A glycosylated rice dreg protease hydrolysate, a preparation method and application thereof

Abstract

The application belongs to the technical field of biochemistry, and discloses a glycosylated rice residue protein enzymolysis product, which is obtained by glycosylation of monosaccharide on rice residue protein enzymolysis product; wherein the degree of enzymolysis of the rice residue protein enzymolysis product is 1% to 12%; and the monosaccharide is any one of galactose and glucose. The glycosylated rice residue protein enzymolysis product has better antioxidant capacity and can be used as an antioxidant to regulate the function of the body.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biochemistry, and particularly relates to a glycosylated rice residue protein enzymatic hydrolysate, a preparation method and application thereof. BACKGROUND

[0002] Rice is one of the most important food crops in the world. The main components of rice are starch (80-90%) and protein (7-8%). Compared with other cereal proteins, rice protein has the characteristics of low allergenicity, high biological value, and comprehensive nutritional value. As a storage protein in cereals, rice protein has poor functional properties, which limits its application in the food industry. Therefore, it is of great significance to improve the functional properties and biological activity of rice protein by enzymatic hydrolysis and glycosylation modification. Current glycosylation modification is time-consuming and uncontrollable, and harmful advanced glycation end products are easily generated. Therefore, it is very important to control the progress of glycosylation by selecting the degree of hydrolysis of rice protein, the type and amount of sugar, and the modification method.

[0003] Peptides and sugars are combined through electrostatic and covalent binding to form peptide-sugar covalent conjugates. Glycopeptides can be prepared by covalently linking the carbonyl group of sugar to the amino group or carboxyl group of amino acid residues, or by chemically / enzymatically hydrolyzing glycoproteins. Compared with glycoproteins, glycopeptides have smaller molecular weights and simpler compositions. Currently, the main methods for synthesizing glycopeptides include chemical synthesis (direct synthesis, liquid phase and solid phase formation, natural chemical linkage), enzymatic synthesis, and chemical-enzymatic synthesis. Peptide-sugar covalent conjugates also have various biological activities, such as antioxidant, antibacterial, immunomodulatory, and anticancer activities. Compared with precursors, peptide-sugar covalent conjugates have stronger thermal stability, resistance to digestive enzymes, and biological activity, and have broad application prospects. SUMMARY

[0004] The present application aims to provide a glycosylated rice residue protein enzymatic hydrolysate with excellent antioxidant effect and resistance to digestive enzyme hydrolysis.

[0005] The above-mentioned objectives are achieved by the following technical solutions.

[0006] The first aspect of the present application provides a glycosylated rice residue protein enzymatic hydrolysate, which is obtained by glycosylating a rice residue protein enzymatic hydrolysate with monosaccharides; wherein the degree of enzymatic hydrolysis of the rice residue protein enzymatic hydrolysate is 1-12%; and the monosaccharide is any one of galactose or glucose.

[0007] In some embodiments, the degree of enzymatic hydrolysis of the rice residue protein enzymatic hydrolysate is 3-10%.

[0008] In some embodiments, the degree of enzymatic hydrolysis of the rice residue protein enzymatic hydrolysate is 6-10%.

[0009] In some embodiments, the degree of hydrolysis of the rice residue protein hydrolysate is 6% to 9%.

[0010] In some embodiments, the degree of hydrolysis of the rice residue protein hydrolysate is 8.5% to 9.5%.

[0011] In some embodiments, the monosaccharide is galactose.

[0012] In some embodiments, the rice residue protein hydrolysate is obtained by enzymatic hydrolysis using neutral protease, flavour protease or trypsin; preferably, the rice residue protein hydrolysate is obtained by enzymatic hydrolysis using trypsin.

[0013] The second aspect of the present application provides a method for preparing a glycosylated rice residue protein hydrolysate, comprising the following steps:

[0014] (1) reacting rice residue raw material and an enzyme to obtain a rice residue protein hydrolysate with a degree of hydrolysis of 1% to 12%;

[0015] (2) mixing the obtained rice residue protein hydrolysate with a monosaccharide to obtain the glycosylated rice residue protein hydrolysate; the monosaccharide is any one of galactose or glucose.

[0016] In some embodiments, in step (2), the concentration of the rice residue protein hydrolysate in the reaction system is 5 mg / mL to 15 mg / mL, and the concentration of the monosaccharide in the reaction system is 15 mg / mL to 25 mg / mL; preferably, the concentration of the rice residue protein hydrolysate in the reaction system is 8 mg / mL to 12 mg / mL, and the concentration of the monosaccharide in the reaction system is 18 mg / mL to 22 mg / mL; more preferably, the concentration of the rice residue protein hydrolysate in the reaction system is 9 mg / mL to 11 mg / mL, and the concentration of the monosaccharide in the reaction system is 19 mg / mL to 21 mg / mL.

[0017] In some embodiments, the temperature of the reaction in step (1) is 45°C to 55°C, and the pH value is 7 to 8.5; preferably, the temperature of the reaction in step (1) is 48°C to 52°C, and the pH value is 7 to 8; and / or,

[0018] The temperature of the reaction in step (2) is 75°C to 85°C, and the reaction time is 80 min to 100 min; preferably, the temperature of the reaction in step (2) is 78°C to 82°C, and the reaction time is 88 min to 92 min.

