Preparation method of rice bran protein carnosine

Abstract

The invention provides a preparation method of rice bran protein carnosine, and belongs to the technical field of food. The preparation method comprises the following steps: uniformly mixing rice bran protein and alkaline protease in water, and performing enzymolysis to obtain hydrolysate; carrying out ultrafiltration separation on the hydrolysate to obtain a C4 component; carrying out ion exchange chromatography separation on the C4 component to obtain two rice bran protein carnopotropic peptides, namely F1 and F2 respectively; the mass concentration of the rice bran protein in water is 2-4%; the addition amount of the alkaline protease in the water is 3500-4500 U / g; the molecular weight interval of ultrafiltration separation is lt; 1 KDa. The preparation method disclosed by the invention is simple in step and efficient, has high free radical scavenging capacity, and can relieve skeletal muscle cell atrophy induced by inflammation and muscle differentiation abnormity caused by inflammation so as to resist muscle injury.

Description

Technical Field

[0001] The invention provides a method for preparing rice bran protein carnosine, and belongs to the technical field of food. Background Art

[0002] Rice bran is the main by-product of rice and a renewable resource. Its current utilization level is low and most of it is wasted.

[0003] Rice bran contains over 90% of essential elements for the human body and is rich in oil and protein that can be extracted. Existing research has explored a variety of applications for rice bran protein, including antioxidant, anti-hypertensive, and anti-muscle damage benefits.

[0004] Chinese patent CN104450839A discloses a method for preparing a rice bran protein peptide with ACE inhibitory activity. Using rice bran as the raw material, the patent first extracts rice bran protein, then uses enzymatic hydrolysis to prepare a rice bran protein hydrolysate. The rice bran ACE inhibitory peptide is then isolated and purified using ultrafiltration, Sephadex G-15, and RP-HPLC. The optimal enzymatic hydrolysis conditions are a substrate concentration of 3.9 mg / mL, an enzyme dosage of 3006 u / g, a pH of 9.1, a hydrolysis temperature of 40°C, and a hydrolysis time of 4 hours. The purified rice bran ACE inhibitory peptide is a tripeptide composed of threonine, isoleucine, and leucine, with a molecular weight of 345.238 Da and an amino acid sequence of Ile-Thr-Leu or Leu-Thr-Ile. The patent utilizes a low substrate concentration but a conventional enzyme dosage, reducing the efficiency and cost of the preparation process. However, the rice bran protein peptide obtained by this preparation method exhibits weak free radical scavenging and anti-muscle damage abilities.

[0005] Chinese patent CN106636274B discloses a method for isolating and preparing a rice bran antioxidant peptide with the sequence KHNRGDEF. This patent utilizes alkaline extraction and acid precipitation and Osborne fractionation to extract defatted rice bran to obtain rice bran glutenin. Ion exchange and gel chromatography techniques are then used to separate and purify the rice bran glutenin. The separated and purified rice bran glutenin is then hydrolyzed with a neutral protease, centrifuged, vacuum concentrated, and freeze-dried. Ion exchange chromatography, gel chromatography, preparative RP-HPLC, and analytical RP-HPLC are then used to separate and purify the antioxidant peptide. This patented preparation method, which involves purification followed by enzymatic hydrolysis and subsequent purification, is complex and cumbersome. Furthermore, the rice bran antioxidant peptides obtained by this preparation method have weak free radical scavenging and anti-muscle damage abilities.

[0006] In his master's thesis, "A Molecular Mechanism of Rice Bran Active Peptides Regulating Muscle Damage through Oxidative Stress," Wu Yixin published the protective effects of rice bran active peptides against muscle damage and their underlying mechanism. The rice bran active peptide used in this article was isolated from rice bran protein by the research team and identified using a MicroTOF-QII tandem mass spectrometer as having a molecular weight of 1002.6 and consisting of eight amino acids (Lys-His-Asn-Arg-Gly-Asp-Glu-Phe). The study demonstrated that the rice bran active peptides could promote activation of the IRS-P13K-AKT pathway, thereby reversing dexamethasone-induced muscle damage. However, the effectiveness of rice bran active peptides with a molecular weight greater than 1000 against muscle damage needs to be improved. Summary of the Invention

[0007] In response to the problems existing in the prior art, the present invention provides a method for preparing rice bran protein carnosine. The preparation method has simple steps and high efficiency, and has high free radical scavenging ability. It can also alleviate inflammation-induced skeletal muscle cell atrophy and alleviate inflammation-induced muscle differentiation abnormalities to resist muscle damage.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing rice bran protein carnosine, comprising the following steps: (1) mixing rice bran protein and alkaline protease in water and performing enzymolysis to obtain a hydrolyzate; (2) ultrafiltration separation of the hydrolyzate obtained in step (1) to obtain a C4 component; (3) subjecting the C4 component obtained in step (2) to ion exchange chromatography to obtain two rice bran protein carnosins, namely F1 and F2; The mass concentration of the rice bran protein in water is 2-4%; The amount of alkaline protease added to water is 3500-4500 U / g; The molecular weight interval of the ultrafiltration separation is <1 KDa.

