Soybean protein isolate nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a soybean protein isolate nano-enzyme as well as a preparation method and application thereof, and belongs to the technical field of artificial nano-enzymes. Dissolving soybean protein isolate in water to obtain a soybean protein isolate solution; mixing with a ZnCl2 solution, stirring to form a suspension, heating, and reacting under stirring; and centrifuging and washing the obtained reaction liquid, and carrying out vacuum freeze drying to obtain the soybean protein isolate nano-enzyme. Soybean protein is used as a plant-derived raw material and has the characteristics of wide source, low cost, high renewability and the like, and meanwhile, the potential sensitization risk of animal-derived protein is avoided; the coordination environment of Zn < 2 + > and protein functional groups is accurately regulated and controlled, so that the active center structure of the natural enzyme is successfully simulated; the obtained zinc-soybean protein isolate material not only shows excellent catalytic efficiency, but also has good environmental stability and thermal stability, and shows wide application prospects in the fields of oxidation resistance, food preservation, environmental governance and the like.

Description

Technical Field

[0001] This invention belongs to the field of artificial nanoenzyme technology, specifically relating to a soybean protein isolate nanoenzyme, its preparation method, and its application. Background Technology

[0002] Natural enzymes are highly efficient biological antioxidants that effectively scavenge reactive oxygen species (ROS), making them valuable in biomedicine, food preservation, and environmental protection. However, natural enzymes suffer from inherent drawbacks such as high production costs, sensitivity to environmental conditions (e.g., temperature, pH), and poor storage stability, severely limiting their large-scale industrial applications. The self-assembly structure of biomolecules and metal ions in natural enzymes provides researchers with new insights for developing efficient and stable artificial enzyme mimics. Studies have shown that by rationally designing the coordination structures of proteins and metal ions, enzyme-like nanopolymers can be constructed, which not only mimic the catalytic function of natural enzymes but also possess higher stability and controllability. However, current research on biomimetic nanozymes mainly relies on animal-derived proteins (such as bovine serum albumin and wool keratin) as the assembly framework. Although these protein-metal nanozymes exhibit excellent catalytic performance and stability, their high raw material costs, complex extraction and purification processes, and potential biocompatibility risks (such as immunogenicity and sensitization) greatly restrict their promotion and application in the industrial field. Therefore, the development of biomimetic nanozymes based on plant-derived proteins has significant research value and application potential. Summary of the Invention

[0003] To address the aforementioned problems in the prior art, this invention provides a soybean protein isolate nanozyme, its preparation method, and its application, featuring an artificial enzyme structure based on plant protein and containing Zn. 2+ By combining amino acid residues rich in coordination sites in soy protein isolate molecules, the conformation of soy protein is induced to recombine, thereby forming a nanosystem with antioxidant-like enzyme active sites.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] This invention provides a method for preparing soybean protein nanozymes, comprising the following steps:

[0006] (1) Precursor preparation: Soy protein isolate was dissolved in water to obtain a soy protein isolate solution;

[0007] (2) Chelation reaction: Take the soy protein isolate solution obtained in step (1) and mix it with ZnCl2 solution, stir magnetically to form a uniform suspension, and then heat and react under stirring;

[0008] (3) Purification treatment: The reaction solution obtained in step (2) is centrifuged at 10,000 rpm for 15 min, and the precipitate is washed to remove free ions;

[0009] (4) Preparation of finished product: The product obtained in step (3) is freeze-dried under vacuum to obtain the soybean protein nanozyme isolate.

[0010] As a preferred embodiment of the present invention, in step (1), the concentration of the soy protein isolate solution is 20-30 mg / mL.

[0011] As a preferred embodiment of the present invention, in step (2), the concentration of the ZnCl2 solution is 100-500 mg / mL, and the volume ratio of the soybean protein isolate solution to the ZnCl2 solution is 1:1.

[0012] As a preferred embodiment of the present invention, in step (2), the heating is carried out at 60°C and the reaction time is 4 hours.

[0013] As a preferred embodiment of the present invention, in step (2), the pH of the suspension is adjusted to 7-8 before heating.

[0014] The present invention also provides a soybean protein nanozyme prepared according to the preparation method described above.

[0015] This invention also provides the application of the soybean protein nanozyme described above in the preparation of antioxidant foods or drugs.

[0016] The present invention also provides an application of the soybean protein isolate nanozyme described above in the preparation of food packaging materials.

