A method for preparing high-F value oligopeptides using rice bran as raw material

Abstract

The present invention discloses a method for preparing high-F-value oligopeptides using rice bran as a raw material: hydrolyzing rice bran protein successively with endopeptidase and exopeptidase to obtain an enzymatic hydrolysate, and obtaining high-F-value oligopeptides after purification, concentration or drying; the endopeptidase is alkaline protease or chymotrypsin with an amino acid sequence as shown in SEQ ID NO: 3; the exopeptidase is selected from flavor protease, papain, and carboxypeptidase with an amino acid sequence as shown in SEQ ID NO: 1 or 2. The present invention uses rice bran protein as a raw material. Rice bran has low cost, high yield, and high protein content. High-F-value oligopeptides are prepared by two-step enzymatic hydrolysis, which has the advantages of low production cost, high safety, and mild conditions. The present invention can improve the utilization rate of by-products of grain and oil processing, turning low-value plant protein into high-value high-F-value oligopeptide products. It reduces resource waste and environmental pollution, and has important significance for the comprehensive development and application of rice bran and the industrial application of high-F-value oligopeptides.

Description

Technical Field

[0001] This invention belongs to the field of protein preparation, specifically relating to a method for preparing high F-value oligopeptides using rice bran as a raw material. Background Technology

[0002] Currently, research on proteins in grain and oil processing byproducts is limited. However, the grain and oil processing industry has historically been a crucial pillar of the national economy, and its byproducts, including rice husks, rice bran, and wheat bran, represent a significant resource. Domestic grain and oil enterprises currently only engage in the general development and utilization of these byproducts, with relatively underdeveloped production technologies. In particular, their understanding of the composition and functions of some byproducts is incomplete, requiring further exploration and development. In fact, grain and oil byproducts contain abundant active substances with various physiological functions. Separating and purifying these substances can allow them to be used as effective functional ingredients in functional foods, which is of great significance for improving the comprehensive utilization rate of grain and oil resources and increasing product added value.

[0003] Rice is one of my country's main grain crops, and rice bran, a byproduct of rice processing, accounts for approximately 5%-8% of the rice's weight. As is well known, rice bran is rich in high-quality plant protein and is the most valuable byproduct of rice processing, comprising about 12%-18% of its weight. It is a low-cost plant protein, with an essential amino acid content of 41.7%, and an amino acid profile close to the FAO / WHO recommended pattern. The unique nutritional value of rice bran protein depends on the comprehensive and balanced amino acid composition of its hydrolysates. Rice bran protein exhibits low allergenicity, anti-cancer activity, and health benefits. Studies have shown that if rice bran is further developed and utilized as a raw material for health foods, its value can be increased approximately 60 times. Based on the characteristics of high protein content, high yield, and low cost, a two-step enzymatic hydrolysis method is considered to prepare high-F-value oligopeptides with physiological activity and nutritional properties for use in functional foods. This can improve the utilization rate of grain and oil processing byproducts and enhance the nutritional and market value of food products.

[0004] Studies have shown that proteins are absorbed as oligopeptides and amino acids, with oligopeptides being more readily absorbed than amino acids. High-F-value oligopeptides are a type of functional oligopeptide that has attracted widespread attention due to their numerous health benefits. Numerous reports on high-F-value oligopeptides with multiple functions have been published in various protein products; for example, high-F-value oligopeptides are significant for preventing hypertension and liver disease. Furthermore, research indicates that high-F-value oligopeptides also have anticoagulant, anti-fatigue, and therapeutic effects on phenylketonuria. High-F-value oligopeptides refer to small peptides composed of 2-10 amino acids, with a molar ratio of branched-chain amino acids to aromatic amino acids greater than 20.

[0005] There are various methods for preparing functional peptides both domestically and internationally, but enzymatic hydrolysis has significant advantages and is currently the most widely used method for preparing bioactive peptides. Enzymatic preparation of bioactive peptides uses animal and plant-derived proteins as substrates, hydrolyzes them with proteases under suitable conditions, and then separates and purifies them to obtain bioactive peptides. This method, as the main method for producing bioactive peptides, has the advantages of low cost, mild production conditions, and the obtained bioactive peptides have high safety, good solubility, stability, and good functional specificity. However, there are still some problems with the enzymatic hydrolysis of grain and oil processing by-products to prepare high-F-value oligopeptides. First, raw materials from different sources require experimentation and optimization based on different protease system combinations. Each enzymatic hydrolysis method has significant limitations and is only applicable to specific protein raw materials. Furthermore, the poor specificity of protease cleavage and low hydrolysis efficiency during enzymatic hydrolysis directly affect the amino acid composition of the hydrolysate, leading to low content of branched active ingredients. The separation and purification process for preparing high-F-value oligopeptides is cumbersome, costly, and results in significant loss of active ingredients. Summary of the Invention

[0006] To address the current problems in the enzymatic preparation of high-F-value oligopeptides and the issue of low utilization rate of grain and oil processing by-products, the present invention aims to provide a method for preparing high-F-value oligopeptide products using rice bran protein as raw material. This method employs specific enzymatic hydrolysis technology to prepare high-F-value oligopeptides, which features high specificity, mild production conditions, high safety and stability of the oligopeptide products, and low energy consumption.

[0007] To achieve the above objectives, the present invention employs the following technical solutions.

[0008] A method for preparing high F-value oligopeptides from rice bran includes the following steps:

[0009] (1) Extracting rice bran protein from rice bran using the alkaline method;

[0010] (2) The rice bran protein was hydrolyzed with endopeptidase to obtain enzymatic hydrolysate I;

[0011] (3) Hydrolyze the enzyme hydrolysate I with an exopeptidase to obtain enzyme hydrolysate II;

[0012] (4) The enzyme hydrolysate II is purified, concentrated or dried to obtain high F-value oligopeptides;

[0013] The endopeptidase is an alkaline protease or a chymotrypsin with an amino acid sequence as shown in SEQ ID NO: 3.

[0014] The exopeptidase is selected from flavor protease, papain, and carboxypeptidase with the amino acid sequence SEQ ID NO: 1 or 2.

