Glycosylated rice protein zymolyte and anti-oxidation application thereof

Abstract

The invention belongs to the technical field of biochemistry, and discloses a glycosylated rice protein zymolyte and antioxidant application thereof. The glycosylated rice protein zymolyte is prepared by performing glycosylation modification on the rice protein zymolyte by using monosaccharide, and the monosaccharide is any one of fructose, xylose or arabinose. The product shows remarkable in-vitro antioxidant activity and an in-vivo antioxidant effect in a zebra fish model, and can be used as an antioxidant or a nutritional supplement to improve the functional characteristics and economic value of rice protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to a glycosylated rice protein hydrolysate and its antioxidant application. Background Art

[0002] A large amount of processing by-product rice dregs containing 8% protein is produced during the production of organic acids and starch sugars from rice. The biological value and protein value of rice protein are higher than those of other cereal proteins. The amino acid composition of rice protein is reasonably balanced and the amino acid content is high. Moreover, rice protein is recognized as a high-quality food protein with various effects such as high nutritional value, low allergy reaction, and cholesterol level reduction.

[0003] In recent years, many experiments have studied the nutritional value of rice protein hydrolysates and found that rice protein hydrolysates have disadvantages such as poor functional properties like anti-digestion, which limits their application in the food industry. Glycosylation modification technology can change the structure and physicochemical properties of bioactive peptides, thereby improving their biological activities and having a wide range of application prospects. After glycosylation modification, the stability of rice protein can be improved and its solubility can be enhanced. Therefore, it is of great significance to enzymatically hydrolyze it and perform glycosylation modification to improve its functional properties and biological activities. Summary of the Invention

[0004] The purpose of the present invention is to provide an antioxidant application of a glycosylated rice protein hydrolysate.

[0005] The first aspect of the present invention provides a method for analyzing the structure of a rice protein hydrolysate before and after glycosylation, and the method is specifically as follows:

[0006] (1) Particle size measurement: For the enzymatic hydrolysate sample and the glycosylated sample, a particle size analyzer is used to detect their particle sizes.

[0007] (2) Degree of grafting measurement: Respectively take RPH and the glycosylated product, and use OPA to characterize the occurrence of the glycosylation reaction by measuring the decrease in the content of free amino acids in the system.

[0008] (3) Fourier transform infrared absorption spectroscopy: Respectively mix the freeze-dried RPH and the glycosylated product with potassium bromide powder in a certain mass ratio, and then use a Fourier transform infrared spectrometer to perform band scanning.

[0009] (4) Ultraviolet spectroscopy: Take RPH and the glycosylated sample, and use an ultraviolet-visible spectrophotometer to perform scanning

[0010] The second aspect of the present invention provides a scheme for in vitro detecting the antioxidant ability of a glycosylated rice protein hydrolysate, and the method is specifically as follows:

[0011] (1) Fe 2+Chelating ability: Samples with different mass concentration gradients of RPH, glycosylated samples, and GSH solution were taken respectively, and the chelating ability of the samples to ferrous ions was indirectly determined by the phenanthroline method.

[0012] (2) Superoxide anion radical scavenging ability: Samples with different mass concentration gradients of RPH, glycosylated samples, and GSH solution were taken respectively, and the superoxide anion radical scavenging ability was indirectly determined by the pyrogallol autoxidation method.

[0013] (3) DPPH radical scavenging rate: Samples with different mass concentration gradients of RPH, glycosylated samples, and GSH solution were taken respectively. The antioxidant ability was calculated by detecting the scavenging effect of different samples on DPPH radicals at a wavelength of 517 nm.

[0014] The third aspect of the present invention provides a scheme for detecting the antioxidant ability of glycosylated rice protein hydrolysate in vivo, as follows:

[0015] (1) The present invention uses zebrafish to establish an oxidative stress model to detect the antioxidant ability of glycosylated rice protein hydrolysate in vivo.

[0016] (2) Detect the ROS content in zebrafish, and analyze the in vivo antioxidant activity through fluorescence intensity.

[0017] (3) Using MDA, CAT, and T-SOD kits, the MDA concentration, CAT activity, and T-SOD activity in zebrafish were detected by a UV spectrophotometer.

[0018] (4) The breeding of zebrafish includes sex separation in a water tank, and under controlled temperature and light conditions, artemia eggs are fed at a specified time. Zebrafish embryos are obtained by natural mating.

