A high mucus permeation-cell absorption type soybean polypeptide nanoparticle and a preparation method and application thereof
Soybean polypeptide nanoparticles were prepared by fractional hydrolysis of soybean protein using protease and alkaline methods, combined with genipin cross-linking. This method solved the problem of nanocarriers being unable to overcome mucus and cell barriers, achieving high mucus penetration and high cell absorption performance, and is suitable for functional foods and biomedicine.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTH CHINA UNIV OF TECH
- Filing Date
- 2023-08-31
- Publication Date
- 2026-06-09
AI Technical Summary
Existing nanocarriers cannot overcome both the mucus barrier and the intestinal epithelial cell barrier simultaneously, which limits the bioavailability of functional factors or drugs. Furthermore, existing zwitterionic polymer carriers are mostly chemically synthesized inorganic materials, which have complicated preparation processes and are not suitable for the food industry.
Soybean protein was hydrolyzed in stages using protease and alkali methods, and high-viscosity, cell-absorbable soybean polypeptide nanoparticles were prepared by covalent cross-linking with genipin. By combining bio-cross-linking agents to regulate particle stiffness, nanoparticles with high safety and good biocompatibility were prepared.
Nanoparticles with high viscosity and high cell absorption performance have been developed, which improves the bioavailability of functional active factors or drugs. The preparation process is simple, conforms to the concept of green processing, and is suitable for industrial production.
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Figure CN117159478B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of functional nanobioproducts, specifically to a high-viscosity-permeability-cell-absorption type soybean polypeptide nanoparticle, a formulation containing the nanoparticle, its preparation method, and its application. Background Technology
[0002] With the development of nanotechnology, nanocarriers have shown great potential in the delivery of functional factors and drugs. Compared with unencapsulated functional factors or drug molecules, nanocarriers can improve the solubility, physiological stability, and absorption and utilization of poorly soluble small molecules, thereby enhancing therapeutic efficacy while reducing oral dosage and minimizing adverse reactions and toxic side effects. Furthermore, nanofunctional factors and nanomedicines exhibit long circulation and good stability in the bloodstream, making them suitable for targeted accumulation and controlled release in in vivo tissues. However, numerous in vitro and in vivo studies have shown that the bioavailability of functional factors or drugs delivered orally via nanocarriers remains limited. In fact, orally administered substances must first penetrate the mucus layer to reach intestinal epithelial cells for absorption through the small intestine, and then be absorbed and transported by the intestinal epithelial cells to effectively enter systemic circulation. Therefore, designing and developing nanocarriers that overcome the mucus barrier and intestinal cell barrier is crucial for improving the bioavailability of loaded functional factors / drugs.
[0003] The mucus layer is a porous network gel structure formed by mucins, exhibiting an electronegative surface. Recent in-depth studies on the structure and properties of the intestinal mucus layer have revealed that regulating the physicochemical properties of nanocarriers (size, shape, surface potential, and hydrophilicity / hydrophobicity, etc.) is an effective means to improve the intestinal absorption performance of nanocarriers. It has been reported that rod-shaped nanoparticles have higher mucus permeability than spherical nanoparticles, and small-sized (<100 nm) nanocarriers can reduce spatial obstruction by mucus and increase permeability. Particles with virus-like electrically neutral surfaces and hydrophilic polymer coatings can diffuse more quickly by avoiding electrostatic and hydrophobic interactions with mucins. However, due to the lipophilicity and electronegativity of cell phospholipid membranes, the physicochemical properties required for effective permeation of nanocarriers into the mucus layer are generally unfavorable for cellular absorption. For example, particles with a high aspect ratio exhibit a faster internalization rate due to their larger surface area in contact with the cell membrane, while extremely small nanocarriers (2-10 nm) cannot be effectively endocytosed. Nanocarriers with positively charged and hydrophobic surfaces are more conducive to their interaction with the cell membrane and are more easily internalized by epithelial cells. Therefore, overcoming both the mucus barrier and the intestinal epithelial cell barrier has become a major challenge in designing delivery systems that target intestinal absorption or target tissues covered with bio-hydrogels.
