Method for extracting tyrosinase inhibitory peptide from potamogeton crispus

Abstract

The invention discloses a method for extracting tyrosinase inhibitory peptide from potamogeton crispus, relates to the technical field of active peptide, and belongs to the patent classification number C07K1 / 14. Comprising the following steps: cleaning potamogeton crispus, drying and crushing to obtain potamogeton crispus powder; adding the potamogeton crispus powder into an ethanol solution, heating, oscillating and extracting, then centrifuging, and collecting supernate to obtain a potamogeton crispus extracting solution; adjusting the pH value of the potamogeton crispus extracting solution to be alkaline, then adding alkaline protease, performing heating enzymolysis, performing enzyme deactivation and centrifugal treatment, and collecting supernate to obtain enzymatic hydrolysate; performing ultrafiltration treatment on the enzymatic hydrolysate, and collecting permeate to obtain ultrafiltration permeate; carrying out nanofiltration treatment on the ultrafiltration permeate liquid, and collecting trapped fluid to obtain nanofiltration concentrated liquid; and freeze-drying the nanofiltration concentrated solution to obtain the tyrosinase inhibitory peptide. According to the method, the tyrosinase inhibitory peptide is concentrated and purified in a manner of combining ultrafiltration and nanofiltration, the method is simple and convenient to operate, and the operation difficulty and the time cost are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of active peptides, belongs to patent classification number C07K1 / 14, and specifically is a method for extracting tyrosinase inhibitory peptides from Potamogeton crispus. Background Art

[0002] Tyrosinase inhibitory peptides, as bioactive substances that effectively inhibit tyrosinase activity, play a key role in numerous fields. In cosmetics, they achieve skin whitening effects by inhibiting melanin production and are a core ingredient in whitening skincare products. In the food industry, they can be used to inhibit enzymatic browning in fruits and vegetables, extending shelf life and preserving food color and quality. In medicine, they are crucial for the treatment of pigmentation disorders such as melasma and freckles, making them a hot topic in drug development. Water chestnut, a submerged plant widely distributed in freshwater bodies, has a short growth cycle, a large biomass, and is rich in various bioactive components, including proteins, polysaccharides, and polyphenols, including peptides with tyrosinase inhibitory activity. Further development of water chestnut and extraction of tyrosinase inhibitory peptides could not only address the aquatic ecological challenges caused by its overgrowth but also provide natural, green active ingredients for related industries, achieving high-value resource utilization.

[0003] Currently, existing technologies for extracting tyrosinase inhibitory peptides from plants have numerous shortcomings. Traditional extraction methods often involve multiple complex steps, such as raw material pretreatment, solvent extraction, and multiple rounds of column chromatography (such as silica gel column chromatography and gel column chromatography). Column chromatography separation requires multiple steps, including column loading, column equilibration, sample loading, and elution. Each round of chromatography requires meticulous operation and control, making the entire process relatively cumbersome, increasing operational difficulty and time costs. Summary of the Invention

[0004] The present invention aims to provide a method for extracting a tyrosinase-inhibiting peptide from Potamogeton crispus, addressing the cumbersome extraction process for the target peptide presented in the aforementioned background art. The present invention utilizes a combination of ultrafiltration and nanofiltration to concentrate and purify the tyrosinase-inhibiting peptide. This method is simple to operate, reducing operational complexity and time costs.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for extracting tyrosinase inhibitory peptides from Potamogeton crispus comprises the following steps:

[0007] S1. Cleaning the water chestnuts, drying them to a constant weight, and crushing them to obtain water chestnut powder;

[0008] S2, adding the water chestnut powder to the ethanol solution, heating and shaking for extraction, and then centrifuging to collect the supernatant to obtain the water chestnut extract;

[0009] S3, adjusting the pH of the extract of Potamogeton crispus to alkaline, then adding alkaline protease, heating and enzymolysis, inactivating the enzyme and centrifuging, collecting the supernatant to obtain an enzymolysis solution;

[0010] S4, subjecting the enzymatic hydrolyzate to ultrafiltration, collecting the permeate to obtain an ultrafiltration permeate;

[0011] S5, filtering the ultrafiltration permeate through nanofiltration, collecting the retentate, and obtaining a nanofiltration concentrate;

[0012] S6. Freeze-drying the nanofiltration concentrate to obtain a tyrosinase inhibitory peptide.

[0013] In the technical solution of the present invention, first clean the water chestnut to remove impurities, dry and crush to increase the contact area, which is conducive to subsequent extraction, then use ethanol solution to heat and vibrate to dissolve the target peptide, collect the supernatant containing the target peptide, rely on the specific catalysis of alkaline protease in an alkaline environment, hydrolyze the protein to release the target peptide, through the screening effect of the ultrafiltration membrane, intercept impurities such as macromolecular pigments, allow the target peptide to pass through the ultrafiltration membrane, finally utilize nanofiltration membrane to pass through small molecule impurities, intercept the target peptide, achieve the purification and concentration of the target peptide, and finally freeze-dry to remove moisture under low temperature vacuum, retain the activity and structural integrity of the peptide to the greatest extent, thereby achieving efficient extraction of tyrosinase inhibitory peptide from water chestnut. Compared with the traditional separation and purification by column chromatography, column chromatography requires multiple steps such as loading column, balancing column, loading, elution, and each round of chromatography requires careful operation and control, and the entire process is relatively cumbersome. Ultrafiltration and nanofiltration are relatively simple, and only the sample needs to be passed through the corresponding membrane assembly to achieve separation, with few operating steps, simple process, time saving and cost saving, and improved enterprise production efficiency.