[0017] The soybean protein isolate nanozyme prepared in this invention possesses antioxidant properties and can be used to prepare antioxidant foods and pharmaceuticals. By precisely mimicking the active site structure and catalytic mechanism of natural enzymes, a zinc-soybean protein isolate artificial enzyme with multiple catalytic activities was constructed using a metal ion-mediated self-assembly method. This nanozyme exhibits various enzymatic activities (such as SOD-like activity, ·OH scavenging activity, and CAT-like activity), effectively scavenging various types of reactive oxygen species.

[0018] The soybean protein nanozyme prepared in this invention is used to prepare food packaging materials: The nanozyme is combined with biodegradable materials to prepare novel fruit packaging materials. The nanozyme activity can significantly reduce the respiration intensity of blueberries by regulating their respiratory metabolic pathways. It can effectively inhibit the hydrolysis of monosaccharides in overripe fruits, thereby maintaining the stability of soluble solids content. Simultaneously, the nanozyme's degradation effect on the endogenous enzyme activity of fruits significantly slows down the softening rate of blueberries.

[0019] In meat product packaging applications, this nanozyme exhibits excellent antibacterial properties. By specifically inhibiting the growth and reproduction of spoilage microorganisms, it can significantly slow down the decomposition rate of amino acids and proteins in meat products, thereby effectively extending the shelf life of meat products.

[0020] Soy protein isolate is a widely available, sustainably supplied, cost-effective, and nutrient-rich plant protein with unique advantages in constructing edible functional materials. Its structure is similar to the protein composition of natural enzymes, rich in hydroxyl, carboxyl, and amino functional groups, and provides numerous potential binding sites for metal ions. This structure can chelate with various transition metal ions to form complexes with enzyme-like activity. 2+ It is a safe and non-toxic essential trace element for the human body. This invention innovatively selects Zn. 2+ Using plant-derived soy protein isolate as a base material, artificial nanozymes are constructed through a biomimetic self-assembly strategy. This design has dual advantages: it overcomes the limitations of traditional animal-derived proteins, avoiding the industrialization bottlenecks of complex extraction processes and high costs; and it fully preserves the antioxidant active site characteristics of natural enzymes, achieving reactive oxygen species scavenging efficiency comparable to that of natural enzymes.

[0021] This invention presents an artificial nanozyme designed based on a biomolecule-metal ion self-assembly strategy. This nanozyme accurately mimics the active site structure and catalytic mechanism of natural enzymes, providing a new approach for developing high-performance enzyme-mimicking materials. A zinc-soybean protein isolate artificial enzyme with multiple catalytic activities was constructed using a metal ion-mediated self-assembly method. This nanozyme exhibits various enzyme activities (such as SOD-like activity, ·OH scavenging activity, and CAT-like activity), effectively scavenging various types of reactive oxygen species. Compared to natural enzymes, it also demonstrates significantly enhanced thermal stability and a wider pH adaptability, showing significant application potential in the field of antioxidants.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention uses soy protein isolate, which is widely available, sustainably supplied, and economically viable, as a biological carrier, combined with the safe and non-toxic essential trace element Zn. 2+ A novel biomimetic nanozyme was constructed through metal coordination. (Zn) 2+ It chelates with the active functional groups such as hydroxyl, carboxyl, and amino groups in soy protein isolate molecules to form stable complexes with enzyme-like activity. This zinc-soy protein isolate catalytic material design has the following significant advantages:

[0024] (1) Raw material advantages: Soy protein, as a plant-based raw material, has the characteristics of wide availability, low cost and strong renewability, while avoiding the potential allergenic risks of animal-derived protein;

[0025] (2) Structural advantages: Through precise control of Zn 2+ The coordination environment with protein functional groups successfully mimicked the active site structure of natural enzymes.

[0026] (3) Performance advantages: The obtained zinc-soy protein isolate material not only exhibits excellent catalytic efficiency, but also has good environmental stability and thermal stability;

[0027] (4) Application advantages: The technical solution of the present invention significantly reduces production costs and simplifies the preparation process, providing a practical solution for the industrial application of nanozymes.

[0028] The innovation of this invention lies in combining economical and environmentally friendly plant proteins with essential trace elements, and constructing a novel catalytic material that combines high performance and low cost through rational design, showing broad application prospects in fields such as anti-oxidation, food preservation, and environmental remediation. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The graph shows the SOD activity test results of solid zinc-soybean protein isolate nanozymes prepared from ZnCl2 solutions of different concentrations in Example 1.

