Epsilon-PL-loaded modified rice bran protein antibacterial nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to epsilon-polylysine epsilon-PL-loaded modified rice bran protein antibacterial nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of preparation of nanoparticles. The preparation method comprises the following steps: extracting rice bran protein from defatted rice bran to prepare a rice bran protein stock solution; under continuous stirring, adding succinic anhydride into the rice bran protein stock solution to obtain acylated rice bran protein; mixing the acylated rice bran protein stock solution, the lactose stock solution and the epsilon-PL stock solution; and centrifuging and freeze-drying to obtain the epsilon-PL-loaded modified rice bran protein antibacterial nanoparticles. The preparation method disclosed by the invention is simple and low in cost, the prepared nanoparticles have smaller particle size and turbidity and higher stability, the hygroscopicity of epsilon-PL can be reduced to a great extent, the bioavailability of epsilon-PL can be improved, and the nanoparticles have an obvious bacteriostatic effect on escherichia coli and staphylococcus aureus.

Description

Technical Field

[0001] This invention belongs to the field of nanoparticle preparation technology, specifically relating to a modified rice bran protein antibacterial nanoparticle loaded with ε-PL, its preparation method, and its application. This invention involves extracting protein from natural rice bran, modifying it, and then encapsulating it with ε-PL to form antibacterial nanoparticles. This improves the encapsulation rate, enhances the stability and bioavailability of ε-PL, and expands its application range in food preservation. Background Technology

[0002] ε-PL possesses broad-spectrum antibacterial activity, inhibiting Gram-positive bacteria, Gram-negative bacteria, fungi, and some other pathogens. Due to its excellent antibacterial activity, good water solubility, high safety, and biodegradability, ε-PL is widely used in the food industry. However, ε-PL is highly hygroscopic and easily absorbs moisture during application; furthermore, its cationic nature readily interacts with anionic components in food, forming insoluble precipitates and reducing its antibacterial performance. These problems severely hinder the application of ε-PL in the food industry.

[0003] Naturally derived biomolecules such as proteins and polysaccharides possess excellent biocompatibility and biodegradability, making them ideal wall materials for delivering bioactive components. They offer unparalleled advantages over synthetic polymers or inorganic materials. Furthermore, protein-polysaccharide complexes, through glycosylation modification, interact with polysaccharides, resulting in superior solubility, emulsification, stability, and antioxidant properties compared to proteins used alone. This makes them the preferred delivery material for bioactive substances. Nanoparticles prepared based on this principle show promising application prospects.

[0004] The invention patent (authorization announcement number: CN202411901436.8, authorization announcement date: March 25, 2025) discloses a protein-polysaccharide ternary composite nanoparticle loaded with cordycepin, its preparation method, and its application. This method uses zein and sodium alginate to combine and load cordycepin into nanoparticles. The resulting nanoparticles are small in size, have good encapsulation effect, high stability, and high bioavailability, achieving a sustained-release effect of cordycepin. However, in existing technologies, the protein structure is compact with few glycosylation binding sites, leading to insufficient reaction between polysaccharides and proteins. This invention first succinates rice bran protein, then mixes it with lactose for glycosylation to obtain a co-modified protein. This co-modified protein is then mixed with an ε-PL solution to prepare ε-PL-loaded modified rice bran protein antibacterial nanoparticles using an efficient and rapid method. The nanoparticles obtained by this method have uniform particle size and excellent dispersibility and stability. The resulting porous structure is more conducive to loading active substances and improving the encapsulation rate. This innovation provides a solid theoretical basis for the application of nanoparticles in the field of food preservation and shows broad application prospects. Summary of the Invention

[0005] This invention provides a method for preparing ε-PL-loaded modified rice bran protein antibacterial nanoparticles. This method not only effectively utilizes abundant rice bran resources, but also endows rice bran protein with better properties through combined modification, significantly improving the encapsulation effect of ε-PL, and has broad application prospects.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a modified rice bran protein antibacterial nanoparticle loaded with ε-PL, the preparation of which includes the following steps:

[0007] Step 1: Prepare rice bran protein;

[0008] Step 2: Dissolve the rice bran protein obtained in Step 1 in deionized water, adjust the pH to alkaline, gradually add succinate to the rapidly stirred rice bran protein RBP suspension, adjust the pH to 8.0, stir to carry out the reaction, and obtain acylated modified rice bran protein RBP-SA after dialyzing and freeze drying.

[0009] Step 3: Dissolve the acylated modified rice bran protein RBP-SA obtained in Step 2 in deionized water to obtain an RBP-SA suspension and a lactose Lac suspension. Mix the RBP-SA suspension and the lactose Lac suspension, stir, adjust the pH to neutral, stir overnight, carry out the heating reaction, cool in an ice-water bath, centrifuge, and freeze-dry the supernatant to obtain acylated co-glycosylated rice bran protein RBP-SA-Lac.

