Preparation method and application of synergistically modified protein nanoparticles

Abstract

The application discloses a preparation method and application of synergistically modified protein nanoparticles, and relates to the field of plant protein processing. The application adopts cod protein as a wall material, adopts curcumin as a hydrophobic polyphenol model, and successfully prepares clear cod protein-curcumin composite nanoparticles through disulfide bond breaking and critical pH. The cod protein-curcumin composite particles prepared by the method have a high embedding rate, and under continuous heating and illumination, the cod protein-curcumin composite particles still have good thermal and light stability, and can be used as a new type of functional ingredient or a carrying system to embed, deliver and carry hydrophobic polyphenol substances such as curcumin. The application obtains stable and clear transparent cod protein-curcumin composite nanoparticles, and the industrial production can be easily realized, so that the application can be widely applied to the fields of functional food, medicine and cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant protein processing, and particularly relates to a preparation method and application of synergistically modified protein nanoparticles. BACKGROUND

[0002] In recent years, with the changes in human lifestyle and ecological environment, chronic diseases such as cardiovascular disease have become increasingly common, which seriously threatens people's health and hinders social development. Research has found that most chronic diseases are related to diet, so the development of new functional foods is more urgent. It is well known that some bioactive substances have nutritional and regulatory functions and can be incorporated into food systems. Curcumin (Cur) is a natural bioactive polyphenol with multiple physiological activities such as hypolipidemic, antioxidant, anticancer and anti-inflammatory. However, curcumin has poor water solubility, unstable chemical properties and low bioavailability, which limits its application as a food ingredient in functional foods.

[0003] To overcome these problems, researchers have found that using delivery systems can improve the stability of bioactive substances such as curcumin, including hydrogels, nanoparticles, emulsions, liposomes and other delivery carriers. Current research has found that once hydrophobic bioactive substances are loaded into certain special nanoparticles, their solubility and stability can be significantly improved, indicating that using nanoparticles as delivery carriers is an effective way to solubilize and stabilize curcumin. Among the many encapsulating wall materials, the biocompatibility and environmental performance of proteins are superior to synthetic polymers and other biological macromolecules. Cod Protein (CP) is a high-quality protein with rich amino acid content and wide application, so it can be an ideal material for protein-based nanocarriers, but it is an underutilized animal protein. However, how to improve the solubility and stability of bioactive substances is a technical bottleneck that needs to be solved for the development of functional foods, and it is also a hot spot in current food science research.

[0004] Based on the above problems, a delivery system capable of protecting and stabilizing curcumin encapsulation is designed, which is of great significance for the protection and stability improvement of other hydrophobic bioactive substances. Despite its considerable advantages, protein-based bioactive substance delivery carriers also have certain disadvantages and problems, such as in protein dispersion, the hydrophobic regions in protein molecules tend to "hide" inside, limiting their interaction with hydrophobic bioactive small molecules. And some methods use complex technology and high cost, which does not meet the modern food industry's green production principle. For example, a kind of amorphous curcumin nanoparticles and its preparation method and application are provided in the Chinese patent with the patent number CN202310244832.7 applied on March 13, 2023, which is prepared by a bottom-up anti-solvent precipitation method, but the anti-solvent method involves organic solvents and complex recovery technology. Similarly, a kind of curcumin polyelectrolyte composite film, its preparation method and application are provided in the Chinese patent with the patent number CN201911011384.6 applied on October 23, 2019, which adopts a solvent evaporation method that is time-consuming and easy to cause pollution. Also, a preparation method of curcumin spray drying microcapsule is provided in the Chinese patent with the patent number CN202211507926.0 applied on November 25, 2022, which adopts a spray drying method to prepare curcumin microcapsules, but the spray drying technology has high energy consumption and low thermal efficiency. The late-stage investment cost is relatively high, which is not suitable for popularization and application. In addition, a preparation method of curcumin microcapsule is provided in the Chinese invention patent with the patent number CN202211117772.4 applied on September 14, 2022, which uses dextrin, sodium alginate, phospholipid, etc. as wall material, curcumin as core material, adds appropriate amount of emulsifier, uses microcapsule granulator, and adopts single nozzle embedding method to prepare curcumin microcapsules. However, this method has complex wall material and limited embedding degree, and the embedding rate is low. Therefore, it is urgent to explore a simple and high-loading embedding method. SUMMARY

[0005] One of the purposes of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a simple, industrialized and high-loading embedding method for hydrophobic polyphenols.