[0009] Furthermore, the enzymatic hydrolysis temperature is 45-50° C. and the time is 2.5-3.5 hours.

[0010] Furthermore, the pH value of the enzymatic hydrolysis is 8.0-9.0.

[0011] Preferably, the mass concentration of the rice bran protein in water is 2-3%.

[0012] Preferably, the amount of alkaline protease added to water is 4000-4500 U / g.

[0013] Furthermore, the conditions for the ion exchange chromatography separation are: 0.02-0.05 mol / L phosphate buffer as phase A, 0.05-1 mol / L NaCl solution as phase B, and elution procedure: 0-45 min 0% phase B; 45-90 min 30% phase B; 95-140 min 70% phase B; 140-215 min 100% phase B, 215-230 min 100% phase A.

[0014] Preferably, the conditions for the ion exchange chromatography separation are: pH 6.8, 0.02 mol / L phosphate buffer as phase A, and 0.6 mol / L NaCl solution as phase B.

[0015] Furthermore, the F1 is the mobile phase for ion exchange chromatography separation of 2500-10000 mL; the F2 is the mobile phase for ion exchange chromatography separation of 17500-22500 mL.

[0016] Preferably, the F1 is the mobile phase for ion exchange chromatography separation of 3750-6250 mL; the F2 is the mobile phase for ion exchange chromatography separation of 20000-22500 mL.

[0017] In a second aspect, the present invention provides a rice bran protein carnosine, characterized in that the rice bran protein carnosine is F1 in the preparation method.

[0018] In a third aspect, the present invention provides a rice bran protein carnosine, characterized in that the rice bran protein carnosine is F2 in the preparation method.

[0019] In a fourth aspect, the present invention provides a food comprising the rice bran protein carnosine according to the third aspect and / or the fourth aspect.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses alkaline protease in conjunction with specific enzymatic hydrolysis conditions and purification methods, making the preparation method simple, efficient, and low-cost. The rice bran protein carnosine produced by this preparation method can alleviate inflammation-induced skeletal muscle cell atrophy and inflammation-induced muscle differentiation abnormalities, thereby resisting muscle damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The graph shows the experimental results of different enzyme addition amounts and enzyme types. The vertical letters ad indicate significant differences, with different letters indicating significant differences, P < 0.05.

[0022] Figure 2 The graph shows the experimental results of different enzymatic hydrolysis times. Different letters in ad indicate significant differences, P < 0.05.

[0023] Figure 3 The graph shows the experimental results of different enzymatic hydrolysis temperatures. Different letters in ac indicate significant differences, P < 0.05.

[0024] Figure 4 The graph shows the experimental results of different enzymatic hydrolysis pH values. Different letters in ac indicate significant differences, P < 0.05.

[0025] Figure 5 The graph shows the experimental results of different substrate concentrations. Different letters in ac indicate significant differences, P < 0.05.

[0026] Figure 6 This is a graph showing the free radical scavenging rates of rice bran protein carnosine prepared by the preparation methods of CN104450839 A and CN106636274 B and the preparation method of the present invention. Different letters in ab indicate significant differences, P<0.05.

[0027] Figure 7 The figure shows the protein bands related to protein degradation of G1 and G2 components.

[0028] Figure 8 The results of the effects of different ultrafiltration components on myocyte protein degradation-related proteins; *: compared with the LPS-treated group, p < 0.05; #: compared with the C3 group, p < 0.05.

[0029] Figure 9 The figure shows the protein bands associated with protein degradation of C4, F1 and F2 components.

[0030] Figure 10 The results of the effects of F1 and F2 components on myocyte protein degradation-related proteins; *: compared with the LPS-treated group, p < 0.05.

[0031] Figure 11 This is the chromatogram of the C4 component.