[0010] Step 4: Prepare RBP-SA-Lac solution and ε-polylysine ε-PL solution separately. Mix the RBP-SA-Lac solution and ε-PL solution, adjust the pH of the mixture, heat the reaction, and freeze-dry to obtain modified rice bran protein antibacterial nanoparticles loaded with ε-PL.

[0011] The above-mentioned modified rice bran protein antibacterial nanoparticles loaded with ε-PL are prepared in the following manner in step 1: defatted rice bran is mixed with deionized water, homogenized, stirred, and the pH is adjusted to alkaline. The rice bran residue is removed by filtration, the supernatant is collected by centrifugation, the pH is adjusted to acidic, the mixture is allowed to stand, the supernatant is removed by centrifugation, the precipitate is diluted with deionized water, the pH is adjusted to 7.0, and the rice bran protein is obtained by freeze-drying.

[0012] In step 2 of the above-mentioned modified rice bran protein antibacterial nanoparticles loaded with ε-PL, the amount of succinate added is 5-20% of the amount of rice bran protein.

[0013] In step 3 of the above-mentioned modified rice bran protein antibacterial nanoparticles loaded with ε-PL, the concentration of RBP-SA suspension is 25 mg / ml, the concentration of Lac suspension is 25 mg / ml, and the volume ratio of the two is 1:1.

[0014] In the above-mentioned modified rice bran protein antibacterial nanoparticles loaded with ε-PL, the heating reaction in step 3 is carried out at 60°C for 12 h.

[0015] In step 4 of the above-mentioned modified rice bran protein antibacterial nanoparticles loaded with ε-PL, the concentration of the RBP-SA-Lac solution is 10 mg / ml, the concentration of the ε-PL solution is 0.15~0.35 mg / ml, and the volume ratio of the two is 1:1.

[0016] In step 4 of the above-mentioned modified rice bran protein antibacterial nanoparticles loaded with ε-PL, the pH of the mixed system is adjusted to 4.5-6.5, the temperature of the heating reaction is 40-80℃, and the time is 30-150 min.

[0017] The above-mentioned application of modified rice bran protein antibacterial nanoparticles loaded with ε-PL in the preparation of antibacterial products.

[0018] The beneficial effects of this invention are:

[0019] This invention achieves the following key beneficial effects by preparing ε-PL-loaded antibacterial nanoparticles through combined modification of rice bran protein:

[0020] 1. The combined modification significantly improved the properties of rice bran protein. Acylation increased the glycosylation binding sites, promoting the full reaction between polysaccharides and proteins; the prepared nanoparticles had more uniform particle size and better dispersibility, resulting in higher bioavailability.

[0021] 2. By encapsulating modified rice bran protein, the hygroscopicity is significantly reduced, effectively alleviating the moisture problem caused by the strong hygroscopicity of ε-PL. At the same time, by utilizing the anionic shielding effect of the protein-polysaccharide complex, the interaction between ε-PL and anionic components in food is reduced, avoiding the formation of insoluble precipitates and ensuring the stability of antibacterial properties.

[0022] 3. These nanoparticles can achieve sustained release of ε-PL in the food industry, prolonging the antibacterial effect. At the same time, due to their good stability and biocompatibility, they can be widely used in food preservation and other fields, providing technical support for the efficient utilization of natural antibacterial agents, and have significant economic value and social significance.

[0023] 4. The obtained nanoparticles have a particle size of 150.8–190.6 nm, a PDI of 0.31–0.40, a turbidity of 0.4–0.54, and an encapsulation efficiency of 53%–72%. They exhibit significant antibacterial activity against S. aureus and E. coli. Attached Figure Description

[0024] Figure 1 Figure 1 shows the particle size and PDI results of the modified rice bran protein antibacterial nanoparticles loaded with ε-PL.

[0025] Figure 2 The figure shows the turbidity and encapsulation efficiency of the modified rice bran protein antibacterial nanoparticles loaded with ε-PL.

[0026] Figure 3 The graph shows the moisture absorption rate of modified rice bran protein antibacterial nanoparticles loaded with ε-PL.

[0027] Figure 4 The graph shows the retention rate of ε-PL-loaded modified rice bran protein antibacterial nanoparticles.

[0028] Figure 5 Antibacterial activity test of modified rice bran protein antibacterial nanoparticles loaded with ε-PL.

[0029] Figure 6 The image shows the morphology of the inhibition zones of modified rice bran protein antibacterial nanoparticles loaded with ε-PL against E. coli and S. aureus.