[0006] The present application can effectively embed curcumin by breaking disulfide bonds and critical pH treatment to maximize protein structure.

[0007] The technical scheme adopted by the present application to achieve the above purpose is to provide a preparation method of protein nanoparticles with broken disulfide bonds and critical pH modification, comprising the following specific steps:

[0008] S1, dispersion treatment: disperse cod protein in water to obtain protein dispersion 1;

[0009] S2, centrifugal treatment: centrifugal treatment of the protein dispersion 1 of step S1, collecting the supernatant to obtain a protein dispersion 2;

[0010] S3, reduction treatment: adding the same volume of sodium pyrosulfite into the protein dispersion 2, stirring, then dialysis, freeze-drying to obtain cod protein powder 1;

[0011] S4, alkalization treatment: mixing the cod protein powder 1 and water to obtain a cod protein powder solution, adjusting the pH of the cod protein powder solution to 9.5-10.5 by NaOH, stirring, and then freeze-drying to obtain the synergistically modified protein nanoparticles.

[0012] Further, the mass concentration of the cod protein in the protein dispersion 1 of step S1 is 5-10%.

[0013] Further, the concentration of sodium pyrosulfite in step S3 is 5-10 mM.

[0014] Further, the molecular weight of the dialysis membrane in step S3 is 3-3.5 kDa.

[0015] Further, the concentration of the cod protein powder solution in step S4 is 3-8 mg / mL.

[0016] Further, the concentration of NaOH in step S4 is 0.8-1.2 M.

[0017] Further, the pH is adjusted to 10 in step S4.

[0018] Further, the stirring time in step S4 is 20-30 min.

[0019] The synergistically modified protein nanoparticles prepared by the above method are provided.

[0020] The synergistically modified protein nanoparticles provided by the application are used as carriers in the preparation of a hydrophobic polyphenol carrying system.

[0021] The application provides a preparation method of cod protein-curcumin composite particles, comprising the following steps:

[0022] The synergistically modified protein nanoparticles are dissolved to obtain a protein dispersion with a concentration of 3-6 mg / mL, then the same volume of a 0.25-20 mg / mL curcumin solution is uniformly mixed for 0.5-1.5 hours, and then the pH is adjusted to neutral to obtain cod protein-curcumin composite nanoparticles.

[0023] Further, the concentration of the protein dispersion is 5 mg / mL, and the concentration of the curcumin solution is 0.25-2.5 mg / mL.

[0024] Further, the uniform mixing time is 1 hour.

[0025] The present application provides a preparation method of cod protein-curcumin complex particles with high curcumin embedding, comprising the following steps:

[0026] 1. Dispersion treatment: cod protein is dispersed in water to obtain a protein dispersion 1;

[0027] 2. Centrifugal treatment: the protein dispersion 1 of step S1 is centrifuged, and the supernatant is collected to obtain a protein dispersion 2;

[0028] 3. Reduction treatment: the same volume of sodium pyrosulfite is added to the protein dispersion 2 and stirred, and then dialyzed and freeze-dried to obtain cod protein powder 1;

[0029] 4. Alkalization treatment: the cod protein powder 1 is mixed with water to obtain a cod protein powder solution, the pH of the cod protein powder solution is adjusted to 9.5-10.5 by NaOH, and stirring is performed to obtain a protein dispersion 5;

[0030] 5. Embedding treatment: the protein dispersion 5 obtained in step S4 is uniformly mixed with an equal volume of a curcumin solution with a concentration of 0.25-20 mg / mL for 0.5-1.5 hours, and then the pH is adjusted to neutral to obtain cod protein-curcumin composite nanoparticles.

[0031] Further, the mass concentration of cod protein in the protein dispersion 1 in step 1 is 5-10%.

