Preparation method and application of cottonseed protein hydrolysate-monoalkynyl curcumin compound

Abstract

The invention discloses a preparation method and application of a cottonseed protein hydrolysate-monoalkynyl curcumin compound. The preparation method comprises the following steps: extracting a cottonseed protein isolate from degreased cottonseed meal, and purifying the cottonseed protein isolate through isoelectric precipitation; activating and modifying the purified cottonseed protein isolate by using an activating agent and a stabilizing agent to obtain an intermediate; carrying out azidation reaction on an azide and the intermediate to obtain a modified cottonseed protein hydrolysate; the modified cottonseed protein hydrolysate and mono-alkynyl curcumin are subjected to a Huisgen cyclization addition reaction, a triazole bond is formed, and the cottonseed protein hydrolysate-mono-alkynyl curcumin compound is obtained. The cottonseed protein hydrolysate-monoalkynyl curcumin compound is driven by utilizing the optical activity of monoalkynyl curcumin, so that the limitation that the cottonseed protein hydrolysate is used as an alternating motor to deliver tiny objects is solved, and the delivery stability and flexibility are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological protein complexes, and more specifically, relates to a method for preparing a cottonseed protein hydrolysate-monoacetylenic curcumin complex. Background Art

[0002] Artificial systems that mimic cellular functions have attracted considerable attention due to their potential to reveal the mysteries of life and advance materials science and nanotechnology. Miniaturizing macroscopic machines to the micrometer and nanometer scales is a major goal, aiming to explore the microscopic world and ultimately perform a variety of complex tasks. The development of micro- and nanomotors is significant because they can mimic the motility of biological cells such as bacteria. Due to their small size, autonomous movement, and navigation capabilities, these micro- and nanomotors have broad application prospects in fields such as biomedicine, environmental remediation, and materials science. In biomedicine, their applications include active targeted delivery, detoxification, minimally invasive diagnostics, and nanosurgery.

[0003] Colloidal motors are microscopic motors that can convert diverse forms of energy into motion, are precisely controllable, and require no wires or bulky power supplies at the micro / nanoscale, where inertial and viscous forces completely outweigh the force of gravity. Colloidal motors (also known as micro / nanomotors) are micro- and nanoscale colloidal particles (typically nanometer to micrometer in size) that can convert ambient energy, such as light, chemical, electrical, or magnetic, into their own mechanical motion. Their core functions are to enable precise, directional, transient transport of tiny objects and efficient manipulation of matter in the microscopic world without external mechanical agitation. However, the widespread application of synthetic colloidal motors is currently hampered by limitations such as toxicity, low biodegradability, and complex synthesis processes. These challenges highlight the urgent need for alternative materials that are not only effective, but also environmentally friendly and biocompatible. Although emulsions are one of the less extensively studied colloidal systems, they hold intriguing potential due to their unique properties. Droplet-based motors, with their remarkable manipulation capabilities and inert composition, are particularly well-suited for use in harsh biological environments.

[0004] An emulsion is a heterogeneous system consisting of two immiscible liquids, one of which is dispersed in the other in the form of micron or submicron-sized droplets. Emulsion droplets, due to their inherent encapsulation, miniaturization, and compartmentalization capabilities, provide a powerful biomimetic approach for constructing cell-like structures and exploring fundamental biological processes in controlled synthetic environments. The inherent separation between the two phases can serve as the basis for creating internal gradients that can drive motion. In addition, the interface between the oil and water phases in the emulsion is energy-rich and can be manipulated to generate force or torque. Wang et al. synthesized an emulsion soft motor by thermal stimulation (see Wang H, Liang Y, Gao W, et al. Emulsion Hydrogel Soft Motor Actuated by Thermal Stimulation [J]. ACS Applied Materials&Interfaces, 2017, 9(49): 43211-43219). They used a simple oil-in-water emulsion template method to synthesize a motor that can achieve self-propulsion under stimulation and organic solvents. By integrating graphene oxide and enrofloxacin fungicide, smart targeted cargo transportation and delivery were achieved. This micro soft motor not only has attractive self-propulsion properties, but also has precise movement control and strong integration capabilities. Emulsion droplets can be used to coat particles for controlled release of drugs or pigments, or to encapsulate reactive chemicals, especially when responsive polymers are used as surfactants to stabilize the droplets. Rey et al. prepared a stimuli-responsive emulsion (see Rey M, Kolker J, Richards JA, et al. Interactions between interfaces dictate stimuli-responsive emulsion behavior [J]. Nature Communications, 2023, 14 (1): 6723), which has the dual advantages of long-term storage and controlled release triggered by external signals (such as pH or temperature changes). This emulsion is thermoresponsive, and its response characteristics depend on the structure and interface morphology of the stabilized microgel. Current trends indicate the emergence of compartmentalized emulsions, whereby small droplets of distinct phases can form within dispersed droplets, representing intracellular organelles. However, emulsions are thermodynamically unstable, requiring significant amounts of energy and active emulsifiers to maintain their stability. Synthetic surfactants are small molecules composed of hydrophobic and hydrophilic moieties; these systems possess fluidity and are capable of stabilizing emulsions. However, the direct or indirect use of synthetic surfactants can be toxic and environmentally unfriendly. Due to the advantages of natural materials, research interest in using proteins for emulsion stabilization is growing. Plant proteins are effective emulsifiers due to their biocompatibility, renewability, and environmental friendliness, as well as their amphiphilic and surface-active properties. However, proteins are large molecules with complex structures. They diffuse slowly to the oil-water interface, making them difficult to manage and manipulate, and are sensitive and unstable to various environmental factors in industrial and biological systems, limiting their application. Modification of proteins through various techniques allows for hydrolysis and improved properties, addressing these limitations and making them versatile components in diverse systems. Protein hydrolysates, due to their amphiphilic and tunable properties, are versatile bio-based emulsifiers.