[0019] In some embodiments, the enzyme is neutral protease, flavour protease or trypsin; preferably, the enzyme is trypsin; and / or,

[0020] The ratio of the rice residue raw material to the enzyme is 1:90-110, and the enzyme activity of the enzyme is 1400 U / mg-1600 U / mg; and / or,

[0021] The reaction in step (1) is carried out in a solvent, preferably, the solvent is distilled water; and / or,

[0022] The reaction in step (2) is carried out in a buffer solution; preferably, the buffer solution is a phosphate buffer solution, and the pH value is 6.5-7.5;

[0023] And / or, the monosaccharide is galactose; and / or,

[0024] The degree of enzymolysis of the rice residue protein enzymolysis product is 3%-10%; preferably, the degree of enzymolysis of the rice residue protein enzymolysis product is 6%-10%; more preferably, the degree of enzymolysis of the rice residue protein enzymolysis product is 8.5%-9.5%.

[0025] The third aspect of the present application provides a use of the glycosylated rice residue protein enzymolysis product as described above in the preparation of an antioxidant.

[0026] The fourth aspect of the present application provides an antioxidant, which comprises the glycosylated rice residue protein enzymolysis product as described above.

[0027] The fifth aspect of the present application provides a pharmaceutical composition, which comprises the glycosylated rice residue protein enzymolysis product as described above, and a pharmaceutically acceptable adjuvant.

[0028] The sixth aspect of the present application provides a nutritional preparation composition, which comprises the glycosylated rice residue protein enzymolysis product as described above, and a pharmaceutically acceptable adjuvant.

[0029] In the present application, it is found that when a rice residue protein enzymolysis product with a specific degree of hydrolysis is glycosylated using a specific monosaccharide, the obtained glycosylated rice residue protein enzymolysis product can effectively improve the ABTS free radical, hydroxyl radical, and superoxide anion scavenging activity in vitro and in a cell environment, significantly reduce the H2O2 content and MDA level of cells after cell damage, and improve the activity of CAT and SOD and the content of GSH, i.e., has a more optimal antioxidant activity, can be used as an antioxidant to regulate the function of the body, and the glycosylated rice residue protein enzymolysis product has anti-digestive enzyme properties, is not easily digested and hydrolyzed by digestive enzymes after entering the body, and has no cytotoxicity and high safety. The present application provides further theoretical basis for the deep development of rice processing by-products. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Different enzymes and different hydrolysis degrees of rice residue protein enzymolysis products were analyzed for their hydroxyl radical and superoxide anion scavenging capacities.

[0031] Figure 2 Effect of different Maillard reaction time on reaction products.

[0032] Figure 3 Comparison of RPH, MRPs on ABTS, hydroxyl radical and superoxide anion scavenging capacity.

[0033] Figure 4 Effect of RPH, RPH-Ga on HepG2 cell survival.

[0034] Figure 5 Effect of RPH, RPH-Ga on H2O2-induced HepG2 cell survival.

[0035] Figure 6 Effect of RPH and RPH-Ga on ROS release in HepG2 cells.

[0036] Figure 7 Effect of RPH and RPH-Ga on MDA in HepG2 cells.

[0037] Figure 8 Effect of RPH and RPH-Ga on antioxidant enzyme activity in HepG2 cells.

[0038] Figure 9 Comparison of GI-RPH, GI-RPH-Ga on ABTS, hydroxyl radical and superoxide anion scavenging capacity.

[0039] Figure 10 Effect of GI-RPH, GI-RPH-Ga on MDA content in HepG2 cells.

[0040] Figure 11 Effect of GI-RPH, GI-RPH-Ga on antioxidant enzyme activity in HepG2 cells. DETAILED DESCRIPTION

[0041] In order to facilitate the understanding of the present application, a more comprehensive description of the present application will be given below. The present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0042] The experimental methods in the following examples, unless otherwise specified, are generally in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. The various common chemical reagents used in the examples are commercially available products.

[0043] The embodiment of the present application provides a glycosylated rice residue protein enzymolysis product, which is obtained by glycosylation of monosaccharide on rice residue protein enzymolysis product; wherein the degree of enzymolysis of the rice residue protein enzymolysis product is 1% to 12%; and the monosaccharide is any one of galactose and glucose.

[0044] When a specific monosaccharide is used to glycosylate the rice residue protein enzymolysis product with a specific degree of hydrolysis, the glycosylated rice residue protein enzymolysis product can effectively improve the ABTS free radical, hydroxyl radical and superoxide anion scavenging activity in vitro and in cells, significantly reduce the H2O2 content and MDA level of cells after cell damage, and improve the activity of CAT and SOD and the content of GSH, that is, the glycosylated rice residue protein enzymolysis product has better antioxidant activity, can be used as an antioxidant to regulate the function of the body, and has anti-digestive enzyme properties, is not easily digested and hydrolyzed by digestive enzymes after entering the body, and has no cytotoxicity and high safety. The glycosylated rice residue protein enzymolysis product provides further theoretical basis for the deep development of rice processing by-products.

[0045] Specifically, the monosaccharide used above is any one of galactose and glucose, and preferably galactose. Since the molecular weight of monosaccharide is small, the structure of RPH after modification by monosaccharide will not cause excessive changes in the peptides in the enzymolysis product to affect the activity, nor will it cause the molecular weight of the glycosylated enzymolysis product to be too large to affect cell absorption and in turn affect the activity.

[0046] In some embodiments, the degree of enzymolysis of the rice residue protein enzymolysis product is 3% to 10%.

[0047] In some embodiments, the degree of enzymolysis of the rice residue protein enzymolysis product is 6% to 10%.

[0048] In some embodiments, the degree of enzymolysis of the rice residue protein enzymolysis product is 6% to 9%.

[0049] In some embodiments, the degree of enzymolysis of the rice residue protein enzymolysis product is 8.5% to 9.5%.

[0050] In some embodiments, the monosaccharide is galactose.

[0051] In some embodiments, the rice residue protein enzymolysis product is obtained by enzymolysis of neutral protease, flavor protease or trypsin; preferably, the rice residue protein enzymolysis product is obtained by enzymolysis of trypsin.

