Method for synergistically extracting dogwood seed oil and antioxidant peptide by coupling supercritical CO2 extraction with deep-eutectic solvent DES

Abstract

The invention belongs to the technical field of food processing, and particularly relates to a method for synergistically extracting dogwood seed oil and antioxidant peptide by coupling supercritical CO2 extraction with deep-eutectic solvent DES. The method comprises the following steps: S1, performing microwave treatment on dogwood seeds, crushing and sieving to obtain dogwood seed powder; s2, the dogwood seed powder is subjected to supercritical CO2 extraction, a eutectic solvent serves as an entrainer, and residues A and dogwood seed oil are obtained after extraction is completed; s3, mixing the residue A with a deep eutectic solvent, and carrying out secondary extraction to obtain a residue B and a filtrate; s4, adding water into the filtrate, uniformly mixing, centrifuging, respectively collecting a water phase and an oil phase, carrying out reduced pressure distillation on the oil phase, and combining with the dogwood seed oil obtained in the step S2; and S5, adding the residue B obtained in the step S3 into water, and adding mixed enzyme to carry out enzymolysis reaction, so as to obtain the dogwood seed antioxidant peptide. According to the method, the dogwood seed oil and the antioxidant peptide can be extracted at the same time, the oil extraction rate of the dogwood seed oil reaches 90% or above, the yield of the antioxidant peptide is 78.7%, and the resource utilization rate is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and in particular relates to a method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by coupling supercritical CO2 extraction with a deep eutectic solvent DES. Background Art

[0002] Cornus officinalis (Fructus Corni) is a common and widely used traditional Chinese medicinal ingredient in my country. Both the Shennong Bencao Jing (Classic of Materia Medica) and the Compendium of Materia Medica list it as a top-grade tonic, boasting benefits such as nourishing the liver and kidneys, astringing semen, and strengthening the body's defenses. Currently, most research on Cornus officinalis focuses on the pulp, while neglecting its seeds. Cornus officinalis seeds, which comprise approximately 80% of the fruit's weight, are a byproduct of pulp processing. Currently, the primary disposal method is incineration or disposal, resulting in significant resource waste. Therefore, how to comprehensively utilize Cornus officinalis seed resources and maximize their value is a key issue facing the Cornus officinalis industry. Cornus officinalis seeds consist of a husk and a kernel. The husk contains a significant amount of cellulose, while the kernel is rich in fat and protein. The fatty acid content of the fat is as high as 97.84%, primarily composed of oleic acid (58.86%), stearic acid (21.00%), palmitic acid (7.10%), and linoleic acid (4.08%). The kernel contains a comprehensive range of amino acids in a well-balanced ratio, making it an ideal plant protein source.

[0003] Deep eutectic solvents (DES) are emerging green solvents. They form eutectic mixtures of hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) through intermolecular forces. They offer advantages such as a low melting point, strong designability, and excellent biocompatibility. In recent years, DES have been applied in the field of vegetable oil extraction, particularly in applications such as high-value-added oils and fats or those with strict requirements on solvent residues (e.g., in food and pharmaceuticals).

[0004] Supercritical CO2 has a strong solubility for non-polar substances, but has a low extraction efficiency for polar substances. By adding a low eutectic solvent as an entrainer, the solubility of polar components (such as polyphenols and alkaloids) can be significantly improved. The low eutectic solvent changes the polarity parameters of supercritical CO2 through hydrogen bonding, expanding its polarity range from non-polar to medium polarity, thereby being compatible with the extraction of more types of compounds. The coupling of the two can construct a composite solvent system to achieve efficient extraction of oils and active ingredients. At present, there has been no report on the simultaneous extraction of Cornus officinalis seed oil and antioxidant peptides using this coupling technology. Summary of the Invention

[0005] In view of the problems and shortcomings in the prior art, the object of the present invention is to provide a method for the synergistic extraction of cornus officinalis seed oil and antioxidant peptides by coupling supercritical CO2 extraction with a deep eutectic solvent DES.