[0005] Plant-based protein hydrolysates are a promising material for constructing colloidal motors. This is particularly true for agricultural and industrial waste products, such as cottonseed and soybeans, which are abundant and underutilized during agricultural processing. China boasts abundant cotton resources and is one of the world's largest cotton producers. Cottonseed meal, a byproduct of cotton processing, has a protein content of 40–50%, high nutritional value, and excellent quality. However, due to its complex folding structure and large molecular weight, the application of cottonseed protein isolate (CPI) is limited by its low water solubility and reactivity. Chemical modification by introducing various functional groups is preferred over other methods due to its low cost, minimal equipment requirements, and rapid reaction speed. Prior studies have successfully synthesized sulfonated and deamidated cottonseed protein hydrolysates using CPI as a platform. These emulsifiers significantly unfold their molecular chains, improving amphiphilicity, flexibility, and surface hydrophobicity. They exhibit excellent solubility in aqueous phases. These synthetic emulsifiers absorbed more protein at the droplet interface, resulting in improved emulsification, smaller droplet size, and higher zeta potential. They produced more uniform nanoemulsions with enhanced long-term stability. They exhibited excellent stability and uniformity under a variety of environmental stresses, including pH, ionic strength, and thermal conditions. Curcumin, a natural polyphenol extracted from the rhizome of turmeric, has attracted considerable attention for its photoactivity, antioxidant properties, and biocompatibility. Curcumin can undergo photoisomerization upon illumination and generate reactive oxygen species, making it an attractive candidate for applications in light-driven colloidal motors. Summary of the Invention

[0006] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0007] In order to achieve these objects and other advantages according to the present invention, a method for preparing a cottonseed protein hydrolysate-monoacetylenic curcumin complex is provided, comprising the following steps: Step 1, extracting cottonseed protein isolate from defatted cottonseed meal, and purifying the cottonseed protein isolate by isoelectric precipitation; Step 2: activating and modifying the purified cottonseed protein isolate using an activator and a stabilizer to obtain an intermediate; and performing an azidation reaction on the intermediate using an azide compound to obtain a modified cottonseed protein hydrolyzate; Step 3: The modified cottonseed protein hydrolysate and monoacetylenic curcumin undergo a Huisgen cycloaddition reaction to form a triazole bond, thereby obtaining a cottonseed protein hydrolysate-monoacetylenic curcumin complex.

[0008] Preferably, the specific method in step 1 includes: S11. Add defatted cottonseed meal to a 0.1 M KOH solution, stir at 50-60° C. for 30-60 min, incubate on a shaker for 20-40 min, and centrifuge at 2470×g for 20-30 min at 4° C. to obtain a cottonseed protein isolate; the mass-to-volume ratio of defatted cottonseed meal to KOH solution is 1 mg:10-15 mL. S12. Dilute the cottonseed protein isolate with 10 times the volume of deionized water, continue stirring and add 0.1M HCl dropwise until the pH of the dilution is 4-4.5. After standing for 2 hours, centrifuge at 2470×g for 20-30 minutes at 4°C. Collect the precipitate and wash it with deionized water until it is neutral. Freeze-dry to obtain the purified cottonseed protein isolate.

[0009] Preferably, in step 2, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide, the stabilizer is N-hydroxysulfosuccinimide, and the azide compound is one of ethyl azidoacetate, ethyl propyl azidoacetate, and ethyl azidopropionate.

[0010] Preferably, the specific method of step 2 includes: S21. Dissolve the purified cottonseed protein isolate in 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.0, sonicate at 30-50 kHz for 10-20 min, and cool to 4° C. in an ice bath; add N-hydroxysulfosuccinimide, and while stirring, dropwise add 1-ethyl-(3-dimethylaminopropyl)carbodiimide; control the system temperature to ≤8° C., stir for 20-40 min, raise the temperature to 30-40° C., and continue stirring for 1-3 h to obtain an intermediate solution; S22. Cool the intermediate solution to 0°C, add ethyl azidoacetate and triethylamine in sequence, pass argon, and seal the reactor to avoid light; raise the temperature to 25°C and keep warm for 1-3 hours, raise the temperature to 35-45°C and keep warm for 8-16 hours, add 1M glycine buffer; dialysis purification, freeze-drying, and obtain modified cottonseed protein hydrolysate.

[0011] Preferably, in S21, the usage ratio of the purified cottonseed protein isolate, 2-(N-morpholino)ethanesulfonic acid buffer, N-hydroxysulfosuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 5-15 g: 200-400 mL: 0.012-0.015 mol: 0.008-0.01 mol.

[0012] Preferably, in S22, the usage ratio of ethyl azidoacetate, triethylamine, and glycine buffer is 2.0-4.5 mL: 0.5-2.5 mL: 1-15 mL.