[0052] Another embodiment of the present application further provides a preparation method of the glycosylated rice residue protein enzymolysis product, comprising the following steps:

[0053] (1) reacting rice residue raw materials and enzymes to obtain a rice residue protein enzymolysis product with a degree of enzymolysis of 1% to 12%;

[0054] (2) mixing the obtained rice residue protein enzymolysis product with monosaccharide to react to obtain the glycosylated rice residue protein enzymolysis product; the monosaccharide is any one of galactose or glucose.

[0055] In some embodiments, in step (2), the concentration of the rice residue protein enzymolysis product in the reaction system is 5 mg / mL-15 mg / mL, and the concentration of the monosaccharide in the reaction system is 15 mg / mL-25 mg / mL; preferably, the concentration of the rice residue protein enzymolysis product in the reaction system is 8 mg / mL-12 mg / mL, and the concentration of the monosaccharide in the reaction system is 18 mg / mL-22 mg / mL; more preferably, the concentration of the rice residue protein enzymolysis product in the reaction system is 9 mg / mL-11 mg / mL, and the concentration of the monosaccharide in the reaction system is 19 mg / mL-21 mg / mL.

[0056] In some embodiments, the temperature of the reaction in step (1) is 45°C-55°C, and the pH value is 7-8.5; preferably, the temperature of the reaction in step (1) is 48°C-52°C, and the pH value is 7-8; and / or,

[0057] The temperature of the reaction in step (2) is 75°C-85°C, and the reaction time is 80 min-100 min; preferably, the temperature of the reaction in step (2) is 78°C-82°C, and the reaction time is 88 min-92 min.

[0058] In some embodiments, the enzyme is neutral protease, flavor protease or trypsin; preferably, the enzyme is trypsin; and / or,

[0059] The ratio of the amount of the rice residue raw material to the amount of the enzyme is 1:90-110, and the enzyme activity of the enzyme is 1400 U / mg-1600 U / mg; and / or,

[0060] The reaction in step (1) is carried out in a solvent, preferably, the solvent is distilled water; and / or,

[0061] The reaction in step (2) is carried out in a buffer solution; preferably, the buffer solution is a phosphate buffer solution, and the pH value is 6.5-7.5;

[0062] and / or, the monosaccharide is galactose; and / or,

[0063] The degree of enzymolysis of the rice residue protein enzymolysis product is 3%-10%; preferably, the degree of enzymolysis of the rice residue protein enzymolysis product is 6%-10%; more preferably, the degree of enzymolysis of the rice residue protein enzymolysis product is 8.5%-9.5%.

[0064] The embodiment of the present application also provides a use of the glycosylated rice residue protein enzymolysis product as described above in the preparation of an antioxidant.

[0065] The present application also provides an antioxidant comprising the glycosylated rice residue protein hydrolysate as described above.

[0066] The present application also provides a pharmaceutical composition comprising the glycosylated rice residue protein hydrolysate as described above, and a pharmaceutically acceptable adjuvant.

[0067] The present application also provides a nutritional preparation composition comprising the glycosylated rice residue protein hydrolysate as described above, and a pharmaceutically acceptable adjuvant.

[0068] The English abbreviations in the following examples represent the meanings shown in Table 1.1 below.

[0069] Table 1.1 English Abbreviation Table

[0070]

[0071] Example 1 Preparation and characterization of glycosylated rice residue protein hydrolysate

[0072] Experimental Methods

[0073] 1 Preparation and enzymatic hydrolysis of rice residue protein

[0074] 1.1 Enzymatic hydrolysis of rice residue protein

[0075] 1 g of lyophilized rice residue protein (crude protein content 80%, Zhejiang Hesheng Biological Technology Co., Ltd.) was weighed out and dispersed in distilled water at a ratio of 1:30 (g / mL) and stirred at 50°C for 60 min to ensure that the protein was fully hydrated. The pH of the protein aqueous solution was adjusted to the optimal pH for enzymatic hydrolysis using 1 mol / L NaOH, and the solution was maintained in a water bath at 50°C for 15 min. Then, trypsin (Biolab), neutral protease (Solabio) and flavourzyme (Solabio) were added to the solution at a ratio of 1:100, and the enzyme was added to the solution at a ratio of 1500 U / mg. The enzymatic hydrolysis was carried out under the optimal conditions for the enzymes, i.e. trypsin (50°C, pH 8), neutral protease (50°C, pH 7) and flavourzyme (50°C, pH 7). Protein hydrolysates with degrees of hydrolysis of 3%, 6% and 9% were prepared, and the degrees of hydrolysis (DH) were determined using the pH-stat method. After the enzymatic hydrolysis was completed, the solution was quickly transferred to a water bath at 90°C and heated for 10 min to inactivate the enzyme. After cooling, the pH was adjusted to 7, and the solution was centrifuged. The supernatant was collected, and the solution was lyophilized and stored at 4°C. The rice residue protein hydrolysate was obtained for further analysis.

[0076] 1.2 Preparation of Maillard glycosylated rice residue protein hydrolysate

[0077] The RPH obtained above was dissolved in phosphate buffer solution at 10 mg / mL (pH 7.0). Then, galactose and glucose were added respectively at a concentration of 20 mg / mL. The solution was slowly stirred for 1 h until complete dissolution. The mixture was heated at 80°C in a water bath for 0, 2 h, 4 h, 6 h, and 8 h, respectively. At the end of glycosylation, the solution was cooled in an ice bath and dialyzed with a 500 Da dialysis bag to remove salt and free sugar. Finally, the solution was freeze-dried and stored at 4°C to obtain the glycosylated rice residue protease hydrolysate (MRPs), including rice residue protease hydrolysate-galactose (RPH-Ga) and rice residue protease hydrolysate-glucose (RPH-Gl) for further analysis.

