A method for separating and purifying high-purity soybean active peptides

Abstract

A method for separating and purifying high-purity soybean active peptides comprises the following steps: (1) extreme enzymatic hydrolysis pretreatment: taking defatted soybean powder, adding 15-25 times the amount of water, then adding 1.0-2.0% of oligosaccharides and 0.5-1.5% of hemicellulase preparation, treating at 105-125°C and 0.12-0.18MPa for 20-30min, then cooling to 45-55°C, adjusting the pH to 7.0-8.0, and obtaining a pretreated soybean protein suspension; (2) dual enzyme synergistic enzymatic hydrolysis: first adding 30% of the pretreated soybean protein suspension to the pretreated soybean protein suspension; 00-7000U / g protein neutral protease, then add 3000-7000U / g protein alkaline protease, enzymolysis at 50-60℃ for 2-4h, during which the pH of the system is maintained at 7.5-8.5 by adding sodium hydroxide solution, and after the enzymolysis is completed, the enzyme is inactivated at 90-100℃ for 10-20min to obtain an enzymolysis solution; (3) ultrafiltration-activated carbon adsorption coupling separation; (4) reverse phase chromatography separation and purification; (5) excipient and packaging: take the active peptide freeze-dried powder, add 15-25% oligofructose, mix well and then package to obtain the soybean active peptide product.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification of active peptides, and in particular to a method for separation and purification of high-purity soybean active peptides. Background Art

[0002] Soybean is a plant protein source with extremely high nutritional value, containing high-quality protein, fatty acids, minerals, vitamins and other nutrients. Soy protein can produce soybean oligopeptides after biotransformation such as enzymatic hydrolysis and fermentation. These bioactive peptides have multiple physiological functions such as anti-oxidation, blood pressure reduction, immune regulation, and anti-tumor, and have broad application prospects in the fields of health food and special medical food.

[0003] In recent years, the preparation technology of soybean oligopeptides has made great progress. Common preparation methods include enzymatic hydrolysis, fermentation and chemical synthesis. Among them, enzymatic hydrolysis is widely used because of its mild conditions, easy absorption of products and few by-products. The key to enzymatic hydrolysis lies in the selection of protease, optimization of hydrolysis conditions and separation and purification of active peptides. Soybean protein isolate was hydrolyzed by Bacillus subtilis alkaline protease at pH 8.5 and 55℃ for 4h. Liquid chromatography-mass spectrometry analysis showed that more than 20 oligopeptides were detected in the hydrolyzate, among which hydrophobic dipeptides and tripeptides were the main ones. Soybean protein was hydrolyzed by papain at pH 7.0 and 50℃ for 3h. The degree of polymerization of the obtained peptides was mainly 2-6. The product can significantly scavenge DPPH free radicals after separation and purification by gel chromatography. These studies have laid the foundation for the efficient preparation of soybean active peptides.

[0004] However, the existing preparation methods of soybean active peptides still have some technical bottlenecks that need to be solved urgently: first, the structure of soybean globulin is tight and the enzymatic hydrolysis sites are not fully exposed, resulting in a low yield of active peptides; second, a single protease is used to hydrolyze soybean protein, the sequence structure of the peptide is single, and the functional activity is poor; third, chemical / physical treatments such as acid-base pH adjustment and high-temperature enzyme inactivation can easily destroy the peptide structure and cause activity loss; fourth, separation and purification methods such as salting out and organic solvent extraction can easily cause protein denaturation, impurities are not completely removed, and the product purity is not high; fifth, there is a lack of efficient and green debittering and solubilization technology, the product has a poor taste and low solubility.

[0005] In view of the above technical problems, the present invention proposes a method for separating and purifying high-purity soybean active peptides. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a separation and purification method for high-purity soybean active peptides. The method uses a physical method (high temperature and high pressure + oligosaccharides) to pretreat soybean protein, so that the protein conformation is stretched and the enzyme cleavage site is fully exposed; neutral proteases and alkaline proteases with complementary pH and temperature adaptability are screened for dual enzyme synergistic hydrolysis, and the hydrophobic region and the hydrophilic region are efficiently cut to obtain a peptide mixture with complementary sequences and a high degree of oligomerization; and then aperture gradient ultrafiltration and reverse phase chromatography are used for separation and purification to remove more than 50% of inactive miscellaneous peptides and pigments, etc., and the yield and purity of active peptides are greatly improved; activated carbon mildly removes bitterness, and oligosaccharide embedding and solubilization significantly improves the sensory quality and solubility of the product. The new method for preparing soybean active peptides of the present invention has carried out systematic optimization and integrated innovation in key links such as protein pretreatment, enzymatic hydrolysis, separation and purification, debittering and solubilization, and has better solved many bottleneck problems existing in the prior art, which is of great significance for the development of high-quality and high-value-added soybean active peptide products. At the same time, the present invention adopts clean production processes such as mild enzymatic hydrolysis and membrane separation, which is green and environmentally friendly and easy to promote industrialization.

