Preparation method of red rice bran protein peptide compound with antioxidant and hypoglycemic effects

Abstract

The invention discloses a preparation method of a red rice bran protein peptide compound with antioxidant and hypoglycemic effects. According to the method, red rice bran protein is extracted through an alkali-solution and acid-isolation method; hydrolyzing the red rice bran protein with alkaline protease to obtain a red rice bran protein hydrolysate; the red rice bran protein hydrolysate prepared by the invention has the functional characteristics of excellent emulsibility, foamability and oil absorbency, and has excellent antioxidant and hypoglycemic functional activities after being subjected to in-vitro simulated gastrointestinal digestion. The red rice bran protein hydrolysate can be used in plant-based food and dairy product substitutes and functional food with a hypoglycemic effect.

Description

Technical Field

[0001] The invention relates to a method for preparing a red rice bran protein peptide complex with antioxidant and blood sugar-lowering effects, and belongs to the field of functional food and low-value-added development of side effects resources. Background Art

[0002] Rice bran's three main components are protein, fat, and fiber. These ingredients can be used as functional ingredients to enhance food quality by providing texture, flavor, and nutritional value. The crude protein content of rice bran ranges from 12% to 22%, second only to oats among cereal by-products, but significantly higher than wheat and corn, indicating its potential as a high-quality plant protein source. Rice bran protein is not only affordable but also boasts outstanding nutritional value. Its amino acid profile meets human metabolic requirements, particularly a balanced ratio of essential amino acids, which closely aligns with the FAO / WHO recommended standard pattern. Lysine is the first limiting amino acid in rice. Compared to other cereal brans, rice bran offers superior protein quality due to its higher lysine content. This balanced amino acid profile contributes to its high biological value and excellent protein digestibility. In vitro digestion tests have shown that its digestibility is often higher than that of other cereal proteins. Furthermore, through appropriate extraction processes, the antigenicity of the protein is significantly reduced, making rice bran protein hypoallergenic, making it suitable for the development of infant formula and foods for those with allergies.

[0003] Enzymatic hydrolysis technology is a process for modifying the amino acid residues of proteins using specific enzymes. In food applications, enzymatic protein hydrolysis, with its excellent nutritional and functional properties, shows broad prospects in areas such as meat imitations, baked goods, functional foods, food packaging biofilms, and as a carrier for bioactive ingredients, making it an attractive alternative in food. Summary of the Invention

[0004] This invention provides a method for preparing a red rice bran protein-peptide complex. Research has found that red rice bran protein, after enzymatic hydrolysis with alkaline protease, exhibits excellent emulsifying, foaming, and oil absorption properties. The hydrolyzate contains multiple functional peptides with excellent antioxidant and hypoglycemic activities. These activities are also maintained after in vitro simulated gastrointestinal digestion. This food additive can be used in baked cakes, plant-based alternatives, dairy products, and functional products, providing more food options for those seeking blood sugar reduction.

[0005] The preparation method of the red rice bran protein peptide complex of the present invention is as follows: 1. Red rice bran is pulverized, sieved, and in red rice bran powder, adds sherwood oil. After stirring at room temperature for 1-3 hour, stratification is removed upper petroleum ether, and then adds sherwood oil to repeat said process. Clarify until the petroleum ether layer, collect the precipitated dryness, and promptly get the red rice bran powder after the defatting; In the red rice bran powder after the defatting, add deionized water, adjust the solution pH to 10-11 after mixing, stir after 2 hours, centrifugal collection supernatant, precipitation adds deionized water and repeats said process 2-3 time, merges supernatant, regulating supernatant pH is that 4-5 carries out acid precipitation, centrifugal, red rice bran protein precipitation washs with deionized water, and freeze drying makes red rice bran protein; The red rice bran is crushed and passed through an 80-mesh sieve; the mass volume ratio of red rice bran to petroleum ether is g:mL=1:4-6; 2. Mix red rice bran protein and deionized water to obtain a red rice bran protein solution, adjust the pH of the solution to 8.5-9, add alkaline protease to the red rice bran protein solution, and perform enzymolysis in a water bath at 48-52° C. in a dark place for 200-250 minutes. After the enzymolysis is completed, boil and inactivate the enzyme, cool and centrifuge, obtain the supernatant, and freeze-dry to obtain a red rice bran protein peptide complex; The added amount of the alkaline protease is 2500 U / g.