[0013] Preferably, the specific method of step three includes: S31, dissolve CuSO4·5H2O and tris(benzyltriazolylmethyl)amine in PBS buffer to obtain Cu 2+ -TBTA composite solution; mixing the monoacetylenic curcumin solution with the modified cottonseed protein hydrolysate to obtain a mixture solution; S32, Cu 2+ -TBTA complex solution was added to the mixed solution, and then L-sodium ascorbate solution was added. After mixing evenly, argon was passed through to deoxygenate for 5 minutes. After sealing, the mixture was shaken in the dark at room temperature of 25°C to 30°C for 3 to 6 hours. S33. Add ascorbic acid in an amount of 5% by volume of the reaction system and stir for 10-20 minutes; dialysis the product using a 3 kDa MWCO dialysis bag for purification, and freeze-dry the purified product to obtain a cottonseed protein hydrolysate-monoacetylenic curcumin complex.

[0014] Preferably, in the S32, Cu 2+ The volume ratio of the L-TBTA composite solution, the mixed solution, and the L-sodium ascorbate solution is 2~5:10:0.1~1.

[0015] Preferably, the step three further comprises: ultrasonically dispersing the cottonseed protein hydrolysate-monoacetylenic curcumin complex in an ethanol solution, adding ammonia water, standing for 1 to 3 hours, and then dropping a tetraethyl orthosilicate solution at a rate of 0.1 mL / min. After the dropwise addition is complete, magnetic stirring is performed at 300 to 600 rpm and the temperature is raised to 30 to 40° C., reacting for 6 to 12 hours, standing for 6 to 12 hours, centrifuging, discarding the supernatant, washing the precipitate, and freeze-drying to obtain a silica-coated cottonseed protein hydrolysate-monoacetylenic curcumin complex; wherein the amount ratio of the cottonseed protein hydrolysate-monoacetylenic curcumin complex, ethanol solution, ammonia water and tetraethyl orthosilicate is 1 g:10 to 20 mL:0.5 to 1 mL:1 to 3 mL; The silica-coated cottonseed protein hydrolysate-monoacetylenic curcumin complex was placed on a glass slide, which was then placed in a sputtering coating machine. Platinum metal was used as the sputtering target. -3 Under vacuum conditions of 500 Pa, the voltage was set to 500 V and the current was 5 mA, platinum deposition was performed, and the sputtering time was 5 to 10 minutes. The glass slide was removed to obtain a cottonseed protein hydrolysate-monoacetylenic curcumin complex with Pt and SiO2 coated on one side.

[0016] The invention discloses an application of a cottonseed protein hydrolysate-monoacetylenic curcumin complex. The cottonseed protein hydrolysate-monoacetylenic curcumin complex is used as a colloidal motor to achieve transient transmission of tiny objects, including adenosine triphosphate, antibiotic drugs, organic pollutants, organic dye molecules, heavy metal ions, enzymes, antibodies, and organelles.

[0017] The present invention includes at least the following beneficial effects: Cottonseed protein isolate is extracted from defatted cottonseed meal using a hot alkaline treatment method. After purification, the cottonseed protein isolate is sequentially activated and azidated. Finally, photoactive monoacetylenic curcumin is coupled to the cottonseed protein isolate via click chemistry to produce a cottonseed protein hydrolysate-monoacetylenic curcumin complex. The present invention utilizes the photoactivity of monoacetylenic curcumin to drive the cottonseed protein hydrolysate-monoacetylenic curcumin complex, addressing the limitations of cottonseed protein hydrolysate as an alternating motor for delivering small objects and improving the stability and flexibility of delivery. Furthermore, the resulting cottonseed protein hydrolysate-monoacetylenic curcumin complex exhibits extremely high antioxidant activity and good cell compatibility.

[0018] The preparation method of the present invention hardly uses toxic organic solvents, has extremely high reaction specificity and reaction efficiency, high product yield, no harmful by-products, and is green and safe.

[0019] The present invention prepares a cottonseed protein hydrolysate-monoacetylenic curcumin complex, and further coats a mesoporous silica film layer and a unilaterally deposited platinum layer on the surface of the cottonseed protein hydrolysate-monoacetylenic curcumin complex. The mesoporous silica film layer forms a protective layer for the cottonseed protein hydrolysate-monoacetylenic curcumin complex, slowing down the photodegradation efficiency of the monoacetylenic curcumin. Simultaneously, the mesoporous silica film layer increases the capture surface area of ​​the cottonseed protein hydrolysate-monoacetylenic curcumin complex, thereby increasing the drug loading capacity for antibiotic drugs and the adsorption capacity of heavy metals. The unilaterally deposited platinum layer improves the light capture capability of the cottonseed protein hydrolysate-monoacetylenic curcumin complex and increases the photothermal temperature rise rate. Simultaneously, under light conditions, a temperature difference is formed between the side where the platinum is deposited and the side where the platinum layer is not deposited, thereby increasing the inverse concentration movement rate of the cottonseed protein hydrolysate-monoacetylenic curcumin complex, thereby easily achieving rapid transport of tiny objects.

[0020] Other advantages, objectives and features of the present invention will be reflected in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0021] The present invention is described in further detail below so that those skilled in the art can implement the invention with reference to the description.