[0007] The object of the present invention is to provide a method for separating and purifying high-purity soybean active peptides, comprising the following steps:

[0008] (1) Extreme enzymatic pretreatment: taking defatted soybean powder, adding 15-25 times the amount of water, then adding 1.0-2.0% of oligosaccharides and 0.5-1.5% of hemicellulase preparation, treating at 105-125° C. and 0.12-0.18 MPa for 20-30 min, then cooling to 45-55° C., adjusting the pH to 7.0-8.0, and obtaining a pretreated soybean protein suspension;

[0009] (2) Dual enzyme synergistic enzymolysis: first add 3000-7000U / g of neutral protease and then add 3000-7000U / g of alkaline protease to the pretreated soybean protein suspension, and perform enzymolysis at 50-60°C for 2-4h. During the process, the pH of the system is maintained at 7.5-8.5 by adding sodium hydroxide solution dropwise. After the enzymolysis is completed, the enzyme is inactivated at 90-100°C for 10-20min to obtain an enzymolysis solution.

[0010] (3) Ultrafiltration-activated carbon adsorption coupled separation: The enzymatic hydrolyzate is passed through 10 kDa and 1 kDa ultrafiltration membranes in sequence, and the permeate <1 kDa is collected and desalted with a macroporous adsorption resin. Then, 5-10% activated carbon is added for adsorption at 40-50°C for 1-3 hours, and the activated carbon is removed through a 0.2-0.8 μm membrane to obtain an active polypeptide solution;

[0011] (4) Reverse phase chromatography separation and purification: The active peptide solution was separated by RP-HPLC using reverse phase silica gel pre-coated with PEG as the stationary phase and a water / acetonitrile gradient system containing 0.05-0.15% TFA as the mobile phase, and the active peptide components with a purity of ≥90% were collected, and vacuum freeze-dried after desalting;

[0012] (5) Excipient and packaging: Take the active peptide freeze-dried powder, add 15-25% oligofructose, mix well and package to obtain the soybean active peptide product.

[0013] Specifically, the oligosaccharides in step (1) are selected from one or more of maltopentaose and xylooligosaccharides.

[0014] Specifically, the enzyme activity of the hemicellulase preparation in step (1) is 8000-12000 U / g.

[0015] Specifically, the neutral protease in step (2) is EC 3.4.24.28.

[0016] Specifically, the alkaline protease in step (2) is EC 3.4.21.62.

[0017] Specifically, the concentration of the sodium hydroxide solution added in step (2) is 2-5 mol / L.

[0018] Specifically, the average pore size of the macroporous adsorption resin in step (3) is The specific surface area of ​​the activated carbon is ≥1000m 2 / g.

[0019] Specifically, the relative molecular mass of the pre-coated PEG in step (4) is 1000-4000, and the average pore size of the reversed phase silica gel is The particle size is 3-7μm.

[0020] Specifically, the polypeptide purity of the obtained soybean active polypeptide is ≥95%, the dipeptide content of the soybean active polypeptide is 47.1%-58.3%, the tripeptide content is 31.2%-33.7%, and the tetrapeptide content and above is 8.0%-21.4%.

[0021] Specifically, the oligofructose in step (5) is selected from one or more of inulin and sucrose oligofructose, and the average degree of polymerization of the oligofructose is 3-7.

[0022] Compared with the prior art, the separation and purification method of high-purity soybean active peptides and the preparation method thereof can achieve the following beneficial effects:

[0023] 1. Extreme pretreatment of soybean protein activates more enzyme cleavage sites, and dual enzyme synergistic hydrolysis efficiently releases oligomeric active peptides. Extreme enzymatic pretreatment refers to the treatment of soybean protein with oligosaccharides such as xylooligosaccharides and maltopentaose and hemicellulase under high temperature (105-125℃) and high pressure (0.12-0.18MPa). The mechanism is that high temperature and high pressure can stretch the tight conformation of soybean globulin and expose more hydrophobic areas; oligosaccharide molecules can be embedded in the hydrophobic cavity of protein and form hydrogen bonds to prevent the hydrophobic area from being buried again; hemicellulase hydrolyzes the glycosylation modification on the surface of protein to further expose the internal peptide bonds. The above synergistic effect significantly increases the action sites of proteases.

[0024] The present invention ingeniously selects alkaline protease and neutral protease with complementary pH tolerance for dual enzyme hydrolysis. The two enzymes are complementary in sequence recognition and substrate preference, and have a synergistic effect on the enzymatic hydrolysis of soybean protein, and can efficiently and specifically cut the hydrophobic region and the hydrophilic region to release oligomeric active peptides, especially dipeptides and tripeptides.

[0025] 2. Membrane separation - resin adsorption effectively desalts and removes impurities, reverse phase chromatography accurately separates and purifies target oligopeptides, and the gradient design of ultrafiltration membrane pore size (10kDa, 1kDa) and the optimization of operating parameters (pressure, temperature) enable the enzymatic hydrolyzate to be effectively removed by two ultrafiltrations, and oligopeptides with a molecular weight of <1kDa can be enriched. Reverse phase silica gel fillers with covalently bonded hydrophilic groups (PEG) are used to separate and purify polypeptides by hydrophobic interactions and hydrogen bonding, and then with the help of an optimized elution system, high purity (>95%) and good uniformity of dipeptides, tripeptides and other active components can be accurately separated.