[0006] Beneficial effects of the present invention: (1) The emulsifying, foaming and oil absorption properties of red rice bran protein were improved to a certain extent through alkaline protease hydrolysis.

[0007] (2) Through alkaline protease hydrolysis, a hydrolyzate with excellent antioxidant and hypoglycemic activities was obtained.

[0008] (3) The hydrolyzate of the present invention still has good antioxidant and hypoglycemic activities after in vitro simulated gastrointestinal digestion, indicating that it still has good functional activity after human digestion. This makes the red rice bran protein hydrolysate not only applicable to food manufacturing, but also has a hypoglycemic effect. Therefore, the present invention is innovative, economical, and practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The superoxide anion free radical scavenging ability of the red rice bran protein peptide complex before and after simulating gastrointestinal digestion; Figure 2 The FRAP iron-reducing ability of the red rice bran protein-peptide complex before and after simulated gastrointestinal digestion; Figure 3 The DPPH scavenging ability of the red rice bran protein peptide complex before and after simulating gastrointestinal digestion; Figure 4 The α-glucosidase inhibitory activity of red rice bran protein peptide complex before and after simulated gastrointestinal digestion; Compared with the RA group in the above figure, p *<0.05 and p **<0.01 indicates significant difference, ns indicates no significance; Figure 5 This is the standard curve of FeSO4·7H2O; Figure 6 Mass spectrum of the digestion product. DETAILED DESCRIPTION

[0010] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following examples and accompanying drawings. However, the present invention is not limited thereto. Unless otherwise specified, the methods in the present examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Example 1: Preparation of red rice bran protein peptide complex 1. Red rice bran is pulverized, cross 80 mesh sieves, press the ratio of solid-liquid ratio g:mL=1:5, in red rice bran powder, add sherwood oil, after stirring 2 hours under room temperature, stratification, remove upper strata sherwood oil, add sherwood oil again and repeat said process, until the sherwood ether layer clarifies, collecting and being deposited in fume hood and volatilize, promptly get the red rice bran powder after the defatting; Press the ratio of solid-liquid ratio g:mL=1:20, in red rice bran powder after the defatting, add deionized water, use NaOH regulator solution pH to 10.5 after mixing, stirred after 2 hours, centrifugal collection supernatant, precipitation added deionized water and repeated said process 3 times, merge supernatant, regulating supernatant pH with 2mol / L HCl is 4.5 and carries out acid precipitation, centrifugal, red rice bran protein precipitation deionized water wash three times, vacuum freeze drying makes red rice bran protein; 2. Red rice bran protein was evenly mixed with deionized water to prepare a red rice bran protein solution with a mass concentration of 5%, and the pH of the solution was adjusted to 8.5. Alkaline protease (2500U / g) was added to the red rice bran protein solution. The solution was enzymatically hydrolyzed in a light-proof, water bath at 50°C for 240 minutes. After the enzymatic hydrolysis was completed, the solution was boiled in a water bath for 10 minutes, cooled to room temperature, centrifuged, and the supernatant was freeze-dried to obtain a red rice bran protein peptide complex.

[0011] Example 2: Determination of properties of red rice bran protein peptide complex (1) The red rice bran protein peptide complex and red rice bran protein were suspended in 0.2 mol / L phosphate buffer (pH 7.0) to a protein concentration of 1 mg / mL; homogenized using a high-speed homogenizer at 10,000 rpm for 90 s; After homogenization, 40 μL of the solution was taken from the bottom of the homogenized mixture and mixed with 4 mL of SDS (0.1%) solution. The absorbance of the solution was measured at a wavelength of 500 nm, and the emulsification ability (EAI) was calculated using the following formula. The experimental results are shown in Table 1; EAI (m 2 / g) = (2×2.303×A0×D) / (w×Φ×10000) Where D represents the dilution factor, w represents the protein content in the sample (g / mL), Φ represents the volume fraction of oil (0.25), and A0 represents the absorbance value.