[0004] In recent years, zwitterionic nanocarriers containing both anionic and cationic groups have demonstrated excellent mucus permeability, cellular uptake, and transepithelial transport efficiency both in vitro and in vivo due to their superhydrophilicity and superior biomimetic properties, opening up new prospects for the field of nanomedicine delivery. Furthermore, the stiffness of nanocarriers has recently been found to play an important role in their physiological performance. Studies have shown that soft particles have better mucus penetration efficiency due to their flexibility in movement within the mucus layer, while hard nanoparticles exhibit rapid endocytosis due to their smaller deformation and lower energy requirements for endocytosis. However, currently reported zwitterionic polymer carriers are mostly chemically synthesized inorganic materials, which greatly limits their application in functional foods, special medical foods, and biopharmaceuticals. Moreover, the carrier preparation process is complex, involves hazardous organic chemicals, and has low efficiency. While the stiffness of carriers is often controlled using chemical cross-linking agents such as glutaraldehyde, these are unsuitable for use in the food industry due to their potential toxicity. Therefore, the preparation of highly mucus-permeable-cellularly-absorbable carriers by controlling stiffness through other methods remains in the exploratory stage. Inspired by the surface properties of zwitterionic particles, we believe that protein-derived peptide structures, as natural zwitterionic electrolytes, may be potential materials for constructing nanocarriers to overcome mucus and epithelial barriers. Based on this, further modification of the carrier structure by combining bio-based crosslinking agents may enable the green, large-scale, and efficient preparation of high-mucus-permeability-cell-absorption nanocarrier particles. Summary of the Invention
[0005] To overcome the aforementioned shortcomings of existing technologies, the present invention aims to provide a high-viscosity-penetration-cell-absorption soybean polypeptide nanoparticle, its preparation method, and its application. This method, guided by mucus penetration and cell absorption efficiency, prepares a high-viscosity-penetration-cell-absorption soybean polypeptide nanoparticle by fractional hydrolysis of soybean protein using protease and alkali methods, coupled with genipin covalent cross-linking.
[0006] The primary objective of this invention is to prepare nanoparticles with high mucus permeability and high cell absorption properties, and to provide a method for preparing high-mucus permeability-cell absorption soybean polypeptide nanoparticles that are safe, biocompatible, and suitable for use in food systems.
[0007] Another object of the present invention is to provide high-viscosity-penetration-cell-absorption soybean polypeptide nanoparticles prepared by the above method.
[0008] Another object of the present invention is to provide the application of the above-mentioned high viscosity-permeability-cell absorption type soybean polypeptide nanoparticles in the encapsulation and delivery of functional active factors or drugs.
[0009] The objective of this invention is achieved by at least one of the following technical solutions.
[0010] A method for preparing highly viscous-permeable, cell-absorbable soybean polypeptide nanoparticles includes the following steps:
[0011] (1) Enzymatic hydrolysis: Soy protein isolate is mixed with water to obtain a protein dispersion. After adjusting the pH of the protein dispersion, pepsin and trypsin are added in sequence. The mixture is stirred at a constant temperature for segmental enzymatic hydrolysis. After the enzymatic hydrolysis reaction is completed, the enzyme is inactivated and centrifuged to obtain soybean protein hydrolysate.
[0012] (2) Alkaline hydrolysis: Adjust the pH of the soybean protein hydrolysate obtained in step (1) to alkaline, and after complete alkaline hydrolysis under constant stirring, adjust it back to neutral and centrifuge to obtain soybean polypeptide dispersion.
[0013] (3) Genipin crosslinking: Genipin was added to the soybean polypeptide dispersion obtained in step (2), and the mixture was reacted for a certain time under constant temperature conditions. The supernatant was then collected by centrifugation.
[0014] (4) Dialysis and freeze-drying: The supernatant obtained in step (3) is dialyzed to obtain soybean polypeptide nanoparticles, namely the high viscosity permeability-cell absorption type soybean polypeptide nanoparticles, which can be freeze-dried to obtain nanoparticle powder.
[0015] Further, in step (1), the mass ratio of soy protein isolate to water in the protein dispersion is 20-60 g / L.
[0016] Further, in step (1), the segmented enzymatic hydrolysis specifically involves: adjusting the pH of the protein dispersion to 2-2.5, adding pepsin for 1-2 hours of enzymatic hydrolysis, then adjusting the pH to 7.0-7.5, adding trypsin and continuing enzymatic hydrolysis for 2-4 hours; the mass of both pepsin and trypsin added is 0.25%-0.75% of the soybean protein isolate, the hydrolysis temperature is 35-40℃, and the stirring rate is 120-180 rpm.
[0017] Further, in step (1), the enzyme inactivation treatment is performed by heating the enzyme, with a heat treatment temperature of 70-75°C and a time of 20-30 min.
[0018] Further, in step (2), the pH of the soybean protein hydrolysate is adjusted to 11.5-12.5, the incubation time is 1.5-2.5h, and then the pH is adjusted back to 7.0-7.5, and the stirring rate is 120-180rpm.
[0019] Further, in step (3), the mass of genipin added is 2% to 10% (w / w) of the protein content in the soybean polypeptide dispersion obtained in step (2), the reaction temperature is 45 to 55°C, and the reaction time is 4 to 12 h.
[0020] Furthermore, in steps (1) to (3), the centrifugation is carried out at a rate of 10,000 to 12,000 g, at a temperature of 4 to 8 °C, and for a time of 20 to 30 min.
[0021] Further, in step (4), the dialysis involves placing the supernatant after centrifugation in step (3) into a dialysis bag with a molecular weight cutoff of 2000-3500 Da, and dialyzing it in deionized water at a temperature of 4-8°C for 24-36 hours, changing the water every 6-10 hours. The soybean polypeptide nanoparticles are the sample in the dialysis bag.
