Preparation method of polysaccharide gel-coated iron nanoszyme and application of prolamine modification thereof

Abstract

The application discloses a preparation method of polysaccharide gel-coated iron nanoscale enzyme and an alcohol-soluble protein modification application thereof, takes carrageenan as a gel matrix, disperses oxidized sodium alginate and histidine in the carrageenan gel, forms a dynamic Schiff base between aldehyde groups of the oxidized sodium alginate and amino groups of the histidine, and forms the iron nanoscale enzyme by complexing with imidazole groups and carboxyl groups in the gel system and in-situ mineralization, so that the preparation process is simple, the condition is mild, the cost is low, the obtained polysaccharide gel-coated iron nanoscale enzyme can modify the structure of zein based on the peroxidase-like activity of the iron nanoscale enzyme, grafts various phenolic compounds on the zein, improves the functional properties and nutritional value of the protein, and solves the problem that natural enzymes such as laccase, peroxidase and tyrosinase cannot catalyze the oxidative modification of the protein in an ethanol-water binary solvent and cannot play a catalytic role due to easy denaturation.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of polysaccharide gel-coated iron nanoszyme and its prolamine modification application. BACKGROUND

[0002] Enzymatic modification is a common method for enhancing the functional properties of biological macromolecules such as proteins and polysaccharides in food. Oxidoreductases such as laccase, horseradish peroxidase, and tyrosinase can catalyze side chain modification or form intermolecular covalent interactions in the presence of phenolic hydroxyl groups, thereby improving functional properties and nutritional value. However, natural enzymes face problems such as complex process, narrow optimal reaction conditions, and easy denaturation during preparation and use. To compensate for the shortcomings of natural enzymes, research on enzyme mimics is increasing. Many materials such as metal particles, metal oxide particles, metal-organic frameworks, and carbon dots can catalyze some redox reactions under mild conditions, thereby exhibiting properties similar to those of oxidases, peroxidases, catalases, and superoxide dismutases. These materials are classified as "nanoszymes" and have the potential to replace natural enzymes.

[0003] Iron plays an important role in the human body, and iron fortifiers including divalent iron salts, trivalent iron salts, various chelated iron, and elemental iron are used in food. Therefore, iron-based materials have high biological safety, low cost, and a wide range of application prospects. The nanoszyme activity of iron-based materials is derived from an electron transfer channel composed of divalent iron and trivalent iron, thereby achieving the conversion of substrates to products. Divalent iron ions are unstable and easily oxidized, so it is necessary to develop materials that can maintain their valence state. In the active center of natural enzymes, histidine is usually coordinated with metal ions, so mimicking natural enzymes and increasing the coordination of histidine with iron ions can help enhance the catalytic activity of materials. Food-grade materials are receiving increasing attention due to their excellent usability, biocompatibility, and biodegradability. Food-grade hydrogels constructed from food-grade materials can serve as templates for nanoszyme synthesis, providing conditions for the structure and property adjustment of nanoszymes by modifying the chemical groups of the materials.

[0004] Prolamine is the main storage protein in grains and a major byproduct after starch extraction. Due to the high content of hydrophobic amino acids, prolamine is water-insoluble, limiting its application. Laccase, peroxidase, and tyrosinase can catalyze the oxidative modification of proteins, but natural enzymes are easily denatured in ethanol-water binary solvents, making it difficult for them to exert catalytic effects. Therefore, developing nanoszymes that are resistant to ethanol-water binary solvents to replace natural enzymes is a new method and approach for prolamine modification. SUMMARY

[0005] The purpose of this invention is to provide a method for preparing polysaccharide gel-coated iron nanozymes and their application in modifying alcohol-soluble proteins, which solves the problem that natural enzymes such as laccase, peroxidase, and tyrosinase are prone to denaturation and cannot exert their catalytic effect when catalyzing the oxidation modification of proteins in ethanol-water binary solvents.

