Preparation method and application of purple rice vinasse protein zymolyte

Abstract

The invention discloses a preparation method and application of purple rice vinasse protein zymolyte, and belongs to the technical field of functional food and biological medicine. The preparation method of the purple rice vinasse protein zymolyte comprises the following steps: directionally extracting purple rice vinasse powder by using an 80% ethanol solution, cellulase and alpha amylase, adding an alkali solution into an extract precipitate for dissolving, and then adding an acid solution for extracting purple rice vinasse crude protein; hydrolyzing the purple rice vinasse crude protein twice, and performing enzyme deactivation, centrifugation and freeze-drying to obtain the purple rice vinasse protein. The preparation method of the purple rice vinasse protein has the advantages of being simple in process, low in cost and suitable for industrial production; the product purple rice vinasse protein has dual functions of resisting oxidation and reducing blood sugar, and can be synergistically applied to medicines for preventing and treating diabetes and related metabolic diseases; the action mechanism is verified through cell experiments, and a scientific basis is provided for developing multifunctional health-care foods or medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of functional foods and biomedicine, and in particular to a preparation method and application of a purple rice distiller's grains protein hydrolysate. Background Art

[0002] Purple rice distiller's grains, a byproduct of traditional Yunnan winemaking, are rich in plant protein and functional ingredients such as anthocyanins and phenolic acids, demonstrating potential for resource utilization. However, current comprehensive utilization of distiller's grains is primarily used as feed and fertilizer, resulting in low added value and untapped nutritional value. Furthermore, limited research exists on the systematic enzymatic hydrolysis products of purple rice distiller's grain proteins, and the structure-activity relationships of these hydrolysis products remain unclear.

[0003] Furthermore, due to the varying amounts of plant protein and other components found in different types of distiller's grains, extraction methods vary significantly. Furthermore, different protease hydrolysis products exhibit varying activities. However, no prior art reports have specifically developed a method for preparing purple rice distiller's grain protein and the synergistic effects of its products. Therefore, it is necessary to develop a method for preparing purple rice distiller's grain protein hydrolysates and their application to address these technical issues. Summary of the Invention

[0004] The main purpose of the present invention is to provide a preparation method and application of purple rice distiller's grains protein hydrolysate to solve the above-mentioned technical problems existing in the prior art.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing a purple rice distiller's grains protein hydrolysate comprises the following steps:

[0007] (1) drying purple rice distiller's grains and pulverizing and sieving to obtain purple rice distiller's grains powder;

[0008] (2) adding 80% ethanol solution to the purple rice distiller's grains powder for crude extraction; after the extraction is completed, centrifuging and removing the supernatant to obtain an extract precipitate A, and air-drying the extract precipitate A for later use;

[0009] (3) adding deionized water to the extract precipitate A obtained in step (2), dispersing the mixture, and then adding cellulase, adjusting the pH to 4.7-4.9, extracting the mixture at a certain temperature, and centrifuging to obtain a supernatant B and an extract precipitate B, which was then air-dried for later use;

[0010] (4) adding deionized water to the extract precipitate B obtained in step (3) and dispersing the mixture, then adding α-amylase, adjusting the pH to 6.0-6.2, extracting the mixture at a certain temperature, and centrifuging to obtain a supernatant C and an extract precipitate C, which was then air-dried for later use;

[0011] (5) adding an alkaline solution to the extract precipitate C of step (3) to dissolve it; after the extract precipitate C is completely dissolved in the alkaline solution, centrifuging to obtain a supernatant D and purple rice wine lees crude protein A;

[0012] (6) after merging the supernatant B in step (3), the supernatant C in step (4) and the supernatant D in step (5), an acid solution is added to perform acid precipitation extraction, and after centrifugation, the precipitate is air-dried to obtain purple rice distiller's grains crude protein B;

[0013] (7) merging purple rice distiller's grains crude protein A and purple rice distiller's grains crude protein B and freeze-drying them to obtain purple rice distiller's grains crude protein;

[0014] (8) dispersing the purple rice distiller's grains crude protein in step (7) in deionized water to obtain a purple rice distiller's grains crude protein suspension having a mass concentration of 4-6%;

[0015] (9) adding trypsin to the purple rice wine lees crude protein suspension in step (8) for primary hydrolysis to obtain a hydrolyzate; subjecting the hydrolyzate to secondary hydrolysis; cooling the suspension to 4-6° C. after the secondary hydrolysis is completed and then centrifuging; taking the supernatant, adjusting the pH of the supernatant to 7 and then performing rotary evaporation and concentration, and freeze-drying the concentrate to obtain a purple rice wine lees protein hydrolyzate.

[0016] Preferably, the drying temperature in step (1) is 60-70° C.; the mesh size of the sieve is 80-10 mesh; and the moisture content of the purple rice distiller's grains powder is 4-5%.

[0017] Preferably, in step (2), the mass volume ratio of purple rice distiller's grains powder to 80% ethanol solution is 1-1.5:10 g / ml; the initial extraction is stirred for 30-35 minutes; and the number of initial extractions is 2-3 times.

[0018] Preferably, in step (3), the mass volume ratio of the extract precipitate A to deionized water is 1-1.2:10 g / ml; the mass ratio of the extract precipitate A to the cellulase is 50-55:1; and the extraction is carried out by stirring at 55-65° C. for 120-130 minutes.

[0019] Preferably, in step (4), the mass volume ratio of the extract precipitate B to deionized water is 1-1.2:10 g / ml; the mass ratio of the extract precipitate B to α-amylase is 45-50:1; and the extraction is carried out by stirring at 70-80° C. for 120-130 minutes.

[0020] Preferably, the alkaline solution in step (5) is a sodium hydroxide solution with a pH of 12; the mass volume ratio of the extract precipitate C to the alkaline solution is 1-1.5:10 g / ml.

[0021] Preferably, the acid solution in step (6) is a 1 mol / L hydrochloric acid solution; the amount of the acid solution is titrated until the pH of the supernatant combined solution is 4.7 to 4.9, which is the isoelectric point of the vinasse protein; and the freeze-drying temperature in step (7) is -40 to -60°C, and the time is 45 to 50 hours.