[0015] In step (1), the extraction step of rice bran protein includes:

[0016] (a) Degrease rice bran to obtain defatted rice bran powder;

[0017] (b) Add defatted rice bran powder to NaOH solution at a certain material-to-liquid ratio, heat to react and extract rice bran protein, separate solid and liquid, repeat the above steps once, and combine the liquids obtained from the two reactions to obtain the extract;

[0018] (c) Adjust the pH of the extract to 5.4 and separate the solid and liquid components to obtain rice bran protein.

[0019] In step (b), the ratio of defatted rice bran powder to NaOH solution is 1:10-50, the concentration of NaOH solution is 0.1-0.5 mol / L, the reaction temperature is 20℃-60℃, and the reaction time is 1-5 h; preferably, the ratio of defatted rice bran powder to NaOH solution is 1:19.6, the concentration of NaOH solution is 0.1 mol / L, the reaction temperature is 40℃, and the reaction time is 4.7 h.

[0020] In step (2), the concentration of rice bran protein extract is 16.5 μg / mL, and the mass-to-volume ratio of endopeptidase to rice bran protein extract is 1%.

[0021] In step (3), the concentration of rice bran protein extract is 16.5 μg / mL, and the mass-volume ratio of exopeptidase to rice bran protein extract is 1%.

[0022] The first hydrolysis step aims to break down the large rice bran protein molecule into polypeptides as much as possible, ensuring that most of the cleaved peptide chains have aromatic amino acids at their C-termini. The second hydrolysis step primarily aims to remove the aromatic amino acids exposed during endopeptidase hydrolysis from the C-termini of the polypeptides. Activated carbon adsorption removes the released aromatic amino acids, resulting in high-F-value oligopeptides. This invention utilizes carboxypeptidase Y obtained from Aspergillus oryzae, which can increase the F-value of the product, especially carboxypeptidase Y obtained through site-directed mutagenesis, which shows an even higher improvement rate.

[0023] The present invention has the following advantages:

[0024] This invention uses rice bran protein as a raw material, which is low-cost, high-yield, and high in protein. High-F-value oligopeptides are prepared through a two-step enzymatic hydrolysis process, offering advantages such as low production cost, high safety, and mild operating conditions. This invention can improve the utilization rate of by-products in grain and oil processing, transforming low-value plant protein into high-value, high-F-value oligopeptide products. It reduces resource waste and environmental pollution, and is of great significance for the comprehensive development and application of rice bran and the industrial application of high-F-value oligopeptides.

[0025] Biological Preservation Information

[0026] Aspergillus oryzae ( AspergillusoryzaeM30011 was deposited on April 15, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 12371. Attached Figure Description

[0027] Figure 1 Protein extraction rate at different solid-liquid ratios;

[0028] Figure 2 The extraction rate of protein under different sodium hydroxide concentrations;

[0029] Figure 3 The extraction rate of protein at different temperatures;

[0030] Figure 4 The extraction rate of protein at different extraction times;

[0031] Figure 5 Agarose gel electrophoresis image of the CPY gene with restriction enzyme sites;

[0032] Figure 6 This is an SDS-PAGE gel electrophoresis image of the crude CPY enzyme solution.

[0033] Figure 7 SDS-PAGE gel electrophoresis image of crude enzyme solution of mutant M517R (lanes 6 and 7 are M517R). Detailed Implementation

[0034] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0035] Example 1: Extraction of Rice Bran Protein

[0036] 1. Defatted rice bran

[0037] Accurately weigh a certain amount of rice bran powder and place it in a 250mL Erlenmeyer flask. Add n-hexane at a rice bran-to-liquid ratio of 1:10. Stir and defatt the mixture at room temperature for 3 hours. Centrifuge (4000×g, 15min) to remove the n-hexane. Add n-hexane again to defatt the mixture and place it in a fume hood to air dry for 12 hours.

[0038] 2. Rice bran protein extraction

[0039] The rice bran protein extraction rate is expressed by the following formula:

[0040] Rice bran protein extraction rate (%) = protein content in extract (%) / protein content in raw material (%).

[0041] (1) Selection of different feed-liquid ratios

[0042] Weigh 5g of defatted rice bran powder and add 0.2mol / L NaOH solution at material-to-liquid ratios of 1:10, 1:20, 1:30, 1:40, and 1:50 respectively. After stirring, place the mixture in a 40℃ constant temperature shaker and extract rice bran protein for 3 hours. Centrifuge (4000×g, 15min), collect the supernatant, and repeat the above steps with NaOH solution to extract the precipitate again. Combine the supernatants obtained from the two extractions (record the volume), adjust the pH to 5.4 with 0.01mol / L HCl solution, centrifuge (4000×g, 15min), discard the supernatant, wash gently three times with distilled water, collect the precipitate, and reconstitute it with PBS solution (pH 7.5, 10mL). Measure the protein concentration using a protein concentration assay kit and calculate the protein extraction rate. The results are as follows: Figure 1 As shown, protein extraction initially increases and then decreases with increasing solid-liquid ratio. The highest protein extraction rate of 39.69% is achieved at a solid-liquid ratio of 1:20. Therefore, a solid-liquid ratio of 1:20 is set.

[0043] (2) Selection of different sodium hydroxide concentrations

[0044] Weigh 5g of defatted rice bran powder and add 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaOH solutions at a material-to-liquid ratio of 1:20, respectively. Stir well and place in a 40℃ constant temperature shaker to extract rice bran protein for 3 hours. Centrifuge (4000×g, 15min), collect the supernatant, and repeat the above steps with NaOH solution to extract the precipitate again. Combine the supernatants obtained from both extractions (record the volume), adjust the pH to 5.4 with 0.01 mol / L HCl solution, centrifuge (4000×g, 15min), discard the supernatant, gently wash three times with distilled water, collect the precipitate, and reconstitute it with PBS solution (pH 7.5, 10mL). Measure the protein concentration using a protein concentration assay kit and calculate the protein extraction rate. Results are as follows: Figure 2 As shown, the protein extraction rate decreased with increasing sodium hydroxide concentration. This may be because high concentrations of alkali can affect proteins, such as through hydrolysis and denaturation. Therefore, the final concentration of sodium hydroxide was determined to be 0.1 mol / L.