[0032] Figure 12 The results of different ultrafiltration fractions regulating myogenic differentiation and related protein expressions; *: compared with the LPS-treated group, p<0.05; #: compared with the C3 group, p<0.05.

[0033] Figure 13 Protein band diagram showing the regulation of myogenic differentiation and related protein expression by C4, F1 and F2 components.

[0034] Figure 14 The results of F1 and F2 components regulating myogenic differentiation and related protein expressions; *: compared with the LPS-treated group, p < 0.05. DETAILED DESCRIPTION

[0035] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific embodiment, but the following embodiment is only a preferred embodiment of the present invention, not all. Based on the embodiment in the embodiment, other embodiments obtained by those skilled in the art without making creative work all fall within the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their source is not specifically limited. The technology and scientific terms used in the embodiment have the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0036] Unless otherwise specified, the concentrations / percentages described in the present invention are all mass concentrations.

[0037] The present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are common commercial products in the technical field.

[0038] The rice bran protein used in the present invention was purchased from Xi'an Nansi Biotechnology Co., Ltd.; alkaline protease was purchased from Beijing Solebow Technology Co., Ltd.; neutral protease was purchased from Beijing Bioatoda Technology Co., Ltd., item number: NM6362C; flavor protease was purchased from McLean Technology Co., Ltd.

[0039] Example 1 A method for preparing rice bran protein carnosine (1) Rice bran protein and alkaline protease were mixed uniformly in water. The mass concentration of rice bran protein in water was 2%, and the amount of alkaline protease added in water was 4000 U / g. Enzymatic hydrolysis was carried out at pH 9 and temperature 50°C for 3 h to obtain a hydrolyzate.

[0040] (2) ultrafiltration separation of the hydrolyzate, collection of components with a molecular weight range of <1 kDa (excluding 1), vacuum concentration, and freeze drying to obtain the C4 component; (3) The C4 component was separated by ion exchange chromatography to obtain two rice bran protein carnosine peptides. F1 was the mobile phase for ion exchange chromatography separation of 3750-6250 mL, and F2 was the mobile phase for ion exchange chromatography separation of 20000-22500 mL (e.g. Figure 11 ), vacuum concentrated and freeze-dried.

[0041] Ion exchange chromatography separation conditions: pH 6.8, 0.02 mol / L phosphate buffer was used as the starting buffer (phase A), and 0.6 mol / L NaCl solution was used as phase B. The column was equilibrated with 0.02 mol / L phosphate buffer (pH 6.5). After equilibration, the sample was added to the instrument and the elution program was run in sequence: 0-45 min A; 45-90 min 30% B; 95-140 min 70% B; 140-215 min 100% B; 215-230 min A.

[0042] 1. Screening of suitable conditions for rice bran enzymatic hydrolysis 1. Screening for suitable enzyme types and addition amounts was performed. The difference from Example 1 was that the enzyme types and addition amounts were changed. The alkaline protease in Example 1 was replaced with neutral protease and flavor protease, respectively. The 4000 U / g in Example 1 was replaced with 2000 U / g, 3000 U / g, 5000 U / g, and 6000 U / g, respectively. Other conditions were the same as in Example 1.

[0043] The degree of hydrolysis (DH) and ABTS free radical scavenging rate were determined.

[0044] ① Determination of the degree of hydrolysis (DH) of rice bran protein using the pH-stat method: The number of peptide bonds cleaved during protein hydrolysis h (mmol / g) Total number of peptide bonds to a given protein h tot The ratio of (mmol / g) is called the degree of hydrolysis, and its calculation formula is as follows:

[0045] B is the volume of NaOH (1M) (mL); N b is the concentration of NaOH (mol / L); under the experimental conditions of pH 9.0 and 50°C, for rice bran protein, 1 / α is 2.23; M P is the mass of protein (g); h tot It is the number of millimoles of peptide bonds per gram of raw material protein. For rice bran protein, this value is 7.8 mmol / g.

[0046] ABTS free radical scavenging rate test: Mix 5 mL of 7 mmol / L ABTS aqueous solution and 88 μL of 140 mmol / L potassium persulfate aqueous solution and store in the dark for 12 h to form an ABTS free radical stock solution.

[0047] The ABTS radical stock solution was diluted with PBS to an initial absorbance of 0.70 ± 0.02 at 734 nm to form the ABTS+ working solution.