[0022] Furthermore, in step (4), the freeze-drying temperature is -40 to -50°C, the freeze-drying vacuum degree is less than 1 mbar, and the freeze-drying time is 20 to 30 hours.
[0023] This invention provides a high-viscosity, cell-absorbable soybean polypeptide nanoparticle prepared by the above-described method.
[0024] The high-viscosity-penetration-cell-absorption soybean polypeptide nanoparticles provided by this invention can be applied to the encapsulation and delivery of functional active factors / drugs. Due to their combination of good mucosity penetration efficiency and good cell absorption performance, they further improve the bioavailability of functional active factors / drugs, providing certain theoretical and technical support for their application in novel functional foods, special medical foods and biomedicine.
[0025] This invention aims to emphasize the integrity of the process. Only soybean polypeptide nanoparticles prepared in strict accordance with the process method of this invention can have the advantages of both high mucus penetration and high cell absorption performance.
[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0027] (1) The method for preparing high-viscosity-permeability-cell absorption type soybean polypeptide nanoparticles provided by the present invention involves enzymatic hydrolysis of soybean protein to extend the structure and expose hydrophobic groups. The exposed hydrophobic groups are removed by a large amount of aggregation and precipitation during the heat inactivation of enzymes. Further alkaline hydrolysis fully degrades the protein subunits into polypeptides, achieving efficient preparation of hydrophilic polypeptides. Finally, the particle stiffness is controlled by covalent cross-linking with the biological cross-linking agent genipin. Soybean polypeptide nanoparticles with high viscosity-permeability and high cell absorption efficiency are successfully prepared. They can be used as nanocarriers for the encapsulation and delivery of functional active factors / drug molecules to improve their bioavailability.
[0028] (2) The method for preparing high viscosity-permeability-cell absorption type soybean polypeptide nanoparticles provided by the present invention has safe and healthy raw materials with good biocompatibility, simple preparation process, easy industrial production, no toxic and harmful reagents involved in the preparation process, which is in line with the concept of green processing and has good development and application prospects. Attached Figure Description
[0029] Figure 1 The particle size distribution diagrams are shown for the four types of soybean polypeptide nanoparticles prepared in Examples 1-3 and Comparative Examples 1-5 of this invention.
[0030] Figure 2 Atomic force microscopy images of the four soybean polypeptide nanoparticles prepared in Examples 1-3 and Comparative Examples 1-5 of this invention.
[0031] Figure 3 The above-mentioned bar chart shows the apparent permeability coefficients of four soybean polypeptide nanoparticles prepared in intestinal mucus in Examples 1-3 and Comparative Examples 1, 3, 4, and 5 of this invention.
[0032] Figure 4 The bar chart shows the cellular uptake of four soybean polypeptide nanoparticles prepared in intestinal epithelial cells in embodiments 1-3 and comparative examples 1, 3, 4, and 5 of this invention. Detailed Implementation
[0033] The following examples further illustrate specific implementations of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.
[0034] Example 1
[0035] A method for preparing highly viscous-permeable, cell-absorbable soybean polypeptide nanoparticles includes the following steps:
[0036] (1) Enzymatic hydrolysis: Soy protein isolate was mixed with water to obtain a protein dispersion with a mass-to-volume ratio of 20 g / L of soy protein isolate to water. The pH of the protein dispersion was adjusted to 2.5, and pepsin was added for 1 h of enzymatic hydrolysis. The mass of pepsin was 0.75% (w / w) of the soy protein isolate. The pH of the hydrolysis solution was adjusted to 7.5, and trypsin was added for 2 h of further enzymatic hydrolysis. The mass of trypsin was 0.75% (w / w) of the soy protein isolate. The reaction temperature was 35℃ and the stirring rate was 120 rpm throughout the entire hydrolysis process. Then, the enzyme was heat-inactivated at 70℃ for 20 min, and centrifuged (centrifugation rate 10000g, centrifugation temperature 8℃, centrifugation time 20 min) to obtain the soy protein hydrolysate.
[0037] (2) Alkaline hydrolysis: Adjust the pH of the soybean protein hydrolysate from step (1) to 11.5, stir and incubate at this pH for 1.5 h at a stirring rate of 120 rpm, then adjust the pH of the reaction solution back to 7.0, centrifuge and take the supernatant (centrifugation rate of 10000 g, centrifugation temperature of 8℃, centrifugation time of 20 min) to obtain soybean polypeptide dispersion.
[0038] (3) Genipin crosslinking: Genipin is added to the soybean polypeptide dispersion described in step (2). The mass of genipin added is 2% of the protein mass in the soybean polypeptide dispersion. The mixture is reacted at a constant temperature of 45°C for 6 hours. After the reaction is completed, the supernatant is centrifuged and centrifuged at 10000g for 20 minutes at 4°C.