[0030] Figure 7 The flowchart shows the preparation process of modified rice bran protein antibacterial nanoparticles loaded with ε-PL. Detailed Implementation

[0031] Example 1

[0032] A method for preparing ε-PL-loaded modified rice bran protein antibacterial nanoparticles, such as... Figure 7 As shown, the specific steps are as follows:

[0033] (1) Extraction of rice bran protein by alkaline dissolution and acid precipitation: 1 kg defatted rice bran: 9000 ml deionized water, homogenized for 5 min, then magnetically stirred at room temperature for 1 h, pH adjusted to 9.5 with 1 M NaOH solution, magnetically stirred at 55℃ for 2 h, filtered to remove rice bran residue, centrifuged at 10000 r / min at room temperature for 30 min to collect the supernatant, pH adjusted to 4.4 with 1 M HCl solution, allowed to stand for 30 min, centrifuged at 10000 r / min at room temperature for 30 min to remove the supernatant, diluted the precipitate with deionized water and adjusted the pH to 7.0 with 1 M NaOH solution, plated and frozen for 24 h, freeze-dried for 48 h to obtain rice bran protein.

[0034] (2) Preparation of acylated rice bran protein: 2 g of rice bran protein was dissolved in 80 ml of deionized water to form a suspension with a concentration of 25 mg / ml. The pH was adjusted to 8.0 with 2 M NaOH. 0.3 g of succinate (SA) was gradually added to the rapidly stirred rice bran protein (RBP) suspension, and the pH was maintained at 8.0 with 1 M NaOH. After the pH stabilized, the solution was stirred at 25 °C for 1 h to complete the reaction. Finally, the solution was dialyzed thoroughly at 4 °C using a 3500 Da dialysis bag for 24 h, plated and frozen at -18 °C for 24 h, and then freeze-dried in a freeze dryer for 48 h to obtain RBP-SA.

[0035] (3) Preparation of acylated and glycosylated rice bran protein: 2 g of RBP-SA was dissolved in 80 ml of deionized water to form an RBP-SA suspension with a concentration of 25 mg / ml; 2 g of lactose Lac was dissolved in 80 ml of deionized water to prepare a Lac suspension with a concentration of 25 mg / ml. 80 ml of RBP-SA suspension and 80 ml of Lac suspension were mixed and stirred at 25°C for 2 h to ensure thorough mixing. The pH was adjusted to 7.0 and then gently stirred overnight at 4°C. After ensuring complete hydration of the mixture, it was heated in a 60°C water bath for 12 h. After the reaction was completed, it was immediately removed and cooled in an ice-water bath. It was centrifuged at 8000 r / min at 4°C for 20 min to obtain the supernatant. The supernatant was poured onto a plate and frozen in a -18°C freezer for 24 h. Then it was freeze-dried in a freeze dryer for 48 h to obtain RBP-SA-Lac.

[0036] (4) Preparation of ε-PL-loaded modified rice bran protein antibacterial nanoparticles: 1 g of RBP-SA-Lac was dissolved in 100 ml of deionized water to obtain an RBP-SA-Lac solution with a mass concentration of 10 mg / ml; 0.015 g of ε-polylysine ε-PL was dissolved in 100 ml of deionized water to obtain an ε-PL solution with a mass concentration of 0.15 mg / ml; 100 ml of RBP-SA-Lac solution and 100 ml of ε-PL solution were thoroughly mixed, and the pH of the mixture was adjusted to 6 with 1 M NaOH. After the pH stabilized, the mixture was heated in a water bath at 60℃ for 120 min to obtain a ε-PL-loaded modified rice bran protein solution. The solution was then plated and frozen in a -18℃ freezer for 24 h and then freeze-dried in a freeze dryer for 48 h to obtain ε-PL-loaded modified rice bran protein nanoparticles.

[0037] (5) The obtained nanoparticles have a particle size of 173.4 nm, a PDI of 0.38, a turbidity of 0.49, and an encapsulation efficiency of 60%. Figure 2As shown, the encapsulation efficiency of the nanoparticles was significantly improved by 10% compared to the control example obtained by unacylated modified protein glycosylation loaded with antimicrobial peptides, and the turbidity decreased by 0.11% compared to the control example. Figure 3 As shown, the moisture absorption rate of the nanoparticles was significantly improved compared to the free ε-PL, decreasing from 85% to 27.9%.

[0038] Example 2

[0039] A method for preparing ε-PL-loaded modified rice bran protein antibacterial nanoparticles, the specific steps of which are as follows:

[0040] (1) Extraction of rice bran protein by alkaline dissolution and acid precipitation: 1 kg defatted rice bran: 9000 ml deionized water, homogenized for 5 min, then magnetically stirred at room temperature for 1 h, pH adjusted to 9.5 with 1 M NaOH solution, magnetically stirred at 55℃ for 2 h, filtered to remove rice bran residue, centrifuged at 10000 r / min at room temperature for 30 min to collect the supernatant, pH adjusted to 4.4 with 1 M HCl solution, allowed to stand for 30 min, centrifuged at 10000 r / min at room temperature for 30 min to remove the supernatant, diluted the precipitate with deionized water and adjusted the pH to 7.0 with 1 M NaOH solution, plated and frozen for 24 h, freeze-dried for 48 h to obtain rice bran protein.