[0048] Then, the mixture was added to a 96-well plate according to the ratio of ABTS+ working solution: rice bran protein peptide solution (mass concentration 2%) = 4:1, and the mixture was allowed to stand at room temperature for 6 minutes. The absorbance value A was measured at 734 nm. 样 At the same time, the absorbance value A was measured at 734 nm according to ABTS+ working solution: ethanol solution = 4:1 control Then the ABTS+ clearance rate Y is:

[0049] like Figure 1 As shown in the figure, the hydrolysis degree and free radical scavenging rate of alkaline protease were higher than those of neutral protease and flavor protease; especially when the addition amount of enzyme was 4000 U / g, the hydrolysis degree and free radical scavenging rate of alkaline protease were significantly higher than those of other addition amounts, and there were significant differences (vertical letters ad, different letters indicate significant differences, P<0.05).

[0050] 2. Screening of enzymatic hydrolysis time was performed. The difference from Example 1 was that the enzymatic hydrolysis time was changed differently. The 3 h in Example 1 was replaced with 1, 2, 4, and 5 h, respectively. Other conditions were the same as Example 1. The ABTS free radical scavenging rate was measured.

[0051] like Figure 2 As shown in the figure, the ABTS free radical scavenging ability reached the maximum at 3 h, and was significantly different from that at other times (different letters in ad indicate significant differences, P < 0.05).

[0052] 3. Enzyme hydrolysis temperature screening was performed. The difference from Example 1 was that the enzymatic hydrolysis temperature was changed to 40, 45, 55 and 60°C instead of 50°C in Example 1, and the ABTS free radical scavenging rates were measured.

[0053] like Figure 3 As shown in the figure, the maximum ABTS free radical scavenging rate was achieved at 50℃, and there was a significant difference compared with other temperatures (different letters ac indicate significant differences, P < 0.05).

[0054] 4. Enzymatic hydrolysis pH screening was performed. The difference from Example 1 was that the pH values ​​of the enzymatic hydrolysis were changed differently. The pH 9 in Example 1 was replaced with pH 8.0, 8.5, 9.5, and 10, respectively. The ABTS free radical scavenging rates were measured.

[0055] like Figure 4As shown in the figure, the maximum ABTS free radical scavenging rate was achieved at pH 9, and there was a significant difference compared with other pH conditions except pH 8.0 (different letters in ab indicate significant differences, P < 0.05).

[0056] 5. Screening of enzymatic substrate concentration was performed. The difference from Example 1 was that the enzymatic substrate concentration was changed differently. The 2% in Example 1 was replaced with 1%, 1.5%, 2.5% and 3%.

[0057] like Figure 5 As shown in the figure, the maximum ABTS free radical scavenging rate was achieved when the substrate concentration was 2.0%-3.0%, and there was a significant difference compared with other substrate concentrations (different letters ac indicate significant differences, P<0.05).

[0058] 2. Preparation method screening According to the preparation methods of patents CN 104450839 A and CN 106636274 B, rice bran protein peptide was prepared, and its free radical scavenging rate was detected by the detection method of the present invention. The results are as follows Figure 6 As shown: After the rice bran protein peptide was prepared by the three methods, the ABTS free radical scavenging rate of the equal amount of rice bran protein peptide obtained by the present invention (Example 1) was the highest at 89%, while the ABTS free radical scavenging rates of the rice bran protein peptide obtained by the other two methods were approximately 72% and 77%, respectively.

[0059] The ABTS free radical scavenging rate of Example 1 was significantly different from that of the rice bran protein peptides prepared from patents CN104450839 A and CN 106636274B (different letters in "ab" indicate significant differences, P<0.05).

[0060] 3. Purification Condition Screening ① The difference from Example 1 is that the molecular weight range of ultrafiltration collection was changed and ion exchange chromatography separation was not performed. The 1 KDa in Example 1 was replaced with >5 KDa (excluding 5), 5-3 KDa, and 3-1 KDa. The component names are shown in Table 1.

[0061] After the ultrafiltration cup was assembled, its tightness was checked and it was connected to a nitrogen bottle. During the ultrafiltration separation process, the hydrolyzate of step (1) of Example 1 was passed through ultrafiltration membranes with molecular weights of 5, 3, and 1 kDa in sequence. The separated components were collected in sequence, vacuum concentrated, and freeze-dried. The obtained components are shown in Table 1.