[0039] (4) Dialysis and freeze-drying: The supernatant from centrifugation in step (3) was placed into a dialysis bag with a molecular weight cutoff of 2000 Da and dialyzed in deionized water at 8°C for 24 hours, with the water changed every 8 hours. The sample from the dialysis bag was collected to obtain soybean polypeptide nanoparticles. Freeze-drying (freeze-drying temperature was -40°C, freeze-drying vacuum degree was less than 1 mbar, and freeze-drying time was 30 hours) yielded the high-viscosity permeability-cell absorption type soybean polypeptide nanoparticle powder product. The sample prepared in this example was named SPNPs-G1, where SPNPs is an abbreviation for soy protein nanoparticles.
[0040] Example 2
[0041] A method for preparing highly viscous-permeable, cell-absorbable soybean polypeptide nanoparticles includes the following steps:
[0042] (1) Enzymatic hydrolysis: Soy protein isolate was mixed with water to obtain a protein dispersion with a mass-to-volume ratio of 40 g / L of soy protein isolate to water. The pH of the protein dispersion was adjusted to 2.3, and pepsin was added for 1.5 h of enzymatic hydrolysis. The mass of pepsin was 0.5% (w / w) of the soy protein isolate. The pH of the hydrolysis solution was adjusted to 7.0, and trypsin was added for 3 h of further enzymatic hydrolysis. The mass of trypsin was 0.5% (w / w) of the soy protein isolate. The reaction temperature was 37℃ and the stirring rate was 150 rpm throughout the entire hydrolysis process. Then, the enzyme was heat-inactivated at 73℃ for 25 min, and centrifuged (centrifugation rate 11000 g, centrifugation temperature 6℃, centrifugation time 25 min) to obtain the soy protein hydrolysate.
[0043] (2) Alkaline hydrolysis: Adjust the pH of the soybean protein hydrolysate from step (1) to 12.0, stir and incubate at this pH for 2 hours at a stirring rate of 150 rpm, then adjust the pH of the reaction solution back to 7.3, centrifuge and take the supernatant (centrifugation rate of 11000g, centrifugation temperature of 6℃, centrifugation time of 25min) to obtain the soybean polypeptide dispersion.
[0044] (3) Genipin crosslinking: Genipin is added to the soybean polypeptide dispersion described in step (2). The mass of genipin added is 5% of the protein mass in the soybean polypeptide dispersion. The mixture is reacted at 50°C for 4 hours. After the reaction is completed, the supernatant is centrifuged and centrifuged at 11000g for 25 minutes at 6°C.
[0045] (4) Dialysis and freeze-drying: The supernatant from centrifugation in step (3) was placed into a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in deionized water at 4°C for 30 h, with the water changed every 6 h. The sample in the dialysis bag was collected to obtain soybean polypeptide nanoparticles. Freeze-drying (freeze-drying temperature was -50°C, freeze-drying vacuum degree was less than 1 mbar, and freeze-drying time was 24 h) yielded the high-viscosity permeability-cell absorption type soybean polypeptide nanoparticle powder product. The sample prepared in this example was named SPNPs-G2, where SPNPs is an abbreviation for soy protein nanoparticles.
[0046] Example 3
[0047] A method for preparing highly viscous-permeable, cell-absorbable soybean polypeptide nanoparticles includes the following steps:
[0048] (1) Enzymatic hydrolysis: Soy protein isolate was mixed with water to obtain a protein dispersion with a mass-to-volume ratio of 60 g / L of soy protein isolate to water. The pH of the protein dispersion was adjusted to 2.0, and pepsin was added for 2 hours of enzymatic hydrolysis. The mass of pepsin was 0.25% (w / w) of the soy protein isolate. The pH of the hydrolysis solution was adjusted to 7.3, and trypsin was added for 4 hours of further enzymatic hydrolysis. The mass of trypsin was 0.25% (w / w) of the soy protein isolate. The reaction temperature was 40℃ and the stirring rate was 180 rpm throughout the entire hydrolysis process. Then, the enzyme was heat-inactivated at 75℃ for 30 minutes, followed by centrifugation (centrifugation rate 12000g, centrifugation temperature 4℃, centrifugation time 30 minutes) to obtain the soy protein hydrolysate.
[0049] (2) Alkaline hydrolysis: Adjust the pH of the soybean protein hydrolysate from step (1) to 12.5, stir and incubate at this pH for 2.5 h at a stirring rate of 180 rpm, then adjust the pH of the reaction solution back to 7.5, centrifuge and take the supernatant (centrifugation rate of 12000 g, centrifugation temperature of 4℃, centrifugation time of 30 min) to obtain the soybean polypeptide dispersion.
[0050] (3) Genipin crosslinking: Genipin is added to the soybean polypeptide dispersion described in step (2). The mass of genipin added is 10% of the protein mass in the soybean polypeptide dispersion. The reaction is carried out at 55℃ for 12 hours. After the reaction is completed, the supernatant is centrifuged and centrifuged at 12000g for 30 minutes at 4℃.