[0045] (3) Selection of different extraction temperatures

[0046] Weigh 5g of defatted rice bran powder and add 0.1mol / L NaOH solution at a ratio of 1:20. Stir well and place in a constant temperature shaker at 20, 30, 40, 50, and 60℃ for 3 hours to extract rice bran protein. Centrifuge (4000×g, 15min), collect the supernatant, add NaOH solution to the precipitate and repeat the above steps for extraction. Combine the supernatants obtained from the two extractions (record the volume), adjust the pH to 5.4 with 0.01mol / L HCl solution, centrifuge (4000×g, 15min), discard the supernatant, wash gently three times with distilled water, collect the precipitate, and reconstitute it with PBS solution (pH 7.5, 10mL). Measure the protein concentration using a protein concentration assay kit and calculate the protein extraction rate. The results are as follows: Figure 3 As shown, when the extraction temperature is between 20℃ and 60℃, it shows a trend of first increasing and then decreasing, and a peak occurs at 40℃. Therefore, 40℃ was selected as the final extraction temperature.

[0047] (4) Selection of different extraction times

[0048] Weigh 5g of defatted rice bran powder and add 0.1mol / L NaOH solution at a ratio of 1:20. After stirring, place the mixture in a 40℃ constant temperature shaker and extract rice bran protein for 1, 2, 3, 4, and 5 hours. Centrifuge (4000×g, 15min), collect the supernatant, and repeat the above steps with NaOH solution to extract the precipitate again. Combine the supernatants obtained from the two extractions (record the volume), adjust the pH to 5.4 with 0.01mol / L HCl solution, centrifuge (4000×g, 15min), discard the supernatant, gently wash three times with distilled water, collect the precipitate, and reconstitute it with PBS solution (pH 7.5, 10mL). Measure the protein concentration using a protein concentration assay kit and calculate the protein extraction rate. The results are as follows: Figure 4 As shown, when the extraction time is between 1 and 5 hours, it shows a trend of first increasing and then decreasing, and a peak appears at 4 hours. Moreover, it is significantly improved compared with other gradients. Therefore, 4 hours was selected as the final extraction time.

[0049] (5) A response surface model was established using three factors: material-to-liquid ratio, extraction temperature, and extraction time. Based on the predicted results of the response surface model, rice bran protein was extracted as follows: 5g of defatted rice bran powder was weighed and added to 0.1mol / L NaOH solution at a ratio of 1:19.6. After stirring, the mixture was placed in a 40℃ constant temperature shaker and shaken for 4.7h to extract rice bran protein. The supernatant was collected by centrifugation at 4000×g for 15min at 25℃. The precipitate was extracted again by adding NaOH solution. The supernatants obtained from the two extractions were combined (volume recorded), and the pH was adjusted to 5.4 with 0.01mol / L HCl solution. The supernatant was discarded by centrifugation at 4000×g for 15min at 25℃. The precipitate was gently washed three times with a small amount of distilled water, and then dissolved in 10mL of 0.2M pH=7.5 PBS solution to obtain rice bran protein extract. The protein concentration of the rice bran protein extract was 16.5μg / mL, and the protein extraction rate was 62.09%.

[0050] Example 2 Preparation of carboxypeptidase CPY and its mutant M517R

[0051] 1. Preparation of carboxypeptidase CPY

[0052] Following the method described in CN106190857A, strain M30011 was inoculated into liquid fermentation medium and cultured at 30℃ and 200 rpm for 48 h. Cells were then collected using sterilized filter paper, minimizing residual culture medium, and pre-cooled with liquid nitrogen before grinding. Total RNA (A260 / A280 ratio of 1.95) was extracted using a kit and cDNA was synthesized.

[0053] Primers as shown in Table 1 were designed and synthesized for PCR amplification. The reaction system was as follows: 10×Taq Buffer 10 μL, dNTP (10 mM) 2.5 μL, primer CPY-EcoRI-F 2 μL, primer CPY-XbaI-R 2 μL, Taq (5 U / μL) 1 μL, target gene 2 μL, and double-distilled water to a final volume of 50 μL. The PCR amplification conditions were: 94℃ pre-denaturation for 3 min, followed by 94℃ denaturation for 30 s, 62.5℃ annealing for 30 s, 72℃ extension for 1 min, 34 cycles, and a final extension at 72℃ for 5 min.

[0054] Table 1. Primer sequences for CPY gene cloning with restriction enzyme sites

[0055]

[0056] The product was subjected to 1% agarose gel electrophoresis, and the results are as follows: Figure 5As shown: The product size is approximately 1600 bp, as expected. The target band was recovered using a gel extraction kit and sequenced, confirming that the obtained target fragment is the CPY gene with restriction enzyme sites. The CPY gene was purified after double digestion with EcoRI and XbaI. The plasmid from the pCold TF vector was extracted, double digested with EcoRI and XbaI, and subjected to 1% agarose gel electrophoresis. The linear plasmid was then recovered, and the CPY gene and linear plasmid were ligated using Exnase II. The plasmid obtained in the above steps was then heat-shocked into… E. coli DH5α competent cells were subjected to colony PCR amplification using primers listed in Table 2, and positive clones were screened. The recombinant plasmid whose product was verified by sequencing was named pCold TF-CPY.

