Cornus officinalis seed antioxidant peptide as well as preparation method and application thereof
Through complex enzymatic lysis and directional purification technology, the problem of low protein utilization of dogwood seeds is solved, and efficient and low-cost preparation of dogwood seeds antioxidant peptides is achieved, which is suitable for large-scale production.
Patent Information
- Application Number
- CN202510625649.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-15
AI Technical Summary
In the prior art, the protein utilization rate of dogwood seeds is low, the traditional enzymatic process is low, the yield and activity of antioxidant peptides are insufficient, the purification process is complex, and it is difficult to achieve large-scale production.
Complex enzymatic lysis technology and directional purification technology, including the synergistic extraction of organic solvents and organic acids, dual enzyme timing hydrolysis and macroporous resin-bound ultrafiltration membrane are used to optimize the preparation method of antioxidant peptides of dogwood seeds.
It improves the dissolution efficiency and yield of antioxidant peptides, reduces production costs, realizes low-temperature operation and efficient large-scale production, and improves antioxidant activity.
Smart Images
Figure BDA0005403863370000061 
Figure BDA0005403863370000062
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of antioxidant peptides, and particularly relates to a cornus officinalis seed antioxidant peptide and a preparation method and application thereof. Background Art
[0002] Cornus officinalis (Cornus officinalis Sieb.Et Zucc.), a plant of the genus Cornus in the family Cornaceae, is a traditional and precious tonic Chinese medicinal herb with a long history of use. First mentioned in the Shennong's Herbal Classic, Cornus officinalis contains a large number of active substances beneficial to the human body, such as ursolic acid, polysaccharides, and vitamins, with tonic, antibacterial, diuretic, and blood-tonifying effects. However, little research has been conducted on Cornus officinalis seeds. Surveys show that Cornus officinalis seeds, the core of the Cornus officinalis fruit after the pulp has been removed, have an annual production of approximately 800,000 kilograms. Currently, they are largely treated as waste and are not effectively utilized. Cornus officinalis seeds are rich in tannins, oils, proteins, and sugars, but current research on them both domestically and internationally is not optimistic. Cornus officinalis seeds have unique nutritional value and medicinal properties.
[0003] Furthermore, studies have shown that compared to protein molecules, active peptides have high absorption efficiency and good biological efficacy. Active peptides are small protein fragments composed of amino acids that are easily absorbed by the human body and have strong targeting properties. They can be obtained through food and synthesis using modern biotechnology. Currently, active peptides are widely used in drug development, health foods, and cosmetics, playing an important role in anti-oxidation, immune enhancement, blood sugar reduction, and blood pressure regulation.
[0004] Cornus officinalis seeds are often discarded as a by-product of traditional Chinese medicine and have low utilization rates, but their protein content is as high as 15-20% and has not been developed. Traditional enzymatic hydrolysis processes (such as single enzyme treatment) are insufficient in the degree of hydrolysis of cornus officinalis seed protein, resulting in low yields of antioxidant peptides (<30%) and weak activity (DPPH clearance <50%). The existing ultrafiltration purification process is not accurate in screening the molecular weight range, which affects the purity of active peptides. The present invention solves the problems of low enzymatic hydrolysis efficiency, insufficient product antioxidant activity, and complex purification process in the prior art by optimizing the composite enzymatic hydrolysis process and directional purification technology, and provides a low-cost, high-yield method for preparing antioxidant peptides suitable for large-scale production. Summary of the Invention
[0005] In view of the problems and shortcomings in the prior art, the present invention aims to provide a cornus officinalis seed antioxidant peptide and a preparation method and application thereof.
[0006] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is as follows:
[0007] The first aspect of the present invention provides a method for preparing cornus officinalis seed antioxidant peptide, comprising the following steps:
[0008] (1) adding an extractant to defatted cornus officinalis seeds, extracting at 30-55° C. to obtain an extract, centrifuging the extract to obtain a precipitate, adding an alkaline solution to the dried and crushed precipitate, extracting at 20-70° C. to obtain an extract, centrifuging the extract to obtain a supernatant, adjusting the pH of the supernatant until a precipitate appears, collecting the precipitate, and freeze-drying the precipitate to obtain cornus officinalis seed protein, wherein the extractant is prepared by adding an organic acid to a mixture of an organic solvent and water;
[0009] (2) adding water and phytase to the cornus officinalis seed protein obtained in step (1), performing enzymatic pretreatment at 10-60° C. to obtain a pretreated enzymatic hydrolyzate, freeze-drying the pretreated enzymatic hydrolyzate to obtain protein powder, dispersing the protein powder in a buffer solution, adding alkaline protease to the buffer solution, performing enzymatic hydrolysis at 25-70° C. under the action of ultrasound, adjusting the pH of the reaction system to 3.0-9.5 after the enzymatic hydrolysis is completed, adding flavor protease, and performing enzymatic hydrolysis at 30-70° C. to obtain an enzymatic hydrolysis reaction solution;
[0010] (3) After the enzyme is inactivated, the enzymatic hydrolysis reaction solution obtained in step (2) is subjected to membrane separation and purification, and the permeate with a molecular weight of less than 3 kDa is collected, and the permeate is debittered and desalted to obtain a purified solution;
[0011] (4) The purified solution obtained in step (3) is mixed with xylose, reacted at 70-130° C. for 10-50 min, and then the reaction is terminated. The reaction solution is vacuum concentrated and dried to obtain the cornus seed antioxidant peptide.