[0032] Further, the concentration of sodium pyrosulfite in step 3 is 5-10 mM.

[0033] Further, the molecular weight of the dialysis membrane in step 3 is 3-3.5 kDa.

[0034] Further, the concentration of the cod protein powder solution in step 4 is 3-8 mg / mL.

[0035] Further, the concentration of NaOH in step 4 is 0.8-1.2 M.

[0036] Further, the pH in step 4 is adjusted to 10.

[0037] Further, the stirring in step 4 is performed for 20-30 min.

[0038] Further, the concentration of the curcumin solution in step 5 is 0.25-2.5 mg / mL.

[0039] Further, the uniform mixing time in step 5 is 1 hour.

[0040] The present application provides cod protein-curcumin complex particles prepared by the above method.

[0041] The cod protein-curcumin complex particles provided by the application are applied to the preparation of functional food, medicine and cosmetics.

[0042] The application has the following beneficial effects:

[0043] 1. The necessity of critical pH in the experiment is verified, which will help to improve the large-scale application of hydrophobic functional substances in production.

[0044] 2. A new encapsulation method suitable for industrial production is developed. The application provides protein nanoparticles modified by breaking disulfide bonds and critical pH, and the application of the protein nanoparticles in a delivery system for hydrophobic polyphenols. Cod protein is used as a wall material, and curcumin is used as a model of hydrophobic polyphenols, and clear cod protein-curcumin composite nanoparticles are successfully prepared.

[0045] 3. The protein nanoparticles are modified by breaking disulfide bonds and alkaline pH, and through specific parameters such as specific reducing agent concentration and specific pH, compared with the cod protein-curcumin combination particles obtained by alkaline treatment alone, the embedding rate of curcumin is significantly improved, especially in high concentration of curcumin, and unexpected technical effects are achieved.

[0046] 4. The embedding preparation method provided by the application is simple and easy to realize industrial production, and expands the application of nanoparticles in the food industry. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The critical point fluorescence spectrum change of the cod protein obtained in Example 1 of the application during the pH shift process is shown in the following table: Figure 1 A is the result of pH 7-12, wherein the interval of pH 9-10.5 is 0.5, Figure 1 B is the result of pH 7-12, wherein the interval of pH 9.6-10.4 is 0.2.

[0048] Figure 2 The critical point circular dichroism spectrum change of the cod protein obtained in Example 1 of the application during the pH shift process is shown in the following table: Figure 2 A is the result of pH 7-12, wherein the interval of pH 9-10.5 is 0.5, Figure 2 B is the result of pH 7-12, wherein the interval of pH 9.6-10.4 is 0.2.

[0049] Figure 3 A is the fluorescence spectrum of the cod protein obtained in Example 2 of the application during the cooperative treatment process, Figure 3 B is the circular dichroism spectrum of the cod protein obtained in Example 2 of the application during the cooperative treatment process.

[0050] Figure 4The embedding rate measurement results of the cod protein-curcumin complex particles obtained in Example 3 of the present application.

[0051] Figure 5 The embedding rate visual appearance of the cod protein-curcumin complex particles obtained in Example 3 of the present application.

[0052] Figure 6 The thermal stability measurement results of the cod protein-curcumin complex particles obtained in Example 3 of the present application, Figure 6 The light stability measurement results of the cod protein-curcumin complex particles obtained.

[0053] Figure 7 The embedding rate measurement results of the cod protein-curcumin complex particles obtained in Example 3 of the present application.

[0054] Figure 8 The embedding rate measurement results of the cod protein-curcumin complex particles obtained in Example 3 of the present application.

[0055] Figure 9 The thermal stability measurement results of the cod protein-curcumin complex particles obtained in Example 3 of the present application, Figure 9 The light stability measurement results of the cod protein-curcumin complex particles obtained.

[0056] Figure 10 The thermal stability measurement results of the cod protein-curcumin complex particles obtained in Example 3 of the present application, Figure 10 The light stability measurement results of the cod protein-curcumin complex particles obtained.