[0057] Table 2. Primer sequences for PCR amplification of the CPY gene

[0058]

[0059] The pCold TF-CPY plasmid was heat-shocked into BL21(DE3) competent cells at 42℃ for 60s. After plating and incubation at 37℃ for 12-16h, bacteria were picked and PCR was performed using the primers in Table 2 under the same conditions. The product was confirmed by agarose gel electrophoresis and sequencing, indicating the acquisition of positive transformants. Positive transformants were then transferred to 50mL of ampicillin-resistant liquid LB medium at a 1% inoculum to expand the culture. The OD was then increased at 37℃ and 200 rpm. 600 The culture was carried out until the culture volume reached between 0.6 and 0.8. IPTG was then added to the fermentation broth to a final concentration of 1.0 mM. The culture was continued at 20°C and 200 rpm for 16 hours with shaking to induce enzyme production. The cells were then collected by centrifugation, resuspended, and subjected to sonication on ice to disrupt the cell walls. The supernatant after centrifugation was the crude enzyme solution. The SDS-PAGE gel electrophoresis image of the crude enzyme solution is shown below. Figure 6 As shown in the figure, the theoretical molecular weight of the target protein is 59.5 kDa. A clear protein accumulation line can be seen around 63 kDa, indicating successful induction and expression of the CPY protease. Its amino acid sequence is shown in SEQ ID NO: 1. The crude enzyme solution was purified using a Ni column to obtain a pure CPY enzyme solution. For small molecule substrates (N-... cbz The activity of 470.6 U / mg of γ-Gly-Phe-Phe is 470.6 U / mg. Store at 4°C for later use.

[0060] 2. Preparation of the carboxypeptidase CPY mutant M517R

[0061] Using the 517Met of carboxypeptidase CPY as the mutation site and the pCold TF-CPY plasmid as the mutation template, two rounds of site-directed mutagenesis were performed using overlapping extension PCR, with the sequences in Table 3 as primers.

[0062] Table 3 Overlapping extension PCR primers

[0063]

[0064] The first round of PCR used the successfully constructed pCold TF-CPY plasmid as a template, and pCold TF-F / M517R-R and M517R-F / pCold TF-R primers were used to perform PCR to obtain the first and second segments containing the mutation site. The second round of PCR used the first and second segment products obtained from the first round of PCR as templates, and pCold TF-F and pCold TF-R primers were used to perform PCR to obtain the full sequence of the mutant. After each PCR step, the PCR products were detected by 1% agarose gel electrophoresis and the gel was excised and recovered.

[0065] The recombinant plasmid pCold TF-CPY-M517R was constructed according to the method in section 1, and then transformed into BL21(DE3) competent cells by heat shock. Positive transformants were induced to produce enzymes. The SDS-PAGE gel electrophoresis image of the crude enzyme solution is shown below. Figure 7 As shown in the figure, a clear protein accumulation is observed around 63 kDa. This indicates successful induction and expression of the recombinant protease. The amino acid sequence of the mutant is shown in SEQ ID NO: 2, with amino acid 517 mutated to arginine. The crude enzyme solution was purified using a Ni column to obtain the pure M517R mutant enzyme solution. This pure enzyme solution is effective for small molecule substrates (N-... cbz The activity of γ-Gly-Phe-Phe is 514.95 U / mg. Store at 4°C for later use.

[0066] Example 3 Preparation of chymotrypsin

[0067] The target gene CHY1, with a nucleotide sequence as shown in SEQ ID NO: 12, was synthesized. Restriction endonuclease recognition sites on the target gene and the multiple cloning site of the vector plasmid pCold TF were analyzed. Two restriction enzyme sites, NdeI and HindIII, were selected, and primers were designed to introduce these sites. The primer sequences are CHY1-F and CHY1-R, as shown in Table 4. The target gene was amplified by PCR. The amplified gene and the plasmid vector pCold TF were digested with restriction enzymes NdeI and HindIII at 37℃ for 3 hours. The digested CHY1 with sticky ends and the linearized pCold TF vector plasmid were detected by 1% (m / v) agarose gel electrophoresis and recovered by gel excision. The digested CHY1 and pCold TF plasmid were placed in a reaction system at a molar ratio of 7:1 and reacted overnight at 16℃ using T4 DNA ligase to obtain the recombinant plasmid pCold TF-CHY1.

[0068] To improve the specificity of CHY for aromatic amino acids, a site-directed mutagenesis was performed on proline at position 308. The pCold TF-CHY1 recombinant plasmid was amplified using overlap extension PCR, with two rounds of amplification performed using the sequences in Table 4 as primers.

[0069] Table 4. Upstream and downstream mutant primers for mutant design

[0070]

[0071] The obtained products were detected by 1% (m / v) agarose gel electrophoresis and recovered by gel excision. pCold TF and the mutant sequence were double-digested separately, and then the double-digested mutant sequence and pCold TF were ligated using T4 DNA ligase to obtain the recombinant plasmid pCold TF-CHY1-P308R. The recombinant plasmid was introduced into BL21(DE3) competent cells, and positive transformants were screened on ampicillin-resistant plates. The transformants were verified by colony PCR and then by sequencing. The verified bacterial culture was induced to produce enzyme by IPTG, and then the cells were sonicated to obtain crude enzyme solution. The bacterial cells were resuspended in 10 mL of 0.2 M, pH 7.5 phosphate-buffered saline (PBS), and the target protein was verified by SDS-PAGE electrophoresis, indicating successful induction and expression of the recombinant protease, namely the chymotrypsin mutant enzyme P308R, whose amino acid sequence is shown in SEQ ID NO: 3, with a mutation at position 308 to arginine. The crude enzyme solution was purified by Ni column to obtain the pure enzyme solution of chymotrypsin mutant enzyme P308R, which has an activity of 50.67 U / mg for small molecule substrate (Leu-Gly-Tyr-Gly-Leu) and was stored at 4℃ for later use.

[0072] Example 4: Preparation of high F-value oligopeptides using rice bran protein as a substrate

[0073] 1. Enzyme solution

[0074] Endopeptidase: nonspecific alkaline protease (AP); chymotrypsin P308R with an amino acid sequence as shown in SEQ ID NO: 3;

[0075] Exopeptidases: nonspecific papain (P) and flavor protease (FP); carboxypeptidase CPY with amino acid sequence as shown in SEQ ID NO:1 and carboxypeptidase M517R with amino acid sequence as shown in SEQ ID NO:2; carboxypeptidase Y (CE) derived from yeast.