[0012] Table 1 Functional properties of red rice bran protein before and after hydrolysis

[0013] Different superscript letters in the same column indicate statistically significant differences ( p < 0.05); From the results in the table, it can be seen that the emulsifying properties of red rice bran protein are significantly improved after alkaline protease hydrolysis compared with red rice bran protein, indicating that the red rice bran protein peptide complex has excellent emulsifying properties and can be applied to the plant-based dairy and meat substitute food industry as well as daily chemical products such as lotions and creams.

[0014] (2) The red rice bran protein peptide complex and red rice bran protein were suspended in 0.2 mol / L phosphate buffer (pH 7.0), and 20 mL of protein solution (10 mg / mL) was taken and homogenized at 10,000 g for 1 minute using a high-speed homogenizer. Then, the mixture was immediately transferred to a 100 mL graduated cylinder. The total volume V1 (mL) at 0 min was recorded. The foaming capacity (FC) was expressed as follows: FC (%) = (V1-20) / 20×100%; The experimental results are shown in Table 1. The red rice bran protein peptide complex exhibits significantly higher foaming properties than red rice bran protein, indicating that protease hydrolysis favors the production of more soluble and flexible protein fragments. Therefore, this complex can be used to replace egg whites in baked goods and desserts.

[0015] (3) 40 mg (m1, g) of red rice bran protein peptide complex or red rice bran protein and 1 mL of oil (m2, g) were mixed in a 2 mL centrifuge tube and homogenized for 2 minutes. After centrifugation at 8000 g for 10 minutes, the mass of the sample was weighed (m3, g). The oil absorption capacity (OAC) was calculated as follows: OAC = (m3-m2-m1) / m1 The experimental results are shown in Table 1. It can be seen from the table that the oil absorption of red rice bran protein increased from 2.1g / g to 3.14g / g after enzymatic hydrolysis, which is a significant improvement. This shows that the red rice bran protein after enzymatic hydrolysis has excellent oil absorption properties and can be used in industries such as the development of oil-absorbing food packaging.

[0016] In summary, red rice bran protein treated with alkaline protease has excellent emulsifying, foaming and oil absorption properties.

[0017] Example 3: Determination of the activity of red rice bran protein peptide complex before and after simulated gastrointestinal digestion 1. Prepare a sample solution RA with a mass concentration of 5% by weight of the red rice bran protein peptide complex using pure water, adjust the pH to 2.5, add pepsin (2500U / 1g protein), and digest at 37°C for 2 hours, taking samples every 1 hour to obtain P1 and P2 samples respectively; after gastric digestion, adjust the pH to 8.5, add trypsin (300U / 1g protein), and digest at 37°C for 4 hours, taking samples every 1 hour to obtain PC1, PC2, PC3, and PC4 samples respectively; heat the samples in a water bath to inactivate the enzyme, and after freeze-drying, test the superoxide anion free radical, FRAP iron reducing ability, DPPH scavenging ability, and α-glucosidase inhibitory activity of the samples at different time periods; 2. Superoxide anion free radical scavenging ability of red rice bran protein peptide complex before and after simulated gastrointestinal digestion Prepare a 1 mg / mL sample solution of the sample from step 1 with deionized water. Accurately weigh 18.92 mg of pyrogallol (pyrogallic acid) and dilute to 50 mL with deionized water to prepare a 3 mmol / L pyrogallol stock solution. Add 0.5 mL of the sample solution to 5 mL of Tris-HCl buffer (50 mmol / L, pH 8.2), mix, and incubate at 25°C for 20 min. Add 0.5 mL of the pyrogallol solution, shake well, and continue incubation for 5 min. After the reaction is complete, add 1 mL of 10 mol / L HCl to terminate the reaction. Measure the absorbance (A0) at 325 nm. The superoxide anion radical scavenging capacity is calculated using the following formula: A1 is the absorbance obtained by replacing the pyrogallol solution with an equal volume of pure water; A2 is the absorbance obtained by replacing the sample solution with an equal volume of pure water. Clearance rate (%) = 1-(A0-A2 / A1) × 100 See the results Figure 1 As can be seen from the figure, during the gastric digestion stage, the superoxide anion free radical scavenging ability of its product decreased, but after gastric and intestinal digestion, the superoxide anion free radical scavenging ability was not significantly different from that before digestion, indicating that it has good stability and still has excellent superoxide anion free radical scavenging ability after gastrointestinal digestion.