[0006] Based on the above purpose, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by coupling supercritical CO2 extraction with a deep eutectic solvent (DES), comprising the following steps:

[0008] S1. Microwave-treating cornus officinalis seeds, drying, crushing, and sieving to obtain cornus officinalis seed powder;

[0009] S2, adding the cornus officinalis seed powder prepared in step S1 to a supercritical CO2 extraction reactor, using a deep eutectic solvent as an entrainer, and simultaneously introducing the deep eutectic solvent and CO2 into the reactor for extraction, and obtaining residue A and cornus officinalis seed oil after the extraction is completed;

[0010] S3, mixing the residue A with the deep eutectic solvent for secondary extraction, and filtering after the extraction to obtain a residue B and a filtrate;

[0011] S4, adding water to the filtrate obtained in step S3, mixing and centrifuging, collecting the aqueous phase and the oil phase respectively, recovering the deep eutectic solvent in the aqueous phase for recycling, and combining the oil phase with the cornus seed oil obtained in step S2 after vacuum distillation to complete the extraction of cornus seed oil;

[0012] S5. Add the residue B obtained in step S3 into water and mix well to obtain a mixture, add a mixed enzyme to the mixture for enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis reaction, filter to obtain a filtrate, and concentrate and dry the filtrate to obtain the cornus seed antioxidant peptide.

[0013] Preferably, in step S2 and step S3, the deep eutectic solvent is a mixture of choline chloride, glycerol and citric acid.

[0014] Preferably, the molar ratio of choline chloride, glycerol and citric acid in the deep eutectic solvent is 1:(1.5-2.5):(0.3-0.7).

[0015] Preferably, in step S2, the flow rate of the deep eutectic solvent is 2 to 5 mL / min; in step S3, the amount of the deep eutectic solvent is 3% to 7% of the molar flow rate of CO2.

[0016] Preferably, in step S2, the CO2 flow rate is 15-20 L / h, the extraction pressure is 350-450 MPa, the extraction temperature is 35-45°C, and the extraction time is 3-5 h.

[0017] Preferably, in step S5, the mixed enzyme is a mixture of alkaline protease and flavor protease.

[0018] Preferably, in step S5, the mass ratio of alkaline protease to flavor protease in the enzyme mixture is 1:1 to 1:3.

[0019] Preferably, in step S5, the enzymatic hydrolysis reaction temperature is 30-50° C., the enzymatic hydrolysis reaction pH is 5-7, and the enzymatic hydrolysis reaction time is 1-2 h.

[0020] Preferably, in step S5, the amount of the enzyme mixture is 0.02% to 0.04% by weight of the residue B.

[0021] Preferably, in step S1, the microwave power is 500-1000 W, the microwave treatment time is 30-60 s, and the microwave treatment temperature is 40-60°C.

[0022] Preferably, in step S1, the sieve mesh number is 40.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The supercritical CO2 extraction coupled with DES extraction method for extracting cornus officinalis seed oil and antioxidant peptides adopted in the present invention can simultaneously extract cornus officinalis seed oil and antioxidant peptides. The cornus officinalis seed oil yield of the present invention is more than 50%, and the antioxidant peptide yield is more than 86%, thereby improving resource utilization.

[0025] (2) The present invention has the advantages of safety, high oil quality, environmental friendliness, and low energy consumption. The prepared cornus officinalis seed oil has a relatively high content of unsaturated fatty acids, a short extraction time, and no problems such as solvent residue. The cornus officinalis seed oil is transparent and bright in color and has a good taste, which maximizes the quality of the cornus officinalis seed oil.

[0026] (3) The antioxidant peptide extracted from the cornus seeds of the present invention can efficiently scavenge DPPH free radicals and ABTS+ free radicals, with a scavenging rate of IC 50 They can reach 3.43mg / mL and 5.59mg / mL respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a process flow chart of the method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with a deep eutectic solvent DES in Example 1 of the present invention;

[0028] Figure 2 This is a graph showing the results of the determination of the DPPH free radical scavenging activity of the cornus seed antioxidant peptide prepared by the method of the present invention;

[0029] Figure 3 This is a graph showing the assay results of the Cornus officinalis seed antioxidant peptide prepared by the method of the present invention for scavenging ABTS+ free radicals. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below through embodiments in conjunction with the accompanying drawings. 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.

[0031] Example 1

[0032] A method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with deep eutectic solvent DES is shown in the process flow chart. Figure 1 The specific steps are as follows:

[0033] S1. Spread cleaned cornus officinalis seeds flatly in a microwave treatment chamber at a microwave power of 500 W, a treatment time of 30 seconds, and a treatment temperature of 50°C. Microwave treatment further destroys cell walls, improving the effectiveness of subsequent treatments. The treated cornus officinalis seeds are oven-dried at low temperature and pulverized through a 40-mesh sieve to obtain cornus officinalis seed powder.