[0078] 1.3 Preparation of glycosylated rice residue protease hydrolysate by simulated gastrointestinal digestion

[0079] The pH of the sample solution was adjusted to 2 with 1 mol / L hydrochloric acid, then 11 mg of pepsin was added, and the solution was incubated at 37°C on a shaker for 1 h to simulate gastric digestion. Further, the pH of the solution was adjusted to 7.5 with 1 mol / mL sodium hydroxide, and 11 mg of trypsin was added, and the solution was incubated at 37°C on a shaker for 2 h. The sample was placed in a boiling water bath for 10 min to stop the digestion. The sample was centrifuged at 16000 g for 10 min, and the supernatant was collected. The solution was freeze-dried and stored at 4°C for further analysis.

[0080] 1.4 Determination of branching degree and determination of browning degree

[0081] The MRPs were diluted to a mass concentration of 10 mg / mL, and 200 μL of the sample was mixed with 4 mL of o-phthaldehyde reagent. It was placed in a 35°C water bath for 2 min, and the absorbance of the sample was measured at 340 nm. The blank control group was 4.0 mL of OPA and 200 μL of the sample before glycosylation. The OPA reagent was prepared by dissolving 40 mg of o-phthaldehyde in 1 mL of methanol, adding 25 mL of 0.1 mol / L borax solution, 2.5 mL of 20% SDS solution, and 100 μL of β-mercaptoethanol, and diluting with deionized water to 50 mL.

[0082] Grafting degree calculation formula:

[0083]

[0084] In the formula, A0 is the absorbance of the blank sample at 340 nm, and A1 is the absorbance of the sample at 340 nm.

[0085] 1.5 Analysis of results

[0086] The branching degree and browning degree were determined as shown in Table 1.2

[0087] Table 1.2 DG of RPH-Ga, RPH-Gl

[0088] No. RPH-Ga RPH-Gl 1 47.53％ 57.53％ 2 46.07％ 57.30％ 3 45.84％ 58.65％ DG 46.48％±0.01 57.83％±0.01

[0089] The Maillard wet-heating method has been widely used to prepare glycosylated protein hydrolysates. The reaction between the carbonyl group of sugar and the free amino group of protein and the formation of Schiff base result in the decrease of free amino group content. Therefore, the decrease of free amino group can prove the combination of sugar with RPHs molecules during glycosylation. As shown in Table 1.2, the DG of RPH-Ga, RPH-Gl, the glycosylated products of rice residue protein hydrolysates, were 46.48% and 57.83%, respectively, indicating that the free amino group of RPHs decreased significantly under the wet-heating reaction conditions. Due to the combination of galactose and glucose, the free amino group of RPHs decreased, resulting in the increase of DG. These results confirmed that sugar was involved in the Maillard reaction.

[0090] Example 2 Study on antioxidant activity of glycosylated rice residue protein hydrolysates

[0091] 2.1 Experimental method

[0092] 2.1.1 ABTS free radical scavenging capacity

[0093] According to the method of conventional ABTS free radical scavenging rate test, the reagents were added according to the combination of Table 2.1. The concentrations of the sample solution to be tested were 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL and 0.625 mg / mL, respectively.

[0094] Table 2.1 ABTS free radical reagent addition amount mL

[0095] Solution name A s (experimental group) A b (blank group) ABTS solution (mL) 3.6 3.6 Sample solution to be tested (mL) 0.4 - Sample solvent solution (mL) - 0.4

[0096] The solution was mixed thoroughly, reacted at room temperature for 5 min in the dark, and the absorbance was measured at 534 nm. Deionized water was used for zero calibration, glutathione was used as a positive control, and the experiment was repeated three times. The ABTS free radical scavenging rate of the sample was calculated according to formula (7).

[0097]

[0098] In the formula, P: ABTS free radical scavenging rate; A b : absorbance of the blank group; A s : absorbance of the experimental group.

[0099] 2.1.2 Hydroxyl radical scavenging capacity

[0100] The conventional hydroxyl radical scavenging rate test method was slightly modified, and the reagents were added according to the combination in Table 2.2 below. The concentrations of the test sample solutions were 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, and 0.625 mg / mL, respectively.

[0101] Table 2.2 Addition amount of hydroxyl radical reaction reagent mL

[0102] Solution name No. 1 (experimental group) No. 2 (blank group) Distilled water (mL) - 0.05 Sample solution to be tested (mL) 0.05 - 1.5 mmol / L o-diazenephthalene solution (mL) 0.05 0.05 1.25 mmol / L copper sulfate solution (mL) 0.05 0.05 Ascorbic acid (mL) 0.02 0.02 pH 7.5 borax buffer (mL) 0.78 0.78 0.02% hydrogen peroxide solution (mL) 0.05 0.05

[0103] Rinse the measuring tube with a small amount of borax buffer solution. Add the sample reagents to the measuring tube, starting with the glycosylated rice residue protein hydrolysate sample, followed by o-phenanthroline, CuSO₄ solution, and then ascorbic acid solution. Add the borax buffer dropwise. After gently tapping the measuring tube, add 30% hydrogen peroxide solution and immediately place it in the ultra-weak luminometer reaction cell. The total volume of the solution in the measuring tube is 1 mL. Place the measuring tube in the reaction cell, secure the lid, and adjust the knob to set the reaction time to 800 seconds. Measure and record the luminescence intensity every 0.1 seconds. The peak luminescence intensity is the average of three measurements. Use distilled water as a blank solution. Calculate the clearance rate.