[0041] (2) Preparation of acylated rice bran protein: 2 g of rice bran protein was dissolved in 80 ml of deionized water to form a suspension with a concentration of 25 mg / ml. The pH was adjusted to 8.0 with 2 M NaOH. 0.3 g of succinate was gradually added to the rapidly stirred RBP suspension, and the pH was maintained at 8.0 with 1 M NaOH. After the pH stabilized, the solution was stirred at 25 °C for 1 h to complete the reaction. Finally, the solution was dialyzed thoroughly at 4 °C using a 3500 Da dialysis bag for 24 h. The plate was then frozen at -18 °C for 24 h and then freeze-dried in a freeze dryer for 48 h to obtain RBP-SA.

[0042] (3) Preparation of acylated and glycosylated rice bran protein: 2 g of RBP-SA was dissolved in 80 ml of deionized water to form an RBP-SA suspension with a concentration of 25 mg / ml; 2 g of lactose was dissolved in 80 ml of deionized water to prepare a Lac suspension with a concentration of 25 mg / ml. 80 ml of RBP-SA suspension and 80 ml of Lac suspension were mixed and stirred at 25°C for 2 h to ensure thorough mixing. The pH was adjusted to 7.0 and then gently stirred overnight at 4°C. After ensuring complete hydration of the mixture, it was heated in a 60°C water bath for 12 h. After the reaction was completed, it was immediately removed and cooled in an ice-water bath. It was centrifuged at 8000 r / min at 4°C for 20 min to obtain the supernatant. The supernatant was poured onto a plate and frozen in a -18°C freezer for 24 h. Then it was freeze-dried in a freeze dryer for 48 h to obtain RBP-SA-Lac.

[0043] (4) Preparation of ε-PL-loaded modified rice bran protein antibacterial nanoparticles: 1 g of RBP-SA-Lac was dissolved in 100 ml of deionized water to obtain an RBP-SA-Lac solution with a mass concentration of 10 mg / ml; 0.025 g of ε-PL was dissolved in 100 ml of deionized water to obtain an ε-PL solution with a mass concentration of 0.25 mg / ml; 100 ml of RBP-SA-Lac solution and 100 ml of ε-PL solution were thoroughly mixed, and the pH of the mixture was adjusted to 4.5 with 1 M HCl. After the pH stabilized, the mixture was heated in a water bath at 60℃ for 120 min to obtain a ε-PL-loaded modified rice bran protein solution. The solution was plated and frozen in a -18℃ freezer for 24 h, and then freeze-dried in a freeze dryer for 48 h to obtain ε-PL-loaded modified rice bran protein antibacterial nanoparticles.

[0044] (5) The obtained nanoparticles had a particle size of 172.63 nm, a PDI of 0.35, a turbidity of 0.54, and an encapsulation efficiency of 53%. Figure 1 As shown, the particle size and PDI of the nanoparticles were significantly lower than those of the control obtained by unacylated modified protein glycosylation loaded with antimicrobial peptides, with a particle size decrease of 50.77 and a PDI decrease of 0.13. Figure 3 As shown, the moisture absorption rate of the nanoparticles was significantly improved compared to the free ε-PL, decreasing from 85% to 30.6%.

[0045] Example 3

[0046] A method for preparing ε-PL-loaded modified rice bran protein antibacterial nanoparticles, the specific steps of which are as follows:

[0047] (1) Extraction of rice bran protein by alkaline dissolution and acid precipitation: 1 kg defatted rice bran: 9000 ml deionized water, homogenized for 5 min, then magnetically stirred at room temperature for 1 h, pH adjusted to 9.5 with 1 M NaOH solution, magnetically stirred at 55℃ for 2 h, filtered to remove rice bran residue, centrifuged at 10000 r / min at room temperature for 30 min to collect the supernatant, pH adjusted to 4.4 with 1 M HCl solution, allowed to stand for 30 min, centrifuged at 10000 r / min at room temperature for 30 min to remove the supernatant, diluted the precipitate with deionized water and adjusted the pH to 7.0 with 1 M NaOH solution, plated and frozen for 24 h, freeze-dried for 48 h to obtain rice bran protein.