[0022] Preferably, in step (9), the mass ratio of trypsin to purple rice wine lees crude protein is 5000 U / g; the primary hydrolysis is to adjust the pH to 8 and perform the primary hydrolysis at 35-38° C.; the secondary hydrolysis is to place the mixture in boiling water and heat it for 15-18 minutes; and the temperature of the rotary evaporation concentration is 35-45° C.

[0023] Preferably, the centrifugal separation in steps (2) to (6) and step (9) is carried out at 8000-8500 r / min for 20-25 min.

[0024] The present invention also provides the use of the purple rice distiller's grains protein hydrolysate prepared by the above-mentioned preparation method, including the use of the purple rice distiller's grains protein hydrolysate in the preparation of antioxidant drugs; the use of the purple rice distiller's grains protein hydrolysate in the preparation of α-glucosidase inhibitors; and the use of the antioxidant drugs or α-glucosidase inhibitors in the preparation of drugs for improving diabetes or oxidative stress-related diseases.

[0025] During the application process, the purple rice wine lees protein hydrolysate was evaluated based on the DPPH free radical scavenging ability, ABTS free radical scavenging ability, iron ion reducing power, and inhibitory activity of α-amylase and α-glucosidase activity. Combined with cell experiments: H2O2-induced HepG2 cell oxidative stress model and glucosamine-induced insulin resistance cell model, the product TE has strong in vitro antioxidant and hypoglycemic activity, providing a basis for the development of purple rice wine lees protein.

[0026] The beneficial effects of the present invention are:

[0027] The method for preparing the purple rice distiller's grains protein hydrolysate of the present invention has the advantages of simple process, low cost and suitability for industrial production; the product, the purple rice distiller's grains protein hydrolysate, has both antioxidant and hypoglycemic functions and can be synergistically applied in drugs for preventing and treating diabetes and related metabolic diseases; its mechanism of action is verified by cell experiments, providing a scientific basis for the development of multifunctional health foods or drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing the effects of the purple rice distiller's grains enzymatic hydrolysate TE and metformin on the viability of HepG2 cells;

[0029] Figure 2The establishment of the H2O2-induced oxidative stress cell model of the present invention: Figure A: Induction at different concentrations; B: Induction at different times Note: * represents a significant difference between the model group and the blank group (*p<0.05);

[0030] Figure 3 The effect of the purple rice distiller's grains protein hydrolysate TE on the oxidase activity in oxidatively damaged cells: A: CAT; B: GSH; C: ROS; D: SOD; E: MDA. Note: Different letters indicate differences between groups (p < 0.05);

[0031] Figure 4 The figure is a flow cytometer graph showing the effect of the purple rice distiller's grains protein of the present invention on apoptosis of oxidatively damaged cells;

[0032] Figure 5 This is the insulin resistance model of HepG2 cells induced by glucosamine (GlcN) of the present invention: (A) optimization at different times; (B) different concentrations. Note: * represents a significant difference between the modeling group and the blank group, *p<0.05;

[0033] Figure 6 Effects of the purple rice distiller's grains enzymatic hydrolysate TE on the insulin resistance cell model. Note: Different letters indicate differences between the groups, p < 0.05;

[0034] Figure 7 This is the Base peak diagram of TE of the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0036] Example 1 Preparation of Purple Rice Distiller's Grains Protein Hydrolysate (TE)

[0037] 1.1 Preparation of crude protein from purple rice distiller's grains

[0038] (1) Pretreatment - Drying, decolorizing and removing impurities from the discarded purple rice wine lees: Take an appropriate amount of purple rice wine lees and dry them in a 60°C oven for 2 hours until the moisture content is 5%. Grind the dried purple rice wine lees through a 100-mesh sieve, take 100g of the purple rice wine lees powder and add it to 100ml of 80% ethanol solution with magnetic stirring for 30 minutes, and repeat this step twice. Centrifuge at 8000r / min for 20 minutes to remove the supernatant, and air-dry the resulting precipitate for later use;

[0039] (2) The material obtained in step (1) was added with 1:10 distilled water and 2% cellulase (the mass ratio of extract precipitate A to cellulase was 50:1), the pH of the solution was adjusted to 4.8, and the extraction was carried out under magnetic stirring at 60°C for 2 h. The extract was centrifuged (8000 rpm, 20 min), and the supernatant and precipitate were collected separately;

[0040] (3) Add 1:10 distilled water and 2% α-amylase (the mass ratio of extract precipitate B to α-amylase is 45:1) to the precipitate obtained in (2), adjust the pH of the solution to 6.0, extract at 75°C for 2 h with magnetic stirring, centrifuge (8000 r / min, 20 min), and collect the supernatant and precipitate separately.

[0041] (4) adding 100 ml of a sodium hydroxide solution having a pH of 12 to the 10 g precipitate obtained in step (3), and obtaining a supernatant and purple rice distiller's grains crude protein after the precipitate is completely dissolved in an alkaline solution by centrifugation;

[0042] (5) combining the supernatant in step (2), the supernatant in step (3), and the supernatant in step (4), and adding 1 mol / L hydrochloric acid solution to perform acid precipitation extraction, wherein the amount of the acid solution is titrated until the pH of the supernatant combined solution is 4.9, the isoelectric point of the lees protein, and after centrifugation, the precipitate is air-dried to obtain purple rice lees crude protein;

[0043] (6) The above purple rice wine lees crude proteins were combined and freeze-dried at -50°C for 50 hours to obtain purple rice wine lees crude protein.

[0044] 1.2 Preparation of purple rice distiller's grains enzymatic hydrolysate (TE)

[0045] 10 g of crude protein from purple rice distiller's grains prepared in Example 1 was dispersed in deionized water to prepare a protein suspension with a mass concentration of 5%. Trypsin (5000 U / g) was added to the suspension and hydrolyzed at pH 8 and 37°C for 2.5 h. After the reaction, the hydrolyzate was heated in boiling water for 15 minutes to allow enzymatic hydrolysis. After cooling and centrifugation (4°C, 8000 rpm, 25 minutes), the pH was adjusted to 7 and concentrated by rotary evaporation (45°C). The concentrate was freeze-dried at -50°C for 50 hours to obtain purple rice distiller's grains protein hydrolysate (TE).