[0012] Preferably, the organic solvent in step (1) is composed of a mixture of ethanol, glycerol and acetone. The organic solvent is composed of a mixture of ethanol, glycerol and acetone.
[0013] The mass ratio of p-toluenesulfonic acid to benzenesulfonic acid in the organic acid of step (1) is 1:(1-7); the volume ratio of ethanol, glycerol and acetone in the organic solvent is (1-5):(4-9):(6-11).
[0014] Preferably, the amount of the organic acid added in step (1) is 2% to 8% of the mass of the mixed solution, the mass ratio of the organic solvent to the organic acid in the extractant is (9 to 11):1, and the volume ratio of the organic solvent to water in the extractant is (4 to 9):(1 to 6).
[0015] Preferably, in step (1), the mass ratio of cornus officinalis seeds to the extractant is 1:15.
[0016] Preferably, the extraction time after adding the extraction liquid in step (1) is 0.5 to 4 hours.
[0017] Preferably, the pH of the alkaline solution in step (1) is 8.0-12.0, and the extraction time after adding the alkaline solution is 2 hours.
[0018] Preferably, the centrifugation condition in step (1) is: centrifugation at 4000 rpm for 20 min.
[0019] Preferably, the pH of the supernatant in step (1) is adjusted to a range of 2.5 to 6.5.
[0020] Preferably, the particle size after the pulverization treatment in step (1) reaches D50=15±2 μm.
[0021] Preferably, the amount of alkaline protease added in step (2) is 1.2% to 3.6% of the cornus seed protein in step (1), the enzymatic hydrolysis time is 2 to 4 hours, and the ultrasonic conditions are: ultrasonic frequency is 20 to 60 kHz, and ultrasonic power is 100 to 500 W.
[0022] Preferably, the amount of flavor protease added in step (2) is 1.0% to 2.5% of the amount of cornus seed protein in step (1), and the amount of phytase added is 0.02% to 1.1% of the amount of cornus seed protein in step (1).
[0023] Preferably, in step (2), the material-liquid ratio of cornus officinalis seed protein to water is 1:10 mg / mL.
[0024] Preferably, in step (2), the enzymatic hydrolysis time of phytase is 1 hour, the enzymatic hydrolysis time of alkaline protease is 3 hours, and the enzymatic hydrolysis time of flavor protease is 4 hours.
[0025] Preferably, the buffer in step (2) is a Tris-HCl buffer with a pH of 5.0 to 12.0.
[0026] Preferably, in step (3), the membrane separation and purification is to filter the enzymatic hydrolysis reaction solution obtained in step (2) using a 50kDa ceramic membrane, a 10kDa hollow fiber membrane, and a 3kDa spiral membrane in sequence. The operating pressure gradients of the ceramic membrane, hollow fiber membrane, and spiral membrane systems are 0.2MPa, 0.5MPa, and 1.0MPa, respectively, and the membrane flux is 80 to 120L / (m 2 ·h).
[0027] Preferably, in step (3), an AB-8 macroporous resin column with a column volume ratio of 1:8 is used for debittering, and Sephadex G-25 is used for desalting.
[0028] Preferably, the mass ratio of the purified liquid in step (4) to xylose is (6-20):1.
[0029] The second aspect of the present invention provides a cornus officinalis seed antioxidant peptide prepared by any method described in the first aspect.
[0030] The third aspect of the present invention provides a use of the cornus officinalis seed antioxidant peptide described in the second aspect in the preparation of antioxidant drugs, foods, and cosmetics.
[0031] Compared with the prior art, the present invention has the following advantages:
[0032] (1) The extractant of the present invention significantly improves the dissolution efficiency of antioxidant peptides through the synergistic effect of organic solvents and organic acids. Organic acids block the key catalytic sites of the oxidation chain reaction by chelating metal ions. Solvent polarity regulation can change the hydrophilic-hydrophobic balance on the polypeptide surface and promote the dissolution of hydrophobic antioxidant peptides. Organic solvents and organic acids achieve high selectivity, high efficiency and functional synergy in the extraction of antioxidant peptides, providing theoretical support for the development of functional foods.
