Extraction method and application of safflower polypeptide

Abstract

The invention relates to the technical field of bioactive substance extraction, and discloses a safflower polypeptide extraction method and application, the safflower polypeptide extraction method comprises the following steps: S1, raw material pretreatment: crushing safflower seed kernels, mixing the crushed safflower seed kernels with an alkaline solution for extraction, and carrying out centrifugal separation to obtain a protein extract; s2, performing multi-stage enzymolysis: S201, adding first compound protease into the protein extracting solution, and performing first-stage enzymolysis; s202, adding second compound protease to carry out second-stage enzymolysis; s3, enzyme deactivation and purification: S301, adjusting the pH value of the enzymatic hydrolysate to an acidic condition to inactivate the enzyme; s302, separating the target polypeptide through a multi-stage ultrafiltration membrane; s303, drying to obtain a finished product of the safflower polypeptide. By controlling the adding sequence and reaction conditions of the alkaline protease, the trypsin and the flavourzyme in stages, the sequence design avoids competitive inhibition of a multi-enzyme system, so that the yield of a target peptide fragment is increased by 40% or above compared with that of a single-enzyme method.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance extraction technology, specifically a method for extracting safflower polypeptides and its application. Background Technology

[0002] Safflower seed kernel protein, as an important raw material for plant-derived bioactive peptides, has attracted widespread attention due to the functional properties (such as antioxidant and anti-inflammatory activities) of its enzymatic hydrolysis products. Currently, conventional extraction methods for safflower peptides mainly rely on single-enzyme hydrolysis or simple combined enzymatic hydrolysis processes, combined with separation techniques such as ultrafiltration and centrifugation to obtain the target product. These methods have reached a certain technical consensus regarding peptide yield, activity retention, and byproduct utilization, and have been applied in fundamental fields such as food additives and skincare product raw materials.

[0003] However, existing methods still have significant limitations in industrial applications: single-enzyme systems result in insufficient protein hydrolysis due to the single cleavage site, leading to insufficient yield of target active peptides (such as those with a molecular weight of 200-500 Da); while in compound enzymatic hydrolysis processes, the simultaneous action of multiple enzymes can easily trigger competitive inhibition, resulting in decreased hydrolysis efficiency and a dispersed molecular weight distribution of the products. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for extracting safflower polypeptides and its application, solving the problem that existing methods, due to the single enzyme system, result in insufficient protein hydrolysis and insufficient yield of target active peptides.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting safflower polypeptides, comprising the following steps:

[0006] S1. Raw material pretreatment: After crushing the safflower kernels, mix them with an alkaline solution for extraction, and then centrifuge to obtain the protein extract.

[0007] S2, Multi-stage enzymatic hydrolysis:

[0008] S201. Add the first complex protease to the protein extract to carry out the first stage of enzymatic hydrolysis.

[0009] S202, Add the second complex protease to carry out the second stage of enzymatic hydrolysis;

[0010] S3. Enzyme inactivation and purification:

[0011] S301. Adjust the pH of the enzyme hydrolysate to acidic conditions to inactivate the enzyme;

[0012] S302, Separation of target peptides via multi-stage ultrafiltration membranes;

[0013] S303, drying to obtain safflower polypeptide product.

[0014] By the technical scheme, the following steps are included: crushing safflower kernel, then extracting protein extract by alkaline solution; then performing enzymatic hydrolysis in stages, i.e., adding first complex protease for preliminary hydrolysis, then adjusting reaction conditions and adding second complex protease for further enzymatic hydrolysis; finally, inactivating the enzyme under acidic conditions, and obtaining the target polypeptide by multi-stage ultrafiltration membrane separation and chromatographic purification; wherein, the enzymatic hydrolysis in stages effectively avoids competitive inhibition between enzymes by sequentially regulating the action time of different proteases and reaction parameters, so that the yield of the target active peptide segment (molecular weight 200-500 Da) is increased by more than 40% compared with the traditional single enzyme method, and the molecular weight distribution is significantly optimized, laying a foundation for subsequent functional application.

[0015] Preferably, the first complex protease in S201 is a mixture of alkaline protease and trypsin, and the enzyme activity ratio is 1:0.5-1:3.

[0016] Preferably, the first-stage enzymatic hydrolysis is performed at pH 6.5-7.5 and temperature 45-58°C, and the enzymatic hydrolysis time is 1.5-4 hours.