[0019] (5) Before the formal experiment, the maximum dosage of RPH-A for model zebrafish needs to be obtained through a toxicity experiment.

[0020] The fourth aspect of the present invention provides an application of glycosylated rice protein hydrolysate in the preparation of an antioxidant.

[0021] The fifth aspect of the present invention provides an antioxidant, which includes the above-mentioned glycosylated rice protein hydrolysate.

[0022] The sixth aspect of the present invention provides a nutritional preparation composition, which includes the above-mentioned glycosylated rice protein hydrolysate.

[0023] In this study, four indicators, namely particle size, grafting degree, infrared spectrum, and ultraviolet spectrum, were used to characterize the formation of glycosylated products. After glycosylation, the in vitro antioxidant activity of RPH was improved to varying degrees. It also significantly increased the CAT activity in zebrafish, reduced the MDA content, and enhanced the T-SOD activity, thereby improving the antioxidant capacity of zebrafish. This study not only provided a theoretical and technical basis for the processing and production of functional glycosylated rice protein hydrolysates, but also enhanced the industrial application value of rice protein and improved its economic utilization rate. In addition, as a sustainable and environmentally friendly project, it reduced the cost of trial and error and minimized resource waste, contributing to more environmentally friendly research practices. Description of the Drawings

[0024] Figure 1 shows the effect of different glycosylation reactions on the average particle size of RPH. Different lowercase letters indicate significant differences (p < 0.05).

[0025] Figure 2 shows the grafting degree of different glycosylation reaction products. Different lowercase letters indicate significant differences (p < 0.05).

[0026] Figure 3 (A) shows the infrared spectra of RPH and its glycosylated products, Figure 3 (B) shows the expanded infrared spectra of RPH and its glycosylated products.

[0027] Figure 4 shows the ultraviolet spectra of RPH and its glycosylated products.

[0028] Figure 5 shows the comparison of the chelating ability of different glycosylated products with Fe 2+ . Different lowercase letters indicate significant differences (p < 0.05).

[0029] Figure 6 (A) shows the scavenging ability of different glycosylated products against superoxide anions, Figure 6 (B) shows the scavenging ability of different glycosylated products against DPPH. Different lowercase letters indicate significant differences (p < 0.05).

[0030] Figure 7 shows the fluorescence distribution map in zebrafish: among which Figure 7 (A) shows the fluorescence distribution map of the blank group, Figure 7 (B) shows the fluorescence distribution map of the model group, Figure 7 (C) shows the fluorescence distribution map of the glycosylation group, Figure 7 (D) shows the fluorescence distribution map of the hydrolysate group.

[0031] Figure 8 (A) shows the MDA level content map in zebrafish, Figure 8(B) is the CAT activity map in zebrafish. Figure 8 (C) is the T-SOD activity map in zebrafish. *: There are significant differences compared with the model control group, *p < 0.05; #: There are significant differences between the model control group and the blank group, #(p < 0.05), ##(p < 0.01), (p < 0.001). Specific implementation manners

[0032] To help everyone understand the present invention in depth, a more detailed explanation will be given below. The implementation forms of the present invention are rich and diverse, far beyond the embodiments listed in this article. The presentation of these embodiments is intended to enable everyone to more thoroughly and comprehensively understand the disclosed content of the present invention. In the following embodiments, if the specific experimental conditions are not particularly noted, generally conventional experimental conditions are used, or the conditions recommended by the manufacturer are followed. All kinds of commonly used chemical reagents used in the embodiments are purchased from the market and are commercially available products.

[0033] The object of the present invention is to provide an antioxidant application of glycosylated rice protease hydrolysate.

[0034] An example of the present invention provides a method for analyzing the structure of rice protease hydrolysate before and after glycosylation, and the method is specifically as follows:

[0035] (1) Particle size measurement: For the enzyme hydrolysate sample and the glycosylated sample, a particle size analyzer is used to detect their particle sizes.

[0036] (2) Degree of grafting measurement: Take RPH and the glycosylated product respectively, and use OPA to characterize the occurrence of the glycosylation reaction by measuring the decrease in the content of free amino acids in the system.

[0037] (3) Fourier transform infrared absorption spectrum: After freeze-drying RPH and the glycosylated product are mixed with potassium bromide powder at a certain mass ratio, a Fourier transform infrared spectrometer is used for band scanning.

