Preparation method of bio-based degradable film containing rice bran protein-chitosan-epsilon-polylysine nanoparticles

Abstract

The invention provides a preparation method of a bio-based degradable thin film containing rice bran protein-chitosan-epsilon-polylysine nanoparticles, and belongs to the technical field of bio-based degradable thin film materials. The preparation method comprises the following steps: extracting rice bran protein from defatted rice bran by an alkali-solution and acid-isolation method; the preparation method comprises the following steps: preparing rice bran protein-chitosan-epsilon-polylysine ternary nanoparticles by adopting a composite thermal aggregation self-assembly method; and adding the nano-particles into the rice bran protein film to obtain a bio-based degradable film product containing the antibacterial nano-particles. The bio-based degradable film prepared by the method is short in preparation period, good in dispersity and high in yield; the epsilon-polylysine is protected, the activity loss caused by environmental factors is reduced, the bioavailability of the epsilon-polylysine is improved, and the antibacterial effect of the epsilon-polylysine is prolonged. When the ternary nano-particles are applied to preparation of a bio-based film, the physical performance of the film is improved, the film is endowed with excellent antibacterial performance, and the ternary nano-particles have wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio-based degradable thin film materials, and particularly relates to a preparation method of a bio-based degradable thin film containing rice bran protein-chitosan-ε-polylysine nanoparticles. Background Art

[0002] With the enhancement of environmental protection awareness and the deepening of the concept of sustainable development, bio-based degradable materials are increasingly widely used in the field of food packaging. Rice bran, as an agricultural by-product, is rich in protein and has high nutritional value and utilization potential. Chitosan, as a natural high molecular polysaccharide, is derived from the exoskeletons of crustaceans and the cell walls of fungi. It has good biocompatibility, carries a positive charge, and can bind to rice bran protein through electrostatic interaction to form a core-shell structure, encapsulating some unstable active substances, such as the food antibacterial agent ε-polylysine with great application potential. ε-Polylysine (ε-PL) is a natural nutritional biopreservative with broad-spectrum antibacterial activity. However, due to problems such as its strong hygroscopicity and easy interaction with food components resulting in a decrease in antibacterial performance, its direct application in food is limited.

[0003] Currently, there are also reports on methods for modifying nanoparticles with proteins to improve their stability and increase their functional properties. For example, in "A Preparation Method of Whey Protein-ε-Polylysine-Arabic Gum Nanoparticles" (CN202110683277.9), through enzymatic hydrolysis and ultrasonic treatment, the nanoparticles have a better encapsulation effect on ε-polylysine. However, there is no report on using a rice bran protein-chitosan composite system prepared by electrostatic interaction to improve... In this patent, a composite thermal aggregation self-assembly method is used to prepare rice bran protein-chitosan-ε-polylysine ternary nanoparticles, and the nanoparticles are applied to the preparation of bio-based degradable thin films, reducing the loss of ε-polylysine activity caused by environmental factors, improving its biological utilization rate, and reducing its hygroscopicity. At the same time, the addition of ε-polylysine makes the nanoparticle structure more compact and has a certain modification effect on the protein structure. Applying the nanoparticles to the preparation of bio-based thin films not only improves the physical properties of the thin films but also endows the thin films with excellent antibacterial properties, having a broader application prospect. Summary of the Invention

[0004] The present invention provides a preparation method of a bio-based degradable thin film containing rice bran protein-chitosan-ε-polylysine nanoparticles, which not only ensures the high biological activity of ε-polylysine but also can endow the degradable bio-based thin film with good antibacterial properties.

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

[0006] A preparation method of a bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles, comprising the following steps:

[0007] 1) Extraction of rice bran protein: Mix defatted rice bran with deionized water, homogenize and stir at room temperature, adjust the pH value of the mixed solution to alkaline, stir, filter to remove rice bran residue and then centrifuge, adjust the pH value of the supernatant obtained after centrifugation to acidic, let it stand and then centrifuge, collect the precipitate obtained after centrifugation and dilute it, adjust its pH value to neutral, and obtain rice bran protein RBP after freeze-drying;

[0008] 2) Preparation of rice bran protein stock solution: Take the rice bran protein RBP obtained in step 1) and dissolve it in deionized water, stir at room temperature, and then hydrate the RPB solution overnight at 4°C to obtain an RBP stock solution;

[0009] 3) Preparation of chitosan stock solution: Dissolve chitosan in an acetic acid solution with a mass-volume concentration of 1%, stir at room temperature until the chitosan CS is completely dissolved to obtain a CS stock solution;