[0017] Preferably, the second complex protease in S202 comprises flavor protease and papain, the enzymatic hydrolysis pH is 6.0-7.0, and the temperature is 50-60°C.

[0018] Preferably, the acid condition in S301 is pH 3.5-4.5, and the inactivation process is controlled at temperature 25-40°C.

[0019] Preferably, the multi-stage ultrafiltration membrane separation in S302 includes:

[0020] The first-stage ultrafiltration membrane has a molecular weight cut-off of 5-15 kDa;

[0021] The second-stage ultrafiltration membrane has a molecular weight cut-off of 1-5 kDa;

[0022] The third-stage ultrafiltration membrane has a molecular weight cut-off of 300-800 Da.

[0023] Preferably, the third-stage ultrafiltration liquid is further purified by anion exchange chromatography, and the eluent is a buffer solution containing 0.05-0.5 mol / L sodium chloride.

[0024] Preferably, it further includes a by-product co-production step:

[0025] S4, adding the residue after centrifugation of S1 to cellulase hydrolysis, and drying to obtain dietary fiber, the water holding capacity of the dietary fiber is ≥7 g / g.

[0026] Preferably, a safflower polypeptide composition comprises:

[0027] Safflower polypeptide, molecular weight range 200-500 Da, purity ≥85%;

[0028] Dietary fiber extracted from safflower seed kernel alkali extraction residue;

[0029] The safflower polypeptide composition can be made into tablets, capsules or oral liquids.

[0030] Preferably, the application of a safflower polypeptide, wherein the safflower polypeptide is used in the preparation of a food, a medicine or a cosmetic having an intestinal barrier repair function, wherein:

[0031] The food includes oral liquids, tablets or capsules;

[0032] The medicine comprises a pharmaceutically acceptable carrier;

[0033] The cosmetic comprises a liposome-encapsulated polypeptide composition.

[0034] The present application provides a safflower polypeptide extraction method and application. It has the following beneficial effects:

[0035] 1. The present application controls the addition sequence and reaction conditions of alkaline protease, trypsin and flavor protease in stages. The first stage of enzyme hydrolysis focuses on cracking the hydrophobic region of macromolecular proteins, and the second stage of enzyme hydrolysis focuses on releasing small molecular hydrophilic peptide segments. This sequence design avoids competitive inhibition of multi-enzyme system, and the yield of target peptide segments is increased by more than 40% compared with single enzyme method.

[0036] 2. The present application uses three-stage ultrafiltration membrane (5-15 kDa→1-5 kDa→300-800 Da) in series with ion exchange chromatography to stepwise intercept different molecular weight components and remove charged impurities. Compared with the traditional centrifugal purification process, the purity of the target polypeptide is increased from 75% to more than 90%, and the molecular weight distribution concentration (200-500 Da ratio) is increased by 25%.

[0037] 3. The present application realizes enzyme inactivation by adjusting the pH of the enzyme hydrolysis solution to 3.5-4.5, instead of traditional high-temperature enzyme inactivation, so that the retention rate of DPPH free radical scavenging activity of polypeptide is increased from 60% to more than 90%.

[0038] 4. The dietary fiber extracted from the alkali extraction residue of the present application is extracted by cellulase directional hydrolysis. The water holding capacity is increased from 5.2 g / g to 7.0-9.2 g / g, and the microstructure (SEM observation) shows that the porosity improves the water adsorption capacity. After being combined with polypeptide, a functional complementary food additive system is formed. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The present application is a flow chart of a safflower polypeptide extraction method. DETAILED DESCRIPTION

[0040] The technical solutions of the present application will be described clearly and completely below in combination with the drawings of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0041] Please refer to the drawings of the present application Figure 1 The present application provides a safflower polypeptide extraction method, comprising the following steps:

[0042] S1, raw material pretreatment: after crushing safflower kernel, mixing with alkaline solution for leaching, centrifugal separation to obtain protein extract;

[0043] S2, multi-stage enzymolysis:

[0044] S201, adding first complex protease to the protein extract for first-stage enzymolysis;

[0045] S202, adding second complex protease for second-stage enzymolysis;

[0046] S3, enzyme inactivation and purification:

[0047] S301, adjusting the pH of the enzymolysis solution to an acidic condition to inactivate the enzyme;

[0048] S302, separating the target polypeptide by multi-stage ultrafiltration membrane;

[0049] S303, drying to obtain safflower polypeptide finished product.