[0014] Preferably, in step S2, the heating temperature is 40-50° C., and the oscillation extraction time is 1-2 h.

[0015] Preferably, in step S3, the pH is adjusted to 7-8.

[0016] Preferably, in step S3, the alkaline protease is Carlberg subtilisin.

[0017] Preferably, in step S3, the enzymatic hydrolysis temperature is 50-60° C., and the enzymatic hydrolysis time is 3-5 h.

[0018] Preferably, in the step S4, during the ultrafiltration treatment, the molecular weight cut-off of the ultrafiltration membrane is 2500-3000 Da.

[0019] Preferably, in step S5, the method for preparing the nanofiltration membrane used in the nanofiltration treatment process comprises the following steps:

[0020] S51, dissolving zinc nitrate hexahydrate in methanol, then adding 2-methylimidazole, stirring to react, centrifuging, washing, and drying to obtain a metal organic framework powder;

[0021] S52. Add the metal organic framework powder to deionized water, stir evenly to obtain a suspension, add graphene oxide to the suspension, ultrasonically oscillate, and then centrifuge, wash and dry to obtain a graphene oxide-metal organic framework composite.

[0022] S53, adding polyethyleneimine to water to dissolve, then adding graphene oxide-metal organic framework composite, and uniformly dispersing by ultrasonic oscillation to obtain an aqueous phase solution; adding trimesoyl chloride to n-hexane solvent, stirring and dissolving to obtain an oil phase solution;

[0023] S54, immersing the polysulfone-based membrane in an aqueous solution, taking it out and then immersing it in an oil solution to perform an interfacial polymerization reaction, and then performing a drying treatment to obtain a nanofiltration membrane.

[0024] In the technical solution of the present invention, by doping a polyamide nanofiltration membrane with a graphene oxide-metal organic framework composite, the graphene oxide-metal organic framework composite is evenly distributed inside and on the surface of the membrane during the membrane preparation process. On the one hand, the porous structure and large number of adsorption sites of the metal organic framework directly increase the membrane's adsorption and retention capacity for tyrosinase inhibitory peptides; on the other hand, the composite structure formed by graphene oxide and the metal organic framework constructs a tortuous and complex mass transfer channel within the membrane. When tyrosinase inhibitory peptide molecules in the solution attempt to pass through the nanofiltration membrane, they need to move along these tortuous channels, greatly increasing the molecules' residence time within the membrane and the length of the mass transfer path. This gives the peptide molecules more opportunities to bind to the adsorption sites within the membrane, while also enhancing the membrane's screening effect on molecules, thereby significantly improving the nanofiltration membrane's retention capacity for tyrosinase inhibitory peptides. In addition, the composite structure can also optimize the membrane's porosity and pore size distribution, achieving a higher retention efficiency while ensuring a certain membrane flux. Through the above effects, not only is the nanofiltration membrane able to concentrate and purify tyrosinase inhibitory peptides, but a higher yield of tyrosinase inhibitory peptides can also be achieved.

[0025] Preferably, in step S52, the mass ratio of the metal organic framework powder to the graphene oxide is 1:0.2-0.5.

[0026] Preferably, in step S53, the mass ratio of polyethyleneimine to graphene oxide-metal organic framework composite is 10:0.5-2.

[0027] In the technical solution of the present invention, in order to achieve a good yield of tyrosinase inhibitory peptides using a nanofiltration membrane, a sufficient amount of graphene oxide-metal organic framework complex must be doped into the nanofiltration membrane. Therefore, the present invention controls the mass ratio of polyethyleneimine to graphene oxide-metal organic framework complex to be less than 10 / 0.5. As the amount of graphene oxide-metal organic framework complex increases further, that is, when the mass ratio of polyethyleneimine to graphene oxide-metal organic framework complex is less than 10 / 2, the present invention team unexpectedly discovered that the flux of the nanofiltration membrane suddenly drops significantly, and the decrease in membrane flux will have a greater impact on the membrane filtration efficiency. This may be because the excessive graphene oxide-metal organic framework complex is excessively accumulated in the membrane, blocking the original nanoscale pore structure, causing the resistance of the solution to pass through the membrane to increase exponentially. Therefore, the present invention simultaneously controls the mass ratio of polyethyleneimine to graphene oxide-metal organic framework complex to be greater than 10:2.