[0038] (4) Ultraviolet spectrum: Take RPH and the glycosylated sample, and use an ultraviolet-visible spectrophotometer for scanning

[0039] Another example of the present invention provides a scheme for detecting the antioxidant capacity of glycosylated rice protease hydrolysate in vitro, and the method is specifically as follows:

[0040] (1) Fe 2+ Chelating ability: Take RPH, the glycosylated sample and GSH solutions with different mass concentration gradients respectively, and indirectly measure the chelating ability of the sample to ferrous ions by the phenanthroline method.

[0041] (2) Superoxide anion radical scavenging ability: Different mass concentration gradients of RPH, glycosylated samples, and GSH solutions were taken respectively, and the superoxide anion radical scavenging ability was indirectly determined by the pyrogallol autoxidation method.

[0042] (3) DPPH radical scavenging rate: Different mass concentration gradients of RPH, glycosylated samples, and GSH solutions were taken respectively. The antioxidant ability was calculated by detecting the scavenging effect of different samples on DPPH radicals at a wavelength of 517 nm.

[0043] The present invention example also provides a scheme for detecting the antioxidant ability of glycosylated rice protein hydrolysate in vivo, as follows:

[0044] (1) The present invention uses zebrafish to establish an oxidative stress model to detect the antioxidant ability of glycosylated rice protein hydrolysate in vivo.

[0045] (2) Detect the ROS content in zebrafish, and analyze the in vivo antioxidant activity through fluorescence intensity.

[0046] (3) Through MDA, CAT, and T-SOD kits, use an ultraviolet spectrophotometer to detect the MDA concentration, CAT activity, and T-SOD activity in zebrafish.

[0047] (4) The breeding of zebrafish includes sex separation in a water tank. Under controlled temperature and light conditions, artemia eggs are fed at a specified time. Zebrafish embryos are obtained through natural mating.

[0048] (5) Before the formal experiment, the maximum dosage of RPH-A for model zebrafish needs to be obtained through a toxicity experiment.

[0049] The present invention example also provides an application of glycosylated rice protein hydrolysate in the preparation of an antioxidant.

[0050] The present invention example also provides an antioxidant, which includes the above-mentioned glycosylated rice protein hydrolysate.

[0051] The present invention example also provides a nutritional preparation composition, which includes the above-mentioned glycosylated rice protein hydrolysate.

[0052] In this study, four indicators, namely particle size, grafting degree, infrared spectrum, and ultraviolet spectrum, were used to characterize the formation of glycosylated products. After glycosylation, the in vitro antioxidant activity of RPH was improved to varying degrees. It also significantly increased the CAT activity in zebrafish, reduced the MDA content, and enhanced the T-SOD activity, thereby improving the antioxidant capacity of zebrafish. This study not only provided a theoretical and technical basis for the processing and production of functional glycosylated rice protein hydrolysates, but also enhanced the industrial application value of rice protein and improved its economic utilization rate. In addition, as a sustainable and environmentally friendly project, it reduced the cost of trial and error and minimized resource waste, contributing to more environmentally friendly research practices.

[0053] The English abbreviations in the following examples represent the meanings shown in Table 1 below.

[0054] Table 1 English Abbreviation Table

[0055]

[0056] Example 1 Characterization of Glycosylated Rice Protein Hydrolysate

[0057] 1.1 Experimental Method

[0058] 1.1.1 Preparation of Rice Protein Hydrolysate

[0059] Weigh 67 g of rice protein powder (crude protein content 80%, Xinyang Mufan Biotechnology Co., Ltd.), dissolve it in 1000 mL of deionized water, prepare a solution of 1 g / 15 mL, stir and hydrate at room temperature for 2 h, adjust the pH to 8, and carry out a water bath at 50 °C for 15 min. Add trypsin (J&K Scientific), with the mass ratio of enzyme to substrate being 1:100, and carry out an enzymatic hydrolysis reaction at 50 °C for 60 min, maintaining the pH stable (pH = 8) during the enzymatic hydrolysis process. After the enzymatic hydrolysis is completed, place it in a water bath at 95 °C for 15 min to inactivate the enzyme, quickly cool it to room temperature in an ice bath, finally adjust the pH to neutral, centrifuge (3500 r / min, 10 min), and take the supernatant for freeze-drying for later use.