[0031] Figure 2 Zn in Example 1 2+ The storage stability test results of zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3 are shown in Figure A, where A is the appearance during storage and B is the particle size, potential and PDI diagram.

[0032] Figure 3 Zn in Example 1 2+ SEM image of zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3;

[0033] Figure 4 Zn in Example 1 2+ TEM image of zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3;

[0034] Figure 5 Zn in Example 1 2+Elemental distribution diagram of zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3;

[0035] Figure 6 Zn in Example 1 2+ XRD pattern of zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3;

[0036] Figure 7 Zn in Example 1 2+ Thermogravimetric analysis of zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3;

[0037] Figure 8 Zn in Example 1 2+ The graph shows the SOD activity test results of the zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3. In the graph, A represents the effect of pH on the activity of the nanozyme ZnSP and natural SOD enzyme, and B represents the activity of ·O2 generated from riboflavin in the presence and absence of the nanozyme ZnSP. - The NBT absorption spectrum after the reaction, C represents the effect of natural SOD enzyme and nanozyme ZnSP on ·O2 at different concentrations. - The elimination efficiency is given by D, where D represents the effect of temperature on the activity of ZnSP nanozyme and natural SOD enzyme.

[0038] Figure 9 Zn in Example 1 2+ The figure shows the ·OH scavenging activity test results of the zinc-soybean protein isolate nanozyme prepared with a concentration of 200 mg / mL and a suspension pH of 7.3. In the figure, A and B are the effects of pH and temperature on the activities of nanozyme and AA, respectively. C is the UV-Vis absorption spectrum of TMB after different concentrations of nanozyme reacted with ·OH. D is the ·OH elimination efficiency of ascorbic acid (AA) and nanozyme at different concentrations.

[0039] Figure 10 Zn in Example 1 2+ The figure shows the CAT enzyme activity test results of the zinc-soybean protein isolate nanozyme prepared with a concentration of 200 mg / mL and a suspension pH of 7.3. In the figure, A and B are the dissolved oxygen yield curves of different concentrations of natural CAT and nanozyme ZnSP as a function of time, respectively. C is the rate of decomposition of H2O2 to generate dissolved O2 by different concentrations of natural CAT and nanozyme. D is the fluorescence spectrum of different concentrations of nanozyme ZnSP system in the presence of H2O2.

[0040] Figure 11 Zn in Example 1 2+The antibacterial performance test results of zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3 are shown in the figure.

[0041] Figure 12 Zn in Example 1 2+ The hemolytic test results of zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3 are shown in the figure. A is the hemolytic image, and B is the relative hemolyticity of water, PBS buffer and nanozyme (20-100 μg / mL).

[0042] Figure 13 Zn in Example 1 2+ The cytotoxicity test results of zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3 are shown in the figure. A and B show the changes in the viability of HepG2 and HeLa cells after incubation with different concentrations of nanozyme for 24 h. Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0044] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0047] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0048] The raw materials used in the following examples are all commercially available and conventional, and are not particularly limited. They will not be described again below.

[0049] Example 1

[0050] Examining Zn 2+ Effects of concentration and pH on the superoxide dismutase (SOD)-like activity of zinc-soybean protein isolate nanoenzymes. Experimental setup: Zn 2+ Concentration gradients (100, 200, 300, 400, 500 mg / mL) and pH gradients (3.0, 5.0, 7.0, 9.0, 11.0) were established, and the specific experimental methods are as follows:

[0051] Examining Zn 2+ Effect of concentration on the superoxide dismutase (SOD)-like activity of zinc-soybean protein isolate nanoenzymes:

[0052] (1) Dissolve soy protein isolate completely in deionized water to prepare a protein solution of 25 mg / mL;

[0053] (2) Chelation reaction: Take 10 mL of soy protein isolate solution and mix it with an equal volume of ZnCl2 solution (concentrations of 50, 100, 200, 300 and 500 mg / mL, respectively) and stir magnetically to form a uniform suspension;

[0054] (3) Condition optimization: The pH was precisely adjusted to 7.0 using 2.0M NaOH solution, and the mixture was continuously stirred for 4 hours under a 60℃ water bath condition;

[0055] (4) Purification treatment: The reaction solution was centrifuged at 10,000 rpm for 15 min and washed three times with deionized water to remove free ions;

[0056] (5) Preparation of finished product: The final product was obtained by vacuum freeze drying to obtain solid zinc-soybean protein nanozyme, which was stored at 4°C in the dark.