[0026] 3. Combine activated carbon decolorization / deodorization with oligosaccharide embedding to retain the activity of peptides to the maximum extent, improve sensory properties and solubility, and use high specific surface area (>1000m 2 / g), suitable pore size Activated carbon can efficiently remove bitter peptides and pigments produced by enzymatic hydrolysis under mild conditions (40-50℃). At the same time, due to its moderate adsorption capacity, most oligomeric antioxidant peptides can be recovered after desorption of activated carbon, with a yield of more than 90%. Adding oligofructose to the final product, using its spatial conformation to combine with the hydrophobic region of the polypeptide molecule to form a quasi-inclusion complex, can prevent the aggregation and denaturation of hydrophobic peptides and significantly improve solubility. At the same time, the sweetness of oligosaccharides can also mask the residual bitterness and improve the taste.

[0027] 4. Screening and enrichment of peptides with high ACE inhibitory activity and structure-activity relationship analysis. ACE catalyzes the hydrolysis of substrate FAPGG to generate FAP and dipeptide Gly-Gly. The generation of Gly-Gly can be tracked by colorimetry to accurately determine ACE activity. The IC50 value (0.3-0.7 mg / mL) of the soybean oligopeptide prepared by the present invention is much lower than the IC50 value (>10 mg / mL) of natural soybean protein, indicating that the screening enriched highly active ACE inhibitory peptides. The polypeptide of the present invention contains hydrophobic dipeptides such as Val-Pro, Ile-Pro, and Leu-Pro, which can react with Zn in the active center of ACE. 2+ Chelating and binding to the surrounding hydrophobic pockets, thereby inhibiting ACE activity, which is consistent with the preparation process of the present invention.

[0028] 5. The polypeptide of the present invention is effective for 2- The scavenging ability (EC50 is 18-25μg / mL) and ORAC value (600-700μmolTE / g) of the peptides are significantly better than those of ordinary soybean oligopeptides, indicating that the screening enriched the peptide components with high antioxidant activity. Studies have shown that small molecule antioxidant peptides mainly exert antioxidant effects through hydrogen atoms (HAT), single electron transfer (SET), lipophilicity and other mechanisms. The polypeptides of the present invention are rich in amino acid residues containing imidazole and phenolic hydroxyl groups such as His, Tyr, and Trp, which can scavenge free radicals through the HAT mechanism; disulfide bond peptides can disproportionate reactive oxygen through the SET mechanism; short peptides with strong hydrophobicity can pass through the cell membrane, scavenge membrane lipid peroxidation free radicals, and block lipid peroxidation chain reactions.

[0029] 6. System optimization and multi-target synergistic enhancement of integrated innovative technologies. The present invention cleverly combines multiple innovative technologies such as extreme pretreatment, double enzymatic hydrolysis, membrane separation-chromatographic purification, activated carbon refining, and oligosaccharide encapsulation. Through the systematic optimization of multiple links such as molecular modification, enzymatic cleavage strategy, separation and purification, inclusion and solubilization, the soybean oligosaccharide active peptides are released, enriched and protected to the maximum extent, achieving synergistic enhancement in multiple health function targets such as solubility, ACE inhibitory activity and antioxidant capacity.

[0030] In summary, the present invention uses soybean protein as raw material, adopts food-grade enzyme preparations and physical separation technology, and can efficiently prepare oligomeric and highly active polypeptide products at room temperature and pressure, which is green, environmentally friendly and low-cost. These oligopeptides have the characteristics of high solubility, high ACE inhibitory activity and high antioxidant capacity, and have great application prospects in the fields of chronic disease management and aging intervention. The multi-technology integration and optimization strategy and multi-target synergistic enhancement mechanism adopted by the present invention have universal significance for the development of other protein-based active peptides. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] A method for separating and purifying high-purity soybean active peptides comprises the following steps:

[0034] (1) Extreme enzymatic pretreatment: Take 500g defatted soybean powder, add 7500mL water (solid-liquid ratio 1:15), then add 10g xylo-oligosaccharide (2%) and 5g hemicellulase preparation (1%, enzyme activity 10000U / g), seal in an autoclave, heat to 125℃, pressure 0.18MPa, maintain for 20min. Then quickly cool to 55℃, adjust pH to 8.0 with 30% NaOH solution, and obtain a pretreated soybean protein suspension.

[0035] (2) Dual enzyme synergistic enzymolysis: 3500U / g protein of neutral protease and 7000U / g protein of alkaline protease were added to the pretreated suspension, and the enzymolysis was carried out at 50°C for 4h. During the enzymolysis, 3mol / L NaOH solution was continuously added to maintain the pH at 8.0. After the enzymolysis was completed, the enzymes were inactivated at 95°C for 20min and cooled to room temperature to obtain an enzymolysis solution. The neutral protease was EC 3.4.24.28 and the alkaline protease was EC 3.4.21.62.

[0036] (3) Ultrafiltration-activated carbon adsorption: The enzymatic hydrolyzate was passed through 10kDa and 1kDa ultrafiltration membranes (operating pressure 2.5MPa, temperature 30°C) in sequence, and the permeate <1kDa was collected. The permeate was treated with a macroporous resin (pore size ) desalination, desalination rate> 98%. The desalted liquid was added with 10% activated carbon (specific surface area 1500m 2 / g) was adsorbed for 1h, and then the activated carbon was removed with a 0.45μm polyethersulfone membrane to obtain a polypeptide solution.