[0028] Preferably, in step S54, the interfacial polymerization reaction time is 5-10 minutes.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention adopts ultrafiltration and nanofiltration to replace traditional column chromatography separation technology, which greatly simplifies the extraction process, reduces the operation steps, significantly saves time and costs, and improves enterprise production efficiency.

[0031] 2. Graphene oxide-metal organic framework composites are doped into the nanofiltration membrane. The porous structure and a large number of adsorption sites of the metal organic framework, combined with the composite structure formed by graphene oxide and the metal organic framework, significantly enhance the membrane's adsorption and retention capacity for tyrosinase inhibitory peptides, thereby greatly improving the yield of tyrosinase inhibitory peptides.

[0032] 3. Control the addition amount of graphene oxide-metal organic framework complex in the nanofiltration membrane to achieve a balance between the yield of tyrosinase inhibitory peptide and membrane flux. DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the implementation regulations described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0034] The main raw material specifications used in the specific examples are as follows:

[0035] Carlberg subtilisin, activity ≥2.4 U / mg; ultrafiltration membrane, polyethersulfone material, molecular weight cutoff 2500-3000 Da; graphene oxide, sheet diameter 100-500 nm, thickness 10-20 nm; polyethyleneimine, MW=600-1000 Da, branched type; polysulfone-based membrane, pore size 0.1 μm, thickness 150 μm.

[0036] Example 1

[0037] A method for extracting tyrosinase inhibitory peptides from Potamogeton crispus comprises the following steps:

[0038] Step S1: Take fresh water chestnuts and rinse them with deionized water to remove surface sediment, impurities, and attached microorganisms. Spread the washed water chestnuts flat on a tray and place them in a 60°C oven to dry to a constant weight. Use a grinder to pulverize the dried water chestnuts and pass them through a 60-mesh sieve to obtain water chestnut powder.

[0039] Step S2: Weigh 30 g of Potamogeton crispus powder and add 1500 mL of 70% ethanol solution. Place the mixture in a 45°C water bath and shake for 1.5 hours. After extraction, transfer the mixture to a centrifuge tube and centrifuge at 4000 rpm for 15 minutes. Aspirate the supernatant to obtain the Potamogeton crispus extract.

[0040] Step S3: Adjust the pH of the Potamogeton crispus extract to 7.8 with 1 mol / L sodium hydroxide solution. Add 1.5 g of Carlberg subtilisin to the extract and stir until completely dissolved. The extract is then placed in a 55°C water bath for enzymatic hydrolysis for 4.5 hours. After completion of the hydrolysis, heat the extract in a 95°C water bath for 10 minutes to inactivate the enzyme. After cooling to room temperature, centrifuge again (4000 rpm for 15 minutes) and collect the supernatant to obtain the hydrolyzate.

[0041] Step S4: Pour the enzymatic hydrolysate into the feed tank of the ultrafiltration device, start the device, adjust the pressure to 0.15 MPa, allow the enzymatic hydrolysate to pass through a 2500Da ultrafiltration membrane, collect the permeate, and obtain the ultrafiltration permeate.

[0042] Step S5: pour the ultrafiltration permeate into the feed tank of the nanofiltration device, start the device, adjust the pressure to 1.5 MPa, allow the solution to pass through the nanofiltration membrane, and collect the retentate, i.e., the nanofiltration concentrate.

[0043] Step S6: The nanofiltration concentrate was transferred to a freeze dryer, frozen at -40°C for 4 hours, and the vacuum pump was started to evacuate to a vacuum degree below 20 Pa and dried for 24 hours to obtain the tyrosinase inhibitory peptide powder.

[0044] Nanofiltration membrane preparation:

[0045] Step S51: Weigh 3.85 g of zinc nitrate hexahydrate and dissolve it in 600 mL of methanol. Stir until completely dissolved. Weigh 9.32 g of 2-methylimidazole and add it to the solution. Stir continuously and allow to react at room temperature for 24 hours. After the reaction, centrifuge the mixture (8000 rpm for 10 minutes), discard the supernatant, and wash the precipitate three times with methanol. Dry it at 60°C for 12 hours to obtain a metal-organic framework powder.

[0046] Step S52: 2.0 g of metal-organic framework powder was added to 200 mL of deionized water and stirred to form a suspension. 0.9 g of graphene oxide was then added to the suspension and ultrasonicated for 1 hour. After ultrasonication, the mixture was centrifuged (8000 rpm for 10 minutes), the supernatant discarded, and the precipitate washed three times with deionized water and dried at 60°C for 12 hours to obtain a graphene oxide-metal-organic framework composite.

[0047] Step S53: Weigh 10 g of polyethyleneimine and dissolve it in 800 mL of deionized water to obtain a polyethyleneimine solution. Weigh 1.5 g of the graphene oxide-metal organic framework complex and add it to the polyethyleneimine solution. Ultrasonicate for 30 minutes to obtain an aqueous solution. Weigh 5 g of trimesoyl chloride and dissolve it in 800 mL of n-hexane. Stir and dissolve to obtain an oily solution.