[0010] 4) Preparation of rice bran protein-chitosan nanoparticles: Take the RBP stock solution obtained in step 2) and the CS stock solution obtained in step 3), mix them in proportion, adjust the pH value of the mixed solution to acidic, continue to stir at room temperature for 40 min to make the two substances fully mixed, then put the mixed solution into a centrifuge tube, place it in a water bath at 80°C and heat for 40 min, take it out and immediately place it in an ice bath, and centrifuge at 10000 r / min for 10 min. Take the supernatant and obtain R-C nanoparticles after freeze-drying;

[0011] 5) Preparation of rice bran protein-chitosan-ε-polylysine nanoparticles: Dissolve the R-C nanoparticles obtained in step 4) in deionized water, add an ε-polylysine solution, stir magnetically, after mixing evenly, slowly adjust the pH value of the mixed system to acidic with 0.1 M HCl solution, continue to stir magnetically at room temperature for 2 h, centrifuge at 6000 r / min for 10 min, take the supernatant to obtain an R-C-ε-PL nanoparticle solution, and obtain R-C-ε-PL nanoparticles after freeze-drying;

[0012] 6) Preparation of film solution: Mix the R-C-ε-PL nanoparticles prepared in step 5) with the RBP solution, adjust the pH of the film solution, add the plasticizer glycerol, heat and stir to obtain a film solution;

[0013] 7) Preparation of a bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles: Take 20 mL of the film solution prepared in step 6) and cast it onto a sterile plastic petri dish with an inner diameter of 9 cm. Place the petri dish containing the film solution horizontally in an oven, take it out after drying, and then place the film in a constant temperature incubator at 25 °C and a relative humidity of 58% for 48 h to obtain the RBP-NPs bio-based degradable film.

[0014] Further, for the above preparation method, the specific steps of step 1) are as follows: Mix defatted rice bran and deionized water at a mass-to-volume ratio of 1:9, homogenize for 4 - 6 min, then stir magnetically at room temperature for 1 - 2 h. Use 1 M NaOH to adjust the pH value of the mixture to 9.5, stir magnetically at 55 °C for 1 - 2 h, filter to remove rice bran residue, then centrifuge at 10000 r / min for 30 min. Use 1 M HCl to adjust the pH value of the supernatant obtained after centrifugation to 4.4, let it stand for 30 - 40 min, then centrifuge at 10000 r / min for 30 min. Collect the precipitate obtained after centrifugation and dilute it, adjust its pH value to neutral, and freeze-dry to obtain rice bran protein RBP.

[0015] Further, for the above preparation method, in step 2), the concentration of the RBP stock solution is 10 mg / mL.

[0016] Further, for the above preparation method, in step 3), the concentration of the CS stock solution is 10 mg / mL.

[0017] Further, for the above preparation method, in step 4), the RBP stock solution and the CS stock solution are mixed at a volume ratio of 2:1.

[0018] Further, for the above preparation method, in step 4), adjust the pH value of the mixed solution to 5.5.

[0019] Further, for the above preparation method, in step 5), the concentration of R-C nanoparticles dissolved in deionized water is 10 mg / mL, and the concentration of the ε-polylysine solution is 0.25 mg / mL.

[0020] Further, for the above preparation method, in step 5), adjust the pH value of the mixed system to 5.

[0021] Further, for the above preparation method, in steps 4) and 5), the freeze-drying conditions are as follows: Pre-freeze at -20 °C for 24 h, then place it in a freeze-dryer and freeze-dry at 0.1 - 0.5 mbar and -60 - 50 °C for 48 h.

[0022] Further, for the above preparation method, in step 6), the concentration of the RBP solution is 60 mg / mL.

[0023] Further, in step 6) of the above preparation method, the addition amount of R-C-ε-PL nanoparticles is 3% to 10% based on the mass of RBP.

[0024] Further, in step 6) of the above preparation method, the pH of the membrane solution is adjusted to 8 to 10.

[0025] Further, in step 6) of the above preparation method, the addition amount of glycerol is 1% to 1.8% of the volume of the membrane solution.

[0026] Further, in step 6) of the above preparation method, the heating temperature is 65 to 85 °C and the time is 20 to 40 min.

[0027] Further, in step 7) of the above preparation method, the drying temperature is 50 to 55 °C and the time is 4.5 to 5 h.

[0028] For the preparation method described in any one of the above, the tensile strength of the bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles is 0.787 to 2.167 MPa, the elongation at break is 26.667 to 64.333%, the opacity is 2.483 to 5.287 A / mm, the diameter of the antibacterial zone against E. coli is 8.9 to 10.56 mm, and the diameter of the antibacterial zone against S. aureus is 9.5 to 10.88 mm.