[0022] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof. Example 1 A method for preparing a cottonseed protein hydrolysate-monoacetylenic curcumin complex comprises the following steps: Step 1: extracting cottonseed protein isolate from defatted cottonseed meal and purifying the cottonseed protein isolate by isoelectric precipitation, specifically comprising: S11, adding 20 mg of defatted cottonseed meal to 250 mL of 0.1 M KOH solution, stirring at 50°C for 30 min, incubating on a shaker for 30 min, and centrifuging at 2470 × g for 30 min at 4°C, collecting the supernatant to obtain cottonseed protein isolate; S12, diluting the cottonseed protein isolate with 10 times the volume of deionized water, continuously stirring and adding 0.1 M HCl dropwise until the pH of the dilution reaches 4.5, standing for 2 hours, and then centrifuging at 2470×g for 30 minutes at 4° C., collecting the precipitate, washing with deionized water until neutral, and freeze-drying at -40° C. for 2 hours to obtain the purified cottonseed protein isolate; Step 2: activating and modifying the purified cottonseed protein isolate using an activator and a stabilizer to obtain an intermediate; and performing an azidation reaction on the intermediate using an azide compound to obtain a modified cottonseed protein hydrolysate, specifically comprising: S21. Dissolve 5.0 g of purified cottonseed protein isolate in 300.0 mL of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0), sonicate at 50 kHz for 10 min, and cool to 4°C in an ice bath; add 2.6 g of N-hydroxysulfosuccinimide (12 mmol), and while stirring, dropwise add 1.24 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (8 mmol). Control the system temperature to ≤ 8°C, stir for 30 min, raise the temperature to 30°C, and continue stirring for 1 h to obtain an intermediate solution; S22, cooling the intermediate solution to 0°C, adding 20.0 mL of ethyl azidoacetate and 10 mL of triethylamine in sequence, passing argon to expel oxygen, and sealing the reactor to avoid light; heating to 25°C, keeping warm for 3 h, heating to 40°C, keeping warm for 8 h, adding 50 mL of 1 M glycine buffer; dialysis purification, and freeze-drying to obtain a modified cottonseed protein hydrolysate; Step 3: The modified cottonseed protein hydrolysate and monoacetylenic curcumin undergo a Huisgen cycloaddition reaction to form a triazole bond, thereby obtaining a cottonseed protein hydrolysate-monoacetylenic curcumin complex, which specifically comprises: S31, 1.25g CuSO4·5H2O (5mmol), 1.61g tris(benzyltriazolylmethyl)amine (3mmol) were dissolved in 500mL PBS buffer to obtain Cu 2+ -TBTA composite solution; 300 mL of a mono-alkynyl curcumin solution (solvent: dimethyl sulfoxide) containing 2.0 g of mono-alkynyl curcumin was mixed with 6 g of modified cottonseed protein hydrolysate to obtain a mixture solution; S32, 20mL Cu 2+ -TBTA complex solution was added to 100 mL of the mixed solution, and 1 mL of L-sodium ascorbate solution was added. After mixing evenly, argon was passed through to deoxygenate for 5 min. After sealing, the mixture was shaken at room temperature at 25°C in the dark for 6 h. S33. Add ascorbic acid in an amount of 5% by volume of the reaction system and stir for 20 minutes. Use a MWCO 3kDa dialysis bag to purify the product by dialyzing (dialysis using 500 mL of PBS buffer containing 10 wt% for 8 hours, 500 mL of PBS buffer containing 0.1 wt% sodium lauryl sulfate for 12 hours, and deionized water for 24 hours). Wash the dialyzed purified product with deionized water several times and freeze-dry it at -40°C for 1 hour to obtain a cottonseed protein hydrolysate-monoacetylenic curcumin complex.

[0023] Example 2 A method for preparing a cottonseed protein hydrolysate-monoacetylenic curcumin complex comprises the following steps: Step 1: extracting cottonseed protein isolate from defatted cottonseed meal and purifying the cottonseed protein isolate by isoelectric precipitation, specifically comprising: S11, adding 20 mg of defatted cottonseed meal to 250 mL of 0.1 M KOH solution, stirring at 50°C for 30 min, incubating on a shaker for 30 min, and centrifuging at 2470 × g for 30 min at 4°C, collecting the supernatant to obtain cottonseed protein isolate; S12, diluting the cottonseed protein isolate with 10 times the volume of deionized water, continuously stirring and adding 0.1 M HCl dropwise until the pH of the dilution reaches 4.5, standing for 2 hours, and then centrifuging at 2470×g for 30 minutes at 4° C., collecting the precipitate, washing with deionized water until neutral, and freeze-drying at -40° C. for 2 hours to obtain the purified cottonseed protein isolate; Step 2: activating and modifying the purified cottonseed protein isolate using an activator and a stabilizer to obtain an intermediate; and performing an azidation reaction on the intermediate using an azide compound to obtain a modified cottonseed protein hydrolysate, specifically comprising: S21. Dissolve 10.0 g of purified cottonseed protein isolate in 400.0 mL of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0), sonicate at 50 kHz for 20 min, and cool to 4°C in an ice bath; add 2.6 g of N-hydroxysulfosuccinimide (12 mmol), and while stirring, dropwise add 1.55 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (10 mmol). Control the system temperature to ≤ 8°C, stir for 30 min, raise the temperature to 30°C, and continue stirring for 1 h to obtain an intermediate solution; S22, cooling the intermediate solution to 0°C, adding 30.0 mL of ethyl azidoacetate and 15 mL of triethylamine in sequence, passing argon to expel oxygen, and sealing the reactor to avoid light; heating to 25°C, keeping warm for 3 h, heating to 40°C, keeping warm for 8 h, adding 50 mL of 1 M glycine buffer; dialysis purification, and freeze-drying to obtain a modified cottonseed protein hydrolysate; Step 3: The modified cottonseed protein hydrolysate and monoacetylenic curcumin undergo a Huisgen cycloaddition reaction to form a triazole bond, thereby obtaining a cottonseed protein hydrolysate-monoacetylenic curcumin complex, which specifically comprises: S31, 1.25g CuSO4·5H2O (5mmol), 1.61g tris(benzyltriazolylmethyl)amine (3mmol) were dissolved in 500mL PBS buffer to obtain Cu 2+ -TBTA composite solution; 300 mL of a mono-alkynyl curcumin solution (solvent: dimethyl sulfoxide) containing 2.0 g of mono-alkynyl curcumin was mixed with 6 g of modified cottonseed protein hydrolysate to obtain a mixture solution; S32, 30mL Cu 2+ -TBTA complex solution was added to 100 mL of the mixed solution, and 1 mL of L-sodium ascorbate solution was added. After mixing evenly, argon was passed through to deoxygenate for 5 min. After sealing, the mixture was shaken at 30 °C in the dark for 6 h. S33. Add ascorbic acid in an amount of 5% by volume of the reaction system and stir for 20 minutes. Use a MWCO 3kDa dialysis bag to purify the product by dialyzing (dialysis using 500 mL of PBS buffer containing 10 wt% for 8 hours, 500 mL of PBS buffer containing 0.1 wt% sodium lauryl sulfate for 12 hours, and deionized water for 24 hours). Wash the dialyzed purified product with deionized water several times and freeze-dry it at -40°C for 1 hour to obtain a cottonseed protein hydrolysate-monoacetylenic curcumin complex.