[0034] S2. Add 250 g of the Cornus officinalis seed powder obtained in step S1 into a supercritical CO2 extraction kettle, use a low eutectic solvent as an entrainer, set the flow rate to 3 mL / min, the CO2 flow rate to 15 L / h, set the pressure to 35 MPa, the temperature to 40°C, and perform dynamic extraction for 4 hours to obtain Cornus officinalis seed powder residue A and Cornus officinalis seed oil; the molar ratio of choline chloride, glycerol, and citric acid in the low eutectic solvent is 1:1.5:0.3.

[0035] S3. The residue A obtained in S2 is further mixed with a deep eutectic solvent, subjected to secondary extraction, and filtered to obtain a residue B and a filtrate, wherein the amount of the deep eutectic solvent used is 5% of the molar flow rate of CO2.

[0036] S4. Add 30 times the volume of water to the filtrate obtained in step S3, centrifuge, collect the aqueous phase and the oil phase respectively, concentrate and distill the aqueous phase under reduced pressure to obtain a deep eutectic solvent mixture, further recover and recycle the oil phase, and merge it with the cornus officinalis seed oil obtained in step S2 after reduced pressure distillation to complete the extraction of cornus officinalis seed oil.

[0037] S5. Add 10 times the weight of clean water to the residue B obtained in S3, control the temperature at 40°C, add a mixed enzyme, adjust the pH to 6, perform enzymatic hydrolysis for 1 hour, instantaneously inactivate the enzyme at high temperature, filter, further rotary concentrate the filtrate, and spray dry the obtained concentrate to obtain cornus seed antioxidant peptide; wherein the mixed enzyme is composed of alkaline protease and flavor protease mixed in a mass ratio of 1:1, and the amount of the mixed enzyme accounts for 0.03% of the weight of the residue B.

[0038] Example 2

[0039] A method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with a deep eutectic solvent DES, comprising the following steps:

[0040] S1. Spread cleaned cornus officinalis seeds flatly in a microwave treatment chamber at a microwave power of 600 W, a treatment time of 60 seconds, and a treatment temperature of 60°C. Microwave treatment further destroys cell walls, improving the effectiveness of subsequent treatments. The treated cornus officinalis seeds are oven-dried at low temperature and pulverized through a 40-mesh sieve to obtain cornus officinalis seed powder.

[0041] S2. Add 250 g of the Cornus officinalis seed powder obtained in step S1 into a supercritical CO2 extraction kettle, use a low eutectic solvent as an entrainer, set the flow rate to 5 mL / min, the CO2 flow rate to 20 L / h, set the pressure to 40 MPa, and the temperature to 45°C. Perform dynamic extraction for 5 hours to obtain Cornus officinalis seed powder residue A and Cornus officinalis seed oil; the molar ratio of choline chloride, glycerol, and citric acid in the low eutectic solvent is 1:2:0.5.

[0042] S3. The residue A obtained in S2 is further mixed with a deep eutectic solvent, subjected to secondary extraction, and filtered to obtain a residue B and a filtrate, wherein the amount of the deep eutectic solvent used is 3% of the molar flow rate of CO2.

[0043] S4. Add 40 times the volume of water to the filtrate obtained in step S3, centrifuge, collect the aqueous phase and the oil phase, respectively, concentrate and distill the aqueous phase under reduced pressure to obtain a deep eutectic solvent mixture, further recover and recycle the oil phase, and merge it with the cornus officinalis seed oil obtained in step S2 after reduced pressure distillation to complete the extraction of cornus officinalis seed oil.

[0044] S5. Add 10 times the weight of clean water to the residue B obtained in S3, control the temperature at 50°C, add a mixed enzyme, adjust the pH to 7, perform enzymatic hydrolysis for 2 hours, instantaneously inactivate the enzyme at high temperature, filter, further rotary concentrate the filtrate, and spray dry the obtained concentrate to obtain cornus seed antioxidant peptide; wherein the mixed enzyme is composed of alkaline protease and flavor protease mixed in a mass ratio of 1:2, and the amount of the mixed enzyme accounts for 0.025% of the weight of the residue B.