[0033] (2) The enzyme preparation for extracting polypeptides from cornus seeds of the present invention can be used to enzymatically hydrolyze cornus seed protein, thereby hydrolyzing cornus seed protein into polypeptides, thereby solving the problem of low utilization rate of cornus seed protein.
[0034] (3) The present invention adopts dual enzyme sequential hydrolysis: alkaline protease preferentially cuts off hydrophobic amino acids, and flavor protease further hydrolyzes hydrophobic peptides, which can significantly increase the exposure of antioxidant active sites.
[0035] (4) The present invention adopts a combined purification technology: selective adsorption of macroporous resin combined with precise retention of ultrafiltration membrane, replacing traditional chromatography, which can reduce costs.
[0036] (5) The preparation method of the cornus seed polypeptide of the present invention has a simple preparation process, low cost and good safety, and is operated at low temperature throughout the process to avoid oxidation and aggregation of peptide chains caused by high temperature. DETAILED DESCRIPTION
[0037] In order to make the purpose, technical solutions and advantages of the present invention more clear and distinct, the present invention is further described in detail below through examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
[0038] Example 1
[0039] A method for preparing cornus officinalis seed antioxidant peptide, comprising the following specific steps:
[0040] (1) Cornus officinalis seeds are defatted by twin-screw low-temperature pressing (temperature not exceeding 45°C, pressure maintained at 50±5MPa) to obtain defatted meal with an oil content not exceeding 5%. The extractant is added to the defatted meal at a mass ratio of cornus officinalis seeds to extractant of 1:15, and the mixture is leached at 40°C for 1.5 hours to obtain an extract, wherein the extractant is formed by forming a mixed liquid of an organic solvent and water, and then an organic acid is added to the mixed liquid, wherein the organic solvent is composed of ethanol, glycerol, and acetone in a volume ratio of 3:5:8, the ratio of organic solvent to water is 7:3, the organic acid is composed of p-toluenesulfonic acid and benzenesulfonic acid in a mass ratio of 1:3, the amount of organic acid added is 5% of the weight of the mixed liquid, and the mass ratio of organic solvent to organic acid is 10:1. The extract was centrifuged (4000 rpm, 20 min) to obtain a precipitate, which was dried and treated with a vortex micronizer to a particle size of D50 = 15 ± 2 μm. The extract was then extracted with a NaOH solution having a pH value of 12.0 at 50° C. for 2 h to obtain an extract. The extract was centrifuged (8000 × g, 15 min) and the supernatant was collected. 12% hydrochloric acid was added to adjust the pH of the supernatant to 4.5 for precipitation. After precipitation, the protein was freeze-dried to obtain the cornus seed protein.
[0041] (2) adding water (the material-liquid ratio of cornus officinalis seed protein to water is 1:10 mg / mL) and phytase to the cornus officinalis seed protein prepared in step (1), and pre-enzymatically hydrolyzing the protein at 40°C for 1 hour to remove phytic acid-bound polypeptides in the cornus officinalis seed protein, and freeze-drying the enzymatic hydrolyzate to obtain protein powder, wherein the amount of phytase added is 0.5% w / w of the cornus officinalis seed protein; dispersing the protein powder in a Tris-HCl buffer solution at a material-liquid ratio of 1:25 (w / v) in pH 9.0, adding alkaline protease to form a reaction system, and hydrolyzing the protein at 55°C with ultrasound assistance (40 kHz, 300 W) for 3 hours, wherein the amount of alkaline protease added is 2.8% (w / w) of the cornus officinalis seed protein, and the alkaline protease preferentially cuts off the hydrophobic amino acid chain; and then adjusting the reaction system to pH 5. 6.5, adding flavor protease and stirring at 50°C for 4 hours to hydrolyze to obtain an enzymatic hydrolysis reaction solution, wherein the amount of flavor protease added is 1.5% (w / w) of the weight of the cornus seed protein. Flavor protease further hydrolyzes the hydrophobic peptides, releasing highly active peptides, and significantly increasing the exposure of antioxidant active sites;
[0042] (3) The enzymatic hydrolysis reaction solution obtained in step (2) was pasteurized (80°C, 10 min) and then filtered through a 50 kDa ceramic membrane (ΔP = 0.2 MPa), a 10 kDa hollow fiber membrane (ΔP = 0.5 MPa), and a 3 kDa spiral membrane (ΔP = 1.0 MPa) to collect the permeate with a molecular weight < 3 kDa; the permeate was debittered by passing through an AB-8 macroporous resin column (column volume ratio 1:8) and then desalted by using Sephadex G-25 to obtain a purified solution;
[0043] (4) The purified solution obtained in step (3) was mixed with xylose in a mass ratio of 15:1, reacted at 110° C. for 30 min, and then immediately terminated in an ice bath. The reaction solution was vacuum concentrated (50° C., −0.09 MPa) to a solid content of 25%, and spray dried (inlet air 180° C. / outlet air 85° C.) to obtain the finished product of the cornus seed antioxidant peptide.