[0104] Clearance = (blank peak - experimental peak) / blank peak

[0105] 2.1.3 Superoxide anion scavenging ability

[0106] The conventional hydroxyl radical scavenging rate test method was slightly modified, and the reagents were added according to the combination in Table 2.3 below. The concentrations of the sample solutions to be tested were 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, and 0.625 mg / mL, respectively.

[0107] Table 2.3 Superoxide anion reaction reagent addition amount mL

[0108] Solution name No. 1 (experimental group) No. 2 (blank group) Distilled water (mL) - 0.1 Sample solution to be tested (mL) 0.1 - 1 mmol / L o-benzotriazole (mL) 0.1 0.1 0.1 mmol / L luminol buffer solution (mL) 0.8 0.8

[0109] Rinse the measuring tube with a small amount of luminol buffer solution. Add the sample reagents (glycosylated rice residue protein hydrolysate sample, pyrogallol, and luminol buffer solution, in that order) to the measuring tube. After gently flicking the measuring tube, immediately place it in the reaction cell of the ultra-weak luminometer. The total volume of the solution in the measuring tube should be 1 mL. The reaction time should be 150 seconds, and the luminescence intensity should be measured every 0.1 seconds. The peak luminescence intensity should be the average of three measurements. Use distilled water as a blank solution. Calculate the clearance rate according to the formula.

[0110] Clearance = (blank peak - sample peak) / blank peak

[0111] 2.1.4 Detection of early, middle and late products of the Maillard reaction at different times

[0112] Early Chromophore Assay: 1 mL of each sample solution was taken. The enzymatic hydrolysate and sugar reaction system was treated with 3 mL of SDS (10% v / v). 1 mL of the solution was diluted 500-fold with distilled water. The absorbance of the sample was measured at 294 nm using a UV-visible spectrophotometer and expressed as A294nm. Each sample solution was measured in triplicate, and the mean and standard deviation were calculated.

[0113] Mid-term chromophore assay: 1 mL of sample solution from each group was treated with 3 mL of SDS (10% v / v) for the enzymatic hydrolysate and sugar reaction system. 1 mL of each solution was diluted 500-fold with distilled water. The absorbance of the sample was measured at 360 nm using a UV-visible spectrophotometer and expressed as A360nm. Each sample solution was assayed in triplicate, and the mean and standard deviation were calculated.

[0114] Late Chromophore Assay: 1 mL of sample solution from each group was taken. The enzymatic hydrolysate and sugar reaction system was treated with 3 mL of SDS (10% v / v). 1 mL of the solution was diluted 100-fold with distilled water. The absorbance of the sample was measured at a wavelength of 420 nm using a UV-visible spectrophotometer and expressed as A420nm. Each sample solution was measured in triplicate, and the mean and standard deviation were calculated.

[0115] Samples were taken at 0, 2, 4, 6, and 8 hours after the hydrolysate reacted with the sugar system to determine the early, middle, and late chromophores, and photos were taken at 0, 1.5, 4, and 8 hours to observe the color changes of the products.

[0116] 2.1.4 Reagent preparation method

[0117] (1) DMEM high-glucose medium: DMEM high-glucose medium was prepared by mixing 10% FBS, 1% sodium penicillin and streptomycin sulfate (100 U / mL) with 89% DMEM liquid medium. The medium was aliquoted and stored at 4°C for later use.

[0118] (2) 1 mg / mL RPH, MRPs stock solution: Accurately weigh 2 mg of RPH, MRPs and fully dissolve them in 2 mL of sterile cell culture water. After aliquoting, store at -20°C in the dark until use.

[0119] 2.1.5 Cell culture

[0120] HepG2 cells were cultured using conventional methods.

[0121] HepG2 cells in logarithmic growth phase were inoculated in culture plates and grouped: blank group (only DMEM complete medium was added), model group (after the same amount of DMEM complete medium was added for 24 h, H2O2 solution was added for stimulation for 4 h, and the final concentration of H2O2 was 1.125 mmol / L), sample group (after the same amount of 100, 200, 400, 800 μg / mL sample was added for intervention for 24 h, H2O2 solution was added for stimulation for 4 h, and the final concentration of H2O2 was 1.125 mmol / L), and positive control group (after the same amount of 10 μg / mL GSH was added for intervention for 24 h, H2O2 solution was added for stimulation for 4 h, and the final concentration of H2O2 was 1.125 mmol / L).

[0122] 2.1.6 Effect of Glycosylated Rice Residue Protein Hydrolysate Component Concentration on H2O2-induced HepG2 Cell Damage

[0123] HepG2 cells in logarithmic phase were inoculated in 96-well culture plates at 1.0×10 5 μL per well, and were placed in a 37℃, 5% CO2 incubator for culture for 24 h. Then, the medium in each well was aspirated, and blank, model, sample, and positive control groups were set up, 6 replicate wells were set up for each concentration. After treatment of the cells, the cell survival rate was determined by CCK8 method.

[0124] 2.1.7 Effect of Glycosylated Rice Residue Protein Hydrolysate Component on H2O2-induced HepG2 Cell Survival

[0125] HepG2 cells in logarithmic phase were inoculated in 96-well culture plates at 1.0×10 5 μL per well, and were placed in a 37℃, 5% CO2 incubator for culture for 24 h. Then, the medium in each well was aspirated, and blank, model, sample, and positive control groups were set up, 6 replicate wells were set up for each concentration. After treatment of the cells, the cell survival rate was determined by CCK8 method.