[0062] Table 1

[0063] ② The difference from Example 1 lies in the separation conditions, which differ from those of Example 1. Gel filtration chromatography was used: Sephadex G50 powder was sequentially expanded, degassed, and loaded onto a column. Elution buffer (20 mM Tris-acetate, pH 6.0) was then equilibrated. After the enzymatic hydrolyzate was added, the elution buffer was pumped through the column at a rate of 1.4 mL / min. Starting at 14 minutes, the filtrate was collected into a centrifuge tube every 15 minutes for 30 minutes. A total of two gel chromatography fractions, designated G1 and G2, were obtained and lyophilized for later use.

[0064] IV. In vitro experiments The obtained separated products (C1-C4, G1-G2, F1-F2) were subjected to LPS-induced cellular skeletal muscle atrophy experiments and LPS-induced muscle differentiation abnormality alleviation experiments.

[0065] 1. LPS-induced cellular skeletal muscle atrophy experiment (1) LPS, as an inflammatory mediator, can induce skeletal muscle inflammatory response. Long-term inflammatory response will further increase the degree of skeletal muscle atrophy and cause irreversible damage to bones. The increase of two specific ubiquitin ligases, Atrogin-1 and MuRF-1, in skeletal muscle is the result of increased myosin catabolism and is also a hallmark event of skeletal muscle atrophy. Therefore, inhibiting these two proteins is crucial for alleviating skeletal muscle atrophy.

[0066] (2) Experimental cells L6 rat myoblasts were purchased from Nanjing Herbal Source Biotechnology Co., Ltd.; LPS was purchased from Sigma.

[0067] Growth medium: DMEM (Gibco, 11965092) containing 10% FBS (Gibco, 10091155) and 1% double-streptomycin (10,000 µg / mL streptomycin and 10,000 U / mL penicillin, Gibco, 15140122).

[0068] (3) Cell culture and passage When the cell growth density in the culture flask reaches 90%, the cells are passaged. After discarding the original growth medium, 1 mL of sterile PBS (pH 7.4) is added to rinse the cells and discarded. Then, 1 mL of 0.25% trypsin is added and the cells are transferred to a CO2 incubator and incubated for 50 s. After observing the cell morphology as small spheres under a microscope, the cell bottle is gently shaken until the cells fall off. Then 2 mL of new growth medium is added, pipetted and transferred to a 15 mL sterile centrifuge tube, placed in a low-speed centrifuge and centrifuged at 700 rpm at room temperature for 5 min, then the supernatant is poured out, 1 mL of growth medium is added and washed evenly, then transferred to 2 culture flasks and the growth medium is supplemented to 5 mL. Gently pipetted to mix into dispersed single cells, and finally placed in a CO2 incubator for culture to a density of 80% for subsequent experiments.

[0069] Sterile PBS: Add 8 g NaCl, 0.2 g KCl, 1.42 g NaH2PO4·H2O, and 0.24 g Na2HPO4·H2O to 1 L of deionized water.

[0070] (4) G1-G2 group cell treatment L6 cells were seeded in 6-well plates. When the cell density reached 80%, the cells were divided into groups. Each group was placed in 2 mL of growth medium, and LPS and G1-G2 were added to reach the following concentrations: control group (no intervention), LPS-treated group (5 μg / mL LPS treatment), LPS+G1 group (5 μg / mL LPS and 0.1 mg / mL G1 component co-treatment), and LPS+G2 group (5 μg / mL LPS and 0.1 mg / mL G2 component co-treatment). Cell samples were collected 24 hours after intervention for subsequent immunoblotting analysis.

[0071] (5) Cell treatment in groups C1-C4 L6 cells were seeded in 6-well plates. When the cell density reached 80%, the cells were grouped and each group was placed in 2 mL of growth medium. LPS and C1-C4 were added to the following concentrations: control group (no intervention), LPS-treated group (treated with 5 μg / mL LPS), LPS+C1 group (co-treated with 5 μg / mL LPS and 0.25 mg / mL C1 component), LPS+C2 group (co-treated with 5 μg / mL LPS and 0.25 mg / mL C2 component), LPS+C3 group (co-treated with 5 μg / mL LPS and 0.25 mg / mL C3 component), and LPS+C4 group (co-treated with 5 μg / mL LPS and 0.25 mg / mL C4 component). Cell samples were collected 24 hours after intervention for subsequent immunoblotting analysis.