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

[0030] 1. Efficient resource utilization and environmental protection: The present invention utilizes the protein resources in rice bran, an agricultural by-product, and through scientific extraction and processing, converts it into a high-value-added food packaging material, achieving effective resource utilization. At the same time, using two natural polymer substances, chitosan and ε-polylysine, not only enriches the composition of the film but also improves its biocompatibility and degradability, meeting the requirements of environmental protection and sustainable development.

[0031] 2. Excellent physical properties: The introduction of nanoparticles enhances the mechanical strength of the film, such as tensile strength and elongation at break, enabling it to meet the physical property requirements of food packaging, and the transparency of the film has also been improved.

[0032] 3. Strong antibacterial properties: As a natural preservative, ε-polylysine has broad-spectrum antibacterial activity and can effectively inhibit the growth and reproduction of microorganisms in food, extending the shelf life of food. Through the embedding technology of nanoparticles, the stability and utilization rate of ε-polylysine are improved, giving full play to its antibacterial properties.

[0033] 4. Simple preparation process and easy for industrial production: The preparation method of the present invention is relatively simple, and the involved equipment and process conditions are relatively mature, which is easy to be popularized and applied in industrial production. The raw materials used in the preparation process are all natural substances, non-toxic and harmless, environmentally friendly, and meet the production requirements of modern food packaging materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a process flow diagram for the preparation of a bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles of the present invention.

[0035] Figure 2 It is the elongation at break and tensile strength of the bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles prepared in Examples 1-5 of the present invention.

[0036] Figure 3 It is the opacity of the bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles prepared in Examples 1-5 of the present invention.

[0037] Figure 4 It is the inhibition zone of the bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles prepared in Examples 1-5 of the present invention and the pure protein film without adding nanoparticles. DETAILED DESCRIPTION OF THE INVENTION

[0038] Example 1

[0039] As Figure 1 , a preparation method of a bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles is as follows:

[0040] 1) Protein extraction: Mix 1 kg of defatted rice bran with 9000 mL of deionized water, homogenize for 5 min, then stir magnetically at room temperature for 1 h, adjust the pH value of the mixed solution to 9.5 with 1 M NaOH, stir magnetically at 55 °C for 1 h, filter to remove rice bran residue, then centrifuge at 10000 r / min for 30 min, adjust the pH value of the supernatant obtained after centrifugation to 4.4 with 1 M HCl, let it stand for 40 min, then centrifuge at 10000 r / min for 30 min, collect the precipitate obtained after centrifugation and dilute it, adjust its pH value to neutral, and freeze-dry to obtain rice bran protein RBP.

[0041] 2) Preparation of stock solutions: Dissolve 1 g of the rice bran protein (RBP) obtained in step 1) in 100 mL of deionized water, and stir magnetically at room temperature for 5 h. Subsequently, hydrate the RPB solution overnight at 4 °C to obtain a 10 mg / mL RBP stock solution. Dissolve 1 g of chitosan in 100 mL of 1% (w / v) acetic acid solution, and stir magnetically at room temperature for 5 h until the CS is completely dissolved to obtain a CS stock solution.

[0042] 3) Preparation of rice bran protein-chitosan binary nanoparticles: Mix 100 mL of the RBP stock solution and 50 mL of the CS stock solution obtained in step 2) evenly, adjust the pH value of the mixed solution to 5.5, and continue stirring at room temperature for 40 min to ensure sufficient mixing of the two substances. Then, place the mixed solution in a centrifuge tube, heat it in a water bath at 80 °C for 40 min, immediately ice-bath it after taking it out, and centrifuge it at 10000 r / min for 10 min. Take the supernatant and freeze-dry it to obtain an R-C binary nanoparticle solution. After pre-freezing at -20 °C for 24 h, place it in a freeze-dryer and freeze-dry it at 0.1 - 0.5 mbar and -60 - 50 °C for 48 h to obtain R-C nanoparticles.

[0043] 4) Preparation of rice bran protein-chitosan-ε-polylysine ternary nanoparticles: Dissolve 1 g of the R-C nanoparticle powder obtained in step 3) in 100 mL of deionized water, dissolve 2.5 mg of ε-polylysine in 10 mL of deionized water, stir magnetically, and after mixing evenly, slowly adjust the pH value of the mixed system to 5 with 0.1 M HCl solution. Continue to stir magnetically at room temperature for 2 h, centrifuge at 6000 r / min for 10 min, take the supernatant to obtain an R-C-ε-PL nanoparticle solution. After pre-freezing at -20 °C for 24 h, place it in a freeze-dryer and freeze-dry it at 0.1 - 0.5 mbar and -60 - 50 °C for 48 h to obtain R-C-ε-PL nanoparticles.