[0057] Figure 11 The embedding rate measurement results of the cod protein-curcumin complex particles obtained in Example 3 of the present application. DETAILED DESCRIPTION

[0058] The present application will be further described below through specific implementation examples.

[0059] Raw material sources

[0060] Low-temperature defatted soybean meal (Shandong YUWANG Industrial Co., Ltd., China); curcumin (98% purity, Shenguo Biotechnology Co., Ltd., China); sodium metabisulfite (Macklin Biochemical Co., Ltd., China); sodium hydroxide and hydrochloric acid (Tianjin Damao Chemical Reagent Co., Ltd., China). Other chemical reagents are all analytical grade.

[0061] The cod protein used in the following examples was obtained by the following extraction method: cod fillets were dispersed in distilled water (1 : 10, w / v), pulverized and adjusted to pH 10.5 with NaOH (1 M), stirred for 1 h, centrifuged at 15800 x g for 30 min, the obtained supernatant was adjusted to pH 4.5 with HC1 (1 M), centrifuged at 10,000 x g for 15 min, and the precipitate was collected. The precipitate was then dissolved in distilled water and neutralized to pH 7.0 with NaOH. The extracted protein was freeze-dried and stored in a refrigerator at -20 °C. The obtained cod protein had a content of 91.0 ± 0.3% (w / w). The cod fillets used were ordinary commercially available cod.

[0062] Detection procedure

[0063] 1. Fluorescence spectrum (FL)

[0064] The sample concentration was 1 mg / mL, the excitation wavelength was set to 290 nm, the excitation and emission slits were both 5.0 nm, the scanning speed was 240 nm / min, and scanning was performed in the range of 300-500 nm. Before measurement, the baseline was zeroed with diluent.

[0065] 2. Circular dichroism spectrum (CD)

[0066] The sample concentration was 0.1 mg / mL, the scanning range was set to 190-260 nm, the scanning speed was 50 nm / min, and scanning was performed 3 times. The determination results were graphically fitted using CDSSTR software to calculate the secondary structure.

[0067] 3. Entrapment efficiency (EE)

[0068] Curcumin standard curve: 95% ethanol was accurately prepared to prepare curcumin standard solutions with mass concentrations of 1, 2.5, 5 and 10 μg / mL, respectively. The absorbance of curcumin at different mass concentrations was measured, and the standard curve was drawn with the mass concentration of curcumin as the horizontal axis and the absorbance as the vertical axis, y = 0.1423x - 0.0021, R 2 = 1. The entrapment efficiency of protein-encapsulated curcumin can be calculated by the following formula:

[0069] EE (%) = (total curcumin content - free curcumin content) x 100 / total curcumin content

[0070] Wherein, the free curcumin content is the amount of curcumin contained in the precipitate after centrifugation. Therefore, the precipitate is completely dissolved in 95% ethanol (w / w), and the obtained solution is centrifuged at 10000 x g for 15 min to remove protein aggregates. The obtained supernatant is measured by ultraviolet-visible spectrophotometer for its absorbance at 420 nm, and the content of curcumin is calculated according to the standard curve.

[0071] 4. Visual appearance

[0072] The visual appearance of the embedding solution was recorded with a camera.

[0073] 5. Thermogravimetry

[0074] The experiment was performed under nitrogen at a rate of 10°C / min from 40°C to 600°C.

[0075] 6. Light stability

[0076] The sample solution was irradiated (0-80 min) at a distance of 20 cm from an incandescent lamp (50 Hz, 30 W) and then subjected to UV spectroscopy. The scanning wavelength was 200-700 nm.

[0077] Example 1

[0078] S1, dispersion treatment: Cod protein powder was uniformly dispersed in water to obtain a protein dispersion 1 with a mass concentration of 10 mg / mL;

[0079] S2, centrifugal treatment: The protein dispersion 1 obtained in step S1 was centrifuged at 10,000 x g to remove impurities and collect the supernatant to obtain a protein dispersion 2;

[0080] S3, alkalization treatment: The pH of the protein dispersion 2 obtained in step S2 was adjusted to 7-12, and then the protein dispersion was kept at the specific pH for 30 min to induce unfolding to obtain a protein dispersion 3.