[0076] The alkaline protease (enzyme activity: 200 U / mg), papain (enzyme activity: 800 U / mg), flavor protease (enzyme activity: 20 U / mg), and carboxypeptidase Y (enzyme activity: 50 U / mg) used were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0077] 2. Different combinations of endopeptidases and exopeptidases and their enzymatic hydrolysis conditions

[0078] (1) AP group: The pH of the rice bran protein extract obtained in Example 1 was adjusted to 9, and alkaline protease AP with an activity of 200 U / mg was added at an enzyme dosage of 1% (E / S, w / v). The mixture was hydrolyzed in a water bath at 50℃ for 4 hours and then boiled for 5 minutes to inactivate the alkaline protease hydrolysate.

[0079] (2) AP+P group: The first step of endopeptidase hydrolysis is the same as that of AP group; the second step of exopeptidase hydrolysis is to adjust the pH of the first step hydrolysate to 7.0, add papain P with an activity of 800 U / mg at 1% enzyme dosage (E / S, w / v), hydrolyze for 4 hours in a 55℃ water bath, and then boil for 5 minutes to inactivate.

[0080] (3) AP+FP group: The first step of endopeptidase hydrolysis is the same as that of AP group; the second step of exopeptidase hydrolysis is to adjust the pH of the first step hydrolysate to 7.0, add flavor protease FP with an activity of 20U / mg at 1% enzyme dosage (E / S, w / v), hydrolyze for 4h in a 50℃ water bath, and then boil for 5min to inactivate.

[0081] (4) P308R+P group: The first step of specific endopeptidase hydrolysis is to adjust the pH of the rice bran protein extract obtained in Example 1 to 7.5, add the purified P308R enzyme solution at 1% enzyme dosage (E / S, w / v), and then hydrolyze for 4 hours in a 50℃ water bath and boil for 5 minutes to inactivate the enzyme, so as to obtain P308R hydrolysate; the second step of hydrolysis conditions are the same as the second step of hydrolysis conditions of AP+P group.

[0082] (5) P308R+FP group: The first step of specific endopeptidase digestion conditions are the same as the first step of P308R+P group; the second step of digestion conditions are the same as the second step of AP+FP group.

[0083] (6) P308R+CPY group: The first step of specific endopeptidase hydrolysis conditions are the same as the first step of hydrolysis conditions of P308R+P group; the second step of hydrolysis conditions are to adjust the pH of the first step hydrolysate to 7.5, add serine carboxypeptidase (CPY) expressed in prokaryotic cells of Aspergillus oryzae M30011 at 1% enzyme dosage (E / S, w / v), and then hydrolyze for 4 hours in a water bath at 40℃ and inactivate by boiling for 5 minutes.

[0084] (7) P308R+M517R group: The first step of specific endopeptidase hydrolysis conditions are the same as those of the first step of P308R+P group; the pH of the first step hydrolysate is adjusted to 7.5, and CPY mutant enzyme M517R is added at 1% enzyme dosage (E / S, w / v). Then, it is hydrolyzed for 4 hours in a water bath at 40℃, and then the M517R enzyme hydrolysate is boiled for 5 minutes to inactivate it.

[0085] (8) P308R+CE group: The first step of specific endopeptidase hydrolysis conditions are the same as those of the first step of P308R+P group; the pH of the first step hydrolysate is adjusted to 7.5, CE is added at 1% enzyme dosage (E / S, w / v) with an activity of 50U / mg, and then hydrolyzed for 4h in a water bath at 40℃. Then the commercial enzyme hydrolysate is boiled for 5min to inactivate it.

[0086] During hydrolysis, continuous stirring was maintained to accelerate the hydrolysis rate. The pH was strictly monitored during hydrolysis and adjusted with 1 mol / L NaOH. After the reaction was complete, the reaction system was immediately placed in a boiling water bath and kept there for 5 minutes to terminate the reaction.

[0087] 3. Determination of F value in enzymatic hydrolysate

[0088] The pH of all the above enzymatic hydrolysates was adjusted to 4.5, and activated carbon was added at a solid-liquid ratio of 1:10. Adsorption was carried out at 25°C for 12 hours, followed by filtration to remove the activated carbon. Ultrafiltration was performed using a 3 kDa ultrafiltration membrane to remove high molecular weight peptide chains, yielding a high F-value oligopeptide solution. The amino acid content of each sample was determined using high-performance liquid chromatography (HPLC). Before measurement, each sample was hydrolyzed with 6M HCl at 110°C for 22 hours. The F-value of each sample was defined as the molar ratio of branched-chain amino acids to aromatic amino acids. All samples were tested in triplicate.

[0089] Table 5. F-values ​​(means±SD) of enzymatic hydrolysates of rice bran protein after treatment with different enzyme combinations.

[0090]

[0091] Note: The mean values ​​represented by the same lowercase letters in the same line are significantly different (P<0.05).

[0092] Table 5 shows the F values ​​of oligopeptide products generated from rice bran protein after treatment with different enzyme combinations. As shown in Table 5, the oligopeptide prepared by hydrolyzing rice bran protein with alkaline protease AP had an F value of 13.1, failing to reach a high F value level (F value > 20). The oligopeptide prepared by combining alkaline protease AP and exopeptidase flavor protease FP had an F value of 28.44, an increase of 117.10% compared to the single enzyme. The product prepared by combining non-specific alkaline protease AP and papain P had an F value of 28.01, an increase of 113.82% compared to the single enzyme.

[0093] The F-value of oligopeptides prepared by hydrolyzing rice bran protein using a combination of alkaline protease AP and flavor protease FP was 28.44, while the F-value of oligopeptides prepared by combining the specific CHY1 mutant enzyme P308R and flavor protease FP was 31.6. This represents an 11.11% increase in F-value compared to the non-specific combination. The F-value of oligopeptides prepared by the non-specific combination of alkaline protease AP and papain P was 28.01, while the F-value of oligopeptides prepared by combining P308R and papain P was 29.54, representing a 5.46% increase in F-value compared to the AP+P combination.