[0126] 2.1.8 Effect of Glycosylated Rice Residue Protein Hydrolysate Component on H2O2-induced MDA Content in HepG2 Cells

[0127] HepG2 cells in logarithmic phase were inoculated in 96-well culture plates at 1.0×10 5HepG2 cells in logarithmic phase were inoculated in 6-well plates at 1.0 x 10

[0128] Centrifuged at 1000 r / min for 5 min, pour out the supernatant, 1 mL PBS was washed once, centrifuged, and the supernatant was poured out. The cells were lysed, and the test operation was performed according to the MDA and BCA protein concentration determination kit instructions.

[0129] 2.1.9 Analysis of the effect of glycosylated rice residue protease hydrolysate components on H2O2-induced intracellular (ROS) content in HepG2 cells

[0130] HepG2 cells in logarithmic phase were inoculated in 6-well plates at 1.0 x 10 5 HepG2 cells in logarithmic phase were inoculated in 6-well plates at 1.0 x 10

[0131] 2.1.10 Effect of glycosylated rice residue protease hydrolysate components on H2O2-induced intracellular antioxidant enzymes and antioxidants in HepG2 cells

[0132] (1) Antioxidant enzyme T-SOD, CAT, GSH-PX activity determination

[0133] HepG2 cells in logarithmic phase were inoculated in 6-well plates at 1.0 x 10 5 HepG2 cells in logarithmic phase were inoculated in 6-well plates at 1.0 x 10

[0134] Centrifuged at 1000 r / min for 5 min, poured out the supernatant, washed once with 1 mL PBS, centrifuged, and poured out the supernatant. Cracked the cells and performed the test operation according to the T-SOD, CAT, GSH-PX assay kit instructions.

[0135] (2) Antioxidant GSH content determination

[0136] The logarithmic phase HepG2 cells were inoculated in 6-hole culture plates at 1.0 x 10 5 After 24 h of culture in a 37℃, 5% CO2 incubator, the culture medium in each hole was aspirated, and the cells were treated in the blank group, model group, sample group, and positive control group. The original culture medium was carefully aspirated, 500 μL trypsin solution was added, and the culture medium plate wall was gently tapped for 1-3 min to make the cells digest and fall off. 1.5 mL DMEM complete culture medium was added to terminate the digestion reaction, and the cell suspension was gently aspirated into a 2 mL centrifuge tube with a pipette gun.

[0137] Centrifuged at 1000 r / min for 5 min, poured out the supernatant, washed once with 1 mL PBS, centrifuged, and poured out the supernatant. Cracked the cells and performed the test operation according to the T-SOD, CAT, GSH-PX assay kit instructions.

[0138] 2.1.11 GI-RPH, GI-RPH-Ga in vitro and in vivo antioxidant analysis

[0139] 2.1.11.1 ABTS, hydroxyl radical, and superoxide anion scavenging capacity analysis of GI-RPH and GI-RPH-Ga: the method is the same as above.

[0140] 2.1.11.2 MAD content and antioxidant enzyme activity analysis of HepG2 cells treated with GI-RPH and GI-RPH-Ga: the method is the same as above.

[0141] 2.2 Result analysis

[0142] 2.2.1 Antioxidant activity analysis of glycosylated enzyme digests prepared from different hydrolysis degrees and different sugars

[0143] The hydroxyl radical and superoxide anion scavenging capacity of the rice residue protein enzyme digests before and after glycosylation were good, such as Figure 1 The hydroxyl radical and superoxide anion scavenging capacity of the trypsin, neutral protease, and flavor protease rice residue protein enzyme digests with a hydrolysis degree of 9% were higher than those of the enzyme digests with a hydrolysis degree of 3% and 6%, and under each hydrolysis degree, the trypsin rice residue protein enzyme digest had better hydroxyl radical and superoxide anion scavenging capacity than the neutral protease and flavor protease rice residue protein enzyme digests. Therefore, the trypsin rice residue protein enzyme digest with a hydrolysis degree of 9% was selected for subsequent antioxidant analysis.

[0144] 2.2.2 Analysis of Maillard reaction products at different times

[0145] like Figure 2 As shown, early Maillard chromophores were measured at 294 nm, mid-Maillard chromophores at 360 nm, and late Maillard chromophores at 420 nm. With increasing reaction time, late Maillard products increased in number. At 4 hours, the product color darkened, and at 8 hours, the color approached caramel. This indicates an increase in late glycosylation products, which may be harmful to the human body. Therefore, sampling was performed at 1.5 hours, when early and mid-Maillard products were more abundant, for subsequent analysis. This sample primarily contained early and mid-Maillard products.

[0146] 2.2.2 Scavenging ability of glycosylated rice residue protein hydrolysate on ABTS, hydroxyl radicals and superoxide anions

[0147] Table 2.4 EC of ABTS scavenging by RPH and MRPs 50

[0148] RPH RPH-Ga RPH-Gl GSH EC50(mg / mL) 2.180 0.400 3.572 0.0276

[0149] Table 2.5 EC of OH scavenging by RPH and MRPs 50

[0150] RPH RPH-Ga RPH-Gl GSH EC50(mg / mL) 6.682 3.312 4.142 3.961

[0151] Table 2.6 RPH, MRPs clearance ˙O 2- EC 50

[0152] RPH RPH-Ga RPH-Gl GSH EC50(mg / mL) 13.293 8.327 11.064 0.929

[0153] Reactive oxygen species (ROS) are a result of normal metabolism in organisms. Oxidative stress is an imbalance in which excess ROS levels exceed those required for normal cell function and disrupt endogenous antioxidant capacity and repair. Excessive oxidation in biological systems can lead to many diseases, including neurodegenerative diseases such as Alzheimer's and Parkinson's. Therefore, measuring the antioxidant activity of drugs can help reveal their mechanisms of action. Figure 3As shown in Table 2.4 to Table 2.6, the ABTS free radical, hydroxyl radical, superoxide anion scavenging activities are positively correlated with the glycosylated rice dreg protein hydrolysate and glutathione content, wherein RPH-Ga can significantly improve the free radical scavenging capacity of RPH, and the EC50 values of ABTS, hydroxyl radical, superoxide anion to RPH-Ga are 0.400 mg / mL, 3.312 mg / mL and 8.327 mg / mL, respectively. These results show that MRPs exhibit good antioxidant activity by ABTS free radical, hydroxyl radical, superoxide anion detection, wherein the hydroxyl radical scavenging capacity of RPH-Ga is equivalent to that of glutathione, a known antioxidant.