[0081] The fluorescence spectrum and the circular dichroism spectrum of the cod protein prepared under different pH conditions in this example were measured. The fluorescence spectrum and the circular dichroism spectrum determination results are shown in Figure 1 and Figure 2 .

[0082] The decrease in fluorescence intensity and the red shift of the wavelength corresponding to the maximum fluorescence intensity (λ max ) indicate that the protein structure is unfolded, and thus the tryptophan residues therein are in a more hydrophilic environment Figure 1 ). When the pH > 9.5, the fluorescence intensity suddenly decreases, indicating that the protein structure is significantly unfolded. As shown in Figure 2 , by continuously increasing the pH, the secondary structure is mainly in the form of α-helix. When the pH > 9.5, the spectrum of CP has a significant blue shift, and the helicity of the α-helix decreases sharply, indicating that the protein structure is mainly unfolded after pH 9.5. According to the above data, it is estimated that the critical pH of CP is after pH 9.5 (in the interval of 9.5-10.5), and the structural changes near pH 10 are further analyzed with an interval of pH 0.2. As shown in Figure 1As shown, compared with other pH intervals, the fluorescence intensity of CP significantly decreased by 12.78% from pH 9.8 to 10, and the red shift was 2 nm, which was significantly higher than 0.2 in other pH intervals, indicating that the degree of protein unfolding was greater at this pH. Similarly, in Figure 2 the spectrum of CP at pH 10 was blue-shifted by 7.5 nm compared with CP at pH 9.8, and the content of α-helix decreased by 49.46%, indicating that at this pH, the degree of unfolding of CP structure began to increase, which was a critical pH. The above results showed that the critical pH of CP structure unfolding during the pH change was pH 10, at which the degree of protein structure unfolding was higher. The above results collectively indicated that when the protein structure unfolded relatively larger, the critical pH of CP during the pH change was pH 10.

[0083] Example 2

[0084] S1, dispersion treatment: uniformly disperse cod protein in water to obtain protein dispersion liquid 1 with a mass concentration of 5 mg / mL;

[0085] S2, centrifugal treatment: centrifuge the protein dispersion liquid 1 obtained in step S1 at 10,000 x g, remove impurities and collect the supernatant to obtain protein dispersion liquid 2;

[0086] S3, reduction treatment: add the same volume of sodium metabisulfite solution (10 mM) dropwise to the protein dispersion liquid 2 and stir for 30 min to induce unfolding, and then freeze-dry the obtained solution after dialysis (dialysis membrane molecular weight: 3.5 kDa) to obtain cod protein powder 1 treated with a reducing agent.

[0087] S4, alkalization treatment: redissolve the cod protein powder 1 (5 mg / mL), adjust the pH of the solution to 10 with 1 M NaOH, and then keep it at this pH for 30 min to induce further unfolding of the protein, and the obtained solution is denoted as protein dispersion liquid 5, and then freeze-dry to obtain the synergistically modified protein nanoparticles.

[0088] Take the cod protein prepared in this example to measure the fluorescence spectrum and circular dichroism spectrum, and take the cod protein treated with reduction and alkalization alone as a control. The fluorescence spectrum and circular dichroism spectrum determination results are shown in Figure 3 .

[0089] As can be seen from the figure, after breaking the disulfide bond and synergistically treating the critical pH, the maximum fluorescence intensity of cod protein decreased significantly (decreased by 59.47% compared with reduction alone, and decreased by 39.01% compared with alkalization alone), and the λmax had a relatively obvious red shift Figure 3 A), indicating that the protein structure unfolded, and tryptophan was exposed to a more hydrophilic microenvironment. In Figure 3In B, the significant decrease of the content of alpha-helix in the protein treated synergistically was accompanied by the increase of the content of random coil, indicating that the protein structure became loose. The above results collectively indicated that after the breaking of disulfide bond and synergistic critical pH treatment, the size of SP particles was significantly reduced, the structure was unfolded, resulting in more hydrophobic sites exposed on the surface of the protein.