[0050] Specifically, step S1 (raw material pretreatment): after shelling and washing safflower kernel, it is crushed to 60-100 mesh by a high-speed pulverizer (such as FL-300 universal pulverizer). The powder is mixed with an alkaline solution (such as 0.05-0.2 mol / L NaOH solution or KOH solution) at a solid-liquid ratio of 1:10-1:25, placed in a constant temperature water bath tank (such as Julabo TW20) with stirring function, the temperature is controlled at 40-60℃, the stirring speed is 200-400 rpm, and the leaching time is 0.5-3 hours to make the protein fully dissolved. After leaching, the supernatant and residue are separated by a horizontal centrifuge (such as GQ105, speed 3000-5000 rpm, time 10-20 minutes) to obtain the protein extract. The protein content of the supernatant is detected by Lowry method to ensure that the protein concentration reaches 35-45 mg / mL.

[0051] Step S2 (multi-stage enzymolysis):

[0052] First stage enzymatic hydrolysis (S201): The protein extract is transferred to a stainless steel enzymatic reactor (e.g. BioFlo 310 type with 10 L capacity) and the pH is adjusted to 6.5-7.5 by adding 1 mol / L HCl or NaOH using an online pH meter (Mettler-Toledo InPro 3250). A first complex protease is added, which comprises a mixture of alkaline protease (e.g. Novozymes Alcalase 2.4L with an enzyme activity of 2.4 AU / g) and trypsin (e.g. Sigma T0303 with an enzyme activity of 2500 U / mg) in a ratio of 1 :0.5-1 :3 of enzyme activity, and the total amount added is 1500-6000 U / g of raw material. The reactor is started with the stirring system (rotation speed 50-100 rpm) and the temperature is maintained at 45-58°C by circulating water through the jacket. The enzymatic hydrolysis is continued for 1.5-4 hours. This stage breaks the hydrophobic regions of the large protein molecules and the degree of hydrolysis is 15-20% (measured by the pH-stat method).

[0053] Second stage enzymatic hydrolysis (S202): The pH of the enzymatic solution is adjusted to 6.0-7.0 by adding 1 mol / L citric acid and a second complex protease is added, which comprises a mixture of flavourzyme (e.g. Novozymes Flavourzyme 500MG with an enzyme activity of 500 LAPU / g) and papain (e.g. Source Leaf Bio S24050 with an enzyme activity of 8000 U / g) in a ratio of 2:1-5:1 of enzyme activity, and the total amount added is 800-1500 U / g of raw material. The temperature is increased to 50-60°C and the solution is stirred in pulses (30 seconds on / 10 seconds off) to prevent local overheating. The enzymatic hydrolysis is continued for 1-2 hours. This stage releases hydrophilic small peptides and the degree of hydrolysis is increased to 25-30% and the average molecular weight of the peptide fragments is reduced to below 500 Da (verified by MALDI-TOF mass spectrometry).

[0054] Step S3 (enzyme inactivation and purification):

[0055] Enzyme inactivation (S301): The pH of the enzymatic solution is adjusted to 3.5-4.5 by slowly adding 10% hydrochloric acid or citric acid using an online pH monitoring system, and the temperature is reduced to 25-40°C by passing circulating cooling water through a plate heat exchanger (e.g. Alfa Laval M6 type) to avoid inactivation of heat-sensitive polypeptides. After enzyme inactivation, the solution is pre-filtered through a plate-and-frame filter (pore size 5 μm) to remove insoluble impurities.

[0056] Membrane separation and purification (S302): A three-stage series ultrafiltration system (Sartocon Slice system, Germany) was used to sequentially pass through membrane modules with molecular weight cut-off of 5-15 kDa (polyether sulfone membrane, operating pressure 0.1-0.8 MPa), 1-5 kDa (ceramic membrane, 0.3-1.2 MPa) and 300-800 Da (polyamide nanofiltration membrane, 0.5-1.5 MPa). The permeate of each stage of membrane was collected after conductivity detection (<100 μS / cm) and entered into the next stage, and the final permeate with molecular weight of 200-500 Da was collected (HPLC analysis, C18 column, acetonitrile-water gradient elution).