[0048] Step S54: Immerse the polysulfone-based membrane in the aqueous solution for 5 minutes, remove it, drain the surface liquid, and quickly immerse it in the oil solution for interfacial polymerization for 9 minutes. After the reaction is completed, place it in a 60°C oven for 2 hours to obtain a nanofiltration membrane.

[0049] Example 2

[0050] A method for extracting tyrosinase inhibitory peptides from Potamogeton crispus comprises the following steps:

[0051] Step S1: Take fresh water chestnuts and rinse them with deionized water to remove surface sediment, impurities, and attached microorganisms. Spread the washed water chestnuts flat on a tray and place them in a 60°C oven to dry to a constant weight. Use a grinder to pulverize the dried water chestnuts and pass them through a 60-mesh sieve to obtain water chestnut powder.

[0052] Step S2: Weigh 30 g of Potamogeton crispus powder and add 1500 mL of 70% ethanol solution. Place the mixture in a 45°C water bath and shake for 1.5 hours. After extraction, transfer the mixture to a centrifuge tube and centrifuge at 4000 rpm for 15 minutes. Aspirate the supernatant to obtain the Potamogeton crispus extract.

[0053] Step S3: Adjust the pH of the Potamogeton crispus extract to 7.5 with 1 mol / L sodium hydroxide solution. Add 1.5 g of Carlberg subtilisin to the extract and stir until completely dissolved. The extract is then placed in a 55°C water bath for enzymatic hydrolysis for 3.5 hours. After completion of the hydrolysis, heat the extract in a 95°C water bath for 10 minutes to inactivate the enzyme. After cooling to room temperature, centrifuge again (4000 rpm for 15 minutes) and collect the supernatant to obtain the hydrolyzate.

[0054] Step S4: Pour the enzymatic hydrolysate into the feed tank of the ultrafiltration device, start the device, adjust the pressure to 0.15 MPa, allow the enzymatic hydrolysate to pass through a 2500Da ultrafiltration membrane, collect the permeate, and obtain the ultrafiltration permeate.

[0055] Step S5: pour the ultrafiltration permeate into the feed tank of the nanofiltration device, start the device, adjust the pressure to 1.5 MPa, allow the solution to pass through the nanofiltration membrane, and collect the retentate, i.e., the nanofiltration concentrate.

[0056] Step S6: The nanofiltration concentrate was transferred to a freeze dryer, frozen at -40°C for 4 hours, and the vacuum pump was started to evacuate to a vacuum degree below 20 Pa and dried for 24 hours to obtain the tyrosinase inhibitory peptide powder.

[0057] Nanofiltration membrane preparation:

[0058] Step S51: Weigh 3.85 g of zinc nitrate hexahydrate and dissolve it in 600 mL of methanol. Stir until completely dissolved. Weigh 9.32 g of 2-methylimidazole and add it to the solution. Stir continuously and allow to react at room temperature for 24 hours. After the reaction, centrifuge the mixture (8000 rpm for 10 minutes), discard the supernatant, and wash the precipitate three times with methanol. Dry it at 60°C for 12 hours to obtain a metal-organic framework powder.

[0059] Step S52: 2.0 g of metal-organic framework powder was added to 200 mL of deionized water and stirred to form a suspension. 0.5 g of graphene oxide was added to the suspension and ultrasonicated for 1 hour. After ultrasonication, the mixture was centrifuged (8000 rpm for 10 minutes), the supernatant discarded, and the precipitate washed three times with deionized water and dried at 60°C for 12 hours to obtain a graphene oxide-metal-organic framework composite.

[0060] Step S53: Weigh 10 g of polyethyleneimine and dissolve it in 800 mL of deionized water to obtain a polyethyleneimine solution. Weigh 0.8 g of the graphene oxide-metal organic framework complex and add it to the polyethyleneimine solution. Ultrasonicate for 30 minutes to obtain an aqueous solution. Weigh 5 g of trimesoyl chloride and dissolve it in 800 mL of n-hexane. Stir and dissolve to obtain an oily solution.

[0061] Step S54: Immerse the polysulfone-based membrane in the aqueous solution for 5 minutes, remove it, drain the surface liquid, and quickly immerse it in the oil solution for interfacial polymerization for 6 minutes. After the reaction is completed, place it in a 60°C oven for 2 hours to obtain a nanofiltration membrane.

[0062] Example 3

[0063] A method for extracting tyrosinase inhibitory peptides from Potamogeton crispus comprises the following steps:

[0064] Step S1: Take fresh water chestnuts and rinse them with deionized water to remove surface sediment, impurities, and attached microorganisms. Spread the washed water chestnuts flat on a tray and place them in a 60°C oven to dry to a constant weight. Use a grinder to pulverize the dried water chestnuts and pass them through a 60-mesh sieve to obtain water chestnut powder.

[0065] Step S2: Weigh 30 g of Potamogeton crispus powder and add 1500 mL of 70% ethanol solution. Place the mixture in a 45°C water bath and shake for 1.5 hours. After extraction, transfer the mixture to a centrifuge tube and centrifuge at 4000 rpm for 15 minutes. Aspirate the supernatant to obtain the Potamogeton crispus extract.