[0044] Example 2
[0045] A method for preparing cornus officinalis seed antioxidant peptide, comprising the following specific steps:
[0046] (1) Cornus officinalis seeds are defatted by twin-screw low-temperature pressing (temperature not exceeding 45°C, pressure maintained at 50±5MPa) to obtain defatted meal with an oil content not exceeding 5%. An extractant is added to the defatted meal at a mass ratio of cornus officinalis seeds to extractant of 1:15, and the mixture is extracted at 30°C for 0.5h. The extractant is prepared by forming a mixed solution of an organic solvent and water, and then adding an organic acid to the mixed solution, wherein the organic solvent is composed of ethanol, glycerol, and acetone in a volume ratio of 1:4:9, and the volume ratio of the organic solvent to water is 4:6; the organic acid is composed of p-toluenesulfonic acid and benzenesulfonic acid in a mass ratio of 1:1, and the amount added is 2% of the mass of the mixed solution, and the mass ratio of the organic solvent to the organic acid is 9:1. The extract was then centrifuged (4000 rpm, 20 min) and the precipitate was dried. The dried defatted meal was processed in a vortex micronizer to a particle size of D50 = 15 ± 2 μm, and then extracted with a NaOH solution with a pH value of 12.0 at 50° C. for 2 h to obtain an extract. The extract was centrifuged (8000 × g, 15 min) and the supernatant was collected. 12% hydrochloric acid was added to adjust the pH of the supernatant to 4.5 for precipitation. After precipitation, the protein was freeze-dried to obtain the cornus officinalis seed protein.
[0047] (2) adding water (the material-liquid ratio of cornus officinalis seed protein to water is 1:10 mg / mL) and phytase to the cornus officinalis seed protein prepared in step (1), and pre-enzymatically hydrolyzing the protein at 40°C for 1 hour to remove phytic acid-bound polypeptides in the cornus officinalis seed protein, and freeze-drying the enzymatic hydrolyzate to obtain protein powder, wherein the amount of phytase added is 0.02% w / w of the cornus officinalis seed protein; dispersing the protein powder in a Tris-HCl buffer solution at a material-liquid ratio of 1:15 (w / v), adding alkaline protease to form a reaction system, and hydrolyzing the protein at 55°C with ultrasound assistance (20 kHz, 150 W) for 3 hours, wherein the amount of alkaline protease added is 1.2% (w / w) of the cornus officinalis seed protein, and the alkaline protease preferentially cuts off the hydrophobic amino acid chain; then adjusting the reaction system to pH 6.5, adding flavor protease and stirring at 50°C for 4 hours to hydrolyze to obtain an enzymatic hydrolysis reaction solution, wherein the amount of flavor protease added is 1.0% (w / w) of the cornus officinalis seed protein. Flavor protease further hydrolyzes the hydrophobic peptides, releasing highly active peptides, and significantly increasing the exposure of antioxidant active sites;
[0048] (3) The enzymatic hydrolysis reaction solution obtained in step (2) was pasteurized (80°C, 10 min) and then filtered through a 50 kDa ceramic membrane (ΔP = 0.2 MPa), a 10 kDa hollow fiber membrane (ΔP = 0.5 MPa), and a 3 kDa spiral membrane (ΔP = 1.0 MPa) to collect the permeate with a molecular weight < 3 kDa; the permeate was debittered by passing through an AB-8 macroporous resin column (column volume ratio 1:8) and then desalted by using Sephadex G-25 to obtain a purified solution;
[0049] (4) The purified solution obtained in step (3) was mixed with xylose in a mass ratio of 15:1, reacted at 110° C. for 30 min, and then immediately terminated in an ice bath. The reaction solution was vacuum concentrated (50° C., −0.09 MPa) to a solid content of 25%, and spray dried (inlet air 180° C. / outlet air 85° C.) to obtain the finished product of the cornus seed antioxidant peptide.