[0072] (6) Cell treatment in C4 and F1-F2 groups L6 cells were seeded in 6-well plates. When the cell density reached 80%, the cells were grouped and each group was placed in 2 mL of growth medium. LPS, C4, and F1-F2 were added to the following concentrations: control group (no intervention), LPS-treated group (treated with 5 μg / mL LPS), LPS+C4 group (co-treated with 5 μg / mL LPS and 0.1 mg / mL C4 component), LPS+F1 group (co-treated with 5 μg / mL LPS and 0.1 mg / mL F1 component), and LPS+F2 group (co-treated with 5 μg / mL LPS and 0.1 mg / mL F2 component). Cell samples were collected 24 hours after intervention for subsequent immunoblotting analysis.

[0073] (7) F1-F2 group cell treatment L6 cells were seeded in 6-well plates. When the cell density reached 80%, the cells were divided into groups. Each group was placed in 2 mL of growth medium, and LPS and F1-F2 were added to reach the following concentrations: control group (no intervention), LPS-treated group (treated with 5 μg / mL LPS), LPS+F1 group (co-treated with 5 μg / mL LPS and 0.1 mg / mL F1 component), and LPS+F2 group (co-treated with 5 μg / mL LPS and 0.1 mg / mL F2 component). Cell samples were collected 24 hours after intervention for subsequent immunoblotting analysis.

[0074] (8) Western Blot The treated 6-well plate was placed on an ice box, washed three times with sterile PBS and discarded. After aspirating the residual PBS, 70 μL of RIPA tissue / cell rapid lysis buffer (R0010, Beijing Solebow Technology Co., Ltd.) was added to each well. The plate was gently shaken to allow the lysis buffer to fully contact the cells. The cells were gently scraped off with a clean cell scraper, and the lysis buffer with cell fragments was transferred to a 1.5 mL EP tube and lysed on ice for 30 minutes.

[0075] After lysis, the EP tubes were centrifuged at 12,000 g for 15 minutes at 4°C. Cell debris was gently removed with a pipette tip to obtain protein samples of each fraction. Protein concentration was determined using a BCA kit (PC0020, Beijing Solebao Technology Co., Ltd.). 4× protein loading buffer (P1015, Beijing Solebao Technology Co., Ltd.) was added to adjust the protein concentration to a uniform level. After vortexing, the samples were denatured by heating at 95°C in a metal bath for 5 minutes. After cooling, they were stored at -80°C until further use. Equal amounts of protein (30 μg) were loaded onto a 12% SDS-PAGE gel and electrophoresed at 80 V for 30 minutes, followed by 120 V for 1 hour. The gels were then transferred to PVDF.

[0076] After transfer, the membrane was blocked with 5% skim milk powder at room temperature (25°C) for 50 minutes. After blocking, the membrane was washed three times with 1% TBST on a shaker for 5 minutes each. The washed membrane was placed in an incubation box and incubated with the primary antibody (1:1000) at 4°C overnight. The membrane was then washed three times with TBST. HRP-conjugated secondary antibody (1:5000) was then added to the incubation box and incubated for 90 minutes. After the secondary antibody incubation was complete, the membrane was washed three times with TBST. Finally, ECL colorimetric solution (Gibco, 34577) was added and the membrane was developed on a gel imaging system to visualize protein bands.

[0077] Image J analysis software is used to quantitatively detect the developed protein bands to obtain the quantitative data of the corresponding proteins, which are then used for statistical analysis.

[0078] (9) Experimental results ①G1-G2: such as Figure 7 As shown in the figure, after LPS induction, the expression of protein degradation-related proteins MuRF-1 and Atrogin-1 in L6 cells was significantly increased, but after intervention with gel filtration chromatography fractions G1 and G2, the trend was not reversed, indicating that gel chromatography fractions G1 and G2 did not show the effect of inhibiting skeletal muscle atrophy.

[0079] ②C1-C4: Figure 8 As shown in Figures B and C, compared with the blank control group, after LPS induction, the expression of Atrogin-1 and MuRF-1 in the LPS-treated group was significantly increased (p < 0.05), indicating that skeletal muscle atrophy occurred. After co-intervention with LPS of rice bran peptide components C1-C4, the expression of Atrogin-1 and MuRF-1 in C4 and C3 was significantly decreased (p < 0.05), and the effect of C4 was better, which was significantly different from that of C3.

[0080] ③C4, F1-F2: Figure 9Compared with the blank control group, after LPS induction, MuRF-1 and Atrogin-1 in the LPS-treated group increased. After F1 and F2 intervention, MuRF-1 decreased compared with C4.

[0081] ④F1-F2: Figure 10 Compared with the blank control group, after LPS induction, the levels of MuRF-1 and Atrogin-1 in the LPS treatment group were significantly increased. After F1 and F2 intervention, the levels of MuRF-1 and Atrogin-1 were decreased, and there were significant differences compared with the LPS treatment group.