[0066] Step S3: Adjust the pH of the Potamogeton crispus extract to 7.5 with 1 mol / L sodium hydroxide solution. Add 1.5 g of Carlberg subtilisin to the extract and stir until completely dissolved. The extract is then placed in a 55°C water bath for 4 hours for enzymatic hydrolysis. After hydrolysis is complete, heat the extract in a 95°C water bath for 10 minutes to inactivate the enzyme. After cooling to room temperature, centrifuge again (4000 rpm for 15 minutes) and collect the supernatant to obtain the hydrolyzate.

[0067] Step S4: Pour the enzymatic hydrolysate into the feed tank of the ultrafiltration device, start the device, adjust the pressure to 0.15 MPa, allow the enzymatic hydrolysate to pass through a 2500Da ultrafiltration membrane, collect the permeate, and obtain the ultrafiltration permeate.

[0068] Step S5: pour the ultrafiltration permeate into the feed tank of the nanofiltration device, start the device, adjust the pressure to 1.5 MPa, allow the solution to pass through the nanofiltration membrane, and collect the retentate, i.e., the nanofiltration concentrate.

[0069] Step S6: The nanofiltration concentrate was transferred to a freeze dryer, frozen at -40°C for 4 hours, and the vacuum pump was started to evacuate to a vacuum degree below 20 Pa and dried for 24 hours to obtain the tyrosinase inhibitory peptide powder.

[0070] Nanofiltration membrane preparation:

[0071] Step S51: Weigh 3.85 g of zinc nitrate hexahydrate and dissolve it in 600 mL of methanol. Stir until completely dissolved. Weigh 9.32 g of 2-methylimidazole and add it to the solution. Stir continuously and allow to react at room temperature for 24 hours. After the reaction, centrifuge the mixture (8000 rpm for 10 minutes), discard the supernatant, and wash the precipitate three times with methanol. Dry it at 60°C for 12 hours to obtain a metal-organic framework powder.

[0072] Step S52: 2.0 g of metal-organic framework powder was added to 200 mL of deionized water and stirred to form a suspension. 0.7 g of graphene oxide was added to the suspension and ultrasonicated for 1 hour. After ultrasonication, the mixture was centrifuged (8000 rpm for 10 minutes), the supernatant discarded, and the precipitate washed three times with deionized water and dried at 60°C for 12 hours to obtain a graphene oxide-metal-organic framework composite.

[0073] Step S53: Weigh 10 g of polyethyleneimine and dissolve it in 800 mL of deionized water to obtain a polyethyleneimine solution. Weigh 1.0 g of the graphene oxide-metal organic framework complex and add it to the polyethyleneimine solution. Ultrasonicate for 30 minutes to obtain an aqueous solution. Weigh 5 g of trimesoyl chloride and dissolve it in 800 mL of n-hexane. Stir and dissolve to obtain an oily solution.

[0074] Step S54: Immerse the polysulfone-based membrane in the aqueous solution for 5 minutes, remove it, drain the surface liquid, and quickly immerse it in the oil solution for interfacial polymerization for 8 minutes. After the reaction is completed, place it in a 60°C oven to dry for 2 hours to obtain a nanofiltration membrane.

[0075] Example 4

[0076] A method for extracting tyrosinase inhibitory peptides from Potamogeton crispus comprises the following steps:

[0077] Step S1: Take fresh water chestnuts and rinse them with deionized water to remove surface sediment, impurities, and attached microorganisms. Spread the washed water chestnuts flat on a tray and place them in a 60°C oven to dry to a constant weight. Use a grinder to pulverize the dried water chestnuts and pass them through a 60-mesh sieve to obtain water chestnut powder.

[0078] Step S2: Weigh 30 g of Potamogeton crispus powder and add 1500 mL of 70% ethanol solution. Place the mixture in a 50°C water bath and shake for 2 hours. After extraction, transfer the mixture to a centrifuge tube and centrifuge at 4000 rpm for 15 minutes. Aspirate the supernatant to obtain the Potamogeton crispus extract.

[0079] Step S3: Adjust the pH of the Potamogeton crispus extract to 8 with 1 mol / L sodium hydroxide solution. Add 1.5 g of Carlberg subtilisin to the extract and stir until completely dissolved. The extract is then placed in a 60°C water bath for enzymatic hydrolysis for 5 h. After completion of the hydrolysis, heat the extract in a 95°C water bath for 10 min to inactivate the enzyme. After cooling to room temperature, centrifuge again (4000 rpm for 15 min), and collect the supernatant to obtain the hydrolyzate.

[0080] Step S4: Pour the enzymatic hydrolysate into the feed tank of the ultrafiltration device, start the device, adjust the pressure to 0.15 MPa, allow the enzymatic hydrolysate to pass through a 2500Da ultrafiltration membrane, collect the permeate, and obtain the ultrafiltration permeate.