[0006] This invention is achieved through the following technical solutions:

[0007] A method for preparing polysaccharide gel-coated iron nanozymes includes the following steps: Under nitrogen purging, sodium oxidized alginate (OSA) is dispersed in a carrageenan aqueous solution, with a carrageenan mass fraction of 0.4-0.6% and an OSA mass fraction of 0.8-1.2%; the mixture is kept at 80°C and stirred for 0.5-1.5 hours; histidine is added and stirred for 20-40 minutes, with a histidine to OSA mass ratio of 0.2:1-1:1; Fe is then added. 3+ and Fe 2+ A mixed solution with an ion molar ratio of 2:1 was stirred until homogeneous. KOH solution was quickly added to bring the pH of the system to 12. Stirring was continued for 10 minutes. After cooling, a gel was formed, resulting in an iron nanozyme coated with a polysaccharide gel. The nanozyme was then freeze-dried for later use.

[0008] The concentration of the KOH solution is 1-2 mol / L.

[0009] The preparation of oxidized sodium alginate (OSA) includes the following steps: sodium alginate (SA) is dispersed in anhydrous ethanol to form a suspension, and an aqueous solution of NaIO4 is added. The molar ratio of NaIO4 to sodium alginate monomer units is 20%-100%. After stirring at room temperature in the dark for 6 hours, ethylene glycol of the same molar ratio as NaIO4 is added to terminate the reaction. Unreacted NaIO4 and ethylene glycol are removed by dialysis. The dialysate is freeze-dried to obtain oxidized sodium alginate (OSA) solid powder with different degrees of oxidation.

[0010] Carrageenan was used as the gel matrix, with sodium alginate oxidized and histidine dispersed within the carrageenan gel. The aldehyde group of sodium alginate oxidized formed a dynamic Schiff base with the amino group of histidine. Iron nanozymes were formed through in-situ mineralization via complexation with imidazole and carboxyl groups in the gel system. The activity of iron nanozymes was regulated by the coordination effect of histidine, mimicking the active site of natural peroxidases. Based on the peroxidase-like activity of iron nanozymes, the structure of zein was modified by grafting various phenolic compounds onto zein, improving the functional properties and nutritional value of the protein.

[0011] Therefore, the application also protects the application of the polysaccharide gel-coated iron nanoszyme obtained by the above preparation method in zein modification. Specifically, the following steps are included: corn zein or wheat zein or sorghum zein is dissolved in a 60-75% (v / v) ethanol aqueous solution, adjusted to pH 2.0-6.0, a phenolic compound is added, adjusted to pH 2.0-6.0, the above polysaccharide gel-coated iron nanoszyme is continuously stirred at 25°C, the iron nanoszyme is recovered by a magnet, the protein solution is dialyzed against a 70% (v / v) ethanol-water solution for 24 hours to remove free polyphenols, and concentrated and lyophilized for standby use.

[0012] In particular, the phenolic compound is selected from any one of tea polyphenols or tannic acid or quercetin.

[0013] The beneficial effects of the application are as follows:

[0014] In the application, carrageenan is used as a gel matrix, oxidized sodium alginate and histidine are dispersed in the carrageenan gel, the aldehyde group of the oxidized sodium alginate and the amino group of the histidine form a dynamic Schiff base, the iron nanoszyme is formed by complexing with the imidazole group and the carboxyl group in the gel system and in-situ mineralization, the preparation process is simple, the conditions are mild, the cost is low, the polysaccharide gel-coated iron nanoszyme obtained can modify the structure of corn zein based on the peroxidase-like activity of the iron nanoszyme, graft various phenolic compounds to the corn zein, and improve the functional properties and nutritional value of the protein, thereby solving the problem that natural enzymes such as laccase, peroxidase and tyrosinase cannot catalyze the oxidative modification of the protein in an ethanol-water binary solvent due to easy denaturation. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the infrared spectrum of the sample and its control; wherein, Carra refers to carrageenan, Carra-OSA-His refers to the hydrogel formed by carrageenan-oxidized sodium alginate-histidine in Example 1. Carra-OSA-His-Fe refers to the iron nanoszyme based on the carrageenan-oxidized sodium alginate-histidine hydrogel.