[0037] (4) Reverse phase chromatography purification: 100 mL of the peptide solution was purified by using a reverse phase C18 silica gel column (pore size) pre-coated with PEG-4000. 7μm) for preparative RP-HPLC separation. Mobile phase: 0.1% TFA in water / acetonitrile, gradient elution; flow rate 1.0mL / min, column temperature 30℃, detection wavelength 214nm. Active peptide components with purity > 95% were collected separately, desalted and freeze-dried at -50℃. The purity of the active peptide reached 95.3%, and the contents of dipeptide, tripeptide, tetrapeptide and above peptide segments were 52.6%, 31.2% and 16.2% respectively.

[0038] (5) Excipient and packaging: Take the active peptide freeze-dried powder, add 25% inulin (DP 3-5), mix for 5 minutes, then sieve, pack into aluminum-plastic bags and seal to obtain the soybean active peptide product.

[0039] Example 2

[0040] A method for separating and purifying high-purity soybean active peptides comprises the following steps:

[0041] (1) Extreme enzymatic pretreatment: 1000 g defatted soybean powder was added with 20 L water (solid-liquid ratio 1:20), then 15 g maltopentaose (1.5%) and 10 g hemicellulase preparation (1%, enzyme activity 8000 U / g) were added, and the mixture was treated at 115°C and 0.15 MPa for 25 min. The mixture was cooled to 50°C and the pH was adjusted to 7.5 to obtain a pretreated soybean protein suspension.

[0042] (2) Dual enzyme synergistic enzymolysis: add 5000U / g neutral protease and 5000U / g alkaline protease to the suspension, and perform enzymolysis at 55°C for 3h. During the enzymolysis, add 5mol / L NaOH liquid to control the pH to 7.5. Inactivate the enzyme at 100°C for 10min, cool and obtain enzymolysis solution. The neutral protease is EC 3.4.24.28 and the alkaline protease is EC 3.4.21.62.

[0043] (3) Ultrafiltration-activated carbon adsorption: The enzymatic hydrolysate was ultrafiltered through 10 kDa and 1 kDa membranes (2.0 MPa, 25 °C), and the permeate < 1 kDa was collected. The desalination rate of the resin is more than 95%. Add 7% activated carbon (1200m 2 / g) was adsorbed for 2h and filtered through a 0.45μm membrane to obtain a polypeptide solution.

[0044] (4) Reverse phase chromatography purification: Take 75 mL of the peptide solution and pre-coat it with PEG-2000 C18 silica gel (pore size 5μm) as the stationary phase, 0.1% TFA water / acetonitrile (gradient) as the mobile phase, flow rate 1.0mL / min, RP-HPLC separation at 30°C. The components with a purity of ≥90% were separated, desalted and freeze-dried, and the purity of the active peptide reached 97.1%, and the contents of dipeptide, tripeptide, tetrapeptide and above peptide segments were 58.3%, 33.7% and 8.0% respectively.

[0045] (5) Excipient and packaging: Take peptide powder, add 20% sucrose oligofructose (DP 5-7), mix well, pass through a 100-mesh sieve, and package by N2 displacement to obtain soybean active peptides.

[0046] Example 3

[0047] A method for separating and purifying high-purity soybean active peptides comprises the following steps:

[0048] (1) Extreme enzymatic pretreatment: Take 200g defatted soybean powder, add 5L water (solid-liquid ratio 1:25), then add 2g maltopentaose, 1g xylo-oligosaccharide (1%) and 1.5g hemicellulase preparation (0.75%, 12000U / g), and treat at 105℃ and 0.12MPa for 30min. Then reduce the temperature to 45℃ and adjust the pH to 7.0 to obtain a pretreated soybean protein suspension.

[0049] (2) Dual enzyme synergistic enzymolysis: 7000U / g neutral protease and 3000U / g alkaline protease were added to the suspension in sequence, and the enzymolysis was carried out at 60°C for 2h. 2mol / LNaOH was added dropwise to control the pH to 8.5. The enzymes were inactivated at 90°C for 15min, and the solution was cooled to obtain an enzymolysis solution. The neutral protease was EC 3.4.24.28, and the alkaline protease was EC 3.4.21.62.

[0050] (3) Ultrafiltration-activated carbon adsorption: The enzymatic hydrolyzate was ultrafiltered through 10kDa and 1kDa membranes (1.5MPa, 20°C) and the permeate was collected. Porous resin desalination, desalination rate ≥ 95%. Desalting solution at 40℃ 5% activated carbon (1000m 2 / g) was adsorbed for 3h and the carbon was removed by 0.22μm membrane to obtain polypeptide liquid.

[0051] (4) Reverse phase chromatography purification: Take 50 mL of peptide solution and coat a C18 column with PEG-1000 ( 3μm), 0.05% TFA water / acetonitrile gradient elution, 0.8mL / min, 25°C separation. The peak with purity>90% was collected, desalted and freeze-dried, and the purity of the active peptide reached 96.6%, and the contents of dipeptide, tripeptide, tetrapeptide and above peptide segments were 47.1%, 31.5% and 21.4% respectively.

[0052] (5) Forming and packaging: Add 15% sucrose oligofructose (DP 3-7) to the peptide powder, mix for 10 minutes, sieve and package in N2 modified atmosphere to obtain the product.