[0081] Step S5: pour the ultrafiltration permeate into the feed tank of the nanofiltration device, start the device, adjust the pressure to 1.5 MPa, allow the solution to pass through the nanofiltration membrane, and collect the retentate, i.e., the nanofiltration concentrate.

[0082] Step S6: The nanofiltration concentrate was transferred to a freeze dryer, frozen at -40°C for 4 hours, and the vacuum pump was started to evacuate to a vacuum degree below 20 Pa and dried for 24 hours to obtain the tyrosinase inhibitory peptide powder.

[0083] Nanofiltration membrane preparation:

[0084] Step S51: Weigh 3.85 g of zinc nitrate hexahydrate and dissolve it in 600 mL of methanol. Stir until completely dissolved. Weigh 9.32 g of 2-methylimidazole and add it to the solution. Stir continuously and allow to react at room temperature for 24 hours. After the reaction, centrifuge the mixture (8000 rpm for 10 minutes), discard the supernatant, and wash the precipitate three times with methanol. Dry it at 60°C for 12 hours to obtain a metal-organic framework powder.

[0085] Step S52: 2.0 g of metal-organic framework powder was added to 200 mL of deionized water and stirred to form a suspension. 1.0 g of graphene oxide was added to the suspension and ultrasonicated for 1 hour. After ultrasonication, the mixture was centrifuged (8000 rpm for 10 minutes), the supernatant discarded, and the precipitate washed three times with deionized water and dried at 60°C for 12 hours to obtain a graphene oxide-metal-organic framework composite.

[0086] Step S53: Weigh 10 g of polyethyleneimine and dissolve it in 800 mL of deionized water to obtain a polyethyleneimine solution. Weigh 2.0 g of the graphene oxide-metal organic framework complex and add it to the polyethyleneimine solution. Ultrasonicate for 30 minutes to obtain an aqueous solution. Weigh 5 g of trimesoyl chloride and dissolve it in 800 mL of n-hexane. Stir and dissolve to obtain an oily solution.

[0087] Step S54: Immerse the polysulfone-based membrane in the aqueous solution for 5 minutes, remove it, drain the surface liquid, and quickly immerse it in the oil solution for interfacial polymerization for 10 minutes. After the reaction is completed, place it in a 60°C oven for 2 hours to obtain a nanofiltration membrane.

[0088] Example 5

[0089] A method for extracting tyrosinase inhibitory peptides from Potamogeton crispus comprises the following steps:

[0090] Step S1: Take fresh water chestnuts and rinse them with deionized water to remove surface sediment, impurities, and attached microorganisms. Spread the washed water chestnuts flat on a tray and place them in a 60°C oven to dry to a constant weight. Use a grinder to pulverize the dried water chestnuts and pass them through a 60-mesh sieve to obtain water chestnut powder.

[0091] Step S2: Weigh 30 g of Potamogeton crispus powder and add 1500 mL of 70% ethanol solution. Place the mixture in a 40°C water bath and shake for 1 hour. After extraction, transfer the mixture to a centrifuge tube and centrifuge at 4000 rpm for 15 minutes. Aspirate the supernatant to obtain the Potamogeton crispus extract.

[0092] Step S3: Adjust the pH of the Potamogeton crispus extract to 7 with 1 mol / L sodium hydroxide solution. Add 1.5 g of Carlberg subtilisin to the extract and stir until completely dissolved. The extract is then placed in a 50°C water bath for enzymatic hydrolysis for 3 h. After completion of the hydrolysis, heat the extract in a 95°C water bath for 10 min to inactivate the enzyme. After cooling to room temperature, centrifuge again (4000 rpm for 15 min), and collect the supernatant to obtain the hydrolyzate.

[0093] Step S4: Pour the enzymatic hydrolysate into the feed tank of the ultrafiltration device, start the device, adjust the pressure to 0.15 MPa, allow the enzymatic hydrolysate to pass through a 3000 Da ultrafiltration membrane, collect the permeate, and obtain the ultrafiltration permeate.

[0094] Step S5: pour the ultrafiltration permeate into the feed tank of the nanofiltration device, start the device, adjust the pressure to 1.5 MPa, allow the solution to pass through the nanofiltration membrane, and collect the retentate, i.e., the nanofiltration concentrate.

[0095] Step S6: The nanofiltration concentrate was transferred to a freeze dryer, frozen at -40°C for 4 hours, and the vacuum pump was started to evacuate to a vacuum degree below 20 Pa and dried for 24 hours to obtain the tyrosinase inhibitory peptide powder.

[0096] Nanofiltration membrane preparation:

[0097] Step S51: Weigh 3.85 g of zinc nitrate hexahydrate and dissolve it in 600 mL of methanol. Stir until completely dissolved. Weigh 9.32 g of 2-methylimidazole and add it to the solution. Stir continuously and allow to react at room temperature for 24 hours. After the reaction, centrifuge the mixture (8000 rpm for 10 minutes), discard the supernatant, and wash the precipitate three times with methanol. Dry it at 60°C for 12 hours to obtain a metal-organic framework powder.