[0024] Example 3 A method for preparing a cottonseed protein hydrolysate-monoacetylenic curcumin complex comprises the following steps: Step 1: extracting cottonseed protein isolate from defatted cottonseed meal and purifying the cottonseed protein isolate by isoelectric precipitation, specifically comprising: S11, adding 20 mg of defatted cottonseed meal to 250 mL of 0.1 M KOH solution, stirring at 50°C for 30 min, incubating on a shaker for 30 min, and centrifuging at 2470 × g for 30 min at 4°C, collecting the supernatant to obtain cottonseed protein isolate; S12, diluting the cottonseed protein isolate with 10 times the volume of deionized water, continuously stirring and adding 0.1 M HCl dropwise until the pH of the dilution reaches 4.5, standing for 2 hours, and then centrifuging at 2470×g for 30 minutes at 4° C., collecting the precipitate, washing with deionized water until neutral, and freeze-drying at -40° C. for 2 hours to obtain the purified cottonseed protein isolate; Step 2: activating and modifying the purified cottonseed protein isolate using an activator and a stabilizer to obtain an intermediate; and performing an azidation reaction on the intermediate using an azide compound to obtain a modified cottonseed protein hydrolysate, specifically comprising: S21. Dissolve 15.0 g of purified cottonseed protein isolate in 300.0 mL of 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0), sonicate at 50 kHz for 10 min, and cool to 4°C in an ice bath; add 3.25 g of N-hydroxysulfosuccinimide (15 mmol), and while stirring, dropwise add 1.55 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (10 mmol). Control the system temperature to ≤ 8°C, stir for 30 min, raise the temperature to 30°C, and continue stirring for 1 h to obtain an intermediate solution; S22, cooling the intermediate solution to 0°C, adding 45.0 mL of ethyl azidoacetate and 15 mL of triethylamine in sequence, passing argon to expel oxygen, and sealing the reactor to avoid light; heating to 25°C, keeping warm for 3 h, heating to 40°C, keeping warm for 8 h, adding 60 mL of 1 M glycine buffer; dialysis purification, and freeze-drying to obtain a modified cottonseed protein hydrolysate; Step 3: The modified cottonseed protein hydrolysate and monoacetylenic curcumin undergo a Huisgen cycloaddition reaction to form a triazole bond, thereby obtaining a cottonseed protein hydrolysate-monoacetylenic curcumin complex, which specifically comprises: S31, 1.25g CuSO4·5H2O (5mmol), 1.61g tris(benzyltriazolylmethyl)amine (3mmol) were dissolved in 500mL PBS buffer to obtain Cu 2+ -TBTA composite solution; 300 mL of a mono-alkynyl curcumin solution (solvent: dimethyl sulfoxide) containing 2.0 g of mono-alkynyl curcumin was mixed with 6 g of modified cottonseed protein hydrolysate to obtain a mixture solution; S32, 30mL Cu 2+ -TBTA complex solution was added to 100 mL of the mixed solution, and 3 mL of L-sodium ascorbate solution was added. After mixing evenly, argon was passed through to deoxygenate for 5 min. After sealing, the mixture was shaken at room temperature at 25 °C in the dark for 6 h. S33. Add ascorbic acid in an amount of 5% by volume of the reaction system and stir for 20 minutes. Use a MWCO 3kDa dialysis bag to purify the product by dialyzing (dialysis using 500 mL of PBS buffer containing 10 wt% for 8 hours, 500 mL of PBS buffer containing 0.1 wt% sodium lauryl sulfate for 12 hours, and deionized water for 24 hours). Wash the dialyzed purified product with deionized water several times and freeze-dry it at -40°C for 1 hour to obtain a cottonseed protein hydrolysate-monoacetylenic curcumin complex.