[0053] These results show that through the integrated process of the present invention, a soybean oligopeptide product with dipeptides and tripeptides as the main components (a total of more than 78%) can be prepared, and the batch-to-batch difference is small and the reproducibility is good. High content of dipeptides and tripeptides helps to improve the intestinal absorption rate and bioavailability of polypeptides, and is easier to exert physiological activities such as lowering blood pressure and anti-oxidation. At the same time, the content of tetrapeptides and above is strictly controlled to keep it at a low level (generally not more than 22%), avoiding the accumulation of large molecular peptides, which helps to improve the solubility and taste of polypeptides. The polypeptide purity of the product is ≥95%, the molecular weight distribution is concentrated in 0.5-1.0kDa, the solubility is >98%, and the ACE inhibitory activity IC50 value is as low as 0.5mg / mL.

[0054] Comparative Example 1

[0055] A method for separating and purifying high-purity soybean active peptides comprises the following steps: taking 500g defatted soybean powder, adding 7500mL water, stirring evenly, adjusting the pH to 8.0, and obtaining a suspension. Adding 3500U / g protein of neutral protease and 7000U / g protein of alkaline protease to the suspension, performing enzymolysis at 50°C for 4h, and dropping 3mol / LNaOH to maintain pH 8.0. Inactivating the enzyme at 95°C for 20min, cooling, and obtaining an enzymolysis solution, wherein the neutral protease is EC 3.4.24.28, and the alkaline protease is EC 3.4.21.62. The remaining steps are the same as those in Example 1.

[0056] The results showed that due to the lack of extreme pretreatment, the enzymatic hydrolysis degree and yield of soybean protein were significantly reduced. The purity of the obtained active peptide product was only 80%, and the molecular weight was also too large. This proved the importance of extreme pretreatment in improving enzymatic hydrolysis efficiency and controlling product molecular weight.

[0057] Comparative Example 2

[0058] A method for separating and purifying high-purity soybean active peptides comprises the following steps: the pretreatment step is the same as in Example 1. Only 7000 U / g alkaline protease EC 3.4.21.62 is added to the pretreatment suspension, and enzymolysis is performed at 50° C. for 4 hours, and the pH is controlled at 8.0. After the enzyme is inactivated, ultrafiltration, activated carbon adsorption, chromatography purification and excipient packaging are performed according to Example 1.

[0059] The results showed that the peptide products obtained by single protease hydrolysis were of heterogeneous composition, with a wide molecular weight distribution (500-5000Da), and a low content of hydrophobic peptides (<30%), which affected the solubility of the product. This proved the advantage of the dual enzyme hydrolysis system in preparing uniform oligomeric hydrophobic peptides.

[0060] Comparative Example 3

[0061] A method for separating and purifying high-purity soybean active peptides comprises the following steps: taking 1000g of pretreated defatted soybean powder suspension, adding 5000U / g neutral protease EC 3.4.24.28 and 5000U / g alkaline protease AEC 3.4.21.62, and performing enzymolysis at 55°C for 3h. The enzymolysis solution is directly used without ultrafiltration. The mixture was desalted by porous resin, and then the soybean polypeptide was prepared according to the steps of activated carbon adsorption, chromatography purification, excipient, etc. in Example 2.

[0062] The results show that ultrafiltration can effectively remove impurities such as macromolecular proteins and polysaccharides remaining in the enzymatic hydrolysate. If this step is missing, the purity of the obtained product will be greatly reduced (<80%), and the impurities will affect the subsequent chromatographic separation effect. This fully illustrates the necessity of ultrafiltration to remove impurities in the preparation process of the present invention.

[0063] Comparative Example 4

[0064] A method for separating and purifying high-purity soybean active peptides comprises the following steps: the preparation process is the same as in Example 2, but in the activated carbon adsorption step, a specific surface area of ​​800m 2 / g of activated carbon, the dosage is reduced to 3% (w / v).

[0065] The results show that the pore size and dosage of activated carbon directly affect its adsorption, decolorization and deodorization effects. If the pore size is too small or the dosage is too low, impurities such as bitter peptides will not be completely removed, and the taste and color of the product will be poor. This shows the importance of optimizing appropriate activated carbon parameters for improving the quality of peptide products.

[0066] Comparative Example 5

[0067] A method for separating and purifying high-purity soybean active peptides comprises the following steps: extreme pretreatment and double enzyme hydrolysis steps are the same as those in Example 3. After ultrafiltration, resin desalting, and activated carbon adsorption, the enzymatic hydrolyzate is replaced by Sephadex G-15 gel column Replace RP-HPLC for separation and purification. Load 20 mL, eluent water, flow rate 0.5 mL / min, detection 280 nm. Collect target components, freeze-dry and package according to Example 3.

[0068] The results showed that compared with reverse phase chromatography, the separation efficiency and resolution of gel filtration columns were relatively low, making it difficult to prepare high-purity (>90%) active peptide components, and the yield of the target product was also reduced. The superiority of the RP-HPLC process using a pre-coated hydrophilic bonded phase for separating oligopeptides was confirmed.

[0069] Comparative Example 6

[0070] A method for separating and purifying high-purity soybean active peptides, wherein the soybean oligopeptide product is prepared completely according to the method of Example 3, the only difference being that oligofructose is not added during the final excipient, and the active peptide freeze-dried powder is directly sieved and packaged.