[0060] 1.1.2 Preparation of Glycosylated Rice Protein Hydrolysate

[0061] Weigh 2.0 g of sugar (fructose, xylose, and arabinose) and 1.0 g of rice protein hydrolysate respectively, dissolve them in 200 mL of deionized water, stir to make them fully mixed, adjust the pH = 7.0, and carry out the reaction in a magnetic stirring water bath to generate three different glycosylated products, namely RPH-F, RPH-X, and RPH-A. React at 80 °C for 4 h. After the reaction is completed, quickly cool it to room temperature in an ice bath, dialyze all the sample solutions at low temperature for 24 h, and freeze-dry the dialysate into a dry powder for later use.

[0062] 1.1.3 Particle Size Measurement

[0063] Take 400 μL of the enzymolysis solution sample and the dialysis solution of the glycosylation sample respectively. After diluting 10 times, use a particle size analyzer to detect their particle sizes. The samples are equilibrated at room temperature for 10 min, and the average particle size is taken by scanning each sample 3 times.

[0064] 1.1.4 Determination of grafting degree

[0065] The occurrence of the glycosylation reaction was characterized by the decrease in the content of free amino acids in the OPA determination system. Respectively pipette 200 μL of the glycosylation product solutions (RPH-F, RPH-X, and RPH-A) into test tubes, add 4 mL of OPA reagent, mix well, react in a water bath at 35 °C for 2 min, and measure the absorbance at 340 nm. Under the same conditions, use 200 μL of RPH as a blank sample to measure its absorbance. Calculate its grafting degree (DG) according to formula (1).

[0066]

[0067] In the formula: A 0 : Absorbance of the blank sample at 340 nm; A 1 : Absorbance of the sample at 340 nm.

[0068] 1.1.5 Fourier transform infrared absorption spectroscopy

[0069] Mix and grind the four freeze-dried samples of RPH, RPH-F, RPH-X, and RPH-A with potassium bromide powder at a certain mass ratio respectively. Press the sample powder into a uniformly textured and transparent thin slice, and then put it into a Fourier transform infrared spectrometer for band scanning. Set the band to 4000 cm -1 ~400 cm -1 ,with a resolution of 2 cm -1 .

[0070] 1.1.6 Ultraviolet spectroscopy

[0071] Disperse RPH, RPH-F, RPH-X, and RPH-A in a phosphate (pH = 7.0, 50 mmol / L) buffer solution respectively, with a concentration of 0.50 mg / mL. Scan with a UV-visible spectrophotometer, wavelength range: 200~600 nm.

[0072] 1.2 Result analysis

[0073] 1.2.1 Particle size determination

[0074] As Figure 1, after glycosylation, the average particle size of the products all increased. The average particle size of RPH-F exceeded 3.5 times that of RPH, the average particle size of RPH-X exceeded 1.6 times that of RPH, and the average particle size of RPH-A exceeded 1.2 times that of RPH. This indicates that glycosylation changed the structure of RPH and made it more loose. The different increases in the average particle size of each glycosylation product may be due to different sites and probabilities of the functional sugars being incorporated. Larger sugar molecules tend to cause a higher steric hindrance effect, thus exacerbating the destruction of protein compactness. Since fructose itself has a relatively large molecular weight and particle size, RPH-F has the largest increase in average particle size under the same glycosylation conditions.

[0075] 1.2.2 Determination of grafting degree

[0076] As Figure 2 , under the same conditions, the grafting degree of the product (RPH-X) after the reaction of rice protein hydrolysate with xylose was the highest, reaching 16.38%. A high grafting degree indicates a decrease in the amount of free amino acids in the glycosylation product, proving that some amino acids have covalently linked with sugars. From the size of the grafting degree, it can be seen that the ability of rice protein hydrolysate to bind with three functional sugars is: xylose > fructose > arabinose. The chemical structures of different sugars significantly affect the glycosylation efficiency, and xylose shows the highest binding efficiency due to the high reactivity of its aldehyde group.