[0048] (2) Preparation of acylated rice bran protein: 2 g of rice bran protein was dissolved in 80 ml of deionized water to form a suspension with a concentration of 25 mg / ml. The pH was adjusted to 8.0 with 2 M NaOH. 0.3 g of succinate was gradually added to the rapidly stirred RBP suspension, and the pH was maintained at 8.0 with 1 M NaOH. After the pH stabilized, the solution was stirred at 25 °C for 1 h to complete the reaction. Finally, the solution was dialyzed thoroughly at 4 °C using a 3500 Da dialysis bag for 24 h. The plate was then frozen at -18 °C for 24 h and then freeze-dried in a freeze dryer for 48 h to obtain RBP-SA.

[0049] (3) Preparation of acylated and glycosylated rice bran protein: 2 g of RBP-SA was dissolved in 80 ml of deionized water to form an RBP-SA suspension with a concentration of 25 mg / ml; 2 g of lactose was dissolved in 80 ml of deionized water to prepare a Lac suspension with a concentration of 25 mg / ml. 80 ml of RBP-SA suspension and 80 ml of Lac suspension were mixed and stirred at 25°C for 2 h to ensure thorough mixing. The pH was adjusted to 7.0 and then gently stirred overnight at 4°C. After ensuring complete hydration of the mixture, it was heated in a 60°C water bath for 12 h. After the reaction was completed, it was immediately removed and cooled in an ice-water bath. It was centrifuged at 8000 r / min at 4°C for 20 min to obtain the supernatant. The supernatant was poured onto a plate and frozen in a -18°C freezer for 24 h. Then it was freeze-dried in a freeze dryer for 48 h to obtain RBP-SA-Lac.

[0050] (4) Preparation of ε-PL-loaded modified rice bran protein antibacterial nanoparticles: 1 g of RBP-SA-Lac was dissolved in 100 ml of deionized water to obtain an RBP-SA-Lac solution with a mass concentration of 10 mg / ml; 0.025 g of ε-PL was dissolved in 100 ml of deionized water to obtain an ε-PL solution with a mass concentration of 0.25 mg / ml; 100 ml of RBP-SA-Lac solution and 100 ml of ε-PL solution were thoroughly mixed; the pH of the mixture was adjusted to 6 with 1 M NaOH; after the pH stabilized, the mixture was heated in a water bath at 60℃ for 120 min to obtain a ε-PL-loaded modified rice bran protein solution; the mixture was plated and frozen in a -18℃ freezer for 24 h; and then freeze-dried in a freeze dryer for 48 h to obtain ε-PL-loaded modified rice bran protein antibacterial nanoparticles.

[0051] (5) The obtained nanoparticles have a particle size of 150.8 nm, a PDI of 0.31, a turbidity of 0.4, and an encapsulation efficiency of 72%. Figure 1 , 2 As shown, the nanoparticles prepared under these conditions exhibited optimal performance in terms of particle size, PDI, turbidity, and encapsulation efficiency. Figure 3 As shown, the moisture absorption rate was significantly improved compared to the free ε-PL, decreasing from 85% to 27.1%. Figure 4 As shown, under the same conditions, the retention rate after long-term storage was significantly improved compared to the control example obtained by loading antimicrobial peptides with unacylated modified protein glycosylation. Figure 6 As shown, the diameter of the antibacterial zone of the nanoparticles prepared under these conditions is larger than that of the control example. Furthermore, the antibacterial effect is superior to that of ε-PL in its free state.

[0052] Example 4

[0053] A method for preparing modified rice bran protein antibacterial nanoparticles effectively loaded with ε-PL, the specific steps of which are as follows:

[0054] (1) Extraction of rice bran protein by alkaline dissolution and acid precipitation: 1 kg defatted rice bran: 9000 ml deionized water, homogenized for 5 min, then magnetically stirred at room temperature for 1 h, pH adjusted to 9.5 with 1 M NaOH solution, magnetically stirred at 55℃ for 2 h, filtered to remove rice bran residue, centrifuged at 10000 r / min at room temperature for 30 min to collect the supernatant, pH adjusted to 4.4 with 1 M HCl solution, allowed to stand for 30 min, centrifuged at 10000 r / min at room temperature for 30 min to remove the supernatant, diluted the precipitate with deionized water and adjusted the pH to 7.0 with 1 M NaOH solution, plated and frozen for 24 h, freeze-dried for 48 h to obtain rice bran protein.

[0055] (2) Preparation of acylated rice bran protein: 2 g of rice bran protein was dissolved in 80 ml of deionized water to form a suspension with a concentration of 25 mg / ml. The pH was adjusted to 8.0 with 2 M NaOH. 0.3 g of succinate was gradually added to the rapidly stirred RBP suspension, and the pH was maintained at 8.0 with 1 M NaOH. After the pH stabilized, the solution was stirred at 25 °C for 1 h to complete the reaction. Finally, the solution was dialyzed thoroughly at 4 °C using a 3500 Da dialysis bag for 24 h. The plate was then frozen at -18 °C for 24 h and then freeze-dried in a freeze dryer for 48 h to obtain RBP-SA.