[0094] The F-values ​​of oligopeptides prepared by hydrolyzing rice bran protein using combinations of P308R with flavor protease FP, papain P, CPY, and M517R were 31.6, 29.54, 29.84, and 43.16, respectively. The P308R and M517R combination showed increases of 36.58%, 46.11%, and 44.64% compared to the first three combinations, respectively. Compared to the commercial P308R and CPY combination (36.54), the F-value was increased by 18.12%, reaching a high level. This indicates that the F-value of the oligopeptide product prepared using the specifically modified enzyme mutants P308R and M517R is significantly better than that of the non-specific alkaline proteases and flavor proteases commonly used in the current market for oligopeptide preparation. sequence list <110> Beijing Technology and Business University <120> A method for preparing high F-value oligopeptides from rice bran. <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 531 <212> PRT <213> Aspergillus oryzae <400> 1 Gly Arg Ala Ala His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly 1 5 10 15 Arg Gly Ser Met Ala Val Pro Pro Leu Gln Gln Val Leu Gly Arg Pro 20 25 30 Glu Glu Gly Met Ser Phe Ser Lys Pro Leu His Ala Phe Gln Glu Gln 35 40 45 Leu Lys Thr Leu Ser Glu Asp Ala Arg Lys Leu Trp Asp Glu Val Ala 50 55 60 Asn Tyr Phe Pro Asp Ser Met Asp His Ser Pro Ile Phe Ser Leu Pro 65 70 75 80 Lys Lys His Thr Arg Arg Pro Asp Ser His Trp Asp His Ile Val Arg 85 90 95 Gly Ser Asp Val Gln Lys Ile Trp Val Asn Asn Ala Asp Gly Glu Lys 100 105 110 Glu Arg Glu Ile Asp Gly Lys Leu Glu Ala Tyr Asp Leu Arg Val Lys 115 120 125 Lys Ala Asp Pro Ser Ala Leu Gly Ile Asp Pro Asn Val Lys Gln Tyr 130 135 140 Thr Gly Tyr Leu Asp Asp Asn Gly Asn Asp Lys His Leu Phe Tyr Trp 145 150 155 160 Phe Phe Glu Ser Arg Asn Asp Pro Lys Asn Asp Pro Val Val Leu Trp 165 170 175 Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Thr Gly Leu Phe Met Glu 180 185 190 Leu Gly Pro Ser Ser Ile Asp Glu Asn Ile Lys Pro Val Tyr Asn Asp 195 200 205 Phe Ser Trp Asn Ser Asn Ala Ser Val Ile Phe Leu Asp Gln Pro Val 210 215 220 Asn Val Gly Tyr Ser Tyr Ser Gly Ser Ala Val Ser Asp Thr Val Ala 225 230 235 240 Ala Gly Lys Asp Val Tyr Ala Leu Leu Ser Leu Phe Phe Lys Gln Phe 245 250 255 Pro Glu Tyr Ala Glu Gln Asp Phe His Ile Ala Gly Glu Ser Tyr Ala 260 265 270 Gly His Tyr Ile Pro Val Phe Ala Ser Glu Ile Leu Ala His Lys Asn 275 280 285 Arg Asn Ile Asn Leu Lys Ser Val Leu Ile Gly Asn Gly Leu Thr Asp 290 295 300 Gly Leu Thr Gln Tyr Gly Tyr Tyr Arg Pro Met Gly Cys Gly Glu Gly 305 310 315 320 Gly Tyr Lys Ala Val Leu Asp Glu Ala Thr Cys Glu Ser Met Asp Asn 325 330 335 Ala Leu Pro Arg Cys Arg Ser Met Ile Glu Ser Cys Tyr Asn Ser Glu 340 345 350 Ser Ala Trp Val Cys Val Pro Ala Ser Ile Tyr Cys Asn Asn Ala Leu 355 360 365 Ile Gly Pro Tyr Gln Arg Thr Gly Gln Asn Val Tyr Asp Val Arg Ser 370 375 380 Lys Cys Glu Asp Glu Ser Asn Leu Cys Tyr Lys Gly Met Gly Tyr Val 385 390 395 400 Ser Glu Tyr Leu Asn Lys Ala Glu Val Arg Glu Ala Val Gly Ala Glu 405 410 415 Val Gly Gly Tyr Asp Ser Cys Asn Phe Asp Ile Asn Arg Asn Phe Leu 420 425 430 Phe His Gly Asp Trp Met Lys Pro Tyr His Arg Leu Val Pro Gly Leu 435 440 445 Leu Glu Gln Ile Pro Val Leu Ile Tyr Ala Gly Asp Ala Asp Tyr Ile 450 455 460 Cys Asn Trp Leu Gly Asn Lys Ala Trp Thr Glu Ala Leu Glu Trp Pro 465 470 475 480 Gly Gln Lys Glu Tyr Ala Ser Ala Glu Leu Glu Asp Leu Lys Ile Glu 485 490 495 Gln Asn Glu His Thr Gly Lys Lys Ile Gly Gln Val Lys Ser His Gly 500 505 510 Asn Phe Thr Phe Met Arg Leu Ser Gly Glu Ser Arg Ala Ile Arg Leu 515 520 525 Leu Pro Cys 530 <210> 2 <211> 531 <212> PRT <213> Aspergillus oryzae <400> 2 Gly Arg Ala Ala His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly 1 5 10 15 Arg Gly Ser Met Ala Val Pro Pro Leu Gln Gln Val Leu Gly Arg Pro 20 25 30 Glu Glu Gly Met Ser Phe Ser Lys Pro Leu His Ala Phe Gln Glu Gln 35 40 45 Leu Lys Thr Leu Ser Glu Asp Ala Arg Lys Leu Trp Asp Glu Val Ala 50 55 60 Asn Tyr Phe Pro Asp Ser Met Asp His Ser Pro Ile Phe Ser Leu Pro 65 70 75 80 It should be noted that there seems to be an incorrect "米曲霉(Aspergillus oryzae)" translation in the original text where the Chinese part is retained in the English translation. It should be fully in English as "Aspergillus oryzae". Also, there is a misspelling in the translation of "Gly Arg Ala Ala His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly" where "28" should be "2". These have been corrected in the translation above.Lys Lys His Thr Arg Arg Pro Asp Ser His Trp Asp His Ile Val Arg 85 90 95 Gly Ser Asp Val Gln Lys Ile Trp Val Asn Asn Ala Asp Gly Glu Lys 100 105 110 Glu Arg Glu Ile Asp Gly Lys Leu Glu Ala Tyr Asp Leu Arg Val Lys 115 120 125 Lys Ala Asp Pro Ser Ala Leu Gly Ile Asp Pro Asn Val Lys Gln Tyr 130 135 140 Thr Gly Tyr Leu Asp Asp Asn Gly Asn Asp Lys His Leu Phe Tyr Trp 145 150 155 160 Phe Phe Glu Ser Arg Asn Asp Pro Lys Asn Asp Pro Val Val Leu Trp 165 170 175 Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Thr Gly Leu Phe Met Glu 180 185 190 Leu Gly Pro Ser Ser Ile Asp Glu Asn Ile Lys Pro Val Tyr Asn Asp 195 200 205 Phe Ser Trp Asn Ser Asn Ala Ser Val Ile Phe Leu Asp Gln Pro Val 210 215 220 Asn Val Gly Tyr Ser Tyr Ser Gly Ser Ala Val Ser Asp Thr Val Ala 225 230 235 240 Ala Gly Lys Asp Val Tyr Ala Leu Leu Ser Leu Phe Phe Lys Gln Phe 245 250 255 Pro Glu Tyr Ala Glu Gln Asp Phe His Ile Ala Gly Glu Ser Tyr Ala 260 265 270 Gly His Tyr Ile Pro Val Phe Ala Ser Glu Ile Leu Ala His Lys Asn 275 280 285 Arg Asn Ile Asn Leu Lys Ser Val Leu