[0057] Drying process (S303): The purified liquid was concentrated to a solid content of 15%-20% by a rotary evaporator (such as Buchi R-300), and then transferred into a vacuum freeze dryer (condensation temperature -50°C, vacuum degree 10 Pa). After 48 hours, white to light yellow polypeptide freeze-dried powder was obtained, with a yield of 18%-22% and a purity of ≥90% (area normalization method).

[0058] The first complex protease in S201 is a mixture of alkaline protease and trypsin, and the ratio of enzyme activity is 1:0.5-1:3.

[0059] Specifically, in actual implementation, 1 kg of safflower seed kernel powder is taken, 0.1 mol / L NaOH solution (material liquid ratio 1:15) is added, and protein extract is obtained after extraction. The pH is adjusted to 7.0, and alkaline protease (Alcalase 2.4L, 10 g) and trypsin (Sigma T0303, 4.4 g) are added at a ratio of total enzyme activity of 3500 U / g of raw material. The ratio of enzyme activity is 2:1. Enzymolysis is carried out at 50°C and a stirring speed of 80 rpm for 2 hours, and the degree of hydrolysis reaches 18%-22% (determined by pH-stat method). SDS-PAGE analysis of the enzyme hydrolysate shows that the macromolecular proteins (>20 kDa) are completely degraded, and the main peptide segments are distributed in 1-5 kDa.

[0060] The first stage of enzyme hydrolysis is carried out at pH 6.5-7.5 and a temperature of 45-58°C, and the enzyme hydrolysis time is 1.5-4 hours.

[0061] Specifically, in actual operation, a high-precision pH control system (such as Emerson pH transmitter) is used to maintain the pH fluctuation range of ±0.1 by automatically adding acid pump (1 mol / L HCl). Temperature control is achieved by a circulating water system (such as Julabo CF41) of a jacketed reactor, with an accuracy of ±0.5°C. For example, under the conditions of pH 7.0 and 50°C, enzyme hydrolysis for 3 hours, the yield of polypeptide is increased by 30% compared with the traditional single enzyme method (only alkaline protease), and the molecular weight distribution of the peptide segments is more concentrated (more than 80% of 200-500 Da).

[0062] The second complex protease in S202 comprises flavourzyme and papain, and the enzymolysis pH is 6.0-7.0 and the temperature is 50-60℃.

[0063] Specifically, in the implementation, Flavourzyme 500MG (Novozymes) and papain (Source Leaf Bio S24050) are mixed in a mass ratio of 3:1, and the total addition amount is 1200 U / g of raw materials. For example, under the condition of pH 6.5 and 55℃, the enzymolysis is carried out for 1.5 hours, the proportion of the generated peptide segment with a molecular weight less than 500 Da reaches 85% (HPLC analysis), and the flavourzyme can effectively remove the bitter peptides and improve the palatability of the product (sensory evaluation score≥8.5 points, full score 10 points).

[0064] The acid condition in S301 is pH 3.5-4.5, and the temperature control during the enzyme inactivation process is 25-40℃.

[0065] Specifically, in the specific operation, a low-temperature enzyme inactivation method is used: the enzymolysis solution is pre-cooled to 30℃ through a plate heat exchanger (Alfa Laval M6), and then 10% citric acid is slowly added to adjust the pH to 4.0, and stirring is maintained for 30 minutes. The comparative experiment shows that the retention rate of the antioxidant activity (DPPH free radical scavenging rate) of the polypeptide under this method is ≥90%, while the retention rate of the traditional boiling method (100℃ enzyme inactivation for 15 minutes) is only 60%.

[0066] The multi-stage ultrafiltration membrane separation in S302 comprises:

[0067] The first-stage ultrafiltration membrane has a molecular weight cut-off of 5-15 kDa;

[0068] The second-stage ultrafiltration membrane has a molecular weight cut-off of 1-5 kDa;

[0069] The third-stage ultrafiltration membrane has a molecular weight cut-off of 300-800 Da.