[0016] Figure 2 is the X-ray photoelectron spectroscopy of the polysaccharide gel of the iron nanoszyme obtained in Example 1.

[0017] Figure 3 is the influence of reaction time on the amount of catalytic product of the iron nanoszyme.

[0018] Figure 4 is the influence of reaction temperature on the amount of catalytic product of the iron nanoszyme.

[0019] Figure 5 is the influence of reaction pH on the amount of catalytic product of the iron nanoszyme with TMB as a substrate.

[0020] Figure 6 Effect of reaction pH on the amount of product generated by iron nanoszyme catalysis under ABTS as substrate.

[0021] Figure 7 Effect of increasing substrate ABTS and H2O2 on the initial reaction rate of iron nanoszyme.

[0022] Figure 8 Double-reciprocal fitting curve of initial reaction rate and substrate (ABTS and H2O2) concentration.

[0023] Figure 9 Effect of increasing substrate TMB and H2O2 on the initial reaction rate of iron nanoszyme.

[0024] Figure 10 Double-reciprocal fitting curve of initial reaction rate and substrate (TMB and H2O2) concentration. DETAILED DESCRIPTION:

[0025] The following is a further description of the present application, but not a limitation of the present application.

[0026] Example 1: Preparation and characterization of iron nanoszyme polysaccharide gel

[0027] 1.1 Preparation of oxidized sodium alginate OSA

[0028] 10 g of sodium alginate SA was dispersed in 50 mL of anhydrous ethanol to form a suspension, and different amounts of NaIO4 were dissolved in 50 mL of water. The molar ratio of NaIO4 to sodium alginate monomer unit was adjusted to 20%, 40%, 60%, 80%, and 100%, respectively. After stirring for 6 h at room temperature in the dark on a magnetic stirrer, the reaction was terminated by adding an equal molar amount of ethylene glycol, and the unreacted NaIO4 and ethylene glycol were removed by dialysis. The dialysate was freeze-dried to obtain OSA solid powder with different degrees of oxidation.

[0029] 1.2 Preparation of iron nanoszyme gel

[0030] In a beaker, 0.5% carrageenan and 1% OSA were dissolved, stirred at 80°C for 1 h at a speed of 600 rpm, and deoxygenated by passing nitrogen gas for 30 min. Thereafter, the experiment was carried out at 80°C under the condition of nitrogen gas. Histidine was added and stirred for 30 min. The ratio of histidine to OSA was 0.2:1, 0.5:1, and 1:1, respectively. When the ratio of histidine to OSA was 0.2:1, the infrared spectrum of the hydrogel formed by carrageenan-oxidized sodium alginate-histidine is shown in FIG. 1. Figure 1Carra-OSA-His. Then a mixed solution of FeCl3and FeCl2(molar ratio 2:1) was added and stirred uniformly. The pH of the system was adjusted to 12 by adding 1.5M KOH solution rapidly. The stirring was continued for 10 min and the gel was formed after cooling. The gel was freeze-dried for later use.

[0031] The samples with the ratio of histidine to OSA of 0.2:1, 0.5:1 and 1:1 were marked as A1, A2 and A3 respectively. The infrared spectrum of sample A1 is shown in Fig. 1. Figure 1 Carra-OSA-His-Fe.

[0032] The control sample with OSA and carrageenan was marked as B.

[0033] The preparation of control sample B included the following steps: 0.5% carrageenan and 1% OSA were dissolved in a beaker, stirred at 80°C for 1 h at a speed of 600 rpm, and deoxygenated by passing nitrogen for 30 min. Thereafter, the experiment was carried out at 80°C with nitrogen passing. A mixed solution of FeCl3and FeCl2(molar ratio 2:1) was added and stirred uniformly. The pH of the system was adjusted to 12 by adding 1.5M KOH solution rapidly. The stirring was continued for 10 min and the gel was formed after cooling. The gel was freeze-dried for later use.

[0034] The control sample with histidine and carrageenan was marked as D.