[0051] (4) Dialysis and freeze-drying: The supernatant from centrifugation in step (3) was placed into a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis was performed in deionized water at 6°C for 36 hours, with the water changed every 6 hours. The sample from the dialysis bag was collected to obtain soybean polypeptide nanoparticles. Freeze-drying (freeze-drying temperature was -50°C, freeze-drying vacuum degree was less than 1 mbar, and freeze-drying time was 30 hours) yielded the high-viscosity permeability-cell absorption type soybean polypeptide nanoparticle powder product. The sample prepared in this example was named SPNPs-G3, where SPNPs is an abbreviation for soy protein nanoparticles.
[0052] Comparative Example 1
[0053] A method for preparing soybean polypeptide nanoparticles specifically includes the following steps:
[0054] (1) Enzymatic hydrolysis: Soy protein isolate was mixed with water to obtain a protein dispersion with a mass-to-volume ratio of 40 g / L of soy protein isolate to water. The pH of the protein dispersion was adjusted to 2.3, and pepsin was added for 1.5 h of enzymatic hydrolysis. The mass of pepsin was 0.5% (w / w) of the soy protein isolate. The pH of the hydrolysis solution was adjusted to 7.0, and trypsin was added for 3 h of further enzymatic hydrolysis. The mass of trypsin was 0.5% (w / w) of the soy protein isolate. The reaction temperature was 37℃ and the stirring rate was 150 rpm throughout the entire hydrolysis process. Then, the enzyme was heat-inactivated at 73℃ for 25 min, and centrifuged (centrifugation rate 11000 g, centrifugation temperature 6℃, centrifugation time 25 min) to obtain the soy protein hydrolysate.
[0055] (2) Alkaline hydrolysis: Adjust the pH of the soybean protein hydrolysate from step (1) to 12.0, and incubate at this pH with stirring for 2 hours at a stirring rate of 150 rpm. Then, adjust the pH of the reaction solution back to 7.3, centrifuge and collect the supernatant (centrifugation rate 11000g, centrifugation temperature 6℃, centrifugation time 25min) to obtain the soybean polypeptide dispersion. Centrifuge and collect the supernatant. The resulting soybean protein nanoparticles, after drying, are used to prepare a sample named SPNPs-1.
[0056] Comparative Example 2
[0057] (1) Mix soy protein isolate with water to obtain a protein dispersion. The mass ratio of soy protein isolate to water in the protein dispersion is 40 g / L. Add genipin to the soy protein dispersion. The mass of genipin added is 5% of the protein mass in the soy protein dispersion. Stir at 150 rpm at 50°C for 4 h. After the reaction is completed, centrifuge the supernatant and centrifuge at 11000 g for 25 min at 6°C.
[0058] (2) The supernatant from centrifugation in step (1) was placed into a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in deionized water at 4°C for 30 h, with the water changed every 6 h. The sample in the dialysis bag was collected to obtain soybean polypeptide nanoparticles. These were then freeze-dried (at -50°C, with a vacuum degree of less than 1 mbar, and for 24 h) to obtain genipin-crosslinked modified soybean protein particle powder. The sample prepared in this comparative example was named SPNPs-2.
[0059] Comparative Example 3
[0060] (1) Enzymatic hydrolysis: Soy protein isolate was mixed with water to obtain a protein dispersion with a mass-to-volume ratio of 40 g / L of soy protein isolate to water. The pH of the protein dispersion was adjusted to 2.3, and pepsin was added for 1.5 h of enzymatic hydrolysis. The mass of pepsin was 0.5% (w / w) of the soy protein isolate. The pH of the hydrolysis solution was adjusted to 7.0, and trypsin was added for 3 h of further enzymatic hydrolysis. The mass of trypsin was 0.5% (w / w) of the soy protein isolate. The reaction temperature was 37℃ and the stirring rate was 150 rpm throughout the entire hydrolysis process. Then, the enzyme was heat-inactivated at 73℃ for 25 min, and centrifuged (centrifugation rate 11000 g, centrifugation temperature 6℃, centrifugation time 25 min) to obtain the soy protein hydrolysate.
[0061] (2) Alkaline hydrolysis: Adjust the pH of the soybean protein hydrolysate from step (1) to 12.0, stir and incubate at this pH for 2 hours at a stirring rate of 150 rpm, then adjust the pH of the reaction solution back to 7.3, centrifuge and take the supernatant (centrifugation rate of 11000g, centrifugation temperature of 6℃, centrifugation time of 25min) to obtain the soybean polypeptide dispersion.
[0062] (3) Genipin crosslinking: Genipin is added to the soybean polypeptide dispersion described in step (2). The mass of genipin added is 0.5% of the protein mass in the soybean polypeptide dispersion. The mixture is stirred at 150 rpm at 50°C for 12 h. After the reaction is completed, the supernatant is centrifuged and centrifuged at 11000 g at 6°C for 25 min.