Ile Gly Asn Gly Leu Thr Asp 290 295 300 Gly Leu Thr Gln Tyr Gly Tyr Tyr Arg Pro Met Gly Cys Gly Glu Gly 305 310 315 320 Gly Tyr Lys Ala Val Leu Asp Glu Ala Thr Cys Glu Ser Met Asp Asn 325 330 335 Ala Leu Pro Arg Cys Arg Ser Met Ile Glu Ser Cys Tyr Asn Ser Glu 340 345 350 Ser Ala Trp Val Cys Val Pro Ala Ser Ile Tyr Cys Asn Asn Ala Leu 355 360 365 Ile Gly Pro Tyr Gln Arg Thr Gly Gln Asn Val Tyr Asp Val Arg Ser 370 375 380 Lys Cys Glu Asp Glu Ser Asn Leu Cys Tyr Lys Gly Met Gly Tyr Val 385 390 395 400 Ser Glu Tyr Leu Asn Lys Ala Glu Val Arg Glu Ala Val Gly Ala Glu 405 410 415 Val Gly Gly Tyr Asp Ser Cys Asn Phe Asp Ile Asn Arg Asn Phe Leu 420 425 430 Phe His Gly Asp Trp Met Lys Pro Tyr His Arg Leu Val Pro Gly Leu 435 440 445 Leu Glu Gln Ile Pro Val Leu Ile Tyr Ala Gly Asp Ala Asp Tyr Ile 450 455 460 Cys Asn Trp Leu Gly Asn Lys Ala Trp Thr Glu Ala Leu Glu Trp Pro 465 470 475 480 Gly Gln Lys Glu Tyr Ala Ser Ala Glu Leu Glu Asp Leu Lys Ile Glu 485 490 495 Gln Asn Glu His Thr Gly Lys Lys Ile Gly Gln Val Lys Ser His Gly 500 505 510 Asn Phe Thr Phe Arg Arg Leu Ser Gly Glu Ser Arg Ala Ile Arg Leu 515 520 525 Leu Pro Cys 530 <210> 3 <211> 359 <212> PRT <213> Artificial Sequence <400> 3 Met Gly Ala Pro Thr Gln Ala Ala Ser Leu His Pro Gln Ile Leu Glu 1 5 10 15 Ala Met Lys Arg Asp Leu Gly Leu Asn Ala Glu Gln Ala Thr Val Arg 20 25 30 Val Ala Arg Glu Ile His Ala Thr Asp Val Ile Glu Gln Leu Arg Ser 35 40 45 Ser Val Ala Phe Ala Gly Ala Trp Ile Asp Ala Asp Val Leu Tyr Ile 50 55 60 Gly Ile Thr Asp Gln Ala Leu Ala Asp Glu Val Thr Ala Ala Gly Ala 65 70 75 80 Thr Pro Ile Val Met Thr Asn Ser Leu Ser Lys Leu Glu Lys Ala Lys 85 90 95 Glu Asp Leu Asp Lys Ile Phe Ile Gly Arg Ala Asn Thr Leu Glu Thr 100 105 110 Ser Ser Asp Thr Ser Ser Gly Ile Ala Ser Tyr Phe Val Asp Val Ala 115 120 125 Ala Asn Lys Leu Val Ile Glu Ala Leu Ala Asp Ser His Gly His Ala 130 135 140 Glu Gln Leu Ala Ala Gln Val Gly Leu Thr Ser Glu Phe Glu Val Arg 145 150 155 160 Thr Val Glu Thr Met Pro Thr Thr Met Ala Thr Val Gln Gly Gly Asp 165 170 175 Val Tyr Tyr Ile Asn Arg Ser Ser Arg Cys Ser Ile Gly Phe Ala Val 180 185 190 Thr Thr Gly Phe Val Ser Ala Gly His Cys Gly Gly Ser Gly Ala Ser 195 200 205 Ala Thr Thr Ser Ser Gly Glu Ala Leu Gly Thr Phe Ser Gly Ser Val 210 215 220 Phe Pro Gly Ser Ala Asp Met Ala Tyr Val Arg Thr Val Ser Gly Thr 225 230 235 240 Val Leu Arg Gly Tyr Ile Asn Gly Tyr Gly Gln Gly Ser Phe Pro Val 245 250 255 Ser Gly Ser Ser Glu Ala Ala Val Gly Ala Ser Ile Cys Arg Ser Gly 260 265 270 Ser Thr Thr Gln Val His Cys Gly Thr Ile Gly Ala Lys Gly Ala Thr 275 280 285 Val Asn Tyr Pro Gln Gly Ala Val Ser Gly Leu Thr Arg Thr Ser Val 290 295 300 Cys Ala Glu Arg Gly Asp Ser Gly Gly Ser Phe Tyr Ser Gly Ser Gln 305 310 315 320 Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asp Cys Ser Arg Gly Gly 325 330 335 Thr Thr Tyr Phe Gln Pro Val Asn Arg Ile Leu Gln Thr Tyr Gly Leu 340 345 350 Thr Leu Val Thr Ala Leu Glu 355 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <400> 4 accctcgagg gatccgaatt ctcatcatca tcacagcagc gg 42 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <400> 5 agcagagatt acctatctag accagactag cctcgggctg 40 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <400> 6 ccggaattct actaccagca gcggcctg 28 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <400> 7 gctctagacc agactagcct cgggctggt 29 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <400> 8 ggaaatttca ccttccggcg tctctccggg gag 33 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <400> 9 ctccccggag agacgccgga aggtgaaatt tcc 33 <210> 10 <211> 42 <212> DNA <213> Artificial Sequence <400> 10 accctcgagg gatccgaatt ctcatcatca tcacagcagc gg 42 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <400> 11 agcagagatt acctatctag accagactag cctcgggctg 40 <210> 12 <211> 1077 <212> DNA <213> Artificial Sequence <400> 12 atgggcgcgc cgacccaggc ggcgagcctg catccgcaga ttctggaagc catgaaacgt 60 gatctgggcc tgaacgcgga acaggcgacc gttcgtgtgg cgcgtgaaat tcatgcgacc 120 gatgtgattg aacagctgcg tagcagcgtg gcgtttgcag gtgcgtggat tgatgcggat 180 gtgctgtata tcggcattac cgatcaggcg ctggccgatg aagttaccgc ggccggcgcg 240 accccgattg tgatgaccaa cagcctgagc aaactggaaa aagcgaaaga agatctggat 300 aaaatcttta ttggccgtgc gaacaccctg gaaaccagca gcgataccag cagcggcatt 360 gcgagctatt ttgtggatgt ggcggcgaac aaactggtga ttgaagccct ggccgatagc 420 catggccatg cggaacagct ggccgcgcag gttggtctga ccagcgaatt tgaagtgcgt 480 accgtggaaa ccatgccgac caccatggcg accgtgcagg gcggtgatgt gtattatatt 540 aaccgtagca gccgttgcag cattggcttt gcggtgacca ccggctttgt gagcgcgggt 600 cattgcggtg gtagcggtgc gagcgcgacc acctctagcg gtgaagcgct gggcaccttt 660 agcggcagcg tgtttccggg cagcgcggat atggcgtatg tgcgcaccgt gagcggtacg 720 gtgctgcgtg gctatattaa cggctatggc cagggcagct ttccggtgag cggcagcagc 780 gaagcggcgg tgggtgcgag catttgtcgt tctggcagca ccacccaggt gcattgcggc 840 accattggcg cgaaaggcgc gaccgtgaac tatccgcagg gcgcggttag cggtctgacc 900 cgtaccagcg tgtgtgcgga accgggcgat agcggtggca gcttttatag cggcagccag 960 gcgcagggtg ttacctctgg tggttctggc gattgtagcc gtggcggcac cacctatttt 1020 cagccggtga accgtattct gcagacctat ggcctgaccc tggtgaccgc gctcgag 1077 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <400> 13 agcgtgtgtg cggaaggcgg cgatagcggt ggcagc 36 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence' <400> 14 gctgccaccg ctatcgccgc cttccgcaca cacgct 36 <210> 15 <211> 25\] <212> DNA <213> Artificial Sequence <400> 15 taggatccat gggcgcgccg accca 25 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <400> 16 taaagcttct cgagcgcggt cacca 25