[0045] Example 3

[0046] A method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with a deep eutectic solvent DES, comprising the following steps:

[0047] S1. Spread cleaned cornus officinalis seeds flatly in a microwave treatment chamber at a microwave power of 1000 W, a treatment time of 60 seconds, and a treatment temperature of 50°C. Microwave treatment further destroys cell walls, improving the effectiveness of subsequent treatments. The treated cornus officinalis seeds are oven-dried at low temperature and pulverized through a 40-mesh sieve to obtain cornus officinalis seed powder.

[0048] S2. Add 250 g of the Cornus officinalis seed powder obtained in step S1 to a supercritical CO2 extraction kettle, use a low eutectic solvent as an entrainer, set the flow rate to 5 mL / min, the CO2 flow rate to 18 L / h, set the pressure to 40 MPa, the temperature to 35°C, and perform dynamic extraction for 4 hours to obtain Cornus officinalis seed powder residue A and Cornus officinalis seed oil; the molar ratio of choline chloride, glycerol, and citric acid in the low eutectic solvent is 1:2.5:0.7.

[0049] S3. The residue A obtained in S2 is further mixed with a deep eutectic solvent, subjected to secondary extraction, and filtered to obtain a residue B and a filtrate, wherein the amount of the deep eutectic solvent used is 4% of the molar flow rate of CO2.

[0050] S4. Add 30 times the volume of water to the filtrate obtained in step S3, centrifuge, collect the aqueous phase and the oil phase respectively, concentrate and distill the aqueous phase under reduced pressure to obtain a deep eutectic solvent mixture, further recover and recycle the oil phase, and merge it with the cornus officinalis seed oil obtained in step S2 after reduced pressure distillation to complete the extraction of cornus officinalis seed oil.

[0051] S5. Add 10 times the weight of clean water to the residue B obtained in S3, control the temperature at 45°C, add a mixed enzyme, adjust the pH to 6, perform enzymatic hydrolysis for 1.5 hours, inactivate the enzyme at instantaneous high temperature, filter, further rotary concentrate the filtrate, and spray dry the obtained concentrate to obtain cornus seed antioxidant peptide; wherein the mixed enzyme is composed of alkaline protease and flavor protease mixed in a mass ratio of 1:2, and the amount of the mixed enzyme accounts for 0.035% of the weight of the residue B.

[0052] Example 4

[0053] A method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with a deep eutectic solvent DES, comprising the following steps:

[0054] S1. Spread cleaned cornus officinalis seeds flatly in a microwave treatment chamber at a microwave power of 900 W, a treatment time of 40 seconds, and a treatment temperature of 40°C. Microwave treatment further destroys cell walls, improving the effectiveness of subsequent treatments. The treated cornus officinalis seeds are oven-dried at low temperature and pulverized through a 40-mesh sieve to obtain cornus officinalis seed powder.

[0055] S2. Add 250 g of the Cornus officinalis seed powder obtained in step S2 to a supercritical CO2 extraction kettle, use a low eutectic solvent as an entrainer, set the flow rate to 4 mL / min, the CO2 flow rate to 16 L / h, set the pressure to 45 MPa, and the temperature to 45°C. Perform dynamic extraction for 3 hours to obtain Cornus officinalis seed powder residue A and Cornus officinalis seed oil; the molar ratio of choline chloride, glycerol, and citric acid in the low eutectic solvent is 1:2:0.5.

[0056] S3. The residue A obtained in S2 is further mixed with a deep eutectic solvent, subjected to secondary extraction, and filtered to obtain a residue B and a filtrate, wherein the amount of the deep eutectic solvent used is 6% of the molar flow rate of CO2.

[0057] S4. Add 40 times the volume of water to the filtrate obtained in step S3, centrifuge, collect the aqueous phase and the oil phase, respectively, concentrate and distill the aqueous phase under reduced pressure to obtain a deep eutectic solvent mixture, further recover and recycle the oil phase, and merge it with the cornus officinalis seed oil obtained in step S2 after reduced pressure distillation to complete the extraction of cornus officinalis seed oil.

[0058] S5. Add 10 times the weight of clean water to the residue B obtained in S3, control the temperature at 30°C, add a mixed enzyme, adjust the pH to 5, perform enzymatic hydrolysis for 1.5 hours, instantaneously inactivate the enzyme at high temperature, filter, further rotary concentrate the filtrate, and spray dry the obtained concentrate to obtain cornus seed antioxidant peptide; wherein the mixed enzyme is composed of alkaline protease and flavor protease mixed in a mass ratio of 1:1, and the amount of the mixed enzyme accounts for 0.03% of the weight of the residue B.