[0090] Example 3

[0091] S1, dispersion treatment: cod protein was uniformly dispersed in water to obtain a protein dispersion solution 1 with a mass concentration of 5 mg / mL;

[0092] S2, centrifugal treatment: the protein dispersion solution 1 obtained in step S1 was centrifuged at 10,000 x g, impurities were removed, and the supernatant was collected to obtain a protein dispersion solution 2;

[0093] S3, reduction treatment: a same volume of sodium metabisulfite solution (10 mM) was added dropwise to the protein dispersion solution 2 with stirring for 30 min to induce unfolding, and the obtained solution was freeze-dried after dialysis (dialysis membrane molecular weight: 3.5 kDa) to obtain a cod protein powder treated with a reducing agent 1.

[0094] S4, alkalization treatment: the cod protein powder 1 was redissolved (5 mg / mL), the pH of the solution was adjusted to 10 with 1 M NaOH, and then the solution was kept at this pH for 30 min to induce further unfolding of the protein, and the obtained solution was denoted as protein dispersion solution 5.

[0095] S5, embedding treatment: the protein dispersion solution 5 obtained in step S4 was uniformly mixed with an equal volume of a curcumin solution with a concentration of 0.25-20 mg / mL for 1 h, and then the pH was adjusted to neutral to obtain cod protein-curcumin composite nanoparticles.

[0096] Comparative Example 1

[0097] S1, dispersion treatment: cod protein was uniformly dispersed in water to obtain a protein dispersion solution 1 with a mass concentration of 5 mg / mL;

[0098] S2, centrifugal treatment: the protein dispersion solution 1 obtained in step S1 was centrifuged at 10,000 x g, impurities were removed, and the supernatant was collected to obtain a protein dispersion solution 2;

[0099] S3, alkalization treatment: the pH of the protein dispersion solution 2 obtained in step S2 was adjusted to 10, and then the solution was kept at a specific pH for 30 min to induce unfolding to obtain a protein dispersion solution 3;

[0100] S4, embedding treatment: the protein dispersion solution 3 obtained in step S3 was uniformly mixed with an equal volume of a curcumin solution with a concentration of 0.25-20 mg / mL for 1 h, and then the pH was adjusted to neutral to obtain cod protein-curcumin composite nanoparticles.

[0101] Comparative Example 2

[0102] S1, dispersion treatment: cod protein was uniformly dispersed in water to obtain a protein dispersion 1 with a mass concentration of 5 mg / mL;

[0103] S2, centrifugal treatment: the protein dispersion 1 obtained in step S1 was centrifuged at 10,000 x g to remove impurities and collect the supernatant to obtain a protein dispersion 2;

[0104] S3, reduction treatment: the same volume of sodium pyrosulfite solution (10 mM) was added dropwise to the protein dispersion 2 and stirred for 30 min to induce unfolding. The obtained solution was dialyzed (dialysis membrane molecular weight: 3.5 kDa) and freeze-dried to obtain a cod protein powder 1 treated with a reducing agent.

[0105] S4, embedding treatment: the cod protein powder 1 obtained in step S3 was uniformly mixed with an equal volume of 0.25-20 mg / mL curcumin solution for 1 h, and then the pH was adjusted to neutral to obtain cod protein-curcumin composite nanoparticles.

[0106] Comparative Example 3

[0107] S1, dispersion treatment: cod protein was uniformly dispersed in water to obtain a protein dispersion 1 with a mass concentration of 5 mg / mL;

[0108] S2, centrifugal treatment: the protein dispersion 1 obtained in step S1 was centrifuged at 10,000 x g to remove impurities and collect the supernatant to obtain a protein dispersion 2;

[0109] S3, reduction treatment: the same volume of sodium pyrosulfite solution (10 mM) was added dropwise to the protein dispersion 2 and stirred for 30 min to induce unfolding. The obtained solution was dialyzed (dialysis membrane molecular weight: 3.5 kDa) and freeze-dried to obtain a cod protein powder 1 treated with a reducing agent.

[0110] S4, alkalization treatment: the cod protein powder 1 was re-dissolved (5 mg / mL), and the pH of the solution was adjusted to 10 with 1 M NaOH, and then the pH was maintained at this value for 30 min to induce further unfolding of the protein. The obtained solution was denoted as protein dispersion 5.