[0071] The results showed that the peptide powder products without oligosaccharides were easy to clump when dissolved, the solubility dropped below 90%, and it was more likely to absorb moisture and clump during storage. This shows that oligofructose plays a positive role as an excipient and solubility promoter in improving the quality of peptide products.

[0072] Next, the effectiveness and superiority of the preparation method of the soybean active polypeptide of the present invention are comprehensively evaluated through experiments. The details are as follows:

[0073] 1. Peptide Solubility Determination

[0074] Experimental conditions: The freeze-dried polypeptide powder was accurately weighed, dissolved in deionized water, and prepared into a 1 mg / mL aqueous solution. Stirred at 25°C for 10 minutes, allowed to stand for 2 hours, and the supernatant was taken to determine the actual dissolved concentration of the polypeptide using the BCA method. Solubility (%) = actual concentration / theoretical concentration × 100%.

[0075] 2. ACE inhibitory activity determination (IC50)

[0076] Experimental method: Using colorimetry, N-hydroxysuccinyl-tripeptide (FAPGG) was used as the substrate. At 37°C and pH 8.3, the inhibition rate of peptides at different concentrations on ACE-catalyzed FAPGG hydrolysis was determined, and the IC50 value (half-maximal inhibitory concentration) was calculated. The smaller the IC50, the stronger the ACE inhibitory activity.

[0077] 3. Eliminate superoxide anion free radicals (O 2- ) Capacity determination (EC50)

[0078] Experimental method: The pyridazine-nitro blue tetrazolium (NBT) photoreduction method was used to determine the ability of peptides at different concentrations to clear O at pH 7.8 and 25°C, with lycopene as the positive control. 2- EC50 value (clearing 50% O 2- The smaller the EC50, the more efficient it is in clearing O 2- The stronger the ability.

[0079] 4. Antioxidant activity assay (ORAC method)

[0080] Experimental method: The oxygen radical absorbance capacity (ORAC) method was used to measure the ability of peptides to capture peroxyl radicals (ROO·) at 37°C and pH 7.4, with Trolox as the control, and the ORAC value (μmolTE / g, i.e., the number of μmol of Trolox per gram of sample) was calculated. The larger the ORAC value, the stronger the antioxidant capacity.

[0081] The test results are shown in Table 1-4, where:

[0082] Table 1 is the solubility test results of soybean active peptides (%)

[0083] Table 2 shows the results of the determination of the inhibitory activity (IC50, mg / mL) of soybean active peptides on ACE

[0084] Table 3 shows the results of soybean active peptides scavenging superoxide anion free radicals (O 2- ) capacity (EC50, μg / mL) determination results

[0085] Table 4 shows the results of the total antioxidant capacity (ORAC value, μmolTE / g) of soybean active peptides

[0086] Note: Solubility data are mean ± standard deviation (n = 3); ACE inhibitory activity, O scavenging 2- The data of capacity and antioxidant activity are mean ± standard error (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001, compared with Example 2 (one-way analysis of variance).

[0087] The test results of the embodiments and comparative examples are shown in Tables 1-4:

[0088] Experiment number Solubility of soybean active peptides (%) Example 1 98.5±0.3 Example 2 99.2±0.2 Example 3 98.9±0.4 Comparative Example 1 90.3±0.8 Comparative Example 2 92.6±1.2 Comparative Example 3 93.1±0.6 Comparative Example 4 94.7±0.5 Comparative Example 5 99.1±0.3 Comparative Example 6 85.2±1.5

[0089] Table 1

[0090] It can be clearly seen from Table 1 that the solubility of the soybean active polypeptides prepared in Examples 1-3 of the present invention is above 98%, among which Example 2 has the highest solubility, reaching 99.2%. In the comparative examples, except for Comparative Example 5 (99.1%), the solubility of the other comparative examples is lower than 95%, especially Comparative Example 6, since no oligosaccharide excipient is added, its solubility is only 85.2%, which is significantly different from the examples.

[0091] The main reasons for the above-mentioned solubility differences include: ① The example adopts the strategy of extreme pretreatment and dual-enzyme synergistic hydrolysis of soy protein, which is more conducive to the release of short peptides with strong hydrophilicity, thereby improving solubility; ② The example introduces oligosaccharides such as oligofructose as excipients in the last step of preparation, which can effectively encapsulate active polypeptides and prevent aggregation, thereby significantly improving solubility; ③ In contrast, Example 1 lacks pretreatment, and Example 2 uses a single enzyme hydrolysis, both of which are not conducive to the formation and release of water-soluble peptides; Example 6 does not add oligosaccharides, resulting in serious peptide aggregation and a significant decrease in solubility.

[0092] Experiment number ACE inhibitory activity IC50 (mg / mL) Example 1 0.35±0.03 Example 2 0.32±0.02 Example 3 0.38±0.05 Comparative Example 1 2.56±0.12 Comparative Example 2 1.83±0.09 Comparative Example 3 1.27±0.08 Comparative Example 4 0.92±0.06 Comparative Example 5 1.41±0.11 Comparative Example 6 2.16±0.14

[0093] Table 2

[0094] It can be clearly seen from the raw data in Table 2 that the IC50 values ​​of the soybean active peptides of Examples 1-3 against ACE are 0.35±0.03, 0.32±0.02 and 0.38±0.05 mg / mL, respectively, and the average values ​​are in the range of 0.32-0.38 mg / mL. Among them, Example 2 has the lowest IC50 (0.32±0.02 mg / mL) and has the strongest inhibitory ability against ACE.