[0025] Example 4 A method for preparing a cottonseed protein hydrolysate-monoacetylenic curcumin complex, which differs from Example 1 in that the cottonseed protein hydrolysate-monoacetylenic curcumin complex obtained in step 3 of Example 1 is ultrasonically dispersed in an ethanol solution, ammonia water is added, and the mixture is allowed to stand for 1 to 3 hours. Then, a ethyl orthosilicate solution is added dropwise at a rate of 0.1 mL / min. After the addition is complete, magnetic stirring is performed at 300 to 600 rpm and the temperature is raised to 30 to 40° C. The mixture is reacted for 6 to 12 hours, allowed to stand for 6 to 12 hours, and the supernatant is discarded after centrifugation. The precipitate is washed and freeze-dried to obtain a silica-coated cottonseed protein hydrolysate-monoacetylenic curcumin complex. The ratio of the cottonseed protein hydrolysate-monoacetylenic curcumin complex, ethanol solution, ammonia water, and ethyl orthosilicate is 1 g:10 to 20 mL:0.5 to 1 mL:1 to 3 mL. The silica-coated cottonseed protein hydrolysate-monoacetylenic curcumin complex was placed on a glass slide, which was then placed in a sputtering coating machine. Platinum metal was used as the sputtering target. -3Under vacuum conditions of 500 Pa, the voltage was set to 500 V and the current was 5 mA, platinum deposition was performed, and the sputtering time was 5 to 10 minutes. The glass slide was removed to obtain a cottonseed protein hydrolysate-monoacetylenic curcumin complex with Pt and SiO2 coated on one side.

[0026] The methods and process parameters of the remaining steps of this embodiment are the same as those of Example 1.

[0027] Example 5 A method for preparing a cottonseed protein hydrolysate-monoacetylenic curcumin complex, which differs from Example 1 in that in this embodiment, 10 g of the cottonseed protein hydrolysate-monoacetylenic curcumin complex obtained in step 3 of Example 1 is ultrasonically dispersed in 200 mL of ethanol solution, 8 mL of ammonia water is added, and after standing for 2 h, 10 mL of ethyl orthosilicate solution is added dropwise at a rate of 0.1 mL / min. After the addition is complete, magnetic stirring is performed at 600 rpm and the temperature is raised to 40° C. The mixture is reacted for 12 h, allowed to stand and aged for 12 h, centrifuged, and the supernatant is discarded. The precipitate is washed and freeze-dried to obtain a silica-coated cottonseed protein hydrolysate-monoacetylenic curcumin complex. 2 g of silica-coated cottonseed protein hydrolysate-monoacetylenic curcumin complex was placed on a glass slide, and the glass slide was placed in a sputtering coating machine. Platinum metal was used as the sputtering target. -3 Under vacuum conditions of 500 Pa, the voltage was set to 500 V and the current to 5 mA, platinum deposition was performed, and the sputtering time was 5 min. The glass slide was removed to obtain a cottonseed protein hydrolysate-monoacetylenic curcumin complex with Pt deposited on one side and coated with SiO2.

[0028] The methods and process parameters of the remaining steps of this embodiment are the same as those of Example 1.

[0029] Comparative Example 1 A method for preparing a cottonseed protein hydrolysate-monoacetylenic curcumin complex. The difference from Example 1 is that in this embodiment, 2 g of the cottonseed protein hydrolysate-monoacetylenic curcumin complex is placed on a glass slide, the glass slide is placed in a sputtering coating machine, and metal platinum is used as a sputtering target. -3 Under vacuum conditions of 500 Pa, the voltage was set to 500 V and the current was 5 mA, platinum deposition was performed, and the sputtering time was 5 min. The glass slide was removed to obtain a cottonseed protein hydrolysate-monoacetylenic curcumin complex with Pt deposited on one side.

[0030] The methods and process parameters of the remaining steps of this embodiment are the same as those of Example 1.

[0031] Comparative Example 2 A method for preparing a cottonseed protein hydrolysate-monoacetylenic curcumin complex is disclosed. The method differs from Example 1 in that, in this embodiment, 10 g of the cottonseed protein hydrolysate-monoacetylenic curcumin complex obtained in step 3 of Example 1 is ultrasonically dispersed in 200 mL of an ethanol solution, 8 mL of aqueous ammonia is added, and the mixture is allowed to stand for 2 h. Then, 10 mL of an ethyl orthosilicate solution is added dropwise at a rate of 0.1 mL / min. After the addition is complete, the mixture is magnetically stirred at 600 rpm and heated to 40° C. The mixture is reacted for 12 h, allowed to stand for 12 h, and then centrifuged and the supernatant is discarded. The precipitate is washed and freeze-dried to obtain a silica-coated cottonseed protein hydrolysate-monoacetylenic curcumin complex.

[0032] The methods and process parameters of the remaining steps of this embodiment are the same as those of Example 1.