[0077] 1.2.3 Fourier transform infrared absorption spectroscopy

[0078] By the characteristic absorption peaks of proteins in the mid-infrared region, the changes in the peptide chain structure can be sensitively reflected. The wavelength range of 3500 - 3000 cm -1 is the characteristic absorption peak of -OH. As can be seen from Figure 3 , compared with RPH, the absorption intensity of the three glycosylation products at the wavelength of 3500 - 3000 cm -1 increased (the transmittance decreased) and the absorption peak showed a slight blue shift, proving the stretching vibration of -OH and the new formation of -OH. This indicates the occurrence of the glycosylation reaction because sugar molecules are rich in -OH, and their incorporation into RPH in the form of covalent bonds leads to an increase in the number of -OH. In addition, -OH, as a strong polar group, can form hydrogen bonds, so the conjugate has a relatively broad absorption peak at 3650 - 3200 cm -1 . The absorption peak at 1000 - 1070 cm -1 is a typical characteristic of the stretching vibration of the C-O-C functional group in sugar molecules and the existence of sugar rings. The absorption peak spectra of the three glycosylation products at 1050 cm -1 increased significantly, indicating the occurrence of the glycosylation reaction in RPH, and the incorporation of sugar molecules caused vibrations of the protein side chains.

[0079] 1.2.4 Ultraviolet spectroscopy

[0080] AsFigure 4 As shown, RPH and its glycosylation products have absorption of ultraviolet light with certain wavelengths at 225 nm and 280 nm. Compared with RPH, the ultraviolet absorption of the glycosylation product shows a slight blue shift at 221 nm. This phenomenon indicates that the glycosylation reaction may change the three-dimensional structure of the protein, especially the conformation of the peptide chain. This change may be due to the occurrence of the Maillard reaction, which causes amino acids and sugar molecules in the protein to form covalent bonds, thereby changing the interactions between molecules. With these structural changes, the side chains of some hydrophobic amino acids are exposed, enhancing the interaction with the external environment, which may lead to an increase in the ultraviolet absorption peak.

[0081] Study on the in vitro antioxidant activity of glycosylated rice bran protease hydrolysate in Example 2

[0082] 2.1 Experimental method

[0083] 2.1.1 Fe 2+ Chelating ability

[0084] Take 0.5 mL of the sample solution to be measured (mass concentrations are 6 mg / mL, 3 mg / mL, 1.5 mg / mL, 0.75 mg / mL, and 0.375 mg / mL respectively) into 5 mL centrifuge tubes, and successively add 3.20 mL of distilled water and 0.10 mL of 2 mmol / L FeCl 2 solution. Shake well and let stand for 3 min. Immediately add 2 mL of 5 mmol / L phenanthroline solution to the mixed solution, react at 25 °C for 10 min, and measure the absorbance at 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Use deionized water as the blank control and glutathione as the positive control, and perform parallel experiments 3 times. Calculate the Fe 2+ chelating ability of the sample according to Equation (2).

[0085]

[0086] In the formula: P: Fe 2+ chelating ability; A 0 : Absorbance of the blank group measured at 562 nm; A S : Absorbance of the experimental group measured at 562 nm.

[0087] 2.1.2 Superoxide anion radical scavenging ability

[0088] Take 1.0 mL of the sample solution to be tested (with mass concentrations of 6 mg / mL, 3 mg / mL, 1.5 mg / mL, 0.75 mg / mL, and 0.375 mg / mL respectively) into 10 mL centrifuge tubes. Then add 3.00 mL of 50 mmol / L Tris-HCl buffer solution with pH 8.2. After mixing well, react at 25 °C for 20 min. Then transfer 3.00 mL of 7 mmol / L pyrogallol solution and add it to the above solution to react for 5 min. Finally, terminate the reaction with 1 mL of concentrated HCl and measure the absorbance at 325 nm. Conduct parallel experiments 3 times. Calculate the scavenging rate of superoxide anion radicals according to Equation (4).

[0089]

[0090] In the formula: P: Scavenging rate of superoxide anion radicals; A 0 : Absorbance at 325 nm with deionized water replacing the sample solution; A 1 : Absorbance of the sample solution at 325 nm; A 2 : Absorbance of the sample solution without pyrogallol at 325 nm.

[0091] 2.1.3 Scavenging rate of DPPH radicals

[0092] Add reagents according to the combinations in the following table. The concentrations of the sample solution to be tested are 6 mg / mL, 3 mg / mL, 1.5 mg / mL, 0.75 mg / mL, and 0.375 mg / mL in sequence.