[0095] In contrast, the IC50 values ​​of Comparative Examples 1-6 were 2.56±0.12, 1.83±0.09, 1.27±0.08, 0.92±0.06, 1.41±0.11 and 2.16±0.14 mg / mL, respectively, with an average range of 0.92-2.56 mg / mL, which was significantly higher than that of the embodiment. Among them, the IC50 of Comparative Example 1 was the highest (2.56±0.12 mg / mL), which may be related to its lack of pretreatment, which is not conducive to the release of ACE inhibitory peptides.

[0096] By comparing the original data, it can be found that the IC50 values ​​of the soybean active peptides prepared in the examples of the present invention for ACE are all lower than 0.5 mg / mL, while the comparative examples are all higher than this value, indicating that the ACE inhibitory activity of the polypeptides of the present invention is significantly better than that of the comparative examples, and the concentration required for half-inhibition of ACE is lower. This is mainly attributed to the unique preparation process of the present invention, including extreme pretreatment to expose more enzyme cleavage sites, dual enzyme synergistic hydrolysis to efficiently release oligomeric active peptides, and a variety of separation and purification methods to remove impurities and interfering components. It can be foreseen that these soybean oligopeptides with high ACE inhibitory activity have broad application prospects in the prevention and adjuvant treatment of cardiovascular diseases such as hypertension.

[0097] Experiment number EC50(μg / mL) Example 1 23.2±1.6 Example 2 18.6±1.2 Example 3 21.5±1.4 Comparative Example 1 48.3±2.5 Comparative Example 2 39.6±2.1 Comparative Example 3 35.8±1.9 Comparative Example 4 26.5±1.7 Comparative Example 5 32.4±2.0 Comparative Example 6 42.1±2.3

[0098] Table 3

[0099] From the raw data in Table 3, it can be seen that the EC50 values ​​of the soybean active polypeptides prepared in Examples 1-3 of the present invention for scavenging O2- are 23.2±1.6, 18.6±1.2 and 21.5±1.4μg / mL, respectively, and the average values ​​are in the range of 18.6-23.2μg / mL. Among them, the EC50 of Example 2 is the lowest, which is only 18.6±1.2μg / mL, indicating that it has the strongest ability to scavenge superoxide anion free radicals, and the lowest polypeptide concentration is required to achieve a 50% scavenging rate.

[0100] In contrast, the EC50 values ​​of Comparative Examples 1-6 were 48.3±2.5, 39.6±2.1, 35.8±1.9, 26.5±1.7, 32.4±2.0 and 42.1±2.3 μg / mL, respectively, with an average range of 26.5-48.3 μg / mL, which was significantly higher than that of the embodiment. Among them, the EC50 of Comparative Example 1 was the highest, at 48.3±2.5 μg / mL, which may be related to its lack of pretreatment, which is not conducive to the release of SOD-like polypeptides. The EC50 of Comparative Example 4 was also high (35.8±1.9 μg / mL), which may be due to the insufficient amount of activated carbon, resulting in the adsorption and loss of some antioxidant peptides.

[0101] By comparing the original data, it can be concluded that the soybean active peptide prepared in the embodiment of the present invention clears O 2- The EC50 values ​​of the control group were all lower than 25 μg / mL, while the control group was higher than this value. This shows that the ability of the polypeptide of the present invention to scavenge superoxide anion free radicals is significantly stronger than that of the control group, and the concentration required to achieve a 50% scavenging rate is lower. The main reason for this difference is that the present invention adopts a unique preparation process, including extreme pretreatment to fully expose the enzyme cleavage site, double enzyme hydrolysis to efficiently release oligomeric antioxidant peptides, membrane separation-adsorption chromatography to effectively remove impurities, and activated carbon refining to maximize the retention of SOD-like polypeptide activity. It can be seen that the high-purity soybean oligopeptides prepared by the present invention have good application potential in scavenging superoxide free radicals and delaying aging.

[0102] Experiment number ORAC value (μmolTE / g) Example 1 652±36 Example 2 685±29 Example 3 620±32 Comparative Example 1 436±45 Comparative Example 2 521±38 Comparative Example 3 335±28 Comparative Example 4 562±41 Comparative Example 5 480±33 Comparative Example 6 408±39

[0103] Table 4

[0104] From the raw data in Table 4, it can be seen that the ORAC values ​​of the soybean active polypeptides prepared in Examples 1-3 of the present invention are 652±36, 685±29 and 620±32 μmolTE / g, respectively, and the average value is in the range of 620-685 μmolTE / g. Among them, the ORAC value of Example 2 is the highest, reaching 685±29 μmolTE / g, indicating that its total antioxidant capacity is the strongest.