[0050] Example 3
[0051] A method for preparing cornus officinalis seed antioxidant peptide, comprising the following specific steps:
[0052] (1) Cornus officinalis seeds are defatted by twin-screw low-temperature pressing (temperature not exceeding 45°C, pressure maintained at 50±5MPa) to obtain defatted meal with an oil content not exceeding 5%. An extractant is added to the defatted meal at a mass ratio of cornus officinalis seeds to extractant of 1:15, and the mixture is extracted at 30°C for 4 hours. The extractant is prepared by forming a mixed liquid with an organic solvent and water, and then adding an organic acid to the mixed liquid, wherein the organic solvent is composed of ethanol, glycerol, and acetone in a volume ratio of 5:9:11, and the volume ratio of the organic solvent to water is 9:1; the organic acid is composed of p-toluenesulfonic acid and benzenesulfonic acid in a mass ratio of 1:7, and the amount added is 8% of the mass of the mixed liquid, and the mass ratio of the organic solvent to the organic acid is 11:1. The extract was then centrifuged (4000 rpm, 20 min) and the precipitate was dried. The dried defatted meal was processed in a vortex micronizer to a particle size of D50 = 15 ± 2 μm, and then extracted with a NaOH solution with a pH value of 12.0 at 50° C. for 2 h to obtain an extract. The extract was centrifuged (8000 × g, 15 min) and the supernatant was collected. 12% hydrochloric acid was added to adjust the pH of the supernatant to 4.5 for precipitation. After precipitation, the protein was freeze-dried to obtain the cornus officinalis seed protein.
[0053] (2) adding water (the material-liquid ratio of cornus officinalis seed protein to water is 1:10 mg / mL) and phytase to the cornus officinalis seed protein prepared in step (1), and pre-treating the protein with phytase at 40°C for 1 hour to remove the phytic acid-bound polypeptides in the cornus officinalis seed protein, and freeze-drying the enzymatic hydrolyzate to obtain protein powder, wherein the amount of phytase added is 1.1% w / w of the cornus officinalis seed protein; dispersing the protein powder in a pH 9.0 Tris-HCl buffer at a material-liquid ratio of 1:35 (w / v), adding alkaline protease to form a reaction system, and hydrolyzing the protein with ultrasound assistance (20 kHz, 150 W) at 55°C for 3 hours, wherein the amount of alkaline protease added is 3.6% (w / w) of the cornus officinalis seed protein, and the alkaline protease preferentially cuts the hydrophobic amino acid chain; and then adjusting the reaction system to pH 6.5, adding flavor protease and stirring at 50°C for 4 hours to hydrolyze to obtain an enzymatic hydrolysis reaction solution, wherein the amount of flavor protease added is 2.5% (w / w) of the weight of the cornus seed protein. Flavor protease further hydrolyzes the hydrophobic peptides, releasing highly active peptides, and significantly increasing the exposure of antioxidant active sites;
[0054] (3) The enzymatic hydrolysis reaction solution obtained in step (2) was pasteurized (80°C, 10 min) and then filtered through a 50 kDa ceramic membrane (ΔP = 0.2 MPa), a 10 kDa hollow fiber membrane (ΔP = 0.5 MPa), and a 3 kDa spiral membrane (ΔP = 1.0 MPa), collecting the permeate < 3 kDa; the permeate was debittered by passing through an AB-8 macroporous resin column (column volume ratio 1:8), and then desalted by using Sephadex G-25 to obtain a purified solution;
[0055] (4) The purified solution obtained in step (3) was mixed with xylose in a mass ratio of 15:1, reacted at 110° C. for 30 min, and then immediately terminated in an ice bath. The reaction solution was vacuum concentrated (50° C., −0.09 MPa) to a solid content of 25%, and spray dried (inlet air 180° C. / outlet air 85° C.) to obtain the finished product of the cornus seed antioxidant peptide.
[0056] Determination of antioxidant peptide indexes in Cornus officinalis seeds:
[0057] (1) Yield determination
[0058] The yield of the antioxidant peptides from the cornus seed meal prepared in the example was determined, and the calculation formula is as follows:
[0059]
[0060] The quality of the antioxidant peptide is the quality of the finished product of the cornus officinalis seed antioxidant peptide in step (4), and the quality of the raw material is the quality of the cornus officinalis seed in step (1).
[0061] (2) DPPH clearance determination
[0062] Weigh 2 mg of DPPH and dissolve it in ethanol solution. Ultrasonication is used to accelerate the dissolution. The DPPH solution is prepared by diluting the volume in a 100 mL volumetric flask and storing it in a refrigerator at 4 ° C in the dark. The cornus seed meal antioxidant peptide prepared in the example is prepared into a sample aqueous solution with a concentration of 2 mg / mL. 2 mL of the sample aqueous solution is mixed with 2 mL of the DPPH solution and shaken. The mixture is reacted in a dark environment for 0.5 h. The absorbance value is measured at 517 nm to obtain the absorbance of the sample group, which is recorded as A i Mix 2 mL of blank sample (distilled water) with 2 mL of DPPH solution, react in a dark environment for 0.5 h, and measure the absorbance at 517 nm to obtain the absorbance of the control group, which is recorded as A0. Mix 2 mL of a 2 mg / mL sample aqueous solution with 2 mL of 95% ethanol, and measure the absorbance at 517 nm to obtain the absorbance of the blank group, which is recorded as Aj. Calculate the DPPH free radical scavenging rate according to the following formula:
[0063]
[0064] Where: A0 = absorbance of control group; A i = absorbance of sample group; A j = absorbance of blank group.