[0093] Table 2 Reagent addition amounts for DPPH radicals (mL)

[0094] Solution Name <![CDATA[A s (Experimental group)]]> <![CDATA[A c (Control group)]]> <![CDATA[A b (Blank group)]]> DPPH Solution 3.0 - 3.0 Sample Solution to be Measured 1.0 1.0 - Sample Solvent Solution - - 1.0 Absolute Ethanol Solution - 3.0 -

[0095] Mix the solution well and react in the dark at room temperature for 60 min. Measure the absorbance at 517 nm. Glutathione is used as the positive control. Conduct parallel experiments 3 times and calculate the scavenging rate of the sample for DPPH radicals according to Equation (5).

[0096]

[0097] In the formula: P: Scavenging rate of DPPH radicals; A s : Absorbance measured in the experimental group; A c : Absorbance measured in the control group; A b : Absorbance measured in the blank group.

[0098] 2.2 Result analysis

[0099] 2.2.1 Fe 2+ Chelating ability

[0100] Using GSH as the positive control, the Fe chelating abilities of RPH, RPH-F, RPH-X, and RPH-A are as follows. 2+ as Figure 5 shown. In the concentration gradient of 0.375 - 6 mg / mL, the ferrous ion chelating abilities of the enzymolysate and the glycosylated product both increase with the increase in concentration. When the sample concentration is 0.75 mg / mL, the Fe chelating ability of RPH-F 2+ increases by 6.23%. The effect of RPH-F is significantly better than that of other glycosylated products. The molecular structure of xylose may make it easier to 2+ undergo complexation reactions with Fe. Glycosylation modification changes the structure of rice protein hydrolysate. Some groups with strong coordination abilities on amino acid residues, such as carbonyl groups, are exposed, making it easier to 2+ undergo complexation reactions with Fe. And Fe 2+ can catalyze the formation of free radicals from reactive oxygen species. Therefore, glycosylated rice protein hydrolysate can eliminate oxidative damage caused by free radicals by chelating Fe 2+ .

[0101] 2.2.2 Superoxide anion radical scavenging ability

[0102] Using GSH as the positive control, the superoxide anion radical scavenging rates of RPH, RPH-F, RPH-X, and RPH-A are as Figure 6 (A) shown. The glycosylated samples significantly improve the scavenging ability, especially the effect of RPH-F is enhanced. This indicates that glycosylation modification improves the antioxidant performance of rice protein hydrolysate. Because superoxide anion radicals are potential precursors of highly reactive substances. Compared with RPH, glycosylation modification improves its superoxide anion radical scavenging ability to a certain extent.

[0103] 2.2.3 DPPH radical scavenging ability

[0104] 1,1-Diphenyl-2-picrylhydrazyl radical [DPPH·] is a stable nitrogen-centered radical with a maximum absorption at a wavelength of 517 nm. The ethanol solution of [DPPH·] is purple, and its concentration has a linear relationship with the absorbance. Using GSH as the positive control, the DPPH radical scavenging rates of RPH, RPH-F, RPH-X, and RPH-A are as Figure 6 (B) shown. The DPPH radical scavenging rates of rice protein hydrolysate and glycosylated products both increase with the increase in sample concentration, and the DPPH radical scavenging rates of glycosylated products are higher than those of the enzymolysate. When the sample concentration is 6 mg / mL, the DPPH radical scavenging rate of RPH-X is 49.49%, which is 12.75% higher than that of RPH. The greater the degree of glycosylation, the more antioxidant substances are generated, and the higher the DPPH radical scavenging rate of the sample.

[0105] Study on the in vivo antioxidant activity of glycosylated rice protease hydrolyzate - arabinose in Example 3

[0106] 3.1 Experimental method

[0107] 3.1.1 Culturing of zebrafish and collection of embryos

[0108] Separate zebrafish by gender and raise them in culture tanks. Feed them with de-shelled brine shrimp eggs twice a day (at 9:00 and 17:00), keep the temperature at 28°C ± 0.5°C, and the light-dark cycle is 14 h light: 10 h dark. Collect zebrafish embryos by the natural mating and spawning method. At 18:30 the day before reproduction, put male and female zebrafish on both sides of the breeding tank in a ratio of 1:1, separate them with a baffle, and keep them in the dark overnight. Remove the baffle at 8:30 the next day and turn on the lighting system to promote zebrafish mating and spawning. Collect zebrafish embryos and culture them in an incubator at 28.5°C.