[0035] The preparation of control sample D included the following steps: 0.5% carrageenan was dissolved in a beaker, stirred at 80°C for 1 h at a speed of 600 rpm, and deoxygenated by passing nitrogen for 30 min. Thereafter, the experiment was carried out at 80°C with nitrogen passing. 1% histidine was added and stirred for 30 min. A mixed solution of FeCl3and FeCl2(molar ratio 2:1) was added and stirred uniformly. The pH of the system was adjusted to 12 by adding 1.5M KOH solution rapidly. The stirring was continued for 10 min and the gel was formed after cooling. The gel was freeze-dried for later use.

[0036] The control sample with only carrageenan was marked as C. The preparation of control sample C included the following steps: 0.5% carrageenan was dissolved in a beaker, stirred at 80°C for 1 h at a speed of 600 rpm, and deoxygenated by passing nitrogen for 30 min. Thereafter, the experiment was carried out at 80°C with nitrogen passing. A mixed solution of FeCl3and FeCl2(molar ratio 2:1) was added and stirred uniformly. The pH of the system was adjusted to 12 by adding 1.5M KOH solution rapidly. The stirring was continued for 10 min and the gel was formed after cooling. The gel was freeze-dried for later use.

[0037] The prepared samples were freeze-dried and then characterized by infrared spectrum and X-ray photoelectron spectroscopy to characterize the structure of the iron-based nanoscale enzyme. Figure 1The infrared spectrum shows that there is a Schiff base in the gel, and iron forms a complex with imidazole and carboxyl groups through coordination bonds, followed by hydrogen bonds between sodium alginate and carrageenan. Figure 2 X-ray photoelectron spectroscopy (XPS) shows that the iron in the iron nanoscale enzyme has two valence states of 2+ and 3+.

[0038] Example 2: Enzymatic properties of the iron nanoscale enzyme polysaccharide gel

[0039] 2.1 Effect of reaction time, temperature, pH, and histidine addition amount on the activity of the iron nanoscale enzyme

[0040] Take 20 μL of the iron nanoscale enzyme prepared in Example 1, add 140 μL of Tris-HCl buffer (0.2 M, pH 7.0) and mix well, then add 20 μL of TMB (20 mM), and finally add 20 μL of H2O2 (20 mM). The final concentration of TMB in the system is 2 mM, and the final concentration of H2O2 is 2 mM. Use an enzyme label instrument to scan the absorbance at 652 nm in kinetic mode to determine the peroxidase properties of the iron nanoscale enzyme. According to the product absorbance coefficient, calculate the initial reaction rate of product generation.

[0041] Determine the peroxidase properties at different times (0-150 min), different temperatures (25-65°C), and different pHs (pH 2-7).

[0042] Figure 3 The results show that the iron nanoscale enzyme catalyzes the oxidation of TMB, and the absorbance of the product oxTMB at the maximum absorption peak of 652 nm increases with time, indicating that it has peroxidase properties.

[0043] Figure 4 The results show that the iron nanoscale enzyme catalyzes the oxidation of TMB, and the absorbance of the product oxTMB at the maximum absorption peak of 652 nm increases with temperature, and then decreases, indicating that the optimal catalytic temperature is around 45°C.

[0044] Figure 5 The results show that the iron nanoscale enzyme catalyzes the oxidation of TMB, and the absorbance of the product oxTMB at the maximum absorption peak of 652 nm decreases with increasing pH, indicating that the catalytic activity of the iron nanoscale enzyme is stronger in acidic conditions, and the enzyme activity decreases by less than 50% in the pH range of 3-5.

[0045] Figure 6 The results show that the iron nanoscale enzyme catalyzes the oxidation of ABTS, and the absorbance of the product oxABTS at the maximum absorption peak of 470 nm decreases with increasing pH, indicating that the catalytic activity of the iron nanoscale enzyme is stronger in acidic conditions.

[0046] Figures 3-6 The results show that increasing the content of histidine in the nanoscale enzyme can significantly increase the generation of the product catalyzed by the nanoscale enzyme.