[0046] Example 2 Qualitative analysis of TE of purple rice distiller's grains enzymatic hydrolysate

[0047] 1. LC-MS / MS instrument parameter settings

[0048] Samples were analyzed using LC-MS / MS equipped with an online nanospray ion source. The system consisted of an EASY-nano LC 1200 connected in series with an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific, USA). 0.1 μL of sample was loaded onto an Acclaim PepMap C18 (75 μm × 25 cm) analytical column. Separation was performed over a 60-min gradient, maintaining a column flow rate of 400 nL / min. The column temperature was set at 40°C, and the electrospray voltage was 2 kV. The gradient started with 2.2% phase B, which increased nonlinearly to 44% over 45 minutes. The gradient then increased to 90% over 3 minutes and was maintained for 12 minutes. Phase A consisted of 0.1% formic acid in water, and phase B consisted of 80% acetonitrile in water.

[0049] The mass spectrometer was in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. Mass spectrometry parameters were as follows: MS scan range m / z 200–1200, resolution 60,000, AGC target value 4e5, and maximum injection time 50 ms; HCD-MS / MS resolution 15,000, AGC target value 5e4, maximum injection time 22 ms, collision energy 30%, and dynamic exclusion time 30 s.

[0050] 2. Database Search

[0051] Tandem mass spectra were analyzed using PEAKS Studio 10.6 (Bioinformatics Solutions Inc., Canada). The database used was the 2024 version of Uniprot-Oryza sativa subsp japonica (43,667 entries). The search parameters were as follows: a fragment ion mass tolerance of 0.02 Da, a parent ion mass tolerance of 10 ppm, and variable modifications including oxidation (M) of 15.99 and deamination (NQ) of 0.98. Protein quantification required at least one unique peptide, and peptide quantifications had to meet a -101 gP ≥ 20.

[0052] 3. Results Analysis

[0053] Figure 7The Base Peak Chromatogram (BPC) intuitively reflects the elution conditions of each component in the sample during the liquid chromatography separation process and the trend of relative content changes. The horizontal axis is the retention time (Retention Time) and the vertical axis is the mass spectrometry signal intensity (Intensity). Each point in the figure represents the strongest ion signal among all ions detected by the mass spectrometer at the corresponding retention time. Overall, a total of 15,046 peptide segments were identified in the purple rice protein hydrolysate TE identified by liquid chromatography-mass spectrometry, of which the top 50 peptide segments were representative. In terms of peptide chain length, the peptide chain length ranged from 4 to 22 amino acids, covering short peptides to relatively long peptides, among which YYGAPSTITTLGGVL with a length of 15 had the highest proportion, indicating that under the action of trypsin, purple rice protein can be cut into peptides of various lengths. In terms of relative molecular mass, the molecular mass of these peptides ranged from 461.23 to 2336.05, indicating that the product contains peptides of various molecular masses, which may have different functional properties. The wide retention time range reflects the varying retention behavior of different peptides on the chromatographic column, which may be related to physicochemical properties such as polarity and hydrophobicity. The -101gP values indicate a certain degree of reliability in peptide identification, with most peptides exhibiting high values, indicating a high degree of confidence in the identification results. In terms of relative abundance, YYGAPSTITTLGGVL accounts for the highest proportion at 1.44%, while among peptides with a chain length of 4, FAVR and LSGW contribute 0.27% and 0.24%, respectively. Furthermore, some peptides, such as PM(+15.99)YPLPR, AAM(+15.99)LDKPR, and GYPM(+15.99)YPLPR, exhibit post-translational modifications, which may affect their stability and bioactivity. The identification of TE peptides provides important foundational data for subsequent research and is crucial for further understanding the composition, properties, and potential functions of TEs from purple rice enzymatic hydrolysates. The top 50 TE peptides by LC-MS analysis of TEs from purple rice distiller's grains enzymatic hydrolysates are shown in Table 1.

[0054] Table 1 Top 50 peptides in TE content of purple rice distiller's grains enzymatic hydrolysate by liquid-mass analysis

[0055]

[0056]

[0057]

[0058] Note: -10lgP: The credibility of the peptide. The larger the value, the better the peptide spectrum matching result. Generally, it is required to be greater than 20. (+15.99) indicates that the peptide may have post-translational modification. For example, PM (+15.99) YPLPR indicates that methionine (M) has undergone oxidation modification, and the mass has increased by 15.99Da.

[0059] Example 3 In vitro antioxidant experiment of purple rice distiller's grains protein

[0060] Based on the DPPH free radical scavenging ability, ABTS free radical scavenging ability and iron reducing power, it was proved that the polyphenol has strong in vitro antioxidant activity and can be used to develop products such as antioxidants; the purple rice wine lees protein hydrolysate prepared in Example 1 was used as the experimental sample.

[0061] (1) Determination of DPPH free radical scavenging ability (DPPH method)

[0062] Purple rice distiller's grains protein hydrolysate was prepared with deionized water to a 1 mg / mL sample solution. DPPH free radical solution was prepared by dissolving DPPH powder in anhydrous ethanol to a 0.1 mM solution (store in the dark and use immediately after preparation). The sample to be tested was diluted with solvent to various concentrations. 100 μL of sample solution was mixed with 100 μL of DPPH solution and reacted in the dark for 30 minutes (25°C). Ascorbic acid was used as a positive control.

[0063] Blank group: 100 μL solvent + 100 μL DPPH solution.

[0064] Control group: 100 μL sample solution + 100 μL solvent.

[0065] The absorbance (A) was measured at 517 nm and the DPPH clearance rate was calculated using the following formula:

[0066]

[0067] The DPPH scavenging rates of 1 mg / ml TE sample and positive ascorbic acid sample were measured to be 81.77±1.43% and 98.73±0.53, respectively. The experimental results showed that purple rice wine lees enzymatic hydrolysate TE has DPPH scavenging ability.