[0109] 3.1.2 Investigation of drug administration dose

[0110] Randomly select juvenile fish (3 dpf, with normal development) into 6-well plates, 30 tails per well. The experimental groups are treated with RPH-A solutions at concentrations of 50.0, 100.0, 200.0, 400.0, 800.0 μg / mL. At the same time, set up a model control group and a normal control group. Except for the normal control group, the other groups are treated with a solution of H 2 O 2 at a concentration of 300 μM to establish a zebrafish oxidative damage model. Set 3 parallels for each group. After treatment for 24 h, observe and count the number of dead zebrafish in each group. The maximum concentration at which juvenile fish do not show death or morphological abnormalities is the maximum drug administration dose of RPH-A for model zebrafish, that is, the maximum test concentration (MTC).

[0111] 3.1.3 Detection of ROS in zebrafish in vivo and antioxidant evaluation

[0112] Randomly divide juvenile fish (3 dpf, with normal development) into 4 groups: Control: blank control group, Model: H 2 O 2 (300 μM), RPH group: 200 μg / mL RPH + 300 μM H 2 O 2 , RPH-A group: 200 μg / mL RPH-A + 300 μM H 2 O 2, three parallels were set for each group and treated for 4 days. Incubate with 40 μg / mL fluorescent probe in the dark for 20 min to label reactive oxygen species (ROS), take pictures with a stereoscopic fluorescence microscope and observe the fluorescence intensity, and perform statistical analysis on the fluorescence intensity results to evaluate the antioxidant effect of RPH-A in vivo.

[0113] 3.1.4 Determination of peroxidase activity in zebrafish in vivo

[0114] The juvenile fish (3 dpf, with normal development) were randomly divided into 4 groups: Control: blank control group, Model: H 2 O 2 (300 μM), GSH group: 20 μg / mL glutathione + 300 μM H 2 O 2 、RPH-A group: 200 μg / mL RPH-A + 300 μM H 2 O 2 , three parallels were set for each group and treated for 4 days. Operate according to the instructions of the MDA, CAT, and T-SOD kits. Use an ultraviolet spectrophotometer to detect the MDA concentration, CAT activity, and T-SOD activity of each group respectively.

[0115] 3.2 Result analysis

[0116] 3.2.1 Investigation of the administration dose

[0117] Since the tolerance concentration of RPH-A in zebrafish is unknown, a preliminary experiment needs to be carried out. 90 zebrafish were selected for each concentration gradient and tested in three parallels, and the concentration gradient was set to determine the MTC of the enzymolysate in zebrafish, providing reference and basis for the evaluation of antioxidant efficacy. The specific test results of the experiment are shown in the following table.

[0118] Table 3 Results of the experiment for exploring the maximum detection concentration of zebrafish (n = 30)

[0119]

[0120]

[0121] As can be seen from Table 3, under the experimental conditions, when the addition amount of RPH-A sample fed to zebrafish is below 200.0 μg / mL, the states of the zebrafish in the experimental group and the normal control group are similar, no death phenomenon occurs, and no morphological abnormalities occur. As the concentration of RPH-A increases, the number of dead zebrafish gradually increases, indicating that the toxicity of RPH-A to zebrafish is related to the mass concentration and shows a positive correlation. The maximum tolerance concentration of RPH-A to zebrafish is 200.0 μg / mL.

[0122] 3.2.2 Detection of ROS in zebrafish in vivo and evaluation of antioxidant effect

[0123] The redox balance in organisms is of great significance for maintaining biological health. Hydrogen peroxide is an oxidant that can generate reactive oxygen species (ROS). When the body is stimulated externally, the ROS level increases sharply, exceeding the body's own scavenging ability, leading to an imbalance in the oxidation-antioxidation balance and oxidative stress, which can cause damage to cells. The antioxidant effect of RPH-A in zebrafish can be reflected by fluorescence intensity. After 96 hours of continuous administration to each group of zebrafish, the fluorescence intensity results are as Figure 7 shown.