[0070] Specifically, in the implementation, the first-stage membrane is made of polyether sulfone (Sartorius Vivaflow 200, molecular weight cut-off 10 kDa), and the operating pressure is 0.3 MPa; the second-stage membrane is a ceramic membrane (Pall Membralox, molecular weight cut-off 3 kDa), and the pressure is 0.6 MPa; and the third-stage membrane is an organic nanofiltration membrane (Dow FilmTec NF270, molecular weight cut-off 500 Da), and the pressure is 1.0 MPa. The permeate of each stage of membrane is detected by conductivity (<100 μS / cm) and then enters the next stage, and finally the component with a molecular weight of 200-500 Da is collected (HPLC verification).

[0071] The third-stage ultrafiltration liquid is further purified by anion exchange chromatography, and the eluent is a buffer solution containing 0.05-0.5 mol / L sodium chloride.

[0072] Specifically, DEAE-Sepharose Fast Flow column (Cytiva, column bed volume 50 L) was used, and the equilibrium buffer was 20 mM Tris-HCl (pH 7.4), and the eluent was a linear gradient containing 0.05-0.5 mol / L NaCl. The elution peak with a conductivity of 12-15 mS / cm (ultraviolet detection wavelength 280 nm) was collected, desalted by a desalting column (HiPrep 26 / 10 Desalting, Cytiva), and then freeze-dried. The final purity of the polypeptide was ≥95% (HPLC area normalization method).

[0073] Also included is a byproduct co-production step:

[0074] S4, the residue after centrifugation of S1 is added to cellulase hydrolysis, and dietary fiber is obtained by drying, and the water holding capacity of the dietary fiber is ≥7 g / g.

[0075] Specifically, the specific method is as follows: the residue is mixed with deionized water at a ratio of 1:5, the pH is adjusted to 4.5-5.5, Genencor Cellic C Tec2 cellulase (50 U / g residue) is added, and enzyme hydrolysis is carried out at 50-65°C for 2-4 hours. After the reaction is completed, the supernatant is obtained by centrifugation, and dietary fiber powder is obtained by spray drying (inlet air temperature 180°C, outlet air temperature 80°C). The water holding capacity of the dietary fiber is 8.5 g / g, the swelling capacity is 4.3 mL / g (determined according to GB 5009.88-2014), and the dietary fiber can be applied to high-fiber biscuits or meal replacement powder. When the addition amount is 10%-30%, the product texture score is optimal.

[0076] A safflower polypeptide composition, comprising:

[0077] Safflower polypeptide, molecular weight range 200-500 Da, purity ≥85%;

[0078] Dietary fiber extracted from safflower kernel alkali extraction residue;

[0079] The safflower polypeptide composition can be made into tablets, capsules or oral liquids.

[0080] Specifically, the mass ratio of polypeptide to dietary fiber in the composition is 1:0.5-1:2, and a disintegrating agent (such as microcrystalline cellulose, FMCPH-101 type) and a lubricant (magnesium stearate, MgSt-R type) can be added to make tablets. For example, the formula is polypeptide 30%, dietary fiber 60%, microcrystalline cellulose 8%, and magnesium stearate 2%, which are uniformly mixed by a V-type mixer (such as Glatt VMA-10), and then compressed into tablets with a rotary tablet press (Fette P3030 type) to obtain tablets with a weight of 500 mg per tablet. The hardness of the tablets is 50-80 N (Erweka TBH-525 test), and the disintegration time is ≤15 minutes (determination method according to Chinese Pharmacopoeia 2020 edition).

[0081] Use of a crocin polypeptide, and use of the crocin polypeptide in preparation of a food, a medicine or a cosmetic having an intestinal barrier repair function, wherein:

[0082] The food comprises an oral liquid, a tablet or a capsule;

[0083] The medicine comprises a pharmaceutically acceptable carrier;

[0084] The cosmetic comprises a liposome-encapsulated polypeptide composition.

[0085] Specifically, the food uses a dosage form and a dosage:

[0086] The crocin polypeptide is mixed with malt dextrin and microcrystalline cellulose to prepare a tablet containing 50 mg of polypeptide per tablet, or is mixed with a fruit juice base to prepare an oral liquid (containing 200 mg of polypeptide per 100 mL). The daily intake is 50-300 mg, and a reduction of 30%-40% in serum LPS level can be achieved after 4 weeks (animal experiment data).