[0047] 2.2 Steady-state kinetics of iron nanoszyme catalyzing TMB and ABTS color development system

[0048] The steady-state kinetics of iron nanoszyme in the TMB / ABTS+H2O2 reaction system was explored. The product concentration was calculated by the molar extinction coefficient corresponding to oxTMB / oxABTS, and the initial reaction rate was calculated. The corresponding parameters were calculated by the Michaelis-Menten equation. In this experiment, the initial reaction rate of iron nanoszyme under different concentrations of TMB / ABTS (0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 4 mM) was determined by fixing the concentration of H2O2 (2 mM); the initial reaction rate of iron nanoszyme under different concentrations of H2O2 (0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM) was determined by fixing the concentration of TMB / ABTS (2 mM).

[0049] Table 1:

[0050]

[0051]

[0052] Table 1 shows the K m and V max calculated according to the Michaelis-Menten equation under different concentrations of TMB / ABTS and different concentrations of H2O2. max The increase of histidine content in nanoszyme can significantly improve the V max of catalytic reaction. The affinity of nanoszyme for H2O2 is higher than that for TMB and ABTS.

[0053] Figure 7 The initial reaction rate of iron nanoszyme increases with the increase of substrates ABTS and H2O2. Figure 8 The double-reciprocal fitting curve of initial reaction rate and substrate (ABTS and H2O2) concentration is shown. Figure 9 The initial reaction rate of iron nanoszyme increases with the increase of substrates TMB and H2O2. Figure 10 The double-reciprocal fitting curve of initial reaction rate and substrate (TMB and H2O2) concentration is shown. Experimental data show that the increase of histidine content in nanoszyme can significantly improve the initial reaction rate of nanoszyme catalyzing products.

[0054] Example 3: Application of iron nanoszyme hydrogel modified prolamin

[0055] 3.1 Prolamin modification process

[0056] Dissolve 1 g zein in 50 mL 70% (v / v) aqueous ethanol solution, adjust to pH 2.0-6.0 with NaOH or HC1. To prepare zein-polyphenol conjugates, dissolve 0.1-0.5 g tea polyphenols / tannic acid / quercetin in 50 mL 70% (v / v) aqueous ethanol solution, adjust to pH 2.0-6.0 with NaOH solution or HC1 solution. Then mix the two solutions together, and add 0.1-0.5 g of iron nanoszyme Al prepared in Example 1, continuously stir the reaction at 25 °C for 2 hours, recover the iron nanoszyme with a magnet, dialyze the protein solution against 70% (v / v) aqueous ethanol solution for 24 hours to remove free polyphenols, concentrate the solution to 1 g / mL, and store at 4 °C until use.

[0057] Freeze-dry for storage. The modified sample without addition of iron nanoszyme is used as a control sample.

[0058] 3.2 Functional properties of modified zein

[0059] Phenolic grafting rate determination: Mix 0.5 mL of diluted sample with 2.5 mL Foline Ciocalteu phenol reagent (0.2 N) at room temperature. After 5 minutes, add 2 mL 7.5% (w / v) sodium carbonate solution, vortex the mixture and let it stand in the dark at room temperature for 2 hours. Measure the absorbance at 760 nm using a UV spectrophotometer.

[0060] Hydrophobicity determination: Dilute the sample to a protein concentration of 0.02-0.40 g / L in 10 mmol / L phosphate buffer (pH 7.0) at a protein concentration of 10 g / L. Then, mix 4 mL of the diluted solution with 20 μΐ^ANS solution (8.0 mmol / L in the same buffer) and measure on a fluorescence spectrometer using 390 / 470 nm excitation / emission wavelengths and 5 nm slits. Plot the intensity values against the protein concentration, and the slope of the regression curve indicates the surface hydrophobicity.

[0061] Table 2

[0062]

[0063] Table 2 shows that the phenolic grafting rate of zein increases with increasing amount of added nanoszyme. The control sample has a very low phenolic grafting rate.

[0064] Table 2 shows that the hydrophobicity of zein decreases with increasing phenolic grafting rate, indicating that the water solubility of zein will be enhanced, which is beneficial for the application of zein in the food industry.