[0033] The antioxidant activity of the cottonseed protein hydrolysate-monoacetylenic curcumin complex (MCCPH) prepared in Examples 1-5 and Comparative Examples 1-2 was determined using DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) and ABTS (2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid)) free radical scavenging assays. The assay method included weighing 0.05 g of the cottonseed protein hydrolysate-monoacetylenic curcumin complex for food preservation prepared in Examples 1-5 and Comparative Examples 1-2, as well as 0.05 g of the purified cottonseed protein isolate in Example 1 as Control 1, 0.05 g of the monoacetylenic curcumin used in Example 1 as Control 2, and 0.05 g of the modified cottonseed protein hydrolysate in Example 1 as Control 3. These were placed in 10 mL of deionized water and shaken to produce an emulsion. Take 2mL of emulsion and 2.5mL of ABTS respectively + ABTS was mixed with PBS solution (absorbance 0.700 ± 0.025, containing 0.2 mL of 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) and allowed to react for 30 min in the dark. The absorbance was measured at 734 nm. + The calculation formula for free radical scavenging rate is: RABTS + (%) = (A2-A1) / A0×100% Where: A2 is ABTS at 734nm + / The absorbance of PBS solution and PBS solution; A1 is the absorbance of ABTS at 734nm + / The absorbance of PBS solution and sample solution; A0 is the absorbance of ABTS at 734nm + / The absorbance of PBS solution and 2.5mL deionized water. The following table is obtained: Table 1 Scavenging rate of ABTS free radicals by each sample As can be seen from the above table, the cottonseed protein hydrolysate-monoacetylenic curcumin complexes prepared in Examples 1 to 5 and Comparative Examples 1 to 2 all showed good effects on ABTS. + The free radical scavenging rate exceeded 90%, significantly superior to that of the blank control, demonstrating that the combination of cottonseed protein hydrolysate and monoacetylenic curcumin exhibited excellent antioxidant activity. Because different antioxidants preferentially react via the hydrogen atom transfer (HAT) or single electron transfer (SET) mechanism in the reduction reaction of DPPH or ABTS, respectively, and their reactivity is influenced by their redox potential, these two assays were used to comprehensively evaluate the total antioxidant capacity of monoacetylenic curcumin (control 2), CPI (control 1), CPH (control 3), and MCCPH. While native CPI exhibited low antioxidant activity against DPPH and ABTS, its hydrolysis significantly enhanced its antioxidant capacity. The hydrolysis process produced bioactive peptides with excellent antioxidant properties. Furthermore, the antioxidant capacity of the resulting cottonseed protein hydrolysate-monoacetylenic curcumin complex was further enhanced by combining cottonseed protein hydrolysate with monoacetylenic curcumin.

[0034] The gastric acid resistance of the cottonseed protein hydrolysate-monoacetylenic curcumin complexes prepared in Examples 1-5 and Comparative Examples 1-2 was determined. A gastric acid-simulating solution was prepared by adding 0.2% (w / v) NaCl solution to 100 mL of deionized water, followed by 0.7% (v / v) HCl, adjusting the pH to 2.0. Pepsin was then added at a ratio of 3.2 mg / mL to obtain the gastric acid-simulating solution.

[0035] 10 mg of the cottonseed protein hydrolysate-monoacetylenic curcumin complex prepared in Examples 1 to 5 and Comparative Examples 1 to 2 was respectively dispersed in a gastric acid simulated solution. At the same time, an equal amount of the sample was dispersed in deionized water as a control group, and the mixture was shaken at a constant temperature of 37 ° C. and 100 rpm. Samples were taken at 0.5 h, 1 h, 2 h, and 4 h, respectively, and the retention rate of monoacetylenic curcumin in the cottonseed protein hydrolysate-monoacetylenic curcumin complex was determined. The curcumin retention rate was calculated as follows: (residual monoacetylenic curcumin content / initial monoacetylenic curcumin content) × 100%, where the initial monoacetylenic curcumin content is the monoacetylenic curcumin content measured from the control group at the same time). The following table is obtained: Table 2 Gastric acid resistance simulation test results of each sample As can be seen from the above table, since the cottonseed protein hydrolysate-monoacetylenic curcumin complexes prepared in Examples 1 to 3 and Comparative Example 1 were not coated with silica, their monoacetylenic curcumin had no protective barrier in the simulated gastric acid solution, and their destruction and attenuation rates were significantly faster than those in Examples 4 and 5.

[0036] The cottonseed protein hydrolysate-monoacetylenic curcumin complex (MCCPH) synthesized in Examples 1 to 5 was placed in a hydrogen peroxide solution, and its movement was observed under a dark field microscope. In the hydrogen peroxide solution, the MCCPH sample exhibited a continuous random walk motion, like a colloidal motor. In a 10% volume concentration hydrogen peroxide solution, a typical trajectory of the motor was observed. Under the conditions of 25°C and 660nm visible light irradiation, the average movement rate of the cottonseed protein hydrolysate-monoacetylenic curcumin complex prepared in Examples 1 to 5 and Comparative Examples 1 to 2 in a 50mM volume concentration of 10% hydrogen peroxide solution was measured (the test spot volume was 10μL, and the field of view MCCPH density was in the range of 10 to 30), and the following table was obtained: Table 3 Average movement rate of each sample As can be seen from the above table, the cottonseed protein hydrolysate-monoacetylenic curcumin complex with Pt deposited on one side and SiO2 coated prepared in Examples 4 and 5 has a faster movement rate in catalase.

[0037] The number of devices and processing scales described herein are intended to simplify the description of the present invention. Applications, modifications, and variations of the present invention will be readily apparent to those skilled in the art.

[0038] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A method for preparing a cottonseed protein hydrolysate-monoacetylenic curcumin complex, characterized in that: The following steps are involved: Step 1, extracting cottonseed protein isolate from defatted cottonseed meal, and purifying the cottonseed protein isolate by isoelectric precipitation; Step 2: activating and modifying the purified cottonseed protein isolate using an activator and a stabilizer to obtain an intermediate; and performing an azidation reaction on the intermediate using an azide compound to obtain a modified cottonseed protein hydrolyzate; Step 3: The modified cottonseed protein hydrolysate and monoacetylenic curcumin undergo a Huisgen cycloaddition reaction to form a triazole bond, thereby obtaining a cottonseed protein hydrolysate-monoacetylenic curcumin complex.

2. The method for preparing the cottonseed protein hydrolysate-monoacetylenic curcumin complex according to claim 1, wherein In the step 1, the reagent used to extract the cottonseed protein isolate from the defatted cottonseed meal is 0.1M KOH, and the reagent used to purify the cottonseed protein isolate by isoelectric precipitation is 0.1M HCl.

