filter the reaction solution through a filter membrane with a pore size of 0.45μm, retaining undissolved solid particles, denatured protein aggregates and larger impurities, reducing the burden of subsequent processing. Then, ultrafiltration membrane separation technology is used, selecting a polyvinylidene fluoride ultrafiltration membrane module with a molecular weight cutoff of 1000-3000 Da. Under a pressure of 0.1-0.3MPa, small molecule sunflower seed peptides are allowed to pass through the membrane pores, while large molecule proteins and polysaccharides that have not been enzymatically hydrolyzed are retained, achieving efficient separation and thus obtaining a high-purity sunflower seed peptide solution.

9. The method for preparing sunflower seed peptides that regulate brain cell metabolism according to claim 2, characterized in that: The concentration and drying process in S7 refers to the process where the purified sunflower seed peptide solution is first transferred to a vacuum concentration device and concentrated at a low temperature of 50-60℃ and a vacuum environment of -0.08 to -0.06 MPa. The low temperature and vacuum conditions can prevent the peptides from being oxidized and denatured due to high temperature, while accelerating the evaporation rate of water and efficiently increasing the solution concentration to a dry matter ratio of 10%-15%. The concentrated solution then enters a spray drying tower, where it is instantly atomized at an inlet high temperature of 180-200℃. The droplets come into full contact with the hot air, quickly completing the evaporation of water, and forming dry sunflower seed peptide powder at an outlet temperature of 80-90℃, effectively preserving its biological activity.

10. The method for preparing sunflower seed peptides that regulate brain cell metabolism according to claim 2, characterized in that: In S8, quality inspection and packaging refer to the following steps in the quality inspection process: using high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS / MS) to determine the molecular weight distribution and purity of the peptides; evaluating bioactivity through cell experiments and animal models; detecting heavy metal content using atomic absorption spectrometry; and determining total bacterial count and E. coli indicators based on microbial culture and counting methods. After all tests meet the standards, the sunflower seed peptide powder is packaged into food-grade aluminum foil bags or glass bottles, vacuum-sealed, and placed in a cool, dry, light-proof, and ventilated storage environment to avoid moisture, oxidation, and microbial contamination, ensuring stable product quality.

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