[0044] 5) Preparation of RBP-NPs film: Dissolve 1.2 g of RBP in 20 mL of deionized water to obtain an RBP solution with a concentration of 60 mg / mL; take 0.072 g of the R-C-ε-PL nanoparticles prepared in step 4) and mix it with the RBP solution, adjust the pH to 9.5, add 0.3 mL of glycerol, and stir at 80 °C for 30 min to obtain a film solution. Measure 20 mL of the film solution and pour it onto a sterile plastic petri dish with an inner diameter of 9 cm. Place the petri dish containing the film solution horizontally in an oven, take it out after drying at 50 °C for 4.5 h, and place the film in a constant temperature incubator at 25 °C and a relative humidity of 58% to obtain the RBP-NPs bio-based biodegradable film product.

[0045] As Figures 2 - 4As shown, the elongation at break of the prepared RBP-NPs film is 42.667%, the tensile strength is 0.787 MPa, the opacity is 3.897 A / mm, the diameter of the inhibition zone against E. coli is 10.28 mm, and the diameter of the inhibition zone against S. aureus is 10.62 mm.

[0046] Example 2

[0047] A preparation method of a bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles, the specific steps are as follows:

[0048] 1) Protein extraction: Mix 1 kg of defatted rice bran with 9000 mL of deionized water, homogenize for 5 min, then stir magnetically at room temperature for 1 h. Use 1 M NaOH to adjust the pH value of the mixed solution to 9.5, stir magnetically at 55 °C for 1 h, filter to remove rice bran residue, then centrifuge at 10000 r / min for 30 min. Use 1 M HCl to adjust the pH value of the supernatant obtained after centrifugation to 4.4, let it stand for 40 min, then centrifuge at 10000 r / min for 30 min. Collect the precipitate obtained after centrifugation and dilute it, adjust its pH value to neutral, and freeze-dry to obtain rice bran protein RBP.

[0049] 2) Preparation of stock solutions: Dissolve 1 g of the rice bran protein (RBP) obtained in step 1) in 100 mL of deionized water, stir magnetically at room temperature for 5 h, and then hydrate the RPB solution overnight at 4 °C to prepare a 10 mg / mL RBP stock solution. Dissolve 1 g of chitosan in 100 mL of 1% (w / v) acetic acid solution, stir magnetically at room temperature for 5 h to completely dissolve CS, and obtain a CS stock solution.

[0050] 3) Preparation of rice bran protein-chitosan binary nanoparticles: Mix 100 mL of the RBP stock solution and 50 mL of the CS stock solution obtained in step 2) evenly, adjust the pH value of the mixed solution to 5.5, continue to stir at room temperature for 40 min to fully mix the two substances, then put the mixed solution into a centrifuge tube, place it in a water bath at 80 °C for 40 min, take it out and immediately ice-bath, and centrifuge at 10000 r / min for 10 min. Take the supernatant and freeze-dry to obtain an R-C binary nanoparticle solution. Pre-freeze at -20 °C for 24 h and then place it in a freeze-dryer, and freeze-dry at 0.1 - 0.5 mbar and -60 - 50 °C for 48 h to obtain R-C nanoparticles.

[0051] 4) Preparation of rice bran protein-chitosan-ε-polylysine ternary nanoparticles: Dissolve 1 g of the R-C nanoparticle powder obtained in step 3) in 100 mL of deionized water. Dissolve 2.5 mg of ε-polylysine in 10 mL of deionized water, stir magnetically, and after mixing evenly, slowly adjust the pH value of the mixed system to 5 with 0.1 M HCl solution. Continue to stir magnetically at room temperature for 2 h, centrifuge at 6000 r / min for 10 min, take the supernatant to obtain the R-C-ε-PL nanoparticle solution. After pre-freezing at -20 °C for 24 h, place it in a freeze dryer and freeze-dry at 0.1 - 0.5 mbar and -60 - -50 °C for 48 h to obtain the R-C-ε-PL nanoparticles.