[0057] The SOD activity of solid zinc-soybean protein isolate nanozymes (ZnSP) prepared from ZnCl2 solutions of different concentrations was tested. Specifically, the SOD activity of the nanozymes was determined by the inhibitory effect of SOD on NBT reduction. Methionine (12 mM), riboflavin (25 μM), NBT (75 μM), and ZnSP of different concentrations were mixed in PBS buffer (25 mM, pH 7.4). The system was irradiated with a constant-intensity ultraviolet light source for 3 minutes at room temperature. Afterwards, the absorbance at 560 nm was measured, and the SOD activity was calculated using equation (1):

[0058]

[0059] Where A0 is the absorbance value without nanozyme, and A is the absorbance value after nanozyme is added.

[0060] The results are as follows Figure 1 As shown, by Figure 1 It can be seen that the solid zinc-soybean protein isolate nanozyme with the highest SOD activity was obtained when the ZnCl2 solution concentration was 200 mg / mL.

[0061] Investigate the effect of pH on the superoxide dismutase (SOD)-like activity of zinc-soybean protein isolate nanozymes:

[0062] (1) Dissolve soy protein isolate completely in deionized water to prepare a protein solution of 25 mg / mL;

[0063] (2) Chelation reaction: Take 10 mL of soy protein isolate solution and mix it with an equal volume of ZnCl2 solution (concentration of 200 mg / mL), and stir magnetically to form a uniform suspension;

[0064] (3) Condition optimization: The pH was precisely adjusted to 4.0, 5.0, 7.0, 9.0 and 11.0 using 2.0M NaOH solution, and stirred continuously for 4 hours under a 60℃ water bath condition;

[0065] (4) Purification treatment: The reaction solution was centrifuged at 10,000 rpm for 15 min and washed three times with deionized water to remove free ions;

[0066] (5) Preparation of finished product: The final product was obtained by vacuum freeze drying to obtain solid zinc-soybean protein nanozyme, which was stored at 4°C in the dark.

[0067] The SOD activity of solid zinc-soybean protein isolate nanozymes prepared at different pH values ​​was tested, and it was found that the SOD activity of the solid zinc-soybean protein isolate nanozymes was the highest at pH 7.0.

[0068] Further, the pH in step (3) was adjusted to 6.3, 6.5, 7.1, 7.3 and 7.5 respectively, and the SOD activity of solid zinc-soybean protein isolate nanozyme was measured. It was found that the SOD activity of the obtained solid zinc-soybean protein isolate nanozyme was the highest when the pH was 7.3.

[0069] In Zn 2+At a concentration of 200 mg / mL, the SOD-like activity reached its peak (66.31%); under near-physiological conditions at pH 7.3, the enzyme activity was optimal (76.32%). Furthermore, the storage stability of zinc-soybean protein isolate (ZnSP) after 7 days of storage at 4°C was evaluated. The results showed that the ZnSP nanozyme was uniformly dispersed in water, appearing as a translucent milky white substance, and exhibited good dispersibility and relative stability during storage. The storage stability of ZnSP was evaluated by measuring the physical changes, particle size, potential, and PDI of the nanozyme after 7 days of storage at 4°C. Figure 2 As shown, Figure A is an appearance diagram of the storage process, and Figure B is a diagram of particle size, potential, and PDI. Figure 2 It can be seen that ZnSP is uniformly dispersed in water, appearing as a translucent milky white substance. The average particle size of ZnSP increased from 412.96 nm to 464.21 nm, and the potential changed from -29.17 mV to -31.39 mV. The PDI showed an increasing trend, but remained around 0.3. This indicates that ZnSP exhibits good dispersibility and relative stability during storage.

[0070] Zn by SEM and TEM 2+ The morphology of the zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3 was characterized as follows: Figures 3-4 As shown, the zinc-soybean protein isolate nanozyme exhibits a layered structure with a relatively smooth surface. To study Zn... 2+ To determine whether the nanozyme integrates well into soy protein isolate, energy dispersive spectroscopy (EDS) elemental mapping analysis was used to obtain the distribution of Zn, N, C, and O on the surface of the nanozyme. Figure 5 As shown in the figure, the distribution of Zn, N, C, and O in the corresponding images of each element is obvious and uniform, indicating that Zn... 2+ Successfully combined with soy protein isolate, and Zn 2+ The binding with nanosheets is more compact. This result suggests that soy protein isolate may serve as a nucleating backbone and stabilizer for nanozymes, Zn 2+ It may bind to groups on soy protein isolate primarily through chelation or self-assembly.