[0154] 2.2.3 Effect of RPH and RPH-Ga on HepG2 cell survival

[0155] It can be seen that, compared with the blank group, RPH and RPH-Ga have no toxic effect on cells and have a promoting effect on cell growth, and the survival rate of HepG2 cells is significantly increased (P<0.01). Figure 4 2.2.4 Effect of RPH and RPH-Ga on H2O2-induced HepG2 cell survival

[0156] It can be seen that, compared with the control group, the cell viability of the H2O2-induced group is <75%, and the cell damage model is constructed. The cell viability is >95% at 100-800 μg / mL, and the cell viability is good, and RPH and RPH-Ga have no cytotoxicity on the H2O2-induced HepG2 cells.

[0157] Figure 5 2.2.5 Total active oxygen (ROS) content analysis

[0158] Under normal physiological conditions, the body will produce a small amount of ROS, which is mainly derived from the active oxygen-containing compounds produced by the body's oxidative metabolism, such as hydrogen peroxide, superoxide anion and hydroxyl radical. When the body is subjected to harmful stimuli, excessive active oxygen free radicals are produced in the body, and the redox balance is destroyed, which will lead to tissue damage. Therefore, the content of ROS in cells is an important indicator of the degree of oxidative damage of cells. At present, the most convenient method for detecting intracellular ROS is the DCFH-DA fluorescent probe method, which often uses normal cells as an internal reference, and the ratio of the number of cells with fluorescence signal intensity change to the total number of cells represents the level of intracellular ROS, and the higher the fluorescence intensity, the higher the percentage, representing the higher the ROS level.

[0159] It can be seen that, compared with the control group, the H2O2-induced group produces significantly more H2O2, and the sample group can significantly reduce the H2O2 content after cell damage, and RPH-Ga inhibits H2O2-induced cells, close to the blank control group. Figure 6 ​​

[0160] 2.2.6 MDA content analysis

[0161] MDA is a decomposition product of lipid peroxides and an important indicator for evaluating lipid peroxidation, which can indirectly reflect the degree of cell damage.

[0162] Depend on Figure 7 As shown in the figure, compared with the blank group, the intracellular MDA content of HepG2 cells in the model group increased significantly to 43 nmol / mgprot after H2O2 stimulation, indicating that H2O2 promoted the lipid peroxidation reaction of the cells, and the higher the MDA content, the more severe the cell damage. Compared with the model group, the intracellular MDA content of HepG2 cells stimulated by H2O2 was significantly reduced by 100, 200, 400, and 800 μg / mL RPH-Ga, which were 34, 32, 17, and 26 nmol / mgprot, respectively (P<0.05), indicating that RPH-Ga can significantly reduce the MDA level of cells and improve the lipid peroxidation reaction of cells. This is consistent with the results of cell morphological changes in the model group cells and the high-dose sample group, and the sample group was protected.

[0163] 2.2.7 Analysis of GSH, SOD, GSH-Px and CAT Contents

[0164] Superoxide dismutase (SOD) is an antioxidant enzyme that scavenges superoxide anions. Its activity indirectly reflects tissue free radical levels and the extent of cellular damage. Catalase (CAT) is an oxidoreductase widely present in biological tissues that catalyzes the decomposition of H2O2 into H2O and O2, thus scavenging H2O2. Reduced glutathione is the most important non-enzymatic antioxidant in the body, scavenging free radicals, detoxifying, and promoting iron absorption. It is a key marker of the body's antioxidant capacity.

[0165] Depend on Figure 8 Compared with the blank group, the activities of CAT and SOD, and the content of GSH in the model group were significantly decreased (P<0.01). Compared with the model group, the activities of CAT and SOD, and the content of GSH were significantly increased after pretreatment with 100 and 200 μg / mL RPH-Ga (P<0.01). This suggests that RPH-Ga, through its antioxidant physiological function, may have scavenged some ROS, reduced the destructive effects of ROS on the cellular antioxidant enzyme system, maintained the normal operation of the cellular antioxidant system, and prevented further oxidative damage to the cells.

[0166] 2.2.8 Scavenging ability of ABTS, DPPH, hydroxyl radicals and superoxide anions by enzyme hydrolysates of glycosylated rice residues during simulated gastrointestinal digestion