[0052] 5) Preparation of RBP-NPs film: Dissolve 1.2 g of RBP in 20 mL of deionized water to obtain an RBP solution with a concentration of 60 mg / mL; Take 0.072 g of the R-C-ε-PL nanoparticles prepared in step 4) and mix with the RBP solution, adjust the pH to 10, add 0.28 mL of glycerol, and stir at 80 °C for 30 min to obtain the film solution. Measure 20 mL of the film solution and pour it onto a sterile plastic Petri dish with an inner diameter of 9 cm. Place the Petri dish containing the film solution horizontally in an oven, take it out after drying at 50 °C for 4.5 h, and put the film into a constant temperature incubator at 25 °C and a relative humidity of 58%, then the RBP-NPs bio-based degradable film product is obtained.

[0053] As Figures 2 - 4 shown, the elongation at break of the prepared RBP-NPs film is 48.333%, the tensile strength is 1.07 MPa, the opacity is 5.25 A / mm, the diameter of the inhibition zone against E. coli is 10.28 mm, and the diameter of the inhibition zone against S. aureus is 10.62 mm.

[0054] Example 3

[0055] A preparation method of a bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles, the specific steps are as follows:

[0056] 1) Protein extraction: Mix 1 kg of defatted rice bran with 9000 mL of deionized water, homogenize for 5 min, then stir magnetically at room temperature for 1 h. Use 1 M NaOH to adjust the pH value of the mixed solution to 9.5, stir magnetically at 55 °C for 1 h, filter to remove the rice bran residue, then centrifuge at 10000 r / min for 30 min. Use 1 M HCl to adjust the pH value of the supernatant obtained after centrifugation to 4.4, let it stand for 40 min, then centrifuge at 10000 r / min for 30 min. Collect the precipitate obtained after centrifugation and dilute it, adjust its pH value to neutral, and obtain rice bran protein RBP after freeze-drying.

[0057] 2) Preparation of stock solutions: Dissolve 1 g of the rice bran protein (RBP) obtained in step 1) in 100 mL of deionized water, stir magnetically at room temperature for 5 h, and then hydrate the RPB solution overnight at 4 °C to obtain a 10 mg / mL RBP stock solution. Dissolve 1 g of chitosan in 100 mL of 1% (w / v) acetic acid solution, stir magnetically at room temperature for 5 h to completely dissolve CS, and obtain a CS stock solution.

[0058] 3) Preparation of rice bran protein-chitosan binary nanoparticles: Mix 100 mL of the RBP stock solution and 50 mL of the CS stock solution obtained in step 2) evenly, adjust the pH value of the mixed solution to 5.5, continue to stir at room temperature for 40 min to fully mix the two substances, then put the mixed solution into a centrifuge tube, heat it in a water bath at 80 °C for 40 min, take it out and immediately place it in an ice bath, and centrifuge at 10000 r / min for 10 min. Take the supernatant and freeze-dry it to obtain an R-C binary nanoparticle solution. After pre-freezing at -20 °C for 24 h, place it in a freeze dryer and freeze-dry it at 0.1 - 0.5 mbar and -60 - -50 °C for 48 h to obtain R-C nanoparticles.

[0059] 4) Preparation of rice bran protein-chitosan-ε-polylysine ternary nanoparticles: Dissolve 1 g of the R-C nanoparticle powder obtained in step 3) in 100 mL of deionized water, dissolve 2.5 mg of ε-polylysine in 10 mL of deionized water, stir magnetically, and after mixing evenly, slowly adjust the pH value of the mixed system to 5 with 0.1 M HCl solution, continue to stir magnetically at room temperature for 2 h, centrifuge at 6000 r / min for 10 min, take the supernatant to obtain an R-C-ε-PL nanoparticle solution. After pre-freezing at -20 °C for 24 h, place it in a freeze dryer and freeze-dry it at 0.1 - 0.5 mbar and -60 - -50 °C for 48 h to obtain R-C-ε-PL nanoparticles.

[0060] 5) Preparation of RBP-NPs film: Dissolve 1.2 g of RBP in 20 mL of deionized water to obtain an RBP solution with a concentration of 60 mg / mL; take 0.072 g of the R-C-ε-PL nanoparticles prepared in step 4) and mix them with the RBP solution, adjust the pH to 9.5, add 0.28 mL of glycerol, and stir at 80 °C for 30 min to obtain a film solution. Measure 20 mL of the film solution and pour it onto a sterile plastic petri dish with an inner diameter of 9 cm. Place the petri dish containing the film solution horizontally in an oven, take it out after drying at 50 °C for 4.5 h, and place the film in a constant temperature incubator at 25 °C and a relative humidity of 58% to obtain the RBP-NPs bio-based biodegradable thin film product.

[0061] As Figures 2 - 4As shown, the elongation at break of the prepared RBP-NPs film is 57.9%, the tensile strength is 1.773 MPa, the opacity is 3.2 A / mm, the diameter of the inhibition zone against E. coli is 10.28 mm, and the diameter of the inhibition zone against S. aureus is 10.62 mm.