[0068] (2) Determination of ABTS free radical scavenging ability (ABTS method)

[0069] The purple rice lees protein hydrolysate was prepared into a sample solution with a concentration of 1 mg / mL using deionized water to prepare ABTS. + Free radical solution: First, mix 7mM ABTS solution with 2.45mM potassium persulfate and react for 12–16 hours in the dark to generate stable ABTS +Stock solution. Before use, dilute the stock solution with PBS buffer (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. Take 10 μL of sample and mix with 190 μL of ABTS. + The working solutions were mixed and reacted for 6 minutes in the dark. The absorbance was then measured at 734 nm. Ascorbic acid was used as a positive control. The ABTS clearance rate was calculated using the following formula based on the absorbance at 734 nm:

[0070]

[0071] The measured ABTS scavenging rates of purple rice wine lees protein hydrolysate sample and positive ascorbic acid sample were 76.40±1.81 and 98.28±0.57, respectively. The experimental results showed that purple rice wine lees protein hydrolysate has the ability to scavenge ABTS free radicals.

[0072] (3) Determination of ferric reducing power (FRAP)

[0073] FRAP working solution preparation: FRAP working solution is composed of 300mM acetate buffer (pH 3.6), 10mM TPTZ (2,4,6-tripyridyl triazine) and 20mM FeCl3 in a ratio of 10:1:1, and is prepared and used immediately. Take 30μL of sample and mix it with 900μL of FRAP working solution. After incubation at 37℃ for 10 minutes, the absorbance is measured on a microplate reader set at a wavelength of 593nm. The results are converted to FeSO4 using the FeSO4 standard curve. 2 + equivalent (μmol / g). This experiment requires strict control of reaction temperature and time to avoid errors caused by TPTZ oxidation. Ascorbic acid was used as a positive control. The total reducing power of purple rice wine lees protein hydrolysate was measured at a concentration of 1 mg / mL. The total reducing power of purple rice wine lees protein hydrolysate was 0.81±0.15 mmol FeSO4 / L, and the total reducing power of ascorbic acid was 1.96±0.03 mmol FeSO4 / L. The experimental results show that purple rice wine lees protein hydrolysate has reducing power.

[0074] Example 4 Inhibitory Effect of Purple Rice Distillers Grains Protein Hydrolysate on α-Amylase and α-Glucosidase

[0075] (1) Determination of α-glucosidase inhibition rate

[0076] Purple rice distiller's grains protein hydrolysate was prepared with deionized water to concentrations of 0.0625, 0.125, 0.25, 0.5, and 1.0 mg / mL. Phosphate buffer (PBS, pH 6.8–7.0) was then prepared. α-Glucosidase (1.0 U / mL) and p-nitrophenyl-α-D-glucopyranoside (pNPG, 5 mM) were dissolved in PBS and used as a buffer. The samples were then serially diluted to concentrations ranging from 62.5 to 1000 μg / mL. Fifty μL of sample solution and 50 μL of α-Glucosidase solution were then added to a 96-well plate. The plates were pre-incubated at 37°C for 10 minutes to simulate enzyme-inhibitor binding. The reaction was then initiated by adding 50 μL of pNPG substrate solution. The reaction was incubated at 37°C in the dark for 20 minutes to allow the enzyme to hydrolyze the substrate to form p-nitrophenol (pNP). After the reaction is completed, 100 μL of Na2CO3 (0.1 M) solution is added to terminate the reaction, and the absorbance (A) of each well is immediately measured at a wavelength of 405 nm using a microplate reader. The blank group is replaced with PBS instead of the enzyme solution, and the control group is replaced with PBS instead of the sample solution. The formula for calculating the α-glucosidase inhibition rate is:

[0077]

[0078] In this experiment, acarbose was set as a positive control, and the half-maximal inhibitory concentration (IC 50 The experimental results showed that the purple rice wine lees protein hydrolysate had a good inhibitory effect on the activity of α-glucosidase. 50 It is 0.264±0.03 mg / mL, and the IC50 value of acarbose is <0.14 μg / mL.

[0079] (2) Determination of α-amylase inhibition rate

[0080] Purple rice distiller's grains protein hydrolysate was prepared with deionized water to a concentration of 1 mg / mL. α-amylase inhibition was determined using the DNS (3,5-dinitrosalicylic acid) method. The following steps were performed: First, phosphate buffered saline (PBS) was prepared at pH 6.9. α-amylase (derived from porcine pancreas, final concentration 1.0 U / mL) was dissolved in PBS and set aside. A 1% soluble starch solution was used as the substrate, which was fully dissolved in a boiling water bath and then cooled to room temperature. The sample to be tested was serially diluted with PBS or an appropriate solvent, along with a positive control, acarbose. 50 μL of sample solution and 50 μL of enzyme solution were added to a 96-well plate, pre-incubated at 37°C for 10 minutes to promote inhibitor-enzyme binding. The reaction was then initiated by adding 100 μL of starch substrate solution and incubated at 37°C for exactly 10 minutes. Immediately after the reaction, 200 μL of DNS reagent was added, and the plate was transferred to a boiling water bath and boiled for 5 minutes to allow the reducing sugar to react with the DNS reagent to form a reddish-brown complex. After cooling to room temperature, the absorbance (A) was measured at a wavelength of 540 nm. The blank group was set up with PBS instead of the enzyme solution, and the control group was set up with PBS instead of the sample solution. The experiments were repeated three times independently, and the results are expressed as mean ± standard deviation. The amount of reducing sugar produced was measured by DNS colorimetry, which indirectly reflects the change in α-amylase activity and thus evaluates the enzyme inhibition effect of the sample. The inhibition rate is calculated as follows:

[0081]

[0082] The inhibition rate of purple rice distiller's grains protein hydrolysate on α-amylase was determined to be 35.87±2.53%, indicating that the purple rice distiller's grains protein hydrolysate has the ability to inhibit α-amylase activity.