[0087] Functional correlation: The polypeptide with a molecular weight of 200-500 Da can pass through the intestinal mucus layer, repair the intestinal epithelial tight junction by up-regulating the expression of ZO-1 protein (verified by Western blot), and improve the abnormal intestinal permeability induced by a high-fat diet (a 35% reduction in lactulose / mannitol excretion ratio).

[0088] Composition of the medicine: The crocin polypeptide is mixed with pharmaceutical excipients (such as hydroxypropyl methylcellulose and magnesium stearate) to prepare enteric-coated capsules. Each capsule contains 100 mg of polypeptide, and is taken orally twice a day.

[0089] Indications and mechanisms: It is used for treating intestinal barrier dysfunction (such as irritable bowel syndrome and inflammatory bowel disease), and the mechanism is:

[0090] Increasing the Caco-2 cell transmembrane electrical resistance (TEER) to 85% of the normal level;

[0091] Reducing the IL-6 secretion amount induced by lipopolysaccharide (LPS) by 50% (ELISA detection).

[0092] Dosage form and transdermal delivery: The crocin polypeptide is prepared into liposomes with lecithin (mass ratio 1:3), the particle size is 80-150 nm (determined by dynamic light scattering), and the encapsulation efficiency is ≥85%. The liposomes are added to an oil-in-water cream base, and the final concentration is 1%-5%.

[0093] Functional verification: Transdermal absorption: Franz diffusion cell test shows that the cumulative penetration amount of polypeptide through the skin is 35% after 8 hours (quantified by HPLC);

[0094] Skin repair: In a UVB-damaged mouse model, the cream containing 1% polypeptide liposomes can restore the epidermal thickness to 85% of the normal group (histological section analysis).

[0095] Antioxidant: DPPH radical scavenging rate is 2 times higher than that of the blank matrix.

[0096] The following will be described in conjunction with specific examples:

[0097] Example 1, when the minimum parameters are implemented, 1 kg of safflower seed kernels is crushed to 60 mesh, and 0.05 mol / L sodium hydroxide solution (material liquid ratio 1:10) is extracted at 40°C for 0.5 hours to obtain an extract with a protein concentration of 32 mg / mL. Then two-stage enzymolysis is carried out: in the first stage, alkaline protease (1500 U / g) and trypsin (750 U / g) are added at pH 6.5 and 45°C, and the enzymolysis is carried out for 1.5 hours, and the degree of hydrolysis is 14.8%; in the second stage, the pH is adjusted to 6.0, and flavor protease (800 U / g) and papain (400 U / g) are added, and the enzymolysis is carried out at 50°C for 1 hour, and the degree of hydrolysis is increased to 22.3%. When the enzyme is inactivated, the pH is adjusted to 3.5, and maintained at 25°C for 30 minutes, and then purified by three-stage ultrafiltration (15 kDa→5 kDa→800 Da membrane), and the final polypeptide yield is 12.5%, the purity is 83%, and the antioxidant activity IC50 value is 1.2 mg / mL. At the same time, the residue after alkali extraction is treated with cellulase to obtain dietary fiber, and the water holding property is 7.0 g / g, and the yield is 15.2%.

[0098] Example 2, under medium parameter conditions, the raw material is crushed to 80 mesh, and 0.1 mol / L sodium hydroxide (material liquid ratio 1:15) is extracted at 50°C for 1.5 hours, and the protein concentration is 40 mg / mL. In the first stage of enzymolysis, alkaline protease (2500 U / g) and trypsin (1250 U / g) are used at pH 7.0 and 50°C for 2.5 hours, and the degree of hydrolysis is 19.6%; in the second stage, flavor protease (1000 U / g) and papain (500 U / g) are used at pH 6.5 and 55°C for 1.5 hours, and the degree of hydrolysis is increased to 28.7%. After the enzyme is inactivated, it is separated by 10 kDa→3 kDa→500 Da ultrafiltration membrane, and the polypeptide yield is 18.7%, the purity is 91%, and the IC50 value is 0.76 mg / mL. The water holding property of the dietary fiber is increased to 8.5 g / g, and the yield is 18.5%.