[0082] (2) Alleviate the abnormal muscle differentiation caused by LPS During the differentiation process of myoblasts, multiple adjacent mononuclear myoblasts will fuse into a mononuclear bundled myotube cell, presenting a relatively neatly arranged muscle bundle morphology. Whether a good muscle bundle morphology can be formed is one of the important indicators to judge whether the myoblasts are well differentiated.

[0083] Fractions C1-C4 and F1-F2 were used to conduct experiments to alleviate the abnormal muscle differentiation caused by LPS.

[0084] 1. Cell differentiation experiment Experimental cells, cell culture and passaging were as described above.

[0085] (1) Treatment of cells in groups C1-C4: When the cell density in the culture flask reached 90%, the cells were digested with trypsin and plated at a rate of 4 × 10 5 When the cell density in the 6-well plate reached about 90%, the cells were plated in 6-well plates and cultured. When the cell density in the 6-well plate reached about 90%, the culture medium was replaced with the corresponding differentiation induction medium (2% horse serum medium, Gibco, 16050130). The cells were divided into groups and each group was placed in 2 mL of differentiation induction medium. LPS and C1-C4 were added to the following concentrations: The blank control group (only using differentiation medium), LPS-treated group (treated with 5 μg / mL LPS), LPS+C1 group (co-treated with 5 μg / mL LPS+0.25 mg / mL C1 component), LPS+C2 group (co-treated with 5 μg / mL LPS+0.25 mg / mL C2 component), LPS+C3 group (co-treated with 5 μg / mL LPS+0.25 mg / mL C3 component), and LPS+C4 group (co-treated with 5 μg / mL LPS+0.25 mg / mL C4 component) were cultured in a 37°C incubator and differentiated for 6 days.

[0086] (2) Treatment of cells in the C4 and F1-F2 groups When the cell density in the culture flask reached 90%, the cells were digested with trypsin and plated at a rate of 4 × 10 5 When the cell density in the 6-well plate reached about 90%, the cells were plated in 2 mL of differentiation induction medium and LPS, C4, and F1-F2 were added to the following concentrations: The blank control group (using differentiation medium only), LPS-treated group (treated with 5 μg / mL LPS), LPS+C4 group (co-treated with 5 μg / mL LPS+0.1 mg / mL C4 component), LPS+F1 group (co-treated with 5 μg / mL LPS+0.1 mg / mL F1 component), and LPS+F2 group (co-treated with 5 μg / mL LPS+0.1 mg / mL F2 component) were cultured in a 37°C incubator and differentiated for 6 days.

[0087] (3) F1-F2 group cell treatment When the cell density in the culture flask reached 90%, the cells were digested with trypsin and plated at a rate of 4 × 10 5 When the cell density in the 6-well plate reached about 90%, the cells were inoculated into a 6-well plate and cultured. When the cell density in the 6-well plate reached about 90%, the culture medium was replaced with the corresponding differentiation induction medium (2% horse serum medium). The cells were divided into groups and each group was placed in 2 mL differentiation induction medium. LPS and F1-F2 were added to the following concentrations: The blank control group (using differentiation medium only), LPS-treated group (treated with 5 μg / mL LPS), LPS+F1 group (co-treated with 5 μg / mL LPS+0.1 mg / mL F1 component), and LPS+F2 group (co-treated with 5 μg / mL LPS+0.1 mg / mL F2 component) were cultured in a 37°C incubator and differentiated for 6 days.

[0088] The myotube morphology was obtained by taking pictures under an inverted microscope (40 times magnification).

[0089] The steps of Western Blot were as described above.

[0090] (3) Experimental results ①C1-C4: Observe the myotube morphology ( Figure 12Figure A shows that, compared with the blank control group, L6 myoblasts failed to form well-defined myotube morphology after LPS induction. However, myotube morphology was restored to some extent after treatment with either rice bran peptide fraction or the other components. This suggests that LPS stimulation inhibits myogenic differentiation and the normal fusion of myocytes into multinucleated myotubes. Rice bran peptide fractions C3 and C4, on the other hand, reversed the LPS-induced myogenic differentiation abnormalities and promoted the differentiation of myoblasts into myotubes. Therefore, intervention with rice bran peptide fractions C3 and C4 can alleviate the LPS-induced myogenic differentiation abnormalities, with fraction C4 having a more pronounced effect.