[0065] The yields and DPPH scavenging rates of cornus officinalis seed antioxidant peptides of Examples 1 to 3 of the present invention are shown in Table 1.
[0066] Table 1 Index determination results of cornus officinalis seed antioxidant peptides in Examples 1 to 3
[0067] serial number Antioxidant peptide yield% DPPH clearance rate (%) Example 1 89.5 97.2 Example 2 78.3 88.6 Example 3 74.1 84.3
[0068] As shown in Table 1, the yield of the cornus officinalis seed antioxidant peptide prepared by the preparation method of the present invention is greater than 74%, and the DPPH scavenging rate is greater than 84%. Among them, the cornus officinalis seed antioxidant peptide obtained in Example 1 has the highest yield, 89.5%, and DPPH scavenging rate, 97.2%. Because the addition ratio of the complex enzyme in Example 1 is appropriate, and the ratio of the organic solvent to the organic acid is appropriate, the substrate hydrolysis efficiency is improved, the release of the antioxidant peptide is promoted, and the yield of the cornus officinalis seed antioxidant peptide and the DPPH scavenging rate are increased.
[0069] In Example 2, the amount of complex enzyme added is small, and the binding sites of the enzyme and the substrate are insufficient, resulting in insufficient protein hydrolysis. Under low enzyme conditions, the enzyme cleavage site is not fully exposed, and the macromolecular protein cannot be effectively degraded into functional active peptides, resulting in a decrease in the yield of antioxidant peptides and the DPPH clearance rate; in Example 3, adding an excessive amount of complex enzyme may cause the enzyme molecules to competitively bind to the substrate or self-hydrolyze, thereby reducing the yield. Excessive enzymatic hydrolysis may also destroy the key functional sites (such as Trp and Tyr residues) of the antioxidant peptides, which will also reduce the yield of antioxidant peptides and the DPPH clearance rate. In Example 2, the excessively high proportion of organic acid will significantly reduce the pH value of the system, resulting in excessive denaturation of the protein and destruction of the key active sites of the antioxidant peptides. The high proportion of organic solvent will form a highly polar environment, making it difficult for low-polarity antioxidant peptides to dissolve, resulting in a decrease in yield and a simultaneous decrease in the DPPH free radical scavenging rate. In Example 3, the proportion of organic acid is too low to effectively break down the fiber-protein complex structure of the seed meal, resulting in insufficient release rate of antioxidant peptides in the cells. The low proportion of organic solvent makes it difficult to dissolve hydrophobic antioxidant peptides, and the residual fat-soluble anti-nutritional factors inhibit their activity. Too high or too low a proportion will lead to a decrease in their yield and DPPH clearance rate.
[0070] Comparative Example 1
[0071] The process is basically the same as Example 1, except that the ratio of the organic solvent to the organic acid in step (1) is 14:1.
[0072] Comparative Example 2
[0073] The process is basically the same as Example 1, except that the ratio of the organic solvent to the organic acid in step (1) is 9:10.
[0074] Comparative Example 3
[0075] The process is basically the same as Example 1, except that no organic solvent is used in step (1).
[0076] Comparative Example 4
[0077] The process is basically the same as Example 1, except that no organic acid is used in step (1).
[0078] The yield and DPPH scavenging rate of the cornus officinalis seed antioxidant peptides prepared in Comparative Examples 1 to 4 were determined according to the method described in the Examples. The results are shown in Table 2 below.