[0167] Table 2.7 EC of GI-RPH, GI-MRPs in scavenging ABTS 50

[0168] GI-RPH GI-RPH-Ga GI-RPH-Gl GSH EC50(mg / mL) 2.323 0.383 1.985 0.0276

[0169] Table 2.8 EC of GI-RPH, GI-MRPs in scavenging ˙OH 50

[0170] GI-RPH GI-RPH-Ga GI-RPH-Gl GSH EC50(mg / mL) 6.700 5.283 6.446 3.961

[0171] Table 2.9 EC of GI-RPH, GI-MRPs in scavenging ˙O 2- 50

[0172] GI-RPH GI-RPH-Ga GI-RPH-Gl GSH EC50(mg / mL) 14.715 6.089 8.301 0.929

[0173] As shown in Tables 2.7-2.9 and Figure 9 , the ABTS, ˙OH and ˙O 2- radical scavenging activities were all positively correlated with the RPH, MRPs and glutathione contents, and the ABTS, ˙OH and ˙O 2- radical scavenging abilities of RPH modified by glucose and galactose in monosaccharides were significantly increased, among which the ABTS, ˙OH and ˙O 2- radical scavenging abilities of RPH modified by galactose in monosaccharides were significantly increased. Since monosaccharides have small molecular weights, the modification of RPH by monosaccharides does not cause excessive changes in the structure of the peptide and affect its activity, nor does it cause the molecular weight of the glycosylated peptide to be too large and affect its cellular absorption, thereby affecting its activity. ABTS, ˙OH and ˙O 2- The EC50 values of GI-RPH, GI-RPH-Ga and GSH were 2.323 mg / mL, 0.383 mg / mL and 0.0276 mg / mL, 6.7 mg / mL, 5.283 mg / mL and 3.961 mg / mL, and 14.715 mg / mL, 6.089 mg / mL and 0.929 mg / mL, respectively. These results showed that GI-RPH-Ga exhibited good antioxidant activity, and that high concentrations of GI-RPH-Ga were comparable to known antioxidants such as glutathione. 2-

[0174] 2.2.9 Effect of GI-RPH, GI-RPH-Ga on MDA in HepG2 cells

[0175] As Figure 10 ​​As shown in the results, compared with the blank group, the intracellular MDA content of HepG2 cells in the model group increased significantly to 44 nmol / mgprot after H2O2 stimulation, indicating that H2O2 promoted the lipid peroxidation reaction of the cells, and the higher the MDA content, the more severe the cell damage. Compared with the model group, the intracellular MDA content of HepG2 cells stimulated by H2O2 was significantly reduced by 100, 200, 400, and 800 μg / mL GI-RPH-Ga, which were 21, 22, 19, and 14 nmol / mgprot, respectively (P<0.05), indicating that GI-RPH-Ga can significantly reduce the MDA level of cells and improve the lipid peroxidation reaction of cells. This is consistent with the results of cell morphological changes in the model group cells and the high-dose sample group, and the sample group was protected.

[0176] 2.2.10 Analysis of the effects of GI-RPH and GI-RPH-Ga on antioxidant enzyme activities in HepG2 cells

[0177] Depend on Figure 11 Compared with the blank group, the activities of CAT and SOD, and the content of GSH in the model group were significantly decreased (P<0.01). Compared with the model group, the activities of CAT and SOD, and the content of GSH were significantly increased after pretreatment with 100 and 200 μg / mL GI-RPH-Ga (P<0.01). This suggests that GI-RPH-Ga, through its antioxidant physiological function, may have scavenged some ROS, reduced the destructive effects of ROS on the cellular antioxidant enzyme system, maintained the normal operation of the cellular antioxidant system, and prevented further oxidative damage to the cells.

[0178] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A glycosylated rice residue protein hydrolysate, characterized in that: The glycosylated rice residue protein hydrolysate is obtained by glycosylation of rice residue protein hydrolysate with monosaccharide; wherein the enzymatic hydrolysis degree of the rice residue protein hydrolysate is 8.5% to 9.5%; and the monosaccharide is galactose; The rice residue protein hydrolysate is obtained by enzymatic hydrolysis with trypsin; The preparation method of the glycosylated rice residue protein hydrolysate comprises the following steps: (1) reacting a rice residue raw material with an enzyme to obtain a rice residue protein hydrolyzate having an enzymatic hydrolysis degree of 8.5% to 9.5%; (2) mixing the obtained rice residue protein hydrolysate with a monosaccharide to react to obtain the glycosylated rice residue protein hydrolysate; The reaction temperature in step (1) is 45° C. to 55° C., and the pH value is 7 to 8.5; The reaction temperature in step (2) is 75° C. to 85° C., and the reaction time is 80 min to 100 min.

2. The glycosylated rice residue protein hydrolysate according to claim 1, wherein The enzymatic hydrolysis degree of the rice residue protein hydrolysate is 9%.

3. The glycosylated rice residue protein hydrolysate according to claim 1, wherein In step (2), the concentration of the rice residue protein hydrolysate in the reaction system is 5 mg / mL to 15 mg / mL, and the concentration of the monosaccharide in the reaction system is 15 mg / mL to 25 mg / mL.

4. The glycosylated rice residue protein hydrolysate according to claim 3, wherein In step (2), the concentration of the rice residue protein hydrolysate in the reaction system is 8 mg / mL to 12 mg / mL, and the concentration of the monosaccharide in the reaction system is 18 mg / mL to 22 mg / mL.

5. The glycosylated rice residue protein hydrolysate according to claim 1, wherein The reaction temperature in step (1) is 48° C. to 52° C., and the pH value is 7 to 8; and / or, The reaction temperature of step (2) is 78° C. to 82° C., and the reaction time is 88 min to 92 min.

6. The glycosylated rice residue protein hydrolysate according to claim 1, wherein The usage ratio of the rice residue raw material to the enzyme is 1:90-110, and the enzyme activity is 1400U / mg-1600U / mg.

7. The glycosylated rice residue protein hydrolysate according to claim 1, wherein The reaction in step (1) is carried out in a solvent.

8. The glycosylated rice residue protein hydrolysate according to claim 1, wherein The reaction in step (2) is carried out in a buffer solution.

9. Use of the glycosylated rice residue protein hydrolysate according to any one of claims 1 to 2 in the preparation of an antioxidant.

10. An antioxidant, characterized in that The antioxidant comprises the glycosylated rice residue protein hydrolysate according to any one of claims 1-2.

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