[0062] Example 4

[0063] A preparation method of a bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles is as follows:

[0064] 1) Protein extraction: Mix 1 kg of defatted rice bran with 9000 mL of deionized water, homogenize for 5 min, then stir magnetically at room temperature for 1 h. Adjust the pH value of the mixture to 9.5 with 1 M NaOH, stir magnetically at 55 °C for 1 h, filter to remove rice bran residue, then centrifuge at 10000 r / min for 30 min. Adjust the pH value of the supernatant obtained after centrifugation to 4.4 with 1 M HCl, let it stand for 40 min, then centrifuge at 10000 r / min for 30 min. Collect the precipitate obtained after centrifugation and dilute it, adjust its pH value to neutral, and freeze-dry to obtain rice bran protein RBP.

[0065] 2) Preparation of stock solutions: Dissolve 1 g of the rice bran protein (RBP) obtained in step 1) in 100 mL of deionized water, stir magnetically at room temperature for 5 h, then hydrate the RPB solution overnight at 4 °C to prepare a 10 mg / mL RBP stock solution. Dissolve 1 g of chitosan in 100 mL of 1% (w / v) acetic acid solution, stir magnetically at room temperature for 5 h until CS is completely dissolved to obtain a CS stock solution.

[0066] 3) Preparation of rice bran protein-chitosan binary nanoparticles: Mix 100 mL of the RBP stock solution and 50 mL of the CS stock solution obtained in step 2) evenly, adjust the pH value of the mixed solution to 5.5, continue to stir at room temperature for 40 min to make the two substances fully mixed. Then put the mixed solution into a centrifuge tube, place it in a water bath at 80 °C for 40 min, take it out and immediately place it in an ice bath, and centrifuge at 10000 r / min for 10 min. Take the supernatant and freeze-dry to obtain an R-C binary nanoparticle solution. Pre-freeze it at -20 °C for 24 h and then place it in a freeze-dryer, and freeze-dry at 0.1 - 0.5 mbar and -60 - -50 °C for 48 h to obtain R-C nanoparticles.

[0067] 4) Preparation of rice bran protein-chitosan-ε-polylysine ternary nanoparticles: Dissolve 1 g of the R-C nanoparticle powder obtained in step 3) in 100 mL of deionized water. Dissolve 2.5 mg of ε-polylysine in 10 mL of deionized water, stir magnetically, and after mixing evenly, slowly adjust the pH value of the mixed system to 5 with 0.1 M HCl solution. Continue to stir magnetically at room temperature for 2 h, centrifuge at 6000 r / min for 10 min, take the supernatant to obtain the R-C-ε-PL nanoparticle solution. Pre-freeze at -20 °C for 24 h and then place it in a freeze dryer, and freeze-dry at 0.1 - 0.5 mbar and -60 - -50 °C for 48 h to obtain the R-C-ε-PL nanoparticles.

[0068] 5) Preparation of RBP-NPs film: Dissolve 1.2 g of RBP in 20 mL of deionized water to obtain an RBP solution with a concentration of 60 mg / mL; Take 0.036 g of the R-C-ε-PL nanoparticles prepared in step 4) and mix it with the RBP solution, adjust the pH to 9.5, add 0.28 mL of glycerol, and stir at 70 °C for 30 min to obtain the film solution. Measure 20 mL of the film solution and pour it onto a sterile plastic petri dish with an inner diameter of 9 cm. Place the petri dish containing the film solution horizontally in an oven, take it out after drying at 50 °C for 4.5 h, and put the film into a constant temperature incubator at 25 °C and a relative humidity of 58%, then the RBP-NPs biobased degradable film product is obtained.

[0069] As Figures 2 - 4 shown, the elongation at break of the prepared RBP-NPs film is 50%, the tensile strength is 1.173 MPa, the opacity is 3.38 A / mm, the inhibition zone diameter against E. coli is 8.9 mm, and the inhibition zone diameter against S. aureus is 9.5 mm.

[0070] Example 5

[0071] A preparation method of a biobased degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles, the specific steps are as follows:

[0072] 1) Protein extraction: Mix 1 kg of defatted rice bran with 9000 mL of deionized water, homogenize for 5 min and then stir magnetically at room temperature for 1 h. Use 1 M NaOH to adjust the pH value of the mixed solution to 9.5, stir magnetically at 55 °C for 1 h, filter to remove the rice bran residue and then centrifuge at 10000 r / min for 30 min. Use 1 M HCl to adjust the pH value of the supernatant obtained after centrifugation to 4.4, let it stand for 40 min and then centrifuge at 10000 r / min for 30 min. Collect the precipitate obtained after centrifugation and dilute it, adjust its pH value to neutral, and obtain rice bran protein RBP after freeze-drying.