[0059] Example 5

[0060] A method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with a deep eutectic solvent DES, comprising the following steps:

[0061] S1. Spread cleaned cornus officinalis seeds flatly in a microwave treatment chamber at a microwave power of 1000 W, a treatment time of 60 seconds, and a treatment temperature of 60°C. Microwave treatment further destroys cell walls, improving the effectiveness of subsequent treatments. The treated cornus officinalis seeds are oven-dried at low temperature and pulverized through a 40-mesh sieve to obtain cornus officinalis seed powder.

[0062] S2. Add 250 g of the Cornus officinalis seed powder obtained in step S1 to a supercritical CO2 extraction kettle, use a low eutectic solvent as an entrainer, set the flow rate to 5 mL / min, the CO2 flow rate to 20 L / h, set the pressure to 40 MPa, and the temperature to 45°C. Perform dynamic extraction for 5 hours to obtain Cornus officinalis seed powder residue A and Cornus officinalis seed oil; the molar ratio of choline chloride, glycerol, and citric acid in the low eutectic solvent is 1:2.5:0.7.

[0063] S3. The residue A obtained in S2 is further mixed with a deep eutectic solvent, subjected to secondary extraction, and filtered to obtain a residue B and a filtrate, wherein the amount of the deep eutectic solvent used is 7% of the molar flow rate of CO2.

[0064] S4. Add 30 times the volume of water to the filtrate obtained in step S3, centrifuge, collect the aqueous phase and the oil phase respectively, concentrate and distill the aqueous phase under reduced pressure to obtain a deep eutectic solvent mixture, further recover and recycle the oil phase, and merge it with the cornus officinalis seed oil obtained in step S2 after reduced pressure distillation to complete the extraction of cornus officinalis seed oil.

[0065] S5. Add 10 times the weight of clean water to the residue B obtained in S3, control the temperature at 40°C, add a mixed enzyme, adjust the pH to 7, perform enzymatic hydrolysis for 2 hours, instantaneously inactivate the enzyme at high temperature, filter, further rotary concentrate the filtrate, and spray dry the obtained concentrate to obtain cornus seed antioxidant peptide; wherein the mixed enzyme is composed of alkaline protease and flavor protease mixed in a mass ratio of 1:1, and the amount of the mixed enzyme accounts for 0.035% of the weight of the residue B.

[0066] Comparative Example 1

[0067] A deep eutectic solvent method for preparing cornus officinalis seed oil comprises the following steps:

[0068] S1. Spread cleaned cornus officinalis seeds flatly in a microwave treatment chamber at a microwave power of 1000 W, a treatment time of 60 seconds, and a treatment temperature of 60°C. Microwave treatment further destroys cell walls, improving the effectiveness of subsequent treatments. The treated cornus officinalis seeds are oven-dried at low temperature and pulverized through a 40-mesh sieve to obtain cornus officinalis seed powder.

[0069] S2. Add 250 g of the Cornus officinalis seed powder obtained in step S1 to 1 L of a deep eutectic solvent for extraction. The extraction time is 3 hours. After the extraction is completed, filter to obtain a residue and a filtrate; wherein the molar ratio of choline chloride, glycerol, and citric acid in the deep eutectic solvent is 1:2.5:0.7.

[0070] S3. Add 30 times the volume of water to the filtrate obtained in step S2, centrifuge, collect the aqueous phase and the oil phase respectively, concentrate and distill the aqueous phase under reduced pressure to obtain a deep eutectic solvent mixture, further recover and recycle the oil phase, and obtain Cornus officinalis seed oil after reduced pressure distillation.

[0071] Comparative Example 2

[0072] A supercritical CO2 extraction method is used to prepare cornus officinalis seed oil, and the specific steps are as follows:

[0073] S1. Spread cleaned cornus officinalis seeds flatly in a microwave treatment chamber at a microwave power of 1000 W, a treatment time of 60 seconds, and a treatment temperature of 60°C. Microwave treatment further destroys cell walls, improving the effectiveness of subsequent treatments. The treated cornus officinalis seeds are oven-dried at low temperature and pulverized through a 40-mesh sieve to obtain cornus officinalis seed powder.