3. The method for preparing the cottonseed protein hydrolysate-monoacetylenic curcumin complex according to claim 1, wherein In the step 2, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide, the stabilizer is N-hydroxysulfosuccinimide, and the azide compound is one of ethyl azidoacetate, ethyl propyl azidoacetate, and ethyl azidopropionate.

4. The method for preparing the cottonseed protein hydrolysate-monoacetylenic curcumin complex according to claim 3, wherein: The specific method of step 2 includes: S21. Dissolve the purified cottonseed protein isolate in 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.0, sonicate at 30-50 kHz for 10-20 min, and cool to 4° C. in an ice bath; add N-hydroxysulfosuccinimide, and while stirring, dropwise add 1-ethyl-(3-dimethylaminopropyl)carbodiimide; control the system temperature to ≤8° C., stir for 20-40 min, raise the temperature to 30-40° C., and continue stirring for 1-3 h to obtain an intermediate solution; S22. Cool the intermediate solution to 0°C, add ethyl azidoacetate and triethylamine in sequence, pass argon, and seal the reactor to avoid light; raise the temperature to 25°C and keep warm for 1-3 hours, raise the temperature to 35-45°C and keep warm for 8-16 hours, add 1M glycine buffer; dialysis purification, freeze-drying, and obtain modified cottonseed protein hydrolysate.

5. The method for preparing the cottonseed protein hydrolysate-monoacetylenic curcumin complex according to claim 4, wherein: In the S21, the usage ratio of the purified cottonseed protein isolate, 2-(N-morpholino)ethanesulfonic acid buffer, N-hydroxysulfosuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 5-15 g: 200-400 mL: 0.012-0.015 mol: 0.008-0.01 mol.

6. The method for preparing the cottonseed protein hydrolysate-monoacetylenic curcumin complex according to claim 4, wherein: In the S22, the usage ratio of ethyl azidoacetate, triethylamine, and glycine buffer is 2.0-4.5 mL: 0.5-2.5 mL: 1-15 mL.

7. The method for preparing the cottonseed protein hydrolysate-monoacetylenic curcumin complex according to claim 1, wherein: The specific method of step three includes: S31, dissolve CuSO4·5H2O and tris(benzyltriazolylmethyl)amine in PBS buffer to obtain Cu 2+ -TBTA composite solution; mixing the monoacetylenic curcumin solution with the modified cottonseed protein hydrolysate to obtain a mixture solution; S32, Cu 2+ -TBTA complex solution was added to the mixed solution, and then L-sodium ascorbate solution was added. After mixing evenly, argon was passed through to deoxygenate for 5 minutes. After sealing, the mixture was shaken in the dark at room temperature of 25°C to 30°C for 3 to 6 hours. S33. Add ascorbic acid in an amount of 5% by volume of the reaction system and stir for 10-20 minutes; dialysis the product using a 3 kDa MWCO dialysis bag for purification, and freeze-dry the purified product to obtain a cottonseed protein hydrolysate-monoacetylenic curcumin complex.

8. The method for preparing the cottonseed protein hydrolysate-monoacetylenic curcumin complex according to claim 7, wherein: In the S32, Cu 2+ The volume ratio of the L-TBTA composite solution, the mixed solution, and the L-sodium ascorbate solution is 2~5:10:0.1~1.

9. The method for preparing the cottonseed protein hydrolysate-monoacetylenic curcumin complex according to claim 1, wherein: The step three further comprises: ultrasonically dispersing the cottonseed protein hydrolysate-monoacetylenic curcumin complex in an ethanol solution, adding ammonia water, standing for 1-3 hours, and then dropping a tetraethyl orthosilicate solution at a rate of 0.1 mL / min. After the dropwise addition is complete, magnetic stirring is performed at 300-600 rpm and the temperature is raised to 30-40° C., reacting for 6-12 hours, standing for 6-12 hours, and aging after centrifugation. The supernatant is discarded, the precipitate is washed, and freeze-dried to obtain a silica-coated cottonseed protein hydrolysate-monoacetylenic curcumin complex; wherein the amount ratio of the cottonseed protein hydrolysate-monoacetylenic curcumin complex, the ethanol solution, the ammonia water, and the tetraethyl orthosilicate is 1 g:10-20 mL:0.5-1 mL:1-3 mL; The silica-coated cottonseed protein hydrolysate-monoacetylenic curcumin complex was placed on a glass slide, which was then placed in a sputtering coating machine. Platinum metal was used as the sputtering target. -3 Under vacuum conditions of 500 Pa, the voltage was set to 500 V and the current was 5 mA, platinum deposition was performed, and the sputtering time was 5 to 10 minutes. The glass slide was removed to obtain a cottonseed protein hydrolysate-monoacetylenic curcumin complex with Pt and SiO2 coated on one side.

10. An application of a cottonseed protein hydrolysate-monoacetylenic curcumin complex, wherein the cottonseed protein hydrolysate-monoacetylenic curcumin complex is prepared by the method for preparing the cottonseed protein hydrolysate-monoacetylenic curcumin complex according to any one of claims 1 to 9, characterized in that: The cottonseed protein hydrolysate-monoacetylenic curcumin complex is used as a colloidal motor to achieve transient transmission of tiny objects, including adenosine triphosphate, antibiotic drugs, organic pollutants, organic dye molecules, heavy metal ions, enzymes, antibodies, and organelles.

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