[0063] (4) Dialysis and freeze-drying: The supernatant from centrifugation in step (3) was placed in a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in deionized water at 4°C for 30 h, with the water changed every 6 h. The sample in the dialysis bag was collected to obtain soybean polypeptide nanoparticles. Freeze-drying (freeze-drying temperature was -50°C, freeze-drying vacuum degree was less than 1 mbar, and freeze-drying time was 24 h) yielded genipin crosslinked modified soybean polypeptide nanoparticle powder. The sample prepared in this comparative example was named SPNPs-3.
[0064] Comparative Example 4
[0065] (1) Enzymatic hydrolysis: Soy protein isolate was mixed with water to obtain a protein dispersion with a mass-to-volume ratio of 40 g / L of soy protein isolate to water. The pH of the protein dispersion was adjusted to 2.3, and pepsin was added for 1.5 h of enzymatic hydrolysis. The mass of pepsin was 0.5% (w / w) of the soy protein isolate. The pH of the hydrolysis solution was adjusted to 7.0, and trypsin was added for 3 h of further enzymatic hydrolysis. The mass of trypsin was 0.5% (w / w) of the soy protein isolate. The reaction temperature was 37℃ and the stirring rate was 150 rpm throughout the entire hydrolysis process. Then, the enzyme was heat-inactivated at 73℃ for 25 min, and centrifuged (centrifugation rate 11000 g, centrifugation temperature 6℃, centrifugation time 25 min) to obtain the soy protein hydrolysate.
[0066] (2) Alkaline hydrolysis: Adjust the pH of the soybean protein hydrolysate from step (1) to 12.0, stir and incubate at this pH for 2 hours at a stirring rate of 150 rpm, then adjust the pH of the reaction solution back to 7.3, centrifuge and take the supernatant (centrifugation rate of 11000g, centrifugation temperature of 6℃, centrifugation time of 25min) to obtain the soybean polypeptide dispersion.
[0067] (3) Genipin crosslinking: Genipin is added to the soybean polypeptide dispersion described in step (2). The mass of genipin added is 15% of the protein mass in the soybean polypeptide dispersion. The mixture is stirred at 150 rpm at 50°C for 12 h. After the reaction is completed, the supernatant is centrifuged and centrifuged at 11000 g at 6°C for 25 min.
[0068] (4) The supernatant from centrifugation in step (3) was placed into a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in deionized water at 4°C for 24 h, with the water changed every 6 h. The sample in the dialysis bag was collected to obtain soybean polypeptide nanoparticles 2. The nanoparticles were then freeze-dried (at a temperature of -40°C, a vacuum degree of less than 1 mbar, and a time of 36 h) to obtain genipin-crosslinked modified soybean polypeptide nanoparticle powder. The sample prepared in this comparative example was named SPNPs-4.
[0069] Comparative Example 5
[0070] (1) Enzymatic hydrolysis: Soy protein isolate was mixed with water to obtain a protein dispersion with a mass-to-volume ratio of 40 g / L of soy protein isolate to water. The pH of the protein dispersion was adjusted to 2.3, and pepsin was added for 1.5 h of enzymatic hydrolysis. The mass of pepsin was 0.5% (w / w) of the soy protein isolate. The pH of the hydrolysis solution was adjusted to 7.0, and trypsin was added for 3 h of further enzymatic hydrolysis. The mass of trypsin was 0.5% (w / w) of the soy protein isolate. The reaction temperature was 37℃ and the stirring rate was 150 rpm throughout the entire hydrolysis process. Then, the enzyme was heat-inactivated at 73℃ for 25 min, and centrifuged (centrifugation rate 11000 g, centrifugation temperature 6℃, centrifugation time 25 min) to obtain the soy protein hydrolysate.
[0071] (2) Alkaline hydrolysis: Adjust the pH of the soybean protein hydrolysate from step (1) to 12.0, stir and incubate at this pH for 2 hours at a stirring rate of 150 rpm, then adjust the pH of the reaction solution back to 7.3, centrifuge and take the supernatant (centrifugation rate of 11000g, centrifugation temperature of 6℃, centrifugation time of 25min) to obtain the soybean polypeptide dispersion.
[0072] (3) Genipin crosslinking: Genipin is added to the soybean polypeptide dispersion described in step (2). The mass of genipin added is 2.5% of the protein mass in the soybean polypeptide dispersion. The mixture is stirred at 150 rpm at 40°C for 1 hour. After the reaction is completed, the supernatant is centrifuged and centrifuged at 11000 g at 6°C for 25 minutes.
[0073] (4) Dialysis and freeze-drying: The supernatant from centrifugation in step (3) was placed in a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in deionized water at 4°C for 30 h, with the water changed every 6 h. The sample in the dialysis bag was collected to obtain soybean polypeptide nanoparticles. Freeze-drying (freeze-drying temperature was -50°C, freeze-drying vacuum degree was less than 1 mbar, freeze-drying time was 24 h) yielded the genipin crosslinked modified soybean polypeptide nanoparticle powder product. The sample prepared in this comparative example was named SPNPs-5.