[0018] 3. FRAP iron reducing ability of red rice bran protein peptide complex before and after simulated gastrointestinal digestion Accurately weigh 0.7750 g of sodium acetate, add 4 mL of glacial acetic acid, and dilute to 250 mL with deionized water to prepare a 0.3 mol / L sodium acetate buffer (pH 3.6). Accurately weigh 0.0781 g of TPTZ and add 25 mL of 40 mmol / L HCl solution to prepare a 10 mmol TPTZ solution. Accurately weigh 0.5406 g of FeCl₃ and dissolve it in 100 mL of pure water to prepare a 20 mmol / L FeCl₃ solution. Accurately weigh 0.0278 g of ferrous sulfate heptahydrate (FeSO₄·7H₂O) and dissolve it in 100 mL of pure water to prepare a 1 mmol / L FeSO₄ standard solution. Mix the sodium acetate buffer, TPTZ solution, and FeCl₃ solution in a volume ratio of 10:1:1 to prepare the FRAP working solution. Take 0.5 mL of 0.2, 0.4, 0.6, 0.8 and 1.0 mmol / L FeSO4 standard solutions respectively, add 4.5 mL of FRAP working solution, react in a dark water bath at 37℃ for 10 minutes, measure the absorbance at a wavelength of 593 nm, and establish a standard curve. Figure 5 ; Prepare a 1 mg / mL sample solution from step 1 with pure water. Add 0.5 mL of the sample solution to 4.5 mL of FRAP working solution. Incubate in a dark water bath at 37°C for 10 minutes. Measure the absorbance at 593 nm. Calculate the iron ion reducing capacity of the sample using the standard curve equation.

[0019] See the results Figure 2 As can be seen from the figure, although the iron ion reducing ability is reduced after gastrointestinal digestion, it still has good iron reducing ability, indicating that the red rice bran protein peptide complex still has good antioxidant properties after digestion.

[0020] 4. DPPH scavenging ability of red rice bran protein peptide complex before and after simulated gastrointestinal digestion Prepare a 1 mg / mL sample solution from the sample in step 1 with pure water. Accurately weigh 0.0394 g of DPPH (M = 394.32 g / mL) and dilute to 100 mL with pure methanol to prepare a 1 mmol / L DPPH stock solution. When using, dilute the stock solution with pure methanol at a ratio of 1:9 (v / v) to obtain a 0.1 mmol / L DPPH reaction solution. Add the DPPH reaction solution to the 1 mg / mL sample solution, oscillate in the dark for 30 minutes, and measure the absorbance at 517 nm to obtain A X The experiment set up blank control group A0 (the sample was replaced by an equal volume of deionized water), sample control group A x0 (The DPPH reaction solution was replaced with an equal volume of pure methanol); VC was used as a positive control.

[0021] Clearance % = [A0-(A x -A x0 )] / A0×100 See the results Figure 3 As can be seen from the figure, the DPPH scavenging ability of the complex increases after gastric digestion and decreases after intestinal digestion, but there is no significant change in the DPPH scavenging ability of the complex, indicating that the complex still has excellent antioxidant activity after gastrointestinal digestion.

[0022] 5. α-glucosidase inhibitory activity of red rice bran protein peptide complex before and after simulated gastrointestinal digestion Prepare the sample from step 1 into a 1 mg / mL sample solution with pure water; prepare α-glucosidase solution (0.5 U / mL) and PNPG solution (5 mmol / L) with phosphate buffered saline (PBS, pH 6.8).

[0023] The following reaction system was set up in a 96-well plate: the experimental group (A1) added 10 μL of sample solution and 40 μL of α-glucosidase solution; the sample blank group (A2) added 10 μL of sample solution and 40 μL of deionized water; the negative control group (A3) added 10 μL of deionized water and 40 μL of α-glucosidase solution; and the negative blank group (A4) added 10 μL of deionized water to 40 μL of deionized water. After reacting at 37°C for 10 min, 50 μL of PNPG solution was added to each group and reacted at 37°C for 30 min. The reaction was terminated by adding 1 mol / L Na2CO3 solution. The absorbance was measured at 405 nm using a microplate reader. Acarbose was used as a positive control.