[0083] Comparative Example 1

[0084] The purple rice distiller's grains crude protein (EJP) obtained in Example 1 was dispersed in deionized water to prepare a protein suspension with a mass concentration of 5%. Alkaline protease (5000 U / g) was added to the suspension. Hydrolysis was carried out for 2.5 hours under the optimal enzyme conditions (alkaline protease pH 9 50°C). After the reaction, the hydrolyzate was heated in boiling water for 15 minutes to allow the enzyme to hydrolyze, cooled and centrifuged (4°C, 8000 r / min, 25 minutes), and then the pH was adjusted to 7. Freeze-dried for later use. Antioxidant and hypoglycemic indices were determined according to Examples 3 and 4.

[0085] Comparative Example 2

[0086] The purple rice distiller's grains crude protein (EJP) prepared in Example 1 was dispersed in deionized water to prepare a protein suspension with a mass concentration of 5%. Papain (5000 U / g) was added to the suspension. Hydrolysis was performed for 2.5 h under optimal enzyme conditions (papain pH 8 at 65°C). After the reaction, the hydrolyzate was heated in boiling water for 15 minutes to allow enzymatic hydrolysis, cooled and centrifuged (4°C, 8000 r / min, 25 minutes), and the pH was adjusted to 7. The mixture was freeze-dried for later use. Antioxidant and hypoglycemic indices were determined according to Examples 3 and 4.

[0087] Comparative Example 3

[0088] The purple rice distiller's grains crude protein EJP prepared in Example 1 was dispersed in deionized water to prepare a protein suspension with a mass concentration of 5%. Neutral protease (5000U / g) was added to the suspension. Hydrolysis was carried out for 2.5h under the optimal enzyme conditions (neutral protease pH 7 45°C). After the reaction, the hydrolyzate was heated in boiling water for 15 minutes to allow the enzyme to hydrolyze, cooled and centrifuged (4°C, 8000r / min, 25 minutes) and then the pH was adjusted to 7. Freeze-dried for later use. Antioxidant and hypoglycemic indicators were determined according to Examples 3 and 4. Specific results are shown in Table 2.

[0089] Table 2 Comparison of EJP activity of purple rice distiller's grains protein hydrolyzed by different enzymes

[0090]

[0091]

[0092] As can be seen from Table 1, the enzymolysis products obtained by hydrolyzing purple rice lees crude protein with different proteases have significant differences in antioxidant capacity and the inhibitory capacity of sugar metabolism related enzymes. From the perspective of antioxidant capacity, the ABTS and DPPH free radical scavenging abilities of alkaline protease (comparative example 1) hydrolysate are the strongest, the reducing power (FRAP value) of papain (comparative example 2) hydrolysate is the strongest, the trypsin (embodiment 1) hydrolysate is second, and the neutral protease (comparative example 3) hydrolysate is the weakest; and in terms of the inhibitory capacity of sugar metabolism related enzymes, the inhibitory effect of trypsin hydrolysate on alpha-amylase and alpha-glucosidase is the most significant, the alkaline protease hydrolysate is second, the papain hydrolysate is again, and the inhibitory capacity of neutral protease hydrolysate is the weakest. Wherein the lees protein product TE using trypsin hydrolysis in Example 1, while possessing good antioxidant capacity, also possesses good ability, therefore it is used as the sample of the selection of the present invention application.

[0093] Example 4 Evaluation of the Antioxidant and Hypoglycemic Effects of Purple Rice Distillers Grains Protein Hydrolysate at the Cellular Level

[0094] HepG2 cells were stored in liquid nitrogen and quickly transferred to a 37°C water bath for thawing upon recovery. After thawing, the cells were centrifuged at 800 rpm / min for 3 minutes, the cryopreservative solution was discarded, and 5 mL of DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% double-antibody was added. Cell clusters were disaggregated by repeated pipetting to obtain a homogenized single-cell suspension. The suspension was transferred to a culture dish, labeled, and cultured in a cell culture incubator at 37°C and 5% CO2. Cell morphology was observed using an inverted microscope. Cells were passaged when they reached 80%-90% growth.

[0095] When subculturing, remove the supernatant from the culture dish, wash twice with PBS buffer, and then add 1 mL of trypsin-EDTA digestion system (0.25%) to digest the cells. Once the cells are mostly suspended, remove 5 mL of complete medium to terminate the digestion. Re-inoculate the cells into a new culture dish at a 1:2 or 1:3 ratio and continue culturing.

[0096] (1) Cell viability determination

[0097] The experimental process of MTT method to determine cell viability is as follows: After counting HepG2 cells using a cell counter, 200 μL of 1.5×10 5 Cells were seeded into 96-well plates at a density of 100 cells / well. Cultured in a 37°C, 5% CO2 incubator for 24 hours to ensure adequate cell attachment. Subsequently, the culture medium was replaced with medium containing different concentrations of sample and treatment agents and incubated for the specified time under the appropriate conditions.

[0098] After treatment, the culture medium was aspirated and 100 μL of MTT solution (final concentration: 0.5 mg / mL) was added to each well. The cells were incubated for another 4 hours. During this time, succinate dehydrogenase in the mitochondria of the living cells reduced MTT to purple formazan crystals. After the incubation period, the supernatant was discarded and 100 μL of DMSO was added to each well to dissolve the formazan crystals. The culture plate was shaken on a low-speed shaker for 10 minutes to homogenize the solution. The absorbance was measured at 490 nm using a microplate reader, and cell viability was calculated using the following formula.

[0099]

[0100] Among them A 实验组 For the treatment group, A 空白孔 Cell-free blank well, A 对照组 These are cells that grow normally without intervention.

[0101] In the experiment, blank wells without cells were set to deduct the interference of H2O2 absorbance value. In the H2O2 treatment group, cells were washed with PBS before MTT incubation to eliminate residual interference.