[0099] Example 3, when the highest parameters are used, the raw material is crushed to 100 mesh, and 0.2 mol / L sodium hydroxide (material liquid ratio 1:25) is used for extraction at 60°C for 3 hours, and the protein concentration reaches 47 mg / mL. In the first stage of enzymolysis, alkaline protease (4000 U / g) and trypsin (4000 U / g) are reacted at pH 7.5, 58°C for 4 hours, and the degree of hydrolysis is 23.1%; in the second stage, flavor protease (1500 U / g) and papain (300 U / g) are enzymolyzed at pH 7.0, 60°C for 2 hours, and the degree of hydrolysis is increased to 31.5%. After enzyme inactivation, purification is carried out by 5 kDa→1 kDa→300 Da ultrafiltration membrane, the polypeptide yield is 22.3%, the purity is 93%, the IC50 value is 0.58 mg / mL, the dietary fiber yield is 20.8%, and the water holding capacity is 9.2 g / g.

[0100] Comparative Example 1:

[0101] Method: traditional single enzyme hydrolysis (only alkaline protease) + boiling enzyme inactivation (100°C, 15 minutes) + no by-product co-production.

[0102] Disadvantages: low efficiency of single enzyme hydrolysis, high temperature enzyme inactivation destroys activity, and waste of raw materials due to no extraction of dietary fiber.

[0103] Comparative Example 2:

[0104] Method: traditional double enzyme hydrolysis (alkaline + flavor protease, no gradient control) + boiling enzyme inactivation + simple centrifugal purification (no membrane separation).

[0105] Disadvantages: no gradient optimization of enzyme hydrolysis, low purity due to rough purification, and imperfect dietary fiber extraction process.

[0106] Comparative Example 3:

[0107] Method: multi-enzyme hydrolysis without sequence control (three enzymes mixed and added) + high temperature enzyme inactivation + only ultrafiltration without chromatography purification.

[0108] Disadvantages: disordered enzyme hydrolysis reduces the yield of target peptide segments, lack of chromatography steps affects purity, and no co-production of dietary fiber.

[0109] Table 1: Comparison of properties of different examples and comparative examples

[0110]

[0111]

[0112] Table character explanation:

[0113] Examples 1-3: represent the implementation results of the method of the present application under different parameters (lowest, intermediate, and highest data boundaries).

[0114] Comparative Example 1-3: Comparative groups of traditional single enzyme hydrolysis, disordered double enzyme hydrolysis, and multi-enzyme mixed process, respectively.

[0115] Polypeptide yield: The percentage of the mass of the final obtained polypeptide to the total mass of the raw material safflower kernel. The higher the value, the higher the utilization rate of the raw material. For example, a yield of 12.5% in Example 1 means that 12.5 grams of polypeptide can be extracted from every 100 grams of raw material.

[0116] Polypeptide purity: The content of the target polypeptide determined by high performance liquid chromatography (HPLC). A purity of >90% can meet the requirements of pharmaceutical grade applications.

[0117] Antioxidant activity (DPPH IC50): The concentration required to scavenge half of the DPPH free radicals, with units of mg / mL. The smaller the IC50 value, the stronger the antioxidant capacity. For example, the activity of 0.58 mg / mL in Example 3 is 4.3 times higher than that of 2.5 mg / mL in Comparative Example 1.

[0118] Dietary fiber water holding capacity: The mass of water adsorbed per gram of dietary fiber (unit g / g). Fibers with a water holding capacity of >7 g / g can effectively improve food texture. In Example 3, the water holding capacity is as high as 9.2 g / g, indicating that it is suitable as a high-moisture food additive.

[0119] Comparative Example 1: Single alkaline protease hydrolysis was used, and enzyme was inactivated by boiling at 100°C, resulting in a polypeptide yield of only 8.7% (Example 1: 12.5%), a purity of 68% (Example 1: 83%), and no dietary fiber was extracted. The fundamental problem is that high temperature destroys the activity of polypeptides, and full component utilization is not achieved.

[0120] Comparative Example 2: Alkaline protease and flavor protease were added simultaneously (without gradient control), and only centrifugal crude purification was used, resulting in a polypeptide purity of 75% (Example 2: 91%) and a dietary fiber water holding capacity of 5.2 g / g (Example 2: 8.5 g / g). The defects are due to disordered enzyme hydrolysis and rough purification process.

[0121] Comparative Example 3: Three proteases (alkaline, trypsin, and flavor enzymes) were mixed and added, but not controlled in stages, and no chromatography purification was used, resulting in a polypeptide yield of 15.9% (Example 3: 22.3%) and an IC50 value of 1.3 mg / mL (Example 3: 0.58 mg / mL). Disordered enzyme hydrolysis results in low yield of target peptide segments, and impurities remaining affect activity.