[0073] 2) Preparation of stock solutions: Dissolve 1 g of the rice bran protein (RBP) obtained in step 1) in 100 mL of deionized water, and stir magnetically at room temperature for 5 h. Subsequently, hydrate the RPB solution overnight at 4 °C to obtain a 10 mg / mL RBP stock solution. Dissolve 1 g of chitosan in 100 mL of 1% (w / v) acetic acid solution, and stir magnetically at room temperature for 5 h until the CS is completely dissolved to obtain a CS stock solution.

[0074] 3) Preparation of rice bran protein-chitosan binary nanoparticles: Mix 100 mL of the RBP stock solution and 50 mL of the CS stock solution obtained in step 2) evenly, adjust the pH value of the mixed solution to 5.5, and continue stirring at room temperature for 40 min to ensure sufficient mixing of the two substances. Then, place the mixed solution in a centrifuge tube, heat it in a water bath at 80 °C for 40 min, immediately ice-bath it after taking it out, and centrifuge it at 10000 r / min for 10 min. Take the supernatant and freeze-dry it to obtain an R-C binary nanoparticle solution. After pre-freezing at -20 °C for 24 h, place it in a freeze-dryer and freeze-dry it at 0.1 - 0.5 mbar and -60 - -50 °C for 48 h to obtain R-C nanoparticles.

[0075] 4) Preparation of rice bran protein-chitosan-ε-polylysine ternary nanoparticles: Dissolve 1 g of the R-C nanoparticle powder obtained in step 3) in 100 mL of deionized water, dissolve 2.5 mg of ε-polylysine in 10 mL of deionized water, stir magnetically, and after mixing evenly, slowly adjust the pH value of the mixed system to 5 with 0.1 M HCl solution. Continue to stir magnetically at room temperature for 2 h, centrifuge at 6000 r / min for 10 min, take the supernatant to obtain an R-C-ε-PL nanoparticle solution. After pre-freezing at -20 °C for 24 h, place it in a freeze-dryer and freeze-dry it at 0.1 - 0.5 mbar and -60 - -50 °C for 48 h to obtain R-C-ε-PL nanoparticles.

[0076] 5) Preparation of the RBP-NPs film: Dissolve 1.2 g of RBP in 20 mL of deionized water to obtain an RBP solution with a concentration of 60 mg / mL; take 0.072 g of the R-C-ε-PL nanoparticles prepared in step 4) and mix it with the RBP solution, adjust the pH to 9.5, add 0.28 mL of glycerol, and stir at 80 °C for 40 min to obtain a film solution. Measure 20 mL of the film solution and pour it onto a sterile plastic Petri dish with an inner diameter of 9 cm. Place the Petri dish containing the film solution horizontally in an oven, take it out after drying at 50 °C for 4.5 h, and place the film in a constant temperature incubator at 25 °C and a relative humidity of 58% to obtain the RBP-NPs biobased degradable film product.

[0077] As Figures 2 - 4As shown, the elongation at break of the prepared RBP-NPs film is 36.667%, the tensile strength is 0.79 MPa, the opacity is 4.84 A / mm, the diameter of the inhibition zone against E. coli is 10.28 mm, and the diameter of the inhibition zone against S. aureus is 10.62 mm.