[0071] XRD diagram as follows Figure 6 As shown, the thermogravimetric analysis diagram is as follows: Figure 7 As shown. Figure 6 and Figure 7 In this context, SP and ZnSP represent soy protein isolate and zinc-soy protein isolate nanozymes, respectively.

[0072] XRD and thermogravimetric analysis showed that, compared with soy protein isolate, zinc-soy protein isolate nanozymes not only exhibit excellent thermal stability over a wide temperature range, but also possess a unique crystal structure. 2+Successful self-assembly on the backbone of soy protein isolate transforms disordered soy protein isolate into an ordered crystal structure.

[0073] In Example 1, Zn 2+ The SOD-type enzyme activity, ·OH scavenging activity, and CAT-type enzyme activity of the zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3 are as follows: Figures 8-10 As shown, the test method for ·OH scavenging activity was as follows: the ability of ZnSP to scavenge ·OH was determined by UV-Vis spectrophotometry using TMB as a probe. ·OH oxidizes TMB to generate the blue product oxTMB, which has an absorption peak at 652 nm. FeSO4 (0.2×10 -3 (moles) and H2O2 (0.2 × 10) -3 (Moles) were reacted in deionized water for 3 minutes to generate ·OH. Nanozyme was added at room temperature and reacted for 5 minutes, followed by the addition of TMB to investigate the ·OH content. The percentage of ·OH removed was calculated according to equation (2):

[0074]

[0075] A0 represents the absorbance of TMB. A1 and A2 represent the absorbance of oxTMB at 652 nm with and without nanozymes, respectively.

[0076] Figure 8 In the diagram, A represents the effect of pH on the activities of nanozyme ZnSP and natural SOD enzyme, and B represents the effect of pH on the activity of ·O2 generated from riboflavin in the presence and absence of nanozyme ZnSP. - The NBT absorption spectrum after the reaction, C represents the effect of natural SOD enzyme and nanozyme ZnSP on ·O2 at different concentrations. - The elimination efficiency is given by D, where D represents the effect of temperature on the activity of nanozyme ZnSP and natural SOD enzyme. Figure 9 In the figure, A and B represent the effects of pH and temperature on the activities of nanozymes and AA, respectively; C represents the UV-Vis absorption spectra of TMB after different concentrations of nanozymes react with ·OH; and D represents the elimination efficiency of ascorbic acid (AA) and nanozymes on ·OH at different concentrations. Figure 10 In the figure, A and B are the dissolved oxygen yield curves of different concentrations of natural CAT and nanozyme ZnSP as a function of time, respectively; C is the rate of decomposition of H2O2 to generate dissolved O2 by different concentrations of natural CAT and nanozyme; and D is the fluorescence spectrum of different concentrations of nanozyme ZnSP system in the presence of H2O2.

[0077] By measuring the activities of SOD-like enzymes, ·OH scavenging activities, and CAT-like enzymes, zinc-soybean protein isolate nanozymes, compared with natural enzymes, not only have multiple antioxidant effects, but also exhibit significantly enhanced thermal stability and wider pH adaptability.

[0078] For Zn in Example 1 2+ The antibacterial properties and biosafety of zinc-soybean protein isolate nanozyme prepared at a concentration of 200 mg / mL and a suspension pH of 7.3 were tested. The specific steps for testing the antibacterial properties were as follows: To test the antibacterial ability of ZnSP against Staphylococcus aureus and Escherichia coli, the nanozyme was diluted to different concentrations (12.5, 25, 50, 75, and 100 μg / mL). Subsequently, 5 mL of bacterial mixture (10 μg / mL) was added. 9 (CFU / mL). A blank control was used without nanozyme. 100 μL of the diluted co-culture solution was extracted and plated onto agar plates. Incubation was performed at 36°C for 24-36 h to allow colony expansion.