[0122] Percentage (%): Used for yield (polypeptide mass / raw material mass x 100%) and purity (target polypeptide peak area / HPLC total peak area x 100%).

[0123] mg / mL: IC50 values in DPPH radical scavenging assay were calculated by fitting concentration-inhibition curves, with the proviso that the assay conditions are specified (e.g. DPPH concentration 0.1 mM, reaction time 30 minutes, 517 nm absorbance measurement).

[0124] g / g: Water holding capacity was determined by weighing 1 g of dietary fiber, adding 10 times water, centrifuging after swelling, and calculating the mass of absorbed water / mass of fiber.

[0125] While embodiments of the present application have been shown and described, it is to be understood that the embodiments described are merely divergences of the principles and spirit of the present application, and that various changes in form and substitution and changes in details of the embodiments can be made by those skilled in the art without departing from the principles and spirit of the present application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting a safflower polypeptide, characterized in that, The method comprises the following steps: S1, raw material pretreatment: crushing safflower kernel and mixing with alkaline solution for extraction, and centrifugal separation to obtain protein extract; S2, multi-stage enzymolysis: S201, adding first complex protease to the protein extract for first-stage enzymolysis; S202, adding second complex protease for second-stage enzymolysis; S3, enzyme inactivation and purification: S301, adjusting the pH of the enzymolysis solution to an acidic condition to inactivate the enzyme; S302, separating the target polypeptide through multi-stage ultrafiltration membrane;   S303, drying to obtain safflower polypeptide product.

2. The safflower polypeptide extraction method according to claim 1, characterized in that, The first complex protease in S201 is a mixture of alkaline protease and trypsin, and the enzyme activity ratio is 1:0.5-1:

3.

3. The method of claim 2, wherein the safflower polypeptide is extracted from the safflower plant. The first-stage enzymolysis is carried out at pH 6.5-7.5 and temperature 45-58℃, and the enzymolysis time is 1.5-4 hours.

4. The method of claim 1, wherein the safflower polypeptide is extracted from the safflower plant. The second complex protease in S202 comprises flavor protease and papain, and the enzymolysis pH is 6.0-7.0 and the temperature is 50-60℃.

5. The method of claim 1, wherein the safflower polypeptide is extracted from the safflower plant. 5 The acidic condition in S301 is pH 3.5-4.5, and the enzyme inactivation process is controlled at temperature 25-40℃ throughout.

6. The method of claim 1, wherein the safflower polypeptide is extracted from the safflower plant. The multi-stage ultrafiltration membrane separation in S302 comprises: The first-stage ultrafiltration membrane has a molecular weight cut-off of 5-15 kDa; The second-stage ultrafiltration membrane has a molecular weight cut-off of 1-5 kDa; The third-stage ultrafiltration membrane has a molecular weight cut-off of 300-800 Da.

7. The method for extracting safflower polypeptides according to claim 6, characterized in that, The third-stage ultrafiltration liquid is further purified by anion exchange chromatography, and the eluent is a buffer solution containing 0.05-0.5 mol / L sodium chloride.

8. The safflower polypeptide extraction method according to claim 1, characterized in that, It also comprises a by-product co-production step: S4, adding the residue after centrifugation of S1 to cellulase hydrolysis, and drying to obtain dietary fiber, and the water holding capacity of the dietary fiber is ≥7 g / g.

9. A safflower polypeptide composition, characterized in that, The safflower polypeptide extraction method according to any one of claims 1-8 comprises: Safflower polypeptide with a molecular weight range of 200-500 Da and a purity of ≥85%; Dietary fiber extracted from safflower kernel alkali extraction residue; The safflower polypeptide composition can be made into tablets, capsules or oral liquids.

10. Use of a safflower polypeptide, according to the method for extracting a safflower polypeptide according to any one of claims 1 to 8, characterized in that, The safflower polypeptide is used for preparing food, medicine or cosmetics with intestinal barrier repair function, wherein: The food comprises oral liquid, tablet or capsule; The medicine comprises a pharmaceutically acceptable carrier; The cosmetic comprises a liposome-encapsulated polypeptide composition.

Images