[0024] Inhibition rate (%) = [1-(A1-A2) / (A3-A4)]×100 The experimental results are shown in Figure 4 As can be seen from the figure, after gastric digestion, its α-glucosidase inhibitory activity did not change significantly. After intestinal digestion, its α-glucosidase inhibitory activity decreased, but there was still good α-glucosidase inhibitory activity, indicating that the complex still has hypoglycemic activity after gastrointestinal digestion.

[0025] In summary, after simulated in vitro gastrointestinal digestion, the red rice bran protein peptide complex still has good antioxidant and hypoglycemic activities.

[0026] Example 4: Identification of peptide fragments of red rice bran protein peptide complex after gastrointestinal digestion Sample PC4, obtained after gastrointestinal digestion in Example 3, was selected for peptide sequence analysis using liquid chromatography-tandem mass spectrometry (LC-MS / MS) equipped with an online nanospray ion source. Chromatographic separation was performed using a reversed-phase C18 capillary column (20 cm × 75 μm ID, 1.9 μm particle size). The injection volume was 5 μL, the column temperature was maintained at 40°C, and the mobile phase flow rate was set at 300 nL / min. A nonlinear gradient elution program was used: the initial phase B ratio was 4%, increasing to 39% over 53 minutes, followed by a rapid increase to 50% over 40 seconds, and then to 95% over another 40 seconds, where it was maintained for 5 minutes and 40 seconds. Mass spectrometry parameters were set as follows: primary mass spectrometry scan range m / z 100-1500, resolution 120,000, automatic gain control (AGC) target value 200%, and maximum ion injection time 100 ms; secondary mass spectrometry was performed in high-energy collision dissociation (HCD) mode with a resolution of 50,000, an AGC target value 200%, a maximum injection time of 86 ms, three collision energy settings of 25%, 30%, and 35%, and a dynamic exclusion time of 30 s. Peptide structural information was analyzed using PEAKS Studio 10.6 software and the Uniprot database. 5761 peptides were identified from the digested products, and their mass spectra were as follows Figure 6 As shown in Table 2, the top 50 peptide sequences were selected according to the relative content. We found that most of the peptide structures contained valine (V), phenylalanine (F) and proline (P), indicating that the red rice bran protein peptide complex has good hypoglycemic activity and antioxidant properties after gastrointestinal digestion. Table 2 The top 50 peptides in terms of content of red rice bran protein complex after gastrointestinal digestion by liquid chromatography-mass spectrometry

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[0029] .

Claims

1. A method for preparing a red rice bran protein peptide complex with antioxidant and hypoglycemic effects, characterized in that: The red rice bran is crushed, sieved and defatted, and the red rice bran protein is extracted. The red rice bran protein is evenly mixed with deionized water to obtain a red rice bran protein solution. The pH of the solution is adjusted to 8.5-9. Alkaline protease is added to the red rice bran protein solution, and enzymatic hydrolysis is performed in a light-proof water bath at 48-52° C. After the enzymatic hydrolysis is completed, the enzyme is inactivated, and the solution is cooled and centrifuged to obtain the supernatant, which is freeze-dried to obtain a red rice bran protein peptide complex.

2. The method for preparing the red rice bran protein peptide complex with antioxidant and hypoglycemic efficacy according to claim 1, wherein: The defatting treatment is to add petroleum ether to the red rice bran powder, stir it at room temperature for 1-3 hours, let it stand for stratification, remove the upper layer of petroleum ether, add petroleum ether again and repeat the above process until the petroleum ether layer is clear, collect the precipitate and dry it to obtain the defatted red rice bran.

3. The method for preparing the red rice bran protein peptide complex with antioxidant and hypoglycemic efficacy according to claim 2, wherein: The red rice bran protein is prepared by adding deionized water to defatted red rice bran powder, mixing and adjusting the pH value of the solution to 10-11, stirring for 2 hours, collecting the supernatant by centrifugation, adding deionized water to the precipitate, repeating the above process 2-3 times, combining the supernatants, adjusting the pH value of the supernatant to 4-5, performing acid precipitation, centrifuging, washing the red rice bran protein precipitate with deionized water, and freeze-drying.

4. The preparation method of the red rice bran protein peptide complex with antioxidant and hypoglycemic efficacy according to claim 2, characterized in that: The amount of alkaline protease added was 2500 U / g.

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