[0105] In contrast, the ORAC values ​​of Comparative Examples 1-6 were 436±45, 521±38, 335±28, 562±41, 480±33 and 408±39 μmolTE / g, respectively, with an average range of 335-562 μmolTE / g, which was significantly lower than that of the embodiment. Among them, the ORAC value of Comparative Example 3 was the lowest, only 335±28 μmolTE / g, which may be due to the fact that it was not ultrafiltered to remove impurities, resulting in residual non-antioxidant components such as impurity proteins, reducing the total antioxidant capacity. The ORAC value of Comparative Example 6 was also low (408±39 μmolTE / g), which may be because it was not embedded with oligosaccharides, causing some antioxidant peptides to be inactivated during storage.

[0106] By comparing the original data, it can be concluded that the ORAC values ​​of the soybean active polypeptides prepared in the embodiments of the present invention are all higher than 600 μmolTE / g, which is 30-100% higher than the comparative example. This shows that the total antioxidant capacity of the polypeptides of the present invention is significantly better than that of the comparative example. This is mainly due to the unique preparation process of the present invention, including extreme pretreatment to fully release antioxidant peptides, double enzymatic hydrolysis to efficiently hydrolyze soybean protein, membrane separation-chromatographic purification to effectively enrich active ingredients, activated carbon refining and oligosaccharide embedding to maximize the retention of the activity of antioxidant peptides. It can be seen that the high-purity soybean oligopeptides prepared by the present invention have great potential in multiple health fields such as scavenging free radicals and anti-oxidative aging.

[0107] Combined with the physicochemical properties and pharmacological activity data, the indicators of Example 2 are the most excellent and can be regarded as the best implementation scheme of the present invention. Its outstanding advantages are: ① Extreme pretreatment + double enzyme hydrolysis to fully release oligomeric active peptides; ② Membrane separation + resin adsorption to effectively desalt and remove impurities; ③ Fine control of activated carbon to efficiently decolorize and deodorize; ④ RP-HPLC separation + oligosaccharide embedding to obtain uniform, stable and high-purity polypeptides.

[0108] In summary, the present invention adopts a multi-technology combination optimization method to prepare soybean active polypeptides, which not only has advanced technology, but also due to the uniqueness of the polypeptide structure composition and physicochemical properties, it exhibits superiority in solubility, ACE inhibitory activity and antioxidant capacity that are difficult to achieve by traditional methods, and has broad application prospects.

[0109] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for separating and purifying high-purity soybean active peptides, characterized in that , including the following steps: (1) Extreme enzymatic pretreatment: Take defatted soybean powder, add 15-25 times the amount of water, then add 1.0-2.0% oligosaccharides and 0.5-1.5% hemicellulase preparation, treat at 105-125°C and 0.12-0.18 MPa for 20-30 min, then cool to 45-55°C, adjust the pH to 7.0-8.0, and obtain a pretreated soybean protein suspension; (2) Dual enzyme synergistic enzymolysis: first add 3000-7000U / g of neutral protease and then add 3000-7000U / g of alkaline protease to the pretreated soybean protein suspension, and perform enzymolysis at 50-60°C for 2-4h. During the process, the pH of the system is maintained at 7.5-8.5 by adding sodium hydroxide solution dropwise. After the enzymolysis is completed, the enzyme is inactivated at 90-100°C for 10-20min to obtain an enzymolysis solution; (3) Ultrafiltration-activated carbon adsorption coupled separation: The enzymatic hydrolyzate is passed through 10 kDa and 1 kDa ultrafiltration membranes in sequence, and the permeate <1 kDa is collected and desalted with a macroporous adsorption resin. Then, 5-10% activated carbon is added for adsorption at 40-50°C for 1-3 hours, and the activated carbon is removed through a 0.2-0.8 μm membrane to obtain an active polypeptide solution; (4) Reverse phase chromatography separation and purification: The active peptide solution was separated by RP-HPLC using reverse phase silica gel pre-coated with PEG as the stationary phase and a water / acetonitrile gradient system containing 0.05-0.15% TFA as the mobile phase, and the active peptide components with a purity of ≥90% were collected, desalted and vacuum freeze-dried; (5) Excipient and packaging: Take the active peptide freeze-dried powder, add 15-25% oligofructose, mix well and package to obtain the soybean active peptide product; The oligosaccharide in step (1) is selected from one or more of maltopentaose and xylooligosaccharides; The enzyme activity of the hemicellulase preparation in step (1) is 8000-12000 U / g; The neutral protease in step (2) is EC 3.4.24.28; The alkaline protease in step (2) is EC 3.4.21.62; The concentration of the sodium hydroxide solution added dropwise in step (2) is 2-5 mol / L; The average pore size of the macroporous adsorption resin in step (3) is 400-800Å, and the specific surface area of ​​the activated carbon is ≥1000m 2 / g; The relative molecular mass of the pre-coated PEG in step (4) is 1000-4000, the average pore size of the reversed-phase silica gel is 80-120Å, and the particle size is 3-7μm.

2. The method according to claim 1, characterized in that The polypeptide purity of the obtained soybean active polypeptide is ≥95%, among which the dipeptide content of the soybean active polypeptide is 47.1%-58.3%, the tripeptide content is 31.2%-33.7%, and the tetrapeptide content and above is 8.0%-21.4%.

3. The method according to claim 1, characterized in that In step (5), the oligofructose is selected from one or more of inulin and sucrose oligofructose, and the average degree of polymerization of the oligofructose is 3-7.