[0056] (3) Preparation of acylated and glycosylated rice bran protein: 2 g of RBP-SA was dissolved in 80 ml of deionized water to form an RBP-SA suspension with a concentration of 25 mg / ml; 2 g of lactose was dissolved in 80 ml of deionized water to prepare a Lac suspension with a concentration of 25 mg / ml. 80 ml of RBP-SA suspension and 80 ml of Lac suspension were mixed and stirred at 25°C for 2 h to ensure thorough mixing. The pH was adjusted to 7.0 and then gently stirred overnight at 4°C. After ensuring complete hydration of the mixture, it was heated in a 60°C water bath for 12 h. After the reaction was completed, it was immediately removed and cooled in an ice-water bath. It was centrifuged at 8000 r / min at 4°C for 20 min to obtain the supernatant. The supernatant was poured onto a plate and frozen in a -18°C freezer for 24 h. Then it was freeze-dried in a freeze dryer for 48 h to obtain RBP-SA-Lac.

[0057] (4) Preparation of ε-PL-loaded modified rice bran protein antibacterial nanoparticles: 1 g of RBP-SA-Lac was dissolved in 100 ml of deionized water to obtain an RBP-SA-Lac solution with a mass concentration of 10 mg / ml; 0.025 g of ε-PL was dissolved in 100 ml of deionized water to obtain an ε-PL solution with a mass concentration of 0.25 mg / ml; 100 ml of RBP-SA-Lac solution and 100 ml of ε-PL solution were thoroughly mixed, and the pH of the mixture was adjusted to 6 with 1 M NaOH. After the pH stabilized, the mixture was heated in a water bath at 40℃ for 120 min to obtain a ε-PL-loaded modified rice bran protein solution. The solution was plated and frozen in a -18℃ freezer for 24 h, and then freeze-dried in a freeze dryer for 48 h to obtain ε-PL-loaded modified rice bran protein antibacterial nanoparticles.

[0058] (5) The obtained nanoparticles have a particle size of 160.1 nm, a PDI of 0.33, a turbidity of 0.44, and an encapsulation efficiency of 63%. Figure 1As shown, the particle size and PDI of the nanoparticles were significantly lower than those of the control obtained from unacylated modified protein glycosylated loaded with antimicrobial peptides, with a particle size decrease of 63.3 and a PDI decrease of 0.15. Figure 3 As shown, the moisture absorption rate was significantly improved compared to the free ε-PL, decreasing from 85% to 29.1%.

[0059] Example 5

[0060] A method for preparing ε-PL-loaded modified rice bran protein antibacterial nanoparticles, the specific steps of which are as follows:

[0061] (1) Extraction of rice bran protein by alkaline dissolution and acid precipitation: 1 kg defatted rice bran: 9000 ml deionized water, homogenized for 5 min, then magnetically stirred at room temperature for 1 h, pH adjusted to 9.5 with 1 M NaOH solution, magnetically stirred at 55℃ for 2 h, filtered to remove rice bran residue, centrifuged at 10000 r / min at room temperature for 30 min to collect the supernatant, pH adjusted to 4.4 with 1 M HCl solution, allowed to stand for 30 min, centrifuged at 10000 r / min at room temperature for 30 min to remove the supernatant, diluted the precipitate with deionized water and adjusted the pH to 7.0 with 1 M NaOH solution, plated and frozen for 24 h, freeze-dried for 48 h to obtain rice bran protein.

[0062] (2) Preparation of acylated rice bran protein: 2 g of rice bran protein was dissolved in 80 ml of deionized water to form a suspension with a concentration of 25 mg / ml. The pH was adjusted to 8.0 with 2 M NaOH. 0.3 g of succinate was gradually added to the rapidly stirred RBP suspension, and the pH was maintained at 8.0 with 1 M NaOH. After the pH stabilized, the solution was stirred at 25 °C for 1 h to complete the reaction. Finally, the solution was dialyzed thoroughly at 4 °C using a 3500 Da dialysis bag for 24 h. The plate was then frozen at -18 °C for 24 h and then freeze-dried in a freeze dryer for 48 h to obtain RBP-SA.