[0065] 3.3 Nutritional value of modified zein

[0066] In vitro digestibility: 10 mL of sample solution (10 g / L) was adjusted to pH 2.0 with 1 mol / L HC1 and 2.0 mg of pepsin was added. The mixture was incubated at 37 °C for 120 min. For pepsin hydrolysis, the sample solution was first digested in pepsin at 37 °C for 60 min, and then heated at 90 °C for 5 min to inactivate the pepsin. The obtained gastric digest was quickly cooled and lyophilized. The lyophilized digest was redissolved in 10 mL of 0.2 mol / L phosphate buffer (pH 8.0), and then digested with 6 mg of trypsin at 37 °C for 60 min. The digestion of enzyme was terminated by adding 10 mL of trichloroacetic acid (200 g / L). The obtained mixture was centrifuged for 20 min. The collected supernatant was diluted with five volumes of water, while the absorbance value was read at 280 nm using a UV spectrophotometer.

[0067] DPPH radical scavenging activity: The antioxidant activity of zein, zein-polyphenol conjugate was measured using DPPH radical method. 2 mL of diluted sample was mixed with 2 mL of DPPH (0.175 mM in methanol). The mixture was then kept in the dark for 1 h, and the residual DPPH concentration was recorded at 517 nm using a spectrophotometer.

[0068] Table 2 shows that the in vitro digestibility of zein was not affected by the grafting of polyphenols.

[0069] Table 2 shows that the antioxidant activity of zein was enhanced with the increase of grafting rate, which has the application prospect as a food functional ingredient.

Claims

1. A method for the preparation of polysaccharide gel-coated iron nanoszyme, characterized by, The method comprises the following steps: under the condition of nitrogen input, oxidized sodium alginate is dispersed in a carrageenan aqueous solution, the mass fraction of the carrageenan is 0.4-0.6%, the mass fraction of the oxidized sodium alginate is 0.8-1.2%, 80 DEG C is kept for 0.5-1.5 hours under stirring, histidine is added and stirred for 20-40 min, the mass ratio of the histidine to the oxidized sodium alginate is 0.2:1-1:1, a mixed solution of Fe 3+ and Fe 2+ with a molar ratio of 2:1 is added, the system is uniformly stirred, KOH solution is quickly added to make the pH of the system reach 12, the stirring is continuously carried out for 10 min, the gel is formed after cooling, a polysaccharide gel coated iron nanoscale enzyme is obtained, and the iron nanoscale enzyme is freeze-dried for standby.

2. The production method according to claim 1, characterized by, The concentration of the KOH solution is 1-2 mol / L.

3. The production method according to claim 1, characterized by, The preparation of oxidized sodium alginate comprises the following steps: Sodium alginate is dispersed in anhydrous ethanol to form a suspension, and an aqueous NaIO4 solution is added, the molar ratio of NaIO4 to monomer units of sodium alginate being 20%-100%, after stirring at room temperature for 6 h in the dark, an equal molar amount of ethylene glycol is added to terminate the reaction, unreacted NaIO4 and ethylene glycol are removed by dialysis, and the dialysate is freeze-dried to obtain oxidized sodium alginate solid powder with different degrees of oxidation.

4. The polysaccharide gel-coated iron nanoszyme prepared by the preparation method according to any one of claims 1-3 is applied to zein modification.

5. Use of the polysaccharide gel-coated iron nanoszyme according to claim 4 for gliadin modification, characterized in that, The following steps are included, corn zein or wheat zein or sorghum zein is dissolved in an aqueous ethanol solution with a volume fraction of 60-75%, the pH is adjusted to 2.0-6.0, a phenolic compound is added, the pH is adjusted to 2.0-6.0, the polysaccharide gel-coated iron nanoszyme is added, continuous stirring is carried out at 25°C, the iron nanoszyme is recovered by a magnet, the protein solution is dialyzed against an aqueous ethanol solution with a volume fraction of 70% for 24 h to remove free polyphenols, and concentration and freeze-drying are carried out for standby use.

6. The polysaccharide gel-coated iron nanoszyme for zein modification according to claim 5, wherein, the phenolic compound is selected from any one of tea polyphenols, tannic acid or quercetin.

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