[0091] After intervention with C3 and C4, the expression levels of MyHC, MyoD, and MyoG were significantly increased compared with those in the LPS-treated group, while the expression of MSTN was significantly decreased (p < 0.05). Figure 12 C, D, E, and F. The levels of MyHC and MyoD proteins in group C4 were significantly higher than those in group C3 (p<0.05).

[0092] ②C4, F1-F2: Compared with the blank control group, after LPS induction, the expression levels of myogenic factors MyHC, MyoD, and MyoG in the LPS-treated group were reduced, and the expression of MSTN was increased. After intervention with rice bran peptide components F1 and F2, the expression levels of MyHC and MyoG were increased compared with C4, and the expression of MSTN was decreased compared with C4. Figure 13 .

[0093] F1 and F2 can promote muscle bundle formation and benefit the recovery of damaged myotubes, and their effects are better than those of C4 component.

[0094] ③F1-F2: Observation Figure 14 As shown in the myotube morphology diagram (A), compared with the blank control group, L6 myoblasts induced by LPS could not form good myotube morphology, but after co-intervention with rice bran peptide components F1 and F2, the myotube morphology was significantly restored, indicating that rice bran peptide components F1 and F2 can promote muscle bundle formation and are beneficial to the recovery of damaged myotubes.

[0095] At the same time Figure 14 C, D, E, F, compared with the blank control group, after LPS induction, the expression levels of myogenic factors MyHC, MyoD, and MyoG were significantly decreased, and the expression of MSTN was significantly increased (p < 0.05). After co-intervention with rice bran peptide component F1, the expression level of MyoG was significantly increased compared with the LPS-induced group, and the expression of MSTN was significantly decreased (p < 0.05). After co-intervention with rice bran peptide component F2, the expression levels of MyHC and MyoD were significantly increased, and the expression of MSTN was significantly decreased compared with the LPS-induced group (p < 0.05).

[0096] These results suggest that LPS prevents myocytes from fusing properly into multinucleated myotubes, inhibiting myogenic differentiation. Rice bran peptide fractions F1 and F2 can, to some extent, reverse this negative effect, alleviating or promoting myoblast-to-myotube differentiation. Therefore, rice bran peptide fractions F1 and F2 may help alleviate abnormal myogenic differentiation caused by inflammation.

[0097] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing rice bran protein carnosine, characterized in that: The following steps are involved: (1) mixing rice bran protein and alkaline protease in water and performing enzymolysis to obtain a hydrolyzate; (2) ultrafiltration separation of the hydrolyzate obtained in step (1) to obtain a C4 component; (3) subjecting the C4 component obtained in step (2) to ion exchange chromatography to obtain two rice bran protein carnosins, namely F1 and F2; The mass concentration of the rice bran protein in water is 2-4%; The amount of alkaline protease added to water is 3500-4500 U / g; The molecular weight interval of the ultrafiltration separation is <1 KDa.

2. The preparation method according to claim 1, characterized in that The temperature of the enzymatic hydrolysis is 45-50° C., and the time is 2.5-3.5 hours.

3. The preparation method according to claim 1, characterized in that The pH value of the enzymatic hydrolysis is 8.0-9.

0.

4. The preparation method according to claim 1, characterized in that The mass concentration of the rice bran protein in water is 2-3%.

5. The preparation method according to claim 1, characterized in that The addition amount of the alkaline protease in water is 4000-4500 U / g.

6. The preparation method according to claim 1, characterized in that The conditions for the ion exchange chromatography separation are: 0.02-0.05 mol / L phosphate buffer as phase A, 0.05-1 mol / L NaCl solution as phase B, and elution procedure: 0-45 min 0% phase B; 45-90 min 30% phase B; 95-140 min 70% phase B; 140-215 min 100% phase B, 215-230 min 100% phase A.

7. The preparation method according to claim 1, characterized in that The F1 is the mobile phase for ion exchange chromatography separation of 2500-10000 mL; the F2 is the mobile phase for ion exchange chromatography separation of 17500-22500 mL.

8. A rice bran protein carnosine, characterized in that The rice bran protein carnosine is F1 in the preparation method according to any one of claims 1 to 7.

9. A rice bran protein carnosine, characterized in that The rice bran protein carnosine is F2 in the preparation method according to any one of claims 1 to 7.

10. A food, characterized in that The method comprises the rice bran protein carnosine according to any one of claims 1 to 7 and / or the rice bran protein carnosine according to claim 8 and / or the rice bran protein carnosine according to claim 9.

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