[0074] The soybean polypeptide nanoparticles prepared in Examples 1-3 and Comparative Examples 1-5 were analyzed as follows.
[0075] Particle size, polymer dispersibility index (PDI), surface potential, and stiffness were determined: The average particle size, PDI (Polydispersity Index), and surface potential of the prepared mucilage-permeable soybean polypeptide nanoparticles were measured using a Malvern Nano-ZS nanoparticle size analyzer. Force curves of the particles were measured using atomic force microscopy, and Young's modulus was obtained by fitting the curves. The Young's modulus value represents the stiffness of the particles. The results are shown in Table 1, and the particle size distribution diagram is shown below. Figure 1 As shown.
[0076] Morphological observation and analysis: The dried nanoparticle powder was dispersed in water, dropped onto a mica sheet, and dried thoroughly. The morphology was then observed using an atomic force microscope. The results are as follows: Figure 2 As shown in the figure, the nanoparticles are all spherical structures. The SPNPs-G1, SPNPs-G2, and SPNPs-G3 soybean peptide nanoparticles prepared in the examples have uniform size and good dispersibility.
[0077] Mucus permeability study: Intestinal mucus was extracted from freshly slaughtered pig small intestine and frozen for later use. Nanoparticles were fluorescently labeled with FITC (Fluorescein Isothiocyanate). The permeability of the prepared protein nanoparticles in intestinal mucus was then studied using a Transwell chamber model. Fluorescence intensity was measured and quantified at 1 hour, and the apparent permeability coefficient P was calculated. app The result is as follows Figure 3 As shown.
[0078] Cellular uptake efficiency study: Caco-2 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1.1% penicillin-streptomycin. After counting the logarithmic-phase cells, they were incubated at 2.5 × 10⁻⁶ cells per cell line. 5FITC-labeled soybean protein / soybean peptide nanoparticles were seeded at a concentration of 1 / mL in 24-well plates and cultured for 21 days to differentiate and construct an intestinal epithelial monolayer cell model. The medium was changed every 2 days during culture. After cell differentiation, the cells were used for quantification of nanoparticle uptake. The culture medium was discarded, and the cells were carefully washed twice with PBS. The FITC-labeled soybean protein / soybean peptide nanoparticles were diluted to 0.5 mg / mL in serum-free DMEM medium and added to the wells. After 4 hours, the cells were lysed, and the intracellular uptake of nanoparticles was quantified using fluorescence. The cell uptake was expressed as μg SPNPs / mg protein, and the protein content in the cell lysate was determined by the BCA method.
[0079] Results Analysis
[0080] 1. Particle size, zeta potential, and morphology characterization
[0081] Table 1
[0082]
[0083]
[0084] As shown in Table 1, the average particle size of the mucus-penetrating-cell-absorption soybean polypeptide nanoparticles prepared in Examples 1-3 was approximately 60 nm, with a PDI of less than 0.3 and a surface potential of approximately -20 mV. The sample prepared in Comparative Example 2 had a particle size of approximately 167 nm, a PDI greater than 0.3, and a surface potential of -35 mV. The soybean polypeptide particles prepared in Comparative Examples 1, 3, 4, and 5 showed no significant differences in particle size, PDI, and surface potential compared to the examples.
[0085] like Figure 1 and Figure 2As shown, the particle size distribution of the mucus-penetrating-cell-absorption soybean polypeptide nanoparticles prepared in Examples 1-3 is a single-peak distribution, with uniform spherical morphology. Compared with the soybean polypeptide particles prepared in the examples, the particle size distribution of the unhydrolyzed sample in Comparative Example 2 is bimodal, exhibiting large aggregates, indicating that direct cross-linking without hydrolysis cannot form uniform monodisperse nanoparticles. The samples prepared in Comparative Examples 1, 3, 4, and 5 show no significant new differences in particle size, potential, and morphology, but the Young's modulus values of the comparative examples differ significantly from those of the examples. Among them, the particles in Comparative Example 1, which are not cross-linked with genipin, have a low Young's modulus value, indicating that the particles have low stiffness and high flexibility. Compared with Examples 1-3, the genipin cross-linking reaction conditions (genipin addition amount, reaction temperature, and time, etc.) in the preparation of nanoparticles in Comparative Examples 3, 4, and 5 exceed the range required by this invention, and the Young's modulus of the prepared particles is either too low or too high, failing to meet the requirements of this invention. These results indicate that hydrolysis combined with genipin crosslinking is an essential condition for the formation of the aforementioned high-viscosity, cell-absorbable soybean peptide nanoparticles; neither can be omitted. Furthermore, genipin crosslinking enables the regulation of the stiffness of the soybean peptide nanoparticles, with the genipin crosslinking reaction conditions having a significant impact on particle stiffness.
[0086] 2. Comparison of mucus permeability and cell absorption efficiency of soybean polypeptide nanoparticles
[0087] In Comparative Example 1, the soybean polypeptide nanoparticles were obtained without genipin crosslinking. The methods in Comparative Examples 3 and 4 all belong to the preparation methods of mucus penetration-cell absorption type soybean polypeptide nanoparticles, but the amount of genipin added and the reaction conditions during the preparation process are not within the scope of protection of this invention.