[0063] (3) Preparation of acylated and glycosylated rice bran protein: 2 g of RBP-SA was dissolved in 80 ml of deionized water to form an RBP-SA suspension with a concentration of 25 mg / ml; 2 g of lactose was dissolved in 80 ml of deionized water to prepare a Lac suspension with a concentration of 25 mg / ml. 80 ml of RBP-SA suspension and 80 ml of Lac suspension were mixed and stirred at 25°C for 2 h to ensure thorough mixing. The pH was adjusted to 7.0 and then gently stirred overnight at 4°C. After ensuring complete hydration of the mixture, it was heated in a 60°C water bath for 12 h. After the reaction was completed, it was immediately removed and cooled in an ice-water bath. It was centrifuged at 8000 r / min at 4°C for 20 min to obtain the supernatant. The supernatant was poured onto a plate and frozen in a -18°C freezer for 24 h. Then it was freeze-dried in a freeze dryer for 48 h to obtain RBP-SA-Lac.

[0064] (4) Preparation of ε-PL-loaded modified rice bran protein antibacterial nanoparticles: 1 g of RBP-SA-Lac was dissolved in 100 ml of deionized water to obtain an RBP-SA-Lac solution with a mass concentration of 10 mg / ml; 0.015 g of ε-PL was dissolved in 100 ml of deionized water to obtain an ε-PL solution with a mass concentration of 0.15 mg / ml; 100 ml of RBP-SA-Lac solution and 100 ml of ε-PL solution were thoroughly mixed, and the pH of the mixture was adjusted to 6 with 1 M NaOH. After the pH stabilized, the mixture was heated in a water bath at 60℃ for 30 min to obtain a ε-PL-loaded modified rice bran protein solution. The solution was then plated and frozen in a -18℃ freezer for 24 h and then freeze-dried in a freeze dryer for 48 h to obtain ε-PL-loaded modified rice bran protein antibacterial nanoparticles.

[0065] (5) The obtained nanoparticles have a particle size of 190.6 nm, a PDI of 0.40, a turbidity of 0.48, and an encapsulation efficiency of 57%. Figure 2 As shown, the encapsulation efficiency of the nanoparticles was significantly higher than that of the control example obtained by loading antimicrobial peptides with unacylated modified protein glycosylation, and the turbidity was significantly lower than that of the control example, with an encapsulation efficiency increase of 7% and a turbidity decrease of 0.12. Figure 3 As shown, the moisture absorption rate was significantly improved compared to the free ε-PL, decreasing from 85% to 27.5%.

[0066] Comparison Example

[0067] (1) Extraction of rice bran protein by alkaline dissolution and acid precipitation: 1 kg defatted rice bran: 9000 ml deionized water, homogenized for 5 min, then magnetically stirred at room temperature for 1 h, pH adjusted to 9.5 with 1 M NaOH solution, magnetically stirred at 55℃ for 2 h, filtered to remove rice bran residue, centrifuged at 10000 r / min at room temperature for 30 min to collect the supernatant, pH adjusted to 4.4 with 1 M HCl solution, allowed to stand for 30 min, centrifuged at 10000 r / min at room temperature for 30 min to remove the supernatant, diluted the precipitate with deionized water and adjusted the pH to 7.0 with 1 M NaOH solution, plated and frozen for 24 h, freeze-dried for 48 h to obtain rice bran protein.

[0068] (2) Preparation of glycosylated rice bran protein: 2 g of RBP was dissolved in 80 ml of deionized water to form an RBP suspension with a concentration of 25 mg / ml; 2 g of lactose was dissolved in 80 ml of deionized water to prepare a Lac suspension with a concentration of 25 mg / ml. 80 ml of RBP suspension and 80 ml of Lac suspension were mixed and stirred at 25°C for 2 h to ensure thorough mixing. The pH was adjusted to 7.0 and then gently stirred overnight at 4°C. After ensuring complete hydration of the mixture, it was heated in a 60°C water bath for 12 h. After the reaction was completed, it was immediately removed and cooled in an ice-water bath. It was centrifuged at 8000 r / min at 4°C for 20 min to obtain the supernatant. The supernatant was poured onto a plate and frozen in a -18°C freezer for 24 h. Then it was freeze-dried in a freeze dryer for 48 h to obtain RBP-Lac.

[0069] (4) Preparation of ε-PL-loaded glycosylated rice bran protein antibacterial nanoparticles: 1 g of RBP-Lac was dissolved in 100 ml of deionized water to obtain an RBP-Lac solution with a mass concentration of 10 mg / ml; 0.015 g of ε-PL was dissolved in 100 ml of deionized water to obtain an ε-PL solution with a mass concentration of 0.15 mg / ml; 100 ml of RBP-Lac solution and 100 ml of ε-PL solution were thoroughly mixed, and the pH of the mixture was adjusted to 6 with 1 M NaOH. After the pH stabilized, the mixture was heated in a water bath at 60℃ for 120 min to obtain an ε-PL-loaded glycosylated rice bran protein solution. The solution was plated and frozen in a -18℃ freezer for 24 h, and then freeze-dried in a freeze dryer for 48 h to obtain ε-PL-loaded glycosylated rice bran protein antibacterial nanoparticles.