[0079] Biosafety experiments: 1. Hemolytic test

[0080] Red blood cell precipitate isolated from mouse serum was washed three times with PBS buffer. Red blood cells were mixed with ZnSP, PBS buffer, and purified water, respectively, and incubated at 37°C for 2 h. The supernatant was centrifuged. Absorbance was measured at 492 nm. The absorbance of the PBS + red blood cell group and the purified water + red blood cell group were used as standards for non-hemolysis and complete hemolysis, respectively, and calculated according to formula (3):

[0081]

[0082] As represents the absorbance of the mixture, An represents the absorbance of the unhemolyzed mixture, and Ap represents the absorbance of the completely hemolyzed mixture.

[0083] The viability of HeLa cells (or HepG2 cells) incubated with nanozymes was determined using the MTT assay. HeLa cells were cultured in 96-well plates at a density of 2 × 10⁶ cells / well. 4 Cells / mL. Replace the substrate with nanozyme solution and incubate for 1 day. Rinse the wells with PBS buffer and add freshly prepared MTT substrate solution (100 μL, 0.5 mg / mL) to each well. Incubate for another 3 hours, remove the MTT substrate, and add DMSO (100 μL) to each well. Finally, the viability of HeLa cells was determined by calculating the absorbance ratio (λ = 570 nm) between ZnSP-incubated HeLa cells and HeLa cells incubated with culture medium only. The results are shown below. Figures 11-13 As shown, where Figure 11 This is a graph showing the results of the antibacterial performance test. Figure 11As can be seen, by evaluating the antibacterial effects on Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), mixing bacteria with different concentrations of nanozymes showed that nanozymes have excellent antibacterial and bactericidal effects (Figure a). Microbial activity decreased rapidly with increasing nanozyme concentration. Even the lowest concentration (12.5 μg / mL) of nanozymes showed a significant bactericidal effect; when the nanozyme concentration reached 100 μg / mL, the bacterial kill rate reached over 90%, with a more pronounced effect on Escherichia coli. The antibacterial effect of nanozymes mainly relies on their inherent oxidoreductase-like activity, which can eliminate non-microorganisms, disrupt bacterial biofilms, and rapidly kill cells. Figure 12 In the image, A represents the hemolytic activity, and B represents the relative hemolytic activity of water, PBS buffer, and nanozyme (20-100 μg / mL). Figure 12 As can be seen from the hemolytic effect of nanozymes studied through erythrocyte separation, the supernatant in the pure water group was bright red due to the destruction of erythrocytes. In the PBS and nanozyme groups, blood cells precipitated, thus the supernatant became clear. The nanozyme group had the highest hemolysis rate (100 μg / mL) at 1.68%, which is below the allowable limit of 5%. Figure 13 In the study, the changes in the viability of HepG2 and HeLa cells after incubation with different concentrations of nanozymes A and B for 24 hours were analyzed. Figure 13 As can be seen from the MTT assay, the toxicity of nanozymes to HepG2 and HeLa cells was evaluated. After incubation with nanozymes for 24 h, cell viability was close to 100% at low concentrations (≤50 μg / mL) and higher than 90% at high concentrations (e.g., 200 μg / mL). These results indicate that nanozymes possess biosafety.

[0084] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing soybean protein isolate nanozymes, characterized in that, The following steps are involved: (1) Precursor preparation: Soy protein isolate was dissolved in water to obtain a soy protein isolate solution; (2) Chelation reaction: Take the soy protein isolate solution obtained in step (1) and mix it with ZnCl2 solution, stir to form a suspension, and then heat and react under stirring; (3) Purification treatment: Centrifuge the reaction solution obtained in step (2) and wash the precipitate; (4) Preparation of finished product: The product obtained in step (3) is freeze-dried under vacuum to obtain the soybean protein nanozyme isolate.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the soy protein isolate solution is 20-30 mg / mL.

3. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the ZnCl2 solution is 100-500 mg / mL, and the volume ratio of the soybean protein isolate solution to the ZnCl2 solution is 1:

1.

4. The preparation method according to claim 1, characterized in that, In step (2), the temperature is raised to 60°C and the reaction time is 4 hours.

5. The preparation method according to claim 1, characterized in that, In step (2), the pH of the suspension is adjusted to 7-8 before heating.

6. A soybean protein nanozyme prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the soybean protein nanozyme according to claim 6 in the preparation of antioxidant foods or pharmaceuticals.

8. The application of the soybean protein nanozyme according to claim 6 in the preparation of food packaging materials.

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