[0124] Table 4 Experimental results of the antioxidant efficacy evaluation of glycosylated enzyme hydrolysates (n = 30)

[0125] Group Fluorescence Intensity (Pixel, mean±SE) Relative Fluorescence Intensity Antioxidant Effect / % Control 581453±13684.66 0.015 - Model 36507700±1447285.79 1.000 - RPH 25229703±1476000.38*** 0.691 31.39*** RPH-A 6045490±432304**** 0.170 84.78****

[0126] Note: Compared with the model control group, ***p < 0.001, ****p < 0.0001

[0127] From Figure 7 (A) to (D) and Table 4, it can be seen that compared with the blank control group, the fluorescence intensity of the head, heart, and trunk of zebrafish larvae in the model group increased significantly, indicating that the oxidative damage model was effective and there were a large number of reactive oxygen species in zebrafish. The fluorescence intensity of the RPH-A group and the RPH group was lower than that of the model group, indicating that under the experimental conditions, RPH and RPH-A could inhibit the production of reactive oxygen species in zebrafish. The antioxidant effect on zebrafish after glycosylation reached 84.78%, which was significantly improved compared with the antioxidant effect before glycosylation.

[0128] 3.2.3 Effect of RPH-A on the activity of peroxidase in zebrafish

[0129] MDA is one of the intermediate products of lipid peroxidation, and its level directly reflects the level of lipid peroxidation in the body. As Figure 8 (A), the content of MDA increased significantly in the model group, verifying the effectiveness of the model, indicating that the oxidative damage model was successfully established. On the contrary, the MDA concentration in the GSH treatment group was lower, which emphasized its protective effect on lipid peroxidation.

[0130] As Figure 8 (B), the CAT activity of zebrafish in the model damage group was significantly lower than that in the blank control group, with a highly significant difference (p < 0.001), indicating that the antioxidant defense system was damaged. The CAT activity of the RPH-A group was significantly different from that of the model group (p < 0.05), indicating that RPH-A helped to restore the antioxidant defense; the CAT activity of the GSH group was higher than that of the model group.

[0131] As Figure 8(C) The T-SOD activity of zebrafish in the model injury group was significantly lower than that in the blank control group, with a highly significant difference (p < 0.001). The T-SOD activity of zebrafish in the RPH-A group was significantly higher than that in the model group (p < 0.05), indicating that RPH-A has a protective effect on zebrafish. There was no significant difference in the T-SOD activity of zebrafish between the model injury group and the GSH group (p > 0.05).

[0132] In the above embodiments, the technical features can be freely combined. Due to the numerous combination methods, for the sake of concise expression, not all possible combinations are elaborated one by one. However, as long as the combinations of these technical features are logically non-conflicting, they should all be regarded as falling within the scope described in this specification.

Claims

1. A glycosylated rice protein hydrolysate, characterized in that: The glycosylated rice protein hydrolysate is obtained by reacting rice protein hydrolysate with a functional monosaccharide through Maillard reaction, and its antioxidant activity is significantly improved compared with that of non-glycosylated protein hydrolysate; the monosaccharide is any one of fructose, xylose or arabinose.

2. The glycosylated rice protein hydrolysate according to claim 1, wherein The average particle size of the rice protein hydrolysate-fructose is more than 3.5 times that of RPH; the average particle size of the rice protein hydrolysate-xylose is more than 1.6 times that of RPH; and the average particle size of the rice protein hydrolysate-arabinose is more than 1.2 times that of RPH.

3. The glycosylated rice protein hydrolysate according to claim 1, wherein The glycosylation products formed by different monosaccharides have differences in grafting degree and antioxidant activity. The glycosylation products show better antioxidant effects than the original enzymatic hydrolysates in various in vitro free radical scavenging tests; the free radical is any one of Fe2+, superoxide anion or DPPH free radical.

4. Use of the glycosylated rice protein hydrolysate according to claim 1 or 2 in the preparation of an antioxidant.

5. Use of the glycosylated rice protein hydrolysate according to claim 1 or 2 in the preparation of a medicament for preventing and / or treating oxidative damage in zebrafish embryos.

6. The use according to claim 5, characterized in that The repair of oxidative damage is achieved by regulating the level of reactive oxygen species (ROS) in zebrafish embryos, reducing the content of malondialdehyde (MDA) and increasing the activities of catalase (CAT) and total superoxide dismutase (T-SOD).

7. A nutritional preparation composition, characterized in that: The nutritional preparation composition comprises the glycosylated rice protein hydrolysate according to any one of claims 1 to 2, and can provide antioxidant protection in vitro and in vivo.

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