[0111] S5, embedding treatment: the protein dispersion 5 obtained in step S4 was uniformly mixed with an equal volume of 5 mg / mL curcumin solution for 0.25-1.5 h, and then the pH was adjusted to neutral to obtain cod protein-curcumin composite nanoparticles.

[0112] The cod protein-curcumin composite nanoparticles prepared in Example 3 were subjected to visual appearance detection, and the results are shown in Figure 5 The solubility of the cod protein-curcumin composite particles was also high.

[0113] The embedding rate of cod protein-curcumin complex nanoparticles prepared in Example 3 and Comparative Examples 1 and 2 was determined. The embedding rate results of Example 3 are shown in Table 1, as compared with the cod protein-curcumin conjugate in Comparative Examples 1 and 2. Figure 4 The embedding rate of cod protein-curcumin complex nanoparticles prepared in Example 3 and Comparative Examples 1 and 2 was determined. The embedding rate results of Example 3 are shown in Table 1, as compared with the cod protein-curcumin conjugate in Comparative Examples 1 and 2. Figure 7 The embedding rate of cod protein-curcumin complex nanoparticles prepared in Example 3 and Comparative Examples 1 and 2 was determined. The embedding rate results of Example 3 are shown in Table 1, as compared with the cod protein-curcumin conjugate in Comparative Examples 1 and 2. Figure 8 The embedding rate of cod protein-curcumin complex nanoparticles prepared in Example 3 and Comparative Examples 1 and 2 was determined. The embedding rate results of Example 3 are shown in Table 1, as compared with the cod protein-curcumin conjugate in Comparative Examples 1 and 2. Figure 4 The embedding rate of cod protein-curcumin complex nanoparticles prepared in Example 3 and Comparative Examples 1 and 2 was determined. The embedding rate results of Example 3 are shown in Table 1, as compared with the cod protein-curcumin conjugate in Comparative Examples 1 and 2. Figure 11 The embedding rate of cod protein-curcumin complex nanoparticles prepared in Example 3 and Comparative Examples 1 and 2 was determined. The embedding rate results of Example 3 are shown in Table 1, as compared with the cod protein-curcumin conjugate in Comparative Examples 1 and 2.

[0114] Table 1 Embedding rate of Example 3 and Comparative Examples 1 and 2

[0115]

[0116] The thermal stability of heating at 40-600℃ and the light stability of 80 min light irradiation of the cod protein-curcumin complex nanoparticles prepared in this Example 3 were determined, as shown in Table 2. The thermal stability and light stability of the cod protein-curcumin conjugate prepared in Comparative Example 1 were determined, and the results are shown in Table 2. The cod protein-curcumin complex nanoparticles prepared by the reduction and alkalization synergistic treatment of Example 3 had a slower degradation rate compared with the cod protein-curcumin conjugate, indicating higher stability. This shows that the protein nanoparticles with broken disulfide bonds and critical pH modification are an effective method for steady-state delivery of hydrophobic polyphenols. Figure 6 The thermal stability of heating at 40-600℃ and the light stability of 80 min light irradiation of the cod protein-curcumin complex nanoparticles prepared in this Example 3 were determined, as shown in Table 2. The thermal stability and light stability of the cod protein-curcumin conjugate prepared in Comparative Example 1 were determined, and the results are shown in Table 2. The cod protein-curcumin complex nanoparticles prepared by the reduction and alkalization synergistic treatment of Example 3 had a slower degradation rate compared with the cod protein-curcumin conjugate, indicating higher stability. This shows that the protein nanoparticles with broken disulfide bonds and critical pH modification are an effective method for steady-state delivery of hydrophobic polyphenols. Figure 9 The thermal stability of heating at 40-600℃ and the light stability of 80 min light irradiation of the cod protein-curcumin complex nanoparticles prepared in this Example 3 were determined, as shown in Table 2. The thermal stability and light stability of the cod protein-curcumin conjugate prepared in Comparative Example 1 were determined, and the results are shown in Table 2. The cod protein-curcumin complex nanoparticles prepared by the reduction and alkalization synergistic treatment of Example 3 had a slower degradation rate compared with the cod protein-curcumin conjugate, indicating higher stability. This shows that the protein nanoparticles with broken disulfide bonds and critical pH modification are an effective method for steady-state delivery of hydrophobic polyphenols. Figure 10 The thermal stability of heating at 40-600℃ and the light stability of 80 min light irradiation of the cod protein-curcumin complex nanoparticles prepared in this Example 3 were determined, as shown in Table 2. The thermal stability and light stability of the cod protein-curcumin conjugate prepared in Comparative Example 1 were determined, and the results are shown in Table 2. The cod protein-curcumin complex nanoparticles prepared by the reduction and alkalization synergistic treatment of Example 3 had a slower degradation rate compared with the cod protein-curcumin conjugate, indicating higher stability. This shows that the protein nanoparticles with broken disulfide bonds and critical pH modification are an effective method for steady-state delivery of hydrophobic polyphenols.