[0102] The MTT assay was used to determine the effect of purple rice wine lees enzymatic hydrolysate TE on the viability of HepG2 cells. The sample concentration gradient was set from 0.25 to 2 mg / ml to determine the optimal loading concentration. Figure 1 As shown by Figure 1 As can be seen, at a concentration of 2 mg / ml, TE reduced the viability of HepG2 cells compared to the blank control. At a concentration of 1 mg / ml, the cell viability increased compared to the blank control, and the growth was better. This may be because the enzymatic hydrolysate is rich in amino acids required for cell growth, providing nutrients to the cells and promoting their growth. However, higher concentrations will increase the extracellular osmotic pressure, leading to water loss in the cells and a decrease in their survival rate. Therefore, we determined the TE loading concentration gradient to be 0.25, 0.5, and 1 mg / ml, corresponding to the low concentration group (S), medium concentration group (M), and high concentration group (L), respectively. In addition, we also determined that the loading concentration of metformin (Metformin), which is used as a positive drug control for insulin resistance in subsequent experiments, is 2.5 mM.

[0103] (2) Establishment and evaluation of oxidative stress cell model

[0104] a. H2O2-induced oxidative damage model Figure 2 As shown in the experimental chart data, the effect of H2O2 on cell viability showed significant concentration and time dependence. In the concentration gradient experiment ( Figure 2 B), the cell viability of the control group was 100%. When the H2O2 concentration was gradually increased from 0.20mM to 1.00mM (treated for 12 hours), the cell viability decreased to about 85% (0.20mM), 70% (0.40mM), 50% (0.80mM) and 35% (1.00mM), indicating that concentrations of 0.80mM and above can significantly induce cell viability to drop to half the inhibition level. In the time gradient experiment ( Figure 2 A, fixed 0.80mM H2O2). As treatment time increased from 4 to 24 hours, cell viability gradually decreased from 75% (4 hours) to 65% (8 hours), 50% (12 hours), and 30% (24 hours). After 12 hours of treatment, cell viability steadily dropped to the critical value of 50%. This range effectively induces oxidative stress damage while avoiding excessive cell death that could lead to irreversible collapse of the model, providing optimal conditions for establishing an oxidative stress cell model. These results demonstrate that 12 hours of 0.80mM H2O2 treatment effectively establishes an oxidative stress cell model, providing controllable injury conditions for subsequent studies.

[0105] b. Antioxidant index detection:

[0106] TE treatment group: cells were pretreated with low (0.25 mg / mL), medium (0.5 mg / mL), and high (1 mg / mL) concentrations of TE for 24 hours, and then H2O2 was added to induce damage.

[0107] Effects of PJP proteolytic enzymes on oxidatively damaged cells Figure 3 The flow cytometric graphs of the effects of TE on oxidatively damaged cells are shown in Figure 4 Result: As shown. Figure 4 It can be seen that the cells in the model group were damaged by oxidation and produced ROS, and the green fluorescence shifted to the right. TE reduced the production of ROS by scavenging free radicals, and the green fluorescence shifted to the left, alleviating the symptoms of oxidatively damaged cells. Figure 3 C) levels in the model group soared to over 80%, while those in the high-concentration TE group (L group) dropped to around 60% (equivalent to the Vc group), indicating that TE directly cleared or inhibited ROS generation. Figure 3 We can see that the high-concentration TE group significantly increased the activity of intracellular antioxidant enzymes, with CAT activity restored to 5.2±0.4U / mg prot (a 60% increase compared to the model group), SOD activity reached 28.0±1.5nmol / mg prot (a 50% increase), and GSH content increased to 34.5±2.1μmol / mg prot; at the same time, ROS levels dropped to 60% (equivalent to the ascorbic acid group), and MDA content decreased to 6.8±0.5nmol / mg prot (a 45% decrease, *p*<0.05). These results indicate that purple rice enzymatic hydrolyzate TE has a protective effect on oxidatively damaged cells.

[0108] (3) Establishment of insulin resistance cell model and evaluation of blood sugar reduction

[0109] a. GlcN-induced insulin resistance model: Figure 5 As shown, HepG2 cells treated with 18 mM glucosamine (GlcN) for 36 hours showed significant differences compared with the blank group, and the difference in cellular glucose consumption was the largest, indicating that the insulin resistance model was successfully established.

[0110] b.TE improves glucose metabolism:

[0111] Treatment groups: Model cells were intervened with 0.25, 0.5, and 1 mg / mL TE for 36 hours.

[0112] Results: As Figure 6, the glucose consumption of the insulin resistance model group (Model) was 4.1±0.3, which was significantly lower than that of the blank control group (Blank, 10.0±0.3, p<0.05), indicating that the insulin resistance model was successfully established. The glucose uptake of the positive control group (Met, metformin) recovered to 7.1±0.3 (p<0.05vs.Model), which is consistent with the known insulin sensitization effect of metformin. In addition, it can be seen that the glucose consumption of the high concentration group (L, 6.7±0.2) was higher and significantly higher than that of the model group (p<0.05), which was equivalent to the positive group, indicating that the PJP enzymatic hydrolysis product TE helps to improve the symptoms of insulin resistance by enhancing the glucose utilization ability of hepatocytes and increasing glucose consumption, and the effect is comparable to that of the traditional hypoglycemic drug metformin.

[0113] The above experiments showed that TE has significant in vitro antioxidant activity. The scavenging rates of 1 mg / ml TE against DPPH and ABTS free radicals were 81.77±1.43% and 76.40±1.81%, respectively, and the iron reducing power was 0.81±0.15 mmolFeSO4 / L. At the same time, the inhibition rate against α-amylase was 35.87±2.53%. In addition, its inhibitory activity against α-glucosidase (IC 50 =0.264±0.03mg / mL). Cell experiments further verified that TE can significantly increase the antioxidant enzyme activity (SOD, CAT) of HepG2 cells, reduce oxidative stress markers (ROS, MDA), and increase glucose consumption to 6.7±0.2mmol / L in the insulin resistance model, which is close to the effect of metformin. It can be seen that TE, a trypsin hydrolyzate of purple rice lees, significantly alleviates oxidative damage and insulin resistance by activating endogenous antioxidant systems (CAT, SOD, GSH) and improving glucose metabolism, providing a cellular level basis for its application in antioxidant and hypoglycemic functional foods or drugs. At the same time, the preparation process is simple and the cost is low. The obtained product has both antioxidant and hypoglycemic dual functions, which can be used in the prevention and treatment of diabetes and the development of antioxidant functional foods or drugs, providing a new way for the high-value utilization of purple rice lees.