Claims

1. A method for preparing high-F value oligopeptides using rice bran as a raw material, characterized in that, It includes the following steps: (1) Extract rice bran protein from rice bran by an alkaline method; (2) Hydrolyze the rice bran protein with endopeptidase to obtain hydrolysate I; (3) Hydrolyze hydrolysate I with exopeptidase to get hydrolysate II; (4) Purify, concentrate or dry hydrolysate II to obtain high-F-value oligopeptides; The endopeptidase is chymotrypsin with the amino acid sequence shown in SEQ ID NO: 3; The exopeptidase is carboxypeptidase with the amino acid sequence shown in SEQ ID NO:

2.

2. The method according to claim 1, wherein The extraction steps of the rice bran protein in step (1) include: (a) Degrease the rice bran to obtain defatted rice bran powder; (b) Add the defatted rice bran powder to a NaOH solution according to a certain solid-liquid ratio, heat and react to extract the rice bran protein, perform solid-liquid separation, repeat the above steps once, and combine the liquids obtained twice, namely the extract; (c) Adjust the pH of the extract to 5.4 and perform solid-liquid separation to obtain rice bran protein.

3. The method according to claim 2, wherein In step (b), the solid-liquid ratio of the defatted rice bran powder to the NaOH solution is 1:10 - 50, the concentration of the NaOH solution is 0.1 - 0.5 mol / L, the reaction temperature is 20°C - 60°C, and the reaction time is 1 - 5 h.

4. The method according to any one of claims 2 or 3, characterized in that, In step (b), the solid-liquid ratio of the defatted rice bran powder to the NaOH solution is 1:19.6, the concentration of the NaOH solution is 0.1 mol / L, the reaction temperature is 40°C, and the reaction time is 4.7 h.

5. The method according to claim 1, wherein In step (2), the mass-volume ratio of the endopeptidase to the rice bran protein is 1%, and the concentration of the rice bran protein is 16.5 μg / mL.

6. The method according to claim 1, wherein In step (3), the mass-volume ratio of the exopeptidase to the rice bran protein is 1%, and the concentration of the rice bran protein is 16.5 μg / mL.

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