[0070] (5) The nanoparticles obtained have a particle size of 223.4 nm, a PDI of 0.48, a turbidity of 0.6, and an encapsulation rate of 50%.

[0071] Compared to the control example obtained by loading antimicrobial peptides onto unacylated, glycosylated rice bran protein, the modified rice bran protein nanoparticles loaded with ε-PL prepared in the examples showed significant advantages: such as... Figure 1 As shown, the particle size ranged from 150.8 to 190.6 nm, significantly smaller than the 223.4 nm of the control example, and the PDI value (0.31–0.40) was lower than the 0.48 of the control example, indicating a more uniform particle size distribution. Examples 1, 3, and 4 showed the most significant effects. Figure 2 As shown, the turbidity (0.4–0.54) was significantly lower than that of the control example (0.6), indicating better dispersibility; the encapsulation efficiency was as high as 53%–72%, far exceeding the 50% of the control example, with Examples 2, 3, and 4 showing the most significant effects; as Figure 3 As shown, the moisture absorption rate of the embodiment is significantly lower than that of the control example and the ε-PL in the free state. Figure 4 As shown, under the same conditions, after a long period of storage, the retention rate of Example 3 was significantly higher than that of the control example, such as... Figure 5 , 6 As shown, the inhibition zone diameters of Example 3 against Staphylococcus aureus and Escherichia coli reached 8.08 mm and 8.75 mm, respectively, which were larger than those of the control examples (6.96 mm and 6.74 mm); at the same time, the antibacterial performance was kept stable.

Claims

1. A modified rice bran protein antibacterial nanoparticle loaded with ε-PL, characterized by, The preparation includes the following steps: Step 1: Prepare rice bran protein; Step 2: Dissolve the rice bran protein obtained in Step 1 in deionized water, adjust the pH to alkaline, gradually add succinate to the rapidly stirred rice bran protein RBP suspension, adjust the pH to 8.0, stir to carry out the reaction, and obtain acylated modified rice bran protein RBP-SA after dialyzing and freeze drying. Step 3: Dissolve the acylated modified rice bran protein RBP-SA obtained in Step 2 in deionized water to obtain an RBP-SA suspension and a lactose Lac suspension. Mix the RBP-SA suspension and the lactose Lac suspension, stir, adjust the pH to neutral, stir overnight, carry out the heating reaction, cool in an ice-water bath, centrifuge, and freeze-dry the supernatant to obtain acylated co-glycosylated rice bran protein RBP-SA-Lac. Step 4: Prepare RBP-SA-Lac solution and ε-polylysine ε-PL solution separately. Mix the RBP-SA-Lac solution and ε-PL solution, adjust the pH of the mixture, heat the reaction, and freeze-dry to obtain modified rice bran protein antibacterial nanoparticles loaded with ε-PL.

2. The ε-PL loaded modified rice bran protein antibacterial nanoparticle according to claim 1, wherein, In step 1, the preparation method of rice bran protein is as follows: defatted rice bran is mixed with deionized water, homogenized, stirred, and the pH is adjusted to alkaline. The rice bran residue is removed by filtration, the supernatant is collected by centrifugation, the pH is adjusted to acidic, the mixture is allowed to stand, the supernatant is removed by centrifugation, the precipitate is diluted with deionized water, the pH is adjusted to 7.0, and the mixture is freeze-dried to obtain rice bran protein.

3. The ε-PL loaded modified rice bran protein antibacterial nanoparticle according to claim 1, wherein, In step 2, the amount of succinic acid added is equivalent to 5-20% of the protein content of rice bran.

4. The ε-PL loaded modified rice bran protein antibacterial nanoparticle according to claim 1, wherein, In step 3, the concentration of RBP-SA suspension is 25 mg / ml, the concentration of Lac suspension is 25 mg / ml, and the volume ratio of the two is 1:

1.

5. The modified rice bran protein antibacterial nanoparticles loaded with ε-PL according to claim 1, characterized in that, In step 3, the heating reaction is carried out at 60°C for 12 hours.

6. The modified rice bran protein antibacterial nanoparticles loaded with ε-PL according to claim 1, characterized in that, In step 4, the concentration of the RBP-SA-Lac solution is 10 mg / ml, the concentration of the ε-PL solution is 0.15~0.35 mg / ml, and the volume ratio of the two is 1:

1.

7. The modified rice bran protein antibacterial nanoparticles loaded with ε-PL according to claim 1, characterized in that, In step 4, the pH of the mixture is adjusted to 4.5–6.5, the temperature of the reaction is heated to 40–80°C, and the time is 30–150 min.

8. The application of the modified rice bran protein antibacterial nanoparticles loaded with ε-PL as described in claim 1 in the preparation of antibacterial products.

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