[0079] Table 2 Index determination results of cornus officinalis seed antioxidant peptides in Example 1 and Comparative Examples 1 to 4
[0080] serial number Antioxidant peptide yield% DPPH clearance rate (%) Example 1 89.5 97.2 Comparative Example 1 70.4 79.5 Comparative Example 2 69.1 75.6 Comparative Example 3 60.1 67.5 Comparative Example 4 58.4 61.6
[0081] It can be seen from Table 2 that the excessive amount of organic solvent used in Comparative Example 1 may change the spatial conformation of the enzyme molecule or destroy the hydrogen bond network of the enzyme active center, resulting in a decrease in the catalytic efficiency of the protease and affecting the efficiency of protein hydrolysis to produce antioxidant peptides. Excessive amount of organic solvent may also cause excessive folding of the hydrophobic region of the protein, reduce the contact area between the enzyme and the substrate binding site, reduce the degree of hydrolysis, and may destroy the hydration layer between the polypeptide molecules, causing the hydrophobic antioxidant peptides to aggregate or precipitate, thereby reducing the yield of soluble peptides. The large amount of organic acid used in Comparative Example 2 may cause the pH of the system to be too low, inhibit enzyme activity or cause excessive acidification and degradation of the peptide chain, resulting in a decrease in the yield of the peptide. The absence of an organic solvent in Comparative Example 3 will result in a significant decrease in the yield of antioxidant peptides and the ability to scavenge free radicals. The reason is that polar organic solvents such as acetone and ethanol reduce the dielectric constant of the solution, weaken the interaction between water molecules and polar groups of the protein, expose hydrophobic regions and induce aggregation and precipitation. Under low temperature (-20°C) conditions, organic solvents can maintain the natural conformation of proteins and are suitable for active enzyme extraction (such as ethanol precipitation method to retain amylase activity); organic solvents may affect the molecular weight distribution of antioxidant peptides by regulating solvation, thereby changing their functional properties (such as free radical scavenging ability). Therefore, not using organic solvents will lead to a decrease in the yield of antioxidant peptides and the ability to scavenge free radicals. In Comparative Example 4, not using organic acids will lead to a significant decrease in the yield of antioxidant peptides and the ability to scavenge free radicals. The reason is that organic acids (such as citric acid and acetic acid) can lower the pH value of the solution and enhance the solubility and stability of antioxidant peptides. Organic acids bind to antioxidant peptides through hydrogen bonds or electrostatic interactions, protecting their active groups (such as sulfhydryl groups and phenolic hydroxyl groups) from oxidative damage, thereby improving the stability and activity of antioxidant peptides. Therefore, not using organic acids will lead to a significant decrease in the yield of antioxidant peptides and the ability to scavenge free radicals.
[0082] Through the analysis of the index data of Comparative Examples 1 to 4 and Example 1, it was concluded that the dosage of the organic solvent and the organic acid of the present invention is better when it is in the range of (9 to 11):1. The effect of using one of them alone is significantly reduced. The organic acid reduces the pH value of the system, enhances the removal efficiency of the organic solvent for fat-soluble impurities, and at the same time stabilizes the peptide structure through hydrogen bonds, reducing the negative impact of the solvent on antioxidant activity. The two have a synergistic effect.
[0083] Comparative Example 5
[0084] The process is basically the same as Example 1, except that the volume ratio of ethanol, glycerol and acetone in the organic solvent in step (1) is 3:5:12.
[0085] Comparative Example 6
[0086] The process is basically the same as Example 1, except that the volume ratio of ethanol, glycerol and acetone in the organic solvent in step (1) is 3:5:5.
[0087] The yield and DPPH scavenging rate of the cornus officinalis seed antioxidant peptides prepared in Comparative Examples 5 and 6 were determined according to the method described in the Examples. The results are shown in Table 3 below.
[0088] Table 3 Index determination results of cornus officinalis seed antioxidant peptides in Example 1, Comparative Examples 5 to 6
[0089] serial number Antioxidant peptide yield% DPPH clearance rate (%) Example 1 89.5 97.2 Comparative Example 5 69.1 75.8 Comparative Example 6 67.0 72.9
[0090] Through the analysis of the index data of Comparative Examples 5 to 6 and Example 1, it can be seen that the appropriate ratio of organic solvents can effectively improve the yield of antioxidant peptides and the DPPH scavenging rate. Comparative Examples 5 and 6 can be concluded that the polarity of the mixed solvent directly affects the solubility of the antioxidant peptide. When the ratio is too high, a polarity mismatch environment will be formed, resulting in hydrophobic antioxidant peptides. The solubility decreases, some peptide segments are lost due to precipitation, the yield decreases, and the oxidation or hydrolysis of active residues such as Trp and Tyr in the antioxidant peptides is accelerated, resulting in a decrease in the DPPH free radical scavenging rate. When the ratio is insufficient, hydrophilic antioxidant peptides are difficult to dissolve effectively, and the residual fat-soluble impurities may inhibit their activity, resulting in a decrease in the yield of antioxidant peptides and the DPPH scavenging rate.
[0091] Comparative Example 7
[0092] The method is basically the same as Example 1, except that the mass ratio of p-toluenesulfonic acid to benzenesulfonic acid in the organic acid in step (1) is 2:3.
[0093] Comparative Example 8
[0094] The method is basically the same as Example 1, except that the mass ratio of p-toluenesulfonic acid to benzenesulfonic acid in the organic acid in step (1) is 1:8.
[0095] The yield and DPPH scavenging rate of the cornus officinalis seed antioxidant peptides prepared in Comparative Examples 7 and 8 were determined according to the method described in the Examples. The results are shown in Table 4 below.