Claims

1. A method for preparing a biodegradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles, characterized in that: The steps include: 1) Extraction of rice bran protein: defatted rice bran is mixed with deionized water, homogenized and stirred at room temperature, the pH value of the mixed solution is adjusted to alkaline, stirred, filtered to remove rice bran residue and centrifuged, the pH value of the supernatant obtained after centrifugation is adjusted to acidic, allowed to stand and then centrifuged, the precipitate obtained after centrifugation is collected and diluted, the pH value thereof is adjusted to neutral, and the rice bran protein RBP is obtained after freeze-drying; 2) Preparation of rice bran protein stock solution: dissolving the rice bran protein RBP obtained in step 1) in deionized water, stirring at room temperature, and then hydrating the RPB solution at 4° C. overnight to prepare the RBP stock solution; 3) Preparation of chitosan stock solution: dissolve chitosan in acetic acid solution with a mass volume concentration of 1%, stir at room temperature to completely dissolve the chitosan CS, and obtain a CS stock solution; 4) Preparation of rice bran protein-chitosan nanoparticles: The RBP stock solution obtained in step 2) and the CS stock solution obtained in step 3) were mixed in proportion, the pH value of the mixed solution was adjusted to acidic, and the mixture was stirred for 40 min at room temperature to allow the two substances to be fully mixed, and then the mixed solution was placed in a centrifuge tube, heated in a water bath at 80° C. for 40 min, and immediately placed in an ice bath after being taken out, and centrifuged at 10000 r / min for 10 min, and the supernatant was freeze-dried to obtain RC nanoparticles; 5) Preparation of rice bran protein-chitosan-ε-polylysine nanoparticles: dissolving the RC nanoparticles obtained in step 4) in deionized water, adding ε-polylysine solution, magnetically stirring, and mixing evenly, slowly adjusting the pH value of the mixed system to acidic with 0.1M HCl solution, continuing magnetic stirring at room temperature for 2 hours, centrifuging at 6000 r / min for 10 minutes, taking the supernatant to obtain RC-ε-PL nanoparticle solution, and freeze-drying to obtain RC-ε-PL nanoparticles; 6) Preparation of membrane solution: Mix the RC-ε-PL nanoparticles prepared in step 5) with the RBP solution, adjust the pH of the membrane solution, add plasticizer glycerol, heat and stir to obtain a membrane solution; 7) Preparation of a bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles: 20 mL of the film solution prepared in step 6) was cast onto a sterile plastic culture dish with an inner diameter of 9 cm, the culture dish containing the film solution was placed horizontally in an oven, taken out after drying, and the film was placed in a constant temperature incubator at a temperature of 25°C and a relative humidity of 58%, and stored for 48 hours to obtain a RBP-NPs bio-based degradable film.

2. The preparation method according to claim 1, characterized in that: The specific steps of step 1) are: defatted rice bran and deionized water are mixed at a mass volume ratio of 1:9, homogenized for 4 to 6 minutes, and then magnetically stirred at room temperature for 1 to 2 hours, the pH value of the mixed solution is adjusted to 9.5 using 1M NaOH, and magnetically stirred at 55°C for 1 to 2 hours, and the rice bran residue is filtered out and then centrifuged at 10000 r / min for 30 minutes, and the pH value of the supernatant obtained after centrifugation is adjusted to 4.4 using 1M HCl, and then centrifuged at 10000 r / min for 30 minutes after standing for 30 to 40 minutes, and the precipitate obtained after centrifugation is collected and diluted, and the pH value thereof is adjusted to neutral, and the rice bran protein RBP is obtained after freeze-drying.

3. The preparation method according to claim 1, characterized in that: In step 2), the concentration of the RBP stock solution is 10 mg / mL.

4. The preparation method according to claim 1, characterized in that: In step 3), the concentration of the CS stock solution is 10 mg / mL.

5. The preparation method according to claim 1, characterized in that: In step 4), the RBP stock solution and the CS stock solution were mixed at a volume ratio of 2:1, and the pH value of the mixed solution was adjusted to 5.

5.

6. The preparation method according to claim 1, characterized in that: In step 5), the concentration of RC nanoparticles dissolved in deionized water is 10 mg / mL, the concentration of ε-polylysine solution is 0.25 mg / mL, and the pH value of the mixed system is adjusted to 5.

7. The preparation method according to claim 1, characterized in that: In step 4) and step 5), the freeze-drying conditions are: pre-freeze at -20°C for 24 hours, place in a freeze dryer, and freeze-dry for 48 hours at 0.1-0.5 mbar and -60--50°C.

8. The preparation method according to claim 1, characterized in that: In step 6), the concentration of the RBP solution is 60 mg / mL; the amount of RC-ε-PL nanoparticles added is 3% to 10% based on the mass of RBP; the pH of the membrane solution is adjusted to 8 to 10; the amount of glycerol added is 1% to 1.8% of the volume of the membrane solution; the heating temperature is 65 to 85° C., and the time is 20 to 40 min.

9. The preparation method according to claim 1, characterized in that: In step 7), the drying temperature is 50-55° C. and the drying time is 4.5-5 hours.

10. The preparation method according to any one of claims 1 to 9, characterized in that: The prepared bio-based degradable film containing rice bran protein-chitosan-ε-polylysine nanoparticles has a film tensile strength of 0.787-2.167 MPa, an elongation at break of 26.667-64.333%, an opacity of 2.483-5.287 A / mm, an inhibition zone diameter of 8.9-10.56 mm for E. coli, and an inhibition zone diameter of 9.5-10.88 mm for S. aureus.

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