[0088] Depend on Figure 3 and Figure 4 ( Figure 3 , Figure 4 Different letters indicate significant differences between groups (p < 0.05). It can be seen that, compared with the uncrosslinked soybean polypeptide nanoparticles SPNPs-1 in Comparative Example 1, the soybean polypeptide nanoparticles (SPNPs-G1, SPNPs-G2, SPNPs-G3) obtained according to the preparation method of this invention all showed significantly improved apparent permeability coefficients of the mucus layer and cell uptake efficiency (p < 0.05). Among them, the P in the mucus layer... app The value can reach up to 12×10 -6 cm / s, for SPNPs-1 mucus layer P app It is 3.2 times the value, and the cellular uptake efficiency can reach up to 340 μg SPNPs / mg protein, which is about 6.0 times the uptake of SPNPs-1 cells.
[0089] Furthermore, compared to Comparative Example 1, although the soybean peptide nanoparticles in Comparative Example 3 showed some improvement in both mucus penetration and cellular uptake efficiency, the effect was far inferior to that of Examples 1-3. The soybean peptide nanoparticles in Comparative Example 4 only showed a significant improvement in cellular uptake efficiency, but their mucus permeability did not change significantly. The soybean peptide nanoparticles in Comparative Example 5 showed no significant improvement in either mucus penetration or cellular uptake efficiency. Therefore, the soybean peptide nanoparticles in Comparative Examples 3, 4, and 5 do not belong to the high mucus penetration-cellular absorption type of soybean peptide nanoparticles.
[0090] In summary, this invention provides a method for improving the mucus permeability and cellular absorption efficiency of soybean polypeptide nanoparticles. This preparation method significantly improves the mucus permeability and cellular absorption efficiency of soybean nanoparticles based on the aforementioned patent. The optimized preparation method yields soybean polypeptide nanoparticles with both high mucus permeability and high cellular absorption performance. The required processing equipment is simple to operate, complies with green processing standards, and has the potential for industrial-scale application.
[0091] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, combinations, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.
Claims
1. A method for preparing high-viscosity-permeable-cell-absorption soybean polypeptide nanoparticles, characterized in that, Includes the following steps: (1) Enzymatic hydrolysis: Soy protein isolate is mixed with water to obtain a protein dispersion, wherein the mass ratio of soy protein isolate to water in the protein dispersion is 20~60 g / L; the pH of the protein dispersion is adjusted to 2~2.5, pepsin is added for 1~2 h of enzymatic hydrolysis, then the pH is adjusted to 7.0~7.5, and trypsin is added for 2~4 h of enzymatic hydrolysis; the mass of pepsin and trypsin added is 0.25%~0.75% of the soy protein isolate, the hydrolysis temperature is 35~40℃, and the stirring rate is 120~180 rpm; after the enzymatic hydrolysis reaction is completed, enzyme inactivation is performed by heating enzyme inactivation at a temperature of 70~75℃ for 20~30 min; the soy protein hydrolysate is obtained by centrifugation. (2) Alkaline hydrolysis: Adjust the pH of the soybean protein hydrolysate obtained in step (1) to 11.5~12.5, and perform alkaline hydrolysis for 1.5-2.5 hours under constant stirring. Then adjust the pH back to 7.0~7.5 and stir at 120~180 rpm. Centrifuge to obtain soybean polypeptide dispersion. (3) Genipin crosslinking: Genipin is added to the soybean polypeptide dispersion obtained in step (2). The mass of genipin added is 2%~10% (w / w) of the protein content in the soybean polypeptide dispersion obtained in step (2). The mixture is reacted at a constant temperature of 45~55℃ for 4~12h, and the supernatant is collected by centrifugation. (4) Dialysis and freeze-drying: The supernatant obtained in step (3) is dialyzed to obtain soybean polypeptide nanoparticles. The dialysis is performed by putting the supernatant after centrifugation in step (3) into a dialysis bag with a molecular weight cutoff of 2000~3500 Da and dialyzing in deionized water at a temperature of 4~8℃ for 24~36h, changing the water every 6~10h. That is, the high viscosity permeability-cell absorption type soybean polypeptide nanoparticles can be freeze-dried to obtain nanoparticle powder. The freeze-drying temperature is -40~-50℃, the freeze-drying vacuum degree is less than 1mbar, and the freeze-drying time is 20~30h. In steps (1) to (3), the centrifugation is carried out at a rate of 10000~12000g, at a temperature of 4~8℃, and for a time of 20~30min.
2. The high-viscosity, cell-absorbable soybean polypeptide nanoparticles prepared by the preparation method according to claim 1.
3. The application of the high viscosity-permeability-cell absorption type soybean polypeptide nanoparticles as described in claim 2 in the preparation of functional active factors or drug encapsulation and delivery carriers.