[0096] Table 4 Index determination results of cornus officinalis seed antioxidant peptides in Example 1, Comparative Examples 7 and 8
[0097] serial number Antioxidant peptide yield% DPPH clearance rate (%) Example 1 89.5 97.2 Comparative Example 7 68.3 71.8 Comparative Example 8 67.0 70.9
[0098] Through the analysis of the index data of Comparative Examples 7-8 and Example 1, an imbalance in the ratio of mixed organic acids will lead to deterioration of chromatographic separation, degradation of low molecular weight peptides due to excessive acid hydrolysis, and a decrease in the proportion of active peptides. In Comparative Example 7, the proportion of p-toluenesulfonic acid is too high. Its high acidity can significantly reduce the pH value of the system, inhibiting protease activity, resulting in incomplete hydrolysis and reduced yield of antioxidant peptides and DPPH clearance rate. In Comparative Example 8, the proportion of benzenesulfonic acid is too high, which may induce intermolecular cross-linking of antioxidant peptides through sulfonic acid groups, destroying their reducing activity, resulting in a decrease in yield and DPPH clearance rate.
Claims
1. A method for preparing cornus officinalis seed antioxidant peptide, characterized in that: The steps include: (1) adding an extractant to defatted cornus officinalis seeds, extracting at 30-55° C. to obtain an extract, centrifuging the extract to obtain a precipitate, adding an alkaline solution to the dried and crushed precipitate, extracting at 20-70° C. to obtain an extract, centrifuging the extract to obtain a supernatant, adjusting the pH of the supernatant until a precipitate appears, collecting the precipitate, and freeze-drying the precipitate to obtain cornus officinalis seed protein, wherein the extractant is prepared by adding an organic acid to a mixture of an organic solvent and water; (2) adding water and phytase to the cornus officinalis seed protein obtained in step (1), performing enzymatic pretreatment at 10-60° C. to obtain a pretreated enzymatic hydrolyzate, freeze-drying the pretreated enzymatic hydrolyzate to obtain protein powder, dispersing the protein powder in a buffer solution, adding alkaline protease to the buffer solution, performing enzymatic hydrolysis at 25-70° C. under the action of ultrasound, adjusting the pH of the reaction system to 3.0-9.5 after the enzymatic hydrolysis is completed, adding flavor protease, and performing enzymatic hydrolysis at 30-70° C. to obtain an enzymatic hydrolysis reaction solution; (3) After the enzyme is inactivated, the enzymatic hydrolysis reaction solution obtained in step (2) is subjected to membrane separation and purification, and the permeate with a molecular weight of less than 3 kDa is collected, and the permeate is debittered and desalted to obtain a purified solution; (4) The purified solution obtained in step (3) is mixed with xylose, reacted at 70-130° C. for 10-50 min, and then the reaction is terminated. The reaction solution is vacuum concentrated and dried to obtain the cornus seed antioxidant peptide.
2. The preparation method according to claim 1, characterized in that The organic acid in step (1) is composed of a mixture of p-toluenesulfonic acid and benzenesulfonic acid, and the organic solvent is composed of a mixture of ethanol, glycerol and acetone.
3. The preparation method according to claim 2, characterized in that The mass ratio of p-toluenesulfonic acid to benzenesulfonic acid in the organic acid of step (1) is 1:(1-7); the volume ratio of ethanol, glycerol and acetone in the organic solvent is (1-5):(4-9):(6-11).
4. The preparation method according to claim 1, characterized in that The amount of the organic acid added in step (1) is 2% to 8% of the mass of the mixed solution, the mass ratio of the organic solvent to the organic acid in the extractant is (9 to 11):1; and the volume ratio of the organic solvent to water in the extractant is (4 to 9):(1 to 6).
5. The preparation method according to claim 4, characterized in that In step (2), the amount of alkaline protease added is 1.2% to 3.6% of the quality of cornus seed protein, the enzymatic hydrolysis time of the alkaline protease is 2 to 4 hours, and the ultrasonic conditions are: ultrasonic frequency is 20 to 60 kHz, and ultrasonic power is 100 to 500 W.
6. The preparation method according to claim 5, characterized in that The amount of flavor protease added in step (2) is 1.0% to 2.5% of the amount of cornus officinalis seed protein in step (1), and the amount of phytase added is 0.02% to 1.1% of the amount of cornus officinalis seed protein in step (1).
7. The preparation method according to claim 6, characterized in that In step (3), the membrane separation and purification is to filter the enzymatic hydrolyzate obtained in step (2) using a 50kDa ceramic membrane, a 10kDa hollow fiber membrane, and a 3kDa spiral membrane in sequence; use an AB-8 macroporous resin column for debittering, and use polysaccharide gel G-25 for desalting.
8. The preparation method according to claim 7, characterized in that In step (4), the mass ratio of the purified liquid to xylose is (6-20):
1.
9. An antioxidant peptide from cornus officinalis seeds prepared by the method according to any one of claims 1 to 8.
10. Use of the cornus officinalis seed antioxidant peptide according to claim 9 in the preparation of antioxidant drugs, foods and cosmetics.