[0117] The embedding rate of cod protein-curcumin complex nanoparticles prepared in Example 3 and Comparative Examples 1 and 2 was determined. The embedding rate results of Example 3 are shown in Table 1, as compared with the cod protein-curcumin conjugate in Comparative Examples 1 and 2. Figure 11and Table 2 below. The results show that the highest embedding rate is 89.66% when the binding time is 1 h, but the embedding rate decreases as the binding time further increases, which is probably because the reaction time is too long, the groups interact to form aggregates, thereby reducing the embedding rate, so the embedding time is most preferably 1 h.

[0118] Table 2 Comparative Example 3 Embedding Rate

[0119]

[0120] In summary, only the preferred embodiments of the present application, but the scope of protection of the present application is not limited to this, any skilled in the art of the technical personnel in the technical range disclosed by the present application, according to the technical solution of the present application and its inventive concept to make equivalent replacement or change, should be covered within the scope of protection of the present application.

Claims

1. A process for the preparation of high curcumin-embedded cod protein-curcumin complex granules, characterized by, The method comprises the following specific steps: S1, dispersion treatment: disperse cod protein in water to obtain a protein dispersion 1; S2, centrifugal treatment: centrifuge the protein dispersion 1 of step S1, and collect the supernatant to obtain a protein dispersion 2; S3, reduction treatment: add the same volume of sodium pyrosulfite into the protein dispersion 2, stir, then dialyze, freeze-dry, and obtain cod protein powder 1; S4, alkalization treatment: mix the cod protein powder 1 with water to obtain a cod protein powder solution, adjust the pH of the cod protein powder solution to 9.5-10.5 by using NaOH, stir, and then freeze-dry to obtain synergistically modified protein nanoparticles; S5, embedding treatment: dissolve the synergistically modified protein nanoparticles obtained in step S4 to obtain a protein dispersion with a concentration of 3-6 mg / mL, then uniformly mix the protein dispersion with the same volume of a curcumin solution with a concentration of 0.25-20 mg / mL for 0.5-1.5 hours, and then adjust the pH to neutral to obtain cod protein-curcumin composite nanoparticles.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass concentration of cod protein in the protein dispersion 1 is 5-10%; in step S3, the concentration of sodium pyrosulfite is 5-10 mM; and in step S3, the molecular weight of the dialysis membrane is 3-3.5 kDa.

3. The preparation method according to claim 1, characterized in that, In step S4, the concentration of the cod protein powder solution is 3-8 mg / mL; in step S4, the concentration of NaOH is 0.8-1.2 M; in step S4, the pH is adjusted to 10; and in step S4, the stirring time is 20-30 min.

4. The preparation method according to claim 1, characterized in that, In step S5, the concentration of the protein dispersion is 5 mg / mL.

5. The method as claimed in claim 1, wherein, The uniform mixing time is 1 hour.

6. The cod protein-curcumin composite nanoparticles prepared by the method of any one of claims 1-5.

7. The cod protein-curcumin composite nanoparticles of claim 6 for use in the preparation of functional food, medicine and cosmetics.

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