[0114] Example 5 Preparation of Purple Rice Distiller's Grains Protein Hydrolysate (TE)

[0115] 1.1 Preparation of crude protein from purple rice distiller's grains

[0116] (1) Pretreatment - Drying, decolorizing and removing impurities from the discarded purple rice wine lees: Take an appropriate amount of purple rice wine lees and dry them in a 70°C oven for 2 hours until the moisture content is 4%. Grind the dried purple rice wine lees through an 80-mesh sieve, take 150g of the purple rice wine lees powder and add it to 100ml of 80% ethanol solution with magnetic stirring for 35 minutes, and repeat this step twice. Centrifuge at 8300r / min for 22 minutes, remove the supernatant, and air-dry the resulting precipitate for later use;

[0117] (2) The material obtained in step (1) was added with 1.1:10 distilled water and 2% cellulase (the mass ratio of extract precipitate A to cellulase was 53:1), the pH of the solution was adjusted to 4.7, and the extraction was performed under magnetic stirring at 65°C for 130 minutes. The extract was centrifuged (8300 rpm, 22 minutes), and the supernatant and precipitate were collected separately;

[0118] (3) Add 1.1:10 distilled water and 2% α-amylase (the mass ratio of extract precipitate B to α-amylase is 50:1) to the precipitate obtained in (2), adjust the pH of the solution to 6.2, extract with magnetic stirring at 70°C for 130 minutes, centrifuge (8300 r / min, 22 minutes), and collect the supernatant and precipitate separately.

[0119] (4) adding 100 ml of a sodium hydroxide solution having a pH of 12 to the 12 g precipitate obtained in step (3), and obtaining a supernatant and purple rice distiller's grains crude protein after centrifugation after the precipitate is completely dissolved in an alkaline solution;

[0120] (5) combining the supernatant in step (2), the supernatant in step (3), and the supernatant in step (4), and adding 1 mol / L hydrochloric acid solution to perform acid precipitation extraction, wherein the amount of the acid solution is titrated until the pH of the supernatant combined solution is 4.7, the isoelectric point of the lees protein, and after centrifugation, the precipitate is air-dried to obtain purple rice lees crude protein;

[0121] (6) The above purple rice wine lees crude proteins were combined and freeze-dried at -40°C for 48 hours to obtain purple rice wine lees crude protein.

[0122] 1.2 Preparation of purple rice distiller's grains enzymatic hydrolysate (TE)

[0123] 10 g of crude protein from purple rice distiller's grains prepared in Example 1 was dispersed in deionized water to prepare a protein suspension with a mass concentration of 5%. Trypsin (5000 U / g) was added to the suspension. Hydrolysis was carried out at pH 8 and 35°C for 2.5 h. After the reaction, the hydrolyzate was heated in boiling water for 16 minutes to allow enzymatic hydrolysis. After cooling and centrifugation (4°C, 8300 rpm, 22 minutes), the pH was adjusted to 7 and the product was concentrated by rotary evaporation (35°C). The concentrate was freeze-dried at -50°C for 50 hours to obtain purple rice distiller's grains protein hydrolysate (TE).

[0124] Example 6 Preparation of Purple Rice Distiller's Grains Protein Hydrolysate (TE)

[0125] 1.1 Preparation of crude protein from purple rice distiller's grains

[0126] (1) Pretreatment - Drying, decolorizing and removing impurities from the discarded purple rice wine lees: Take an appropriate amount of purple rice wine lees and dry them in a 65°C oven for 2 hours until the moisture content is 4.5%. Grind the dried purple rice wine lees through a 90-mesh sieve, take 120g of the purple rice wine lees powder and add it to 100ml of 80% ethanol solution with magnetic stirring for 33 minutes, and repeat this step three times. Centrifuge at 8500r / min for 25 minutes to remove the supernatant, and air-dry the resulting precipitate for later use;

[0127] (2) The material obtained in step (1) was added with 1.2:10 distilled water and 2% cellulase (the mass ratio of extract precipitate A to cellulase was 55:1), the pH of the solution was adjusted to 4.9, and the extraction was performed under magnetic stirring at 55°C for 125 minutes. The extract was centrifuged (8500 rpm, 25 minutes), and the supernatant and precipitate were collected separately;

[0128] (3) Add 1.2:10 distilled water and 2% α-amylase (the mass ratio of extract precipitate B to α-amylase is 47:1) to the precipitate obtained in (2), adjust the pH of the solution to 6.1, extract at 80°C with magnetic stirring for 125 minutes, centrifuge (8500 r / min, 25 minutes), and collect the supernatant and precipitate separately.

[0129] (4) adding 100 ml of a sodium hydroxide solution having a pH of 12 to the 15 g precipitate obtained in step (3), and obtaining a supernatant and purple rice distiller's grains crude protein after centrifugation after the precipitate is completely dissolved in an alkaline solution;

[0130] (5) combining the supernatant in step (2), the supernatant in step (3), and the supernatant in step (4), and adding 1 mol / L hydrochloric acid solution to perform acid precipitation extraction, wherein the amount of the acid solution is titrated to a pH value of the supernatant combined solution equal to the isoelectric point of vinasse protein of 4.8, and after centrifugation, taking the precipitate and air-drying it to obtain purple rice vinasse crude protein;

[0131] (6) The above purple rice wine lees crude proteins were combined and freeze-dried at -60°C for 45 hours to obtain purple rice wine lees crude protein.