[0098] Step S52: 2.0 g of metal-organic framework powder was added to 200 mL of deionized water and stirred to form a suspension. 0.4 g of graphene oxide was then added to the suspension and ultrasonicated for 1 hour. After ultrasonication, the mixture was centrifuged (8000 rpm for 10 minutes), the supernatant discarded, and the precipitate washed three times with deionized water and dried at 60°C for 12 hours to obtain a graphene oxide-metal-organic framework composite.

[0099] Step S53: Weigh 10 g of polyethyleneimine and dissolve it in 800 mL of deionized water to obtain a polyethyleneimine solution. Weigh 0.5 g of the graphene oxide-metal organic framework complex and add it to the polyethyleneimine solution. Ultrasonicate for 30 minutes to obtain an aqueous solution. Weigh 5 g of trimesoyl chloride and dissolve it in 800 mL of n-hexane. Stir and dissolve to obtain an oily solution.

[0100] Step S54: Immerse the polysulfone-based membrane in the aqueous solution for 5 minutes, remove it, drain the surface liquid, and quickly immerse it in the oil solution for interfacial polymerization for 5 minutes. After the reaction is completed, place it in a 60°C oven for 2 hours to obtain a nanofiltration membrane.

[0101] Comparative Example 1

[0102] The difference between Comparative Example 1 and Example 1 is that no graphene oxide-metal organic framework composite is added to the aqueous solution during the preparation of the nanofiltration membrane, and the other steps are the same.

[0103] Comparative Example 2

[0104] The difference between Comparative Example 2 and Example 1 is that the graphene oxide-metal organic framework composite added to the aqueous solution in the nanofiltration membrane preparation process is replaced by a metal organic framework, and the remaining steps are the same.

[0105] Comparative Example 3

[0106] The difference between Comparative Example 3 and Example 4 is that the mass ratio of polyethyleneimine to graphene oxide-metal organic framework composite is 10:3, and the other steps are the same.

[0107] Comparative Example 4

[0108] The difference between Comparative Example 4 and Example 4 is that the mass ratio of polyethyleneimine to graphene oxide-metal organic framework composite is 10:4, and the other steps are the same.

[0109] Performance testing:

[0110] 1. Tyrosinase inhibition activity test:

[0111] Tyrosinase inhibitory activity was determined using the DOPA (L-3,4-dihydroxyphenylalanine) colorimetric assay. 100 μL of a 0.5 mg / mL tyrosinase inhibitory peptide sample solution was mixed with 200 μL of a 0.2 mg / mL tyrosinase solution. After preincubation in a 37°C water bath for 10 minutes, the reaction was initiated by adding 200 μL of a 2.5 mM DOPA solution. Phosphate buffer (pH 6.8) was used as a blank control. The absorbance was measured every 30 seconds at a wavelength of 475 nm using a microplate reader, and the absorbance change was recorded for a total of 5 minutes. Kojic acid was used as a positive control, and the inhibition rate was calculated according to the formula:

[0112] Inhibition rate (%) = [1-(A sample-A sample blank) / (A control-A control blank)] × 100%.

[0113] Where A sample is the absorbance of the sample reacting with tyrosinase and DOPA, A sample blank is the absorbance of the sample reacting with DOPA (without tyrosinase), A control is the absorbance of the phosphate buffer reacting with tyrosinase and DOPA, and A control blank is the absorbance of the phosphate buffer reacting with DOPA (without tyrosinase). The results are shown in Table 1.

[0114] 2. Tyrosinase inhibitory peptide yield test

[0115] The tyrosinase inhibitory peptide content before and after extraction was determined using the Folin-phenol method, based on the initial mass of the water chestnut raw material. A certain amount of water chestnut raw material was weighed, and tyrosinase inhibitory peptide powder was prepared according to the methods of the Examples and Comparative Examples. Appropriate amounts of water chestnut raw material and extracted peptide powder were dissolved in deionized water and brought to volume. The protein content was determined using the Folin-phenol method, and the tyrosinase inhibitory peptide content was converted based on the peptide purity (determined by high-performance liquid chromatography).

[0116] Yield (%) = (mass of tyrosinase inhibitory peptide extracted / mass of tyrosinase inhibitory peptide theoretically contained in the raw material) × 100%.

[0117] The theoretical mass of tyrosinase inhibitory peptides in the raw materials was estimated based on the protein content of the raw materials and the proportion of peptides in the protein. The results are shown in Table 1.

[0118] 3. Nanofiltration membrane retention rate test

[0119] A solution containing 500 mg / L of tyrosinase inhibitory peptide was prepared as the feed solution. Nanofiltration experiments were performed at an operating pressure of 0.8 MPa. The retentate and permeate were collected. The concentrations of tyrosinase inhibitory peptide in the feed solution, retentate, and permeate were determined by high-performance liquid chromatography (HPLC). Retention rate (%) = [1 - (C permeate / C feed)] × 100%, where C permeate and C feed represent the concentrations of tyrosinase inhibitory peptide in the permeate and feed solutions (mg / L), respectively. The results are shown in Table 1.