[0132] 1.2 Preparation of purple rice distiller's grains enzymatic hydrolysate (TE)

[0133] 10 g of crude protein from purple rice distiller's grains prepared in Example 1 was dispersed in deionized water to prepare a protein suspension with a mass concentration of 5%. Trypsin (5000 U / g) was added to the suspension. Hydrolysis was carried out at pH 8 and 38°C for 2.5 h. After the reaction, the hydrolyzate was heated in boiling water for 18 minutes to allow enzymatic hydrolysis. After cooling and centrifugation (4°C, 8500 rpm, 25 minutes), the pH was adjusted to 7 and rotary evaporation was performed (40°C). The concentrate was freeze-dried at -50°C for 50 hours to obtain purple rice distiller's grains protein hydrolysate (TE).

[0134] The purple rice distiller's grains protein hydrolysate (TE) prepared in Examples 5 and 6 also has the above-mentioned effects.

[0135] The above detailed description of the specific embodiments of the invention is intended to be illustrative only, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications or substitutions to the invention are also within the scope of the present invention. Therefore, equivalent changes, modifications, and improvements made without departing from the spirit and scope of the present invention are also encompassed within the scope of the present invention.

Claims

1. A method for preparing a purple rice distiller's grains protein hydrolysate, characterized in that: The steps include: (1) drying purple rice distiller's grains and pulverizing and sieving to obtain purple rice distiller's grains powder; (2) adding 80% ethanol solution to the purple rice distiller's grains powder for crude extraction; after the extraction is completed, centrifuging and removing the supernatant to obtain an extract precipitate A, and air-drying the extract precipitate A for later use; (3) adding deionized water to the extract precipitate A obtained in step (2), dispersing the mixture, and then adding cellulase, adjusting the pH to 4.7-4.9, extracting the mixture at a certain temperature, and centrifuging to obtain a supernatant B and an extract precipitate B, which was then air-dried for later use; (4) adding deionized water to the extract precipitate B obtained in step (3) and dispersing the mixture, then adding α-amylase, adjusting the pH to 6.0-6.2, extracting the mixture at a certain temperature, and centrifuging to obtain a supernatant C and an extract precipitate C, which was then air-dried for later use; (5) adding an alkaline solution to the extract precipitate C of step (3) to dissolve it; after the extract precipitate C is completely dissolved in the alkaline solution, centrifuging to obtain a supernatant D and purple rice wine lees crude protein A; (6) after merging the supernatant B in step (3), the supernatant C in step (4) and the supernatant D in step (5), an acid solution is added to perform acid precipitation extraction, and after centrifugation, the precipitate is air-dried to obtain purple rice distiller's grains crude protein B; (7) merging purple rice distiller's grains crude protein A and purple rice distiller's grains crude protein B and freeze-drying them to obtain purple rice distiller's grains crude protein; (8) dispersing the purple rice distiller's grains crude protein in step (7) in deionized water to obtain a purple rice distiller's grains crude protein suspension having a mass concentration of 4-6%; (9) adding trypsin to the purple rice distiller's grains crude protein suspension in step (8) for primary hydrolysis to obtain a hydrolyzate; The hydrolyzate is subjected to secondary hydrolysis; after the secondary hydrolysis is completed, it is cooled to 4-6° C. and then centrifuged; the supernatant is taken, the pH of the supernatant is adjusted to 7, and then concentrated by rotary evaporation; the concentrate is freeze-dried to obtain the purple rice wine lees protein hydrolysate.

2. The method for preparing the purple rice distiller's grains protein hydrolysate as claimed in claim 1, wherein The drying temperature in step (1) is 60-70° C.; the mesh size of the sieve is 80-100 mesh; and the moisture content of the purple rice distiller's grains powder is 4-5%.

3. The method for preparing the purple rice distiller's grains protein hydrolysate as claimed in claim 1, wherein In step (2), the mass volume ratio of the purple rice distiller's grains powder to the 80% ethanol solution is 1-1.5:10 g / ml; the initial extraction is carried out by stirring for 30-35 minutes; and the number of initial extractions is 2-3 times.

4. The method for preparing the purple rice distiller's grains protein hydrolysate as claimed in claim 1, wherein In step (3), the mass volume ratio of the extract precipitate A to deionized water is 1-1.2:10 g / ml; the mass ratio of the extract precipitate A to the cellulase is 50-55:1; and the extraction is carried out at 55-65° C. with stirring for 120-130 minutes.

5. The method for preparing the purple rice distiller's grains protein hydrolysate as claimed in claim 1, wherein In step (4), the mass volume ratio of the extract precipitate B to deionized water is 1-1.2:10 g / ml; the mass ratio of the extract precipitate B to α-amylase is 45-50:1; and the extraction is carried out by stirring at 70-80° C. for 120-130 minutes.

6. The method for preparing the purple rice distiller's grains protein hydrolysate as claimed in claim 1, wherein The alkaline solution in step (5) is a sodium hydroxide solution with a pH of 12; the mass volume ratio of the extract precipitate C to the alkaline solution is 1-1.5:10g / ml.

7. The method for preparing the purple rice distiller's grains protein hydrolysate according to claim 1, wherein The acid solution in step (6) is a 1 mol / L hydrochloric acid solution; the amount of the acid solution is titrated until the pH of the supernatant combined solution is 4.7 to 4.9, which is the isoelectric point of the lees protein; the freeze-drying temperature in step (7) is -40 to -60°C, and the time is 45 to 50 hours.

8. The method for preparing the purple rice distiller's grains protein hydrolysate as claimed in claim 1, wherein In step (9), the mass ratio of trypsin to purple rice wine lees crude protein is 5000 U / g; the primary hydrolysis is to adjust the pH to 8 and perform the primary hydrolysis at 35-38° C.; the secondary hydrolysis is to place the mixture in boiling water and heat it for 15-18 minutes; and the temperature of the rotary evaporation concentration is 35-45° C.

9. The method for preparing the purple rice distiller's grains protein hydrolysate as claimed in claim 1, wherein The centrifugal separation in steps (2) to (6) and (9) is carried out at 8000-8500 r / min for 20-25 min.

10. An application of a purple rice distiller's grains protein hydrolysate prepared according to any one of claims 1 to 9, characterized in that: Application of the purple rice distiller's grains protein hydrolysate in the preparation of antioxidant drugs; application of the purple rice distiller's grains protein hydrolysate in the preparation of α-glucosidase inhibitors.

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