[0120] 4. Nanofiltration membrane flux test

[0121] Using pure water as the test solution, the nanofiltration membrane was installed in a filtration device at room temperature (25°C). The operating pressure was adjusted to 0.8 MPa. After 15 minutes of stable operation, the volume of water permeating the membrane over a 10-minute period was collected using a graduated cylinder. The membrane flux was calculated using the formula: Membrane flux (L / (m²・h)) = V / (A × t). Where V is the collected water volume (L), A is the effective filtration area of the membrane (m²), and t is the collection time (h). Each experiment was repeated three times, and the average value was calculated. The results are shown in Table 1.

[0122] Table 1:

[0123] Enzyme inhibition rate (%) Yield (%) Retention rate (%) Membrane flux (L / (m²・h)) Example 1 87.3 74.6 98.2 21.6 Example 2 86.6 73.5 97.5 23.5 Example 3 87.0 74.2 97.8 22.9 Example 4 87.6 75.4 98.3 19.2 Example 5 86.1 74.0 97.1 25.7 Comparative Example 1 65.3 32.6 51.3 32.1 Comparative Example 2 72.6 53.4 72.5 24.6 Comparative Example 3 87.8 75.7 98.5 10.3 Comparative Example 4 87.9 78.0 98.8 8.7

[0124] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting tyrosinase inhibitory peptides from Potamogeton crispus, characterized in that: The following steps are involved: S1. Cleaning the water chestnuts, drying them to a constant weight, and crushing them to obtain water chestnut powder; S2, adding the water chestnut powder to the ethanol solution, heating and shaking for extraction, and then centrifuging to collect the supernatant to obtain the water chestnut extract; S3, adjusting the pH of the extract of Potamogeton crispus to alkaline, then adding alkaline protease, heating and enzymolysis, inactivating the enzyme and centrifuging, collecting the supernatant to obtain an enzymolysis solution; S4, subjecting the enzymatic hydrolyzate to ultrafiltration, collecting the permeate to obtain an ultrafiltration permeate; S5, filtering the ultrafiltration permeate through nanofiltration, collecting the retentate, and obtaining a nanofiltration concentrate; S6. Freeze-drying the nanofiltration concentrate to obtain a tyrosinase inhibitory peptide.

2. The method for extracting tyrosinase inhibitory peptide from Potamogeton crispus according to claim 1, characterized in that: In step S2, the heating temperature is 40-50° C., and the oscillation extraction time is 1-2 h.

3. The method for extracting tyrosinase inhibitory peptide from Potamogeton crispus according to claim 1, characterized in that: In step S3, the pH is adjusted to 7-8.

4. The method for extracting tyrosinase inhibitory peptide from Potamogeton crispus according to claim 1, characterized in that: In step S3, the alkaline protease is Carlberg subtilisin.

5. The method for extracting tyrosinase inhibitory peptide from Potamogeton crispus according to claim 1, characterized in that: In step S3, the enzymolysis temperature is 50-60° C., and the enzymolysis time is 3-5 h.

6. The method for extracting tyrosinase inhibitory peptide from Potamogeton crispus according to claim 1, characterized in that: In the step S4, during the ultrafiltration treatment, the molecular weight cut-off of the ultrafiltration membrane is 2500-3000 Da.

7. The method for extracting tyrosinase inhibitory peptide from Potamogeton crispus according to claim 1, characterized in that: In step S5, the method for preparing the nanofiltration membrane used in the nanofiltration treatment process includes the following steps: S51, dissolving zinc nitrate hexahydrate in methanol, then adding 2-methylimidazole, stirring to react, centrifuging, washing, and drying to obtain a metal organic framework powder; S52. Add the metal organic framework powder to deionized water, stir evenly to obtain a suspension, add graphene oxide to the suspension, ultrasonically oscillate, and then centrifuge, wash and dry to obtain a graphene oxide-metal organic framework composite. S53, adding polyethyleneimine to water to dissolve, then adding graphene oxide-metal organic framework composite, and uniformly dispersing by ultrasonic oscillation to obtain an aqueous phase solution; adding trimesoyl chloride to n-hexane solvent, stirring and dissolving to obtain an oil phase solution; S54, immersing the polysulfone-based membrane in an aqueous solution, taking it out and then immersing it in an oil solution to perform an interfacial polymerization reaction, and then performing a drying treatment to obtain a nanofiltration membrane.

8. The method for extracting tyrosinase inhibitory peptide from Potamogeton crispus according to claim 7, characterized in that: In step S52, the mass ratio of the metal organic framework powder to the graphene oxide is 1:0.2-0.

5.

9. The method for extracting tyrosinase inhibitory peptide from Potamogeton crispus according to claim 7, characterized in that: In the step S53, the mass ratio of polyethyleneimine to graphene oxide-metal organic framework composite is 10:0.5-2.

10. The method for extracting tyrosinase inhibitory peptide from Potamogeton crispus according to claim 7, characterized in that: In step S54, the interfacial polymerization reaction time is 5-10 minutes.