[0074] S2. Weigh 250.00 g of the cornus officinalis seed powder obtained in step S1, add the cornus officinalis seed powder into a supercritical CO2 extraction kettle, set the CO2 flow rate to 20 L / h, set the pressure to 45 MPa, and the temperature to 45°C, and perform dynamic extraction for 3 hours. After the extraction, cornus officinalis seed oil is obtained.

[0075] Comparative Example 3

[0076] A method for ultrasonically extracting antioxidant peptides from cornus officinalis seeds, comprising the following steps:

[0077] The method is basically the same as Example 1, except that: in step S5, ultrasonic treatment is used to extract cornus seed antioxidant peptides, and the specific steps are: adding 10 times the weight of clean water to the residue B obtained in S3, setting the ultrasonic power to 1000 W, the time to 20 min, and the initial temperature to 40°C. After the ultrasonication is completed, centrifugation is performed, the supernatant is taken, concentrated, and dried to obtain cornus seed antioxidant peptides.

[0078] 1. The oil yield of cornus officinalis seed oil and the yield of cornus officinalis seed antioxidant peptides of the present invention:

[0079] (1) Determination of Cornus officinalis seed oil yield

[0080] The oil yield of the cornus officinalis seed oil obtained in Examples 1 to 5 and Comparative Examples 1 to 2 was calculated using the following formula:

[0081]

[0082] The oil yield corresponds to the mass of the cornus officinalis seed oil after combining the steps S4 of Examples 1 to 5, and the mass of the cornus officinalis seed oil prepared in Comparative Examples 1 and 2, respectively. The total amount of raw materials is the mass of the cornus officinalis seed powder.

[0083] (2) Determination of the yield of antioxidant peptides from Cornus officinalis seeds

[0084] The yield of cornus officinalis seed antioxidant peptides obtained in Examples 1-5 and Comparative Example 3 was calculated. 1 mL of the supernatant from step S5 of Examples 1-5 and Comparative Example 3 was mixed with an equal volume of 15 g / 100 mL trichloroacetic acid solution. The mixture was allowed to stand for 10 minutes, then centrifuged at 8000 rpm for 5 minutes. The antioxidant peptide content in the supernatant was determined using the biuret method. The protein mass of cornus officinalis seeds (raw material protein mass) was determined using the Kjeldahl method. The calculation formula is as follows:

[0085]

[0086] The calculated cornus officinalis seed oil yield and cornus officinalis seed antioxidant peptide yield are shown in Table 1 below.

[0087] Table 1 Cornus officinalis seed oil yield and Cornus officinalis seed antioxidant peptide yield

[0088] sample Cornus officinalis seed oil yield (%) Yield of antioxidant peptides from cornus officinalis seeds (%) Example 1 51.3 87.8 Example 2 52.2 86.5 Example 3 50.7 89.4 Example 4 51.4 88.2 Example 5 50.2 87.6 Comparative Example 1 15.2 / Comparative Example 2 39.3 / Comparative Example 3 / 64.5

[0089] As shown in Table 1, the method of the present invention can simultaneously extract cornus officinalis seed oil and cornus officinalis seed antioxidant peptides, with the cornus officinalis seed oil yield exceeding 50% and the cornus officinalis seed antioxidant peptide yield exceeding 86%, thereby improving resource utilization.

[0090] 2. Cornus officinalis seed oil index test of the present invention:

[0091] The physical and chemical properties of the cornus officinalis seed oils prepared in Examples 1-5 and Comparative Examples 1-2 were tested. The performance indicators and testing methods included: acid value according to GB / T 5530-2005; peroxide value according to GB / T 5538-2005; saponification value according to GB / T 5534-2024; fatty acid content according to GB / T 22223-2008; and tocopherol content according to GB / T 26635-2011. Specific test results are shown in Table 2 below.

[0092] Table 2 Test results of physical and chemical indicators of Cornus officinalis seed oil

[0093]

[0094] As shown in Table 2, the unsaturated fatty acid content of the cornus officinalis seed oil extracted by the present invention is higher than 80%, and the total amount of tocopherol is higher than that of the control group, which helps to reduce the trans fatty acid content, improve arteriosclerosis and thrombosis, and regulate blood lipid levels, indicating that the method of the present invention can better retain the active ingredients.

[0095] 3. Determination of the antioxidant capacity of the cornus seed antioxidant peptide prepared by the present invention

[0096] (1) DPPH free radical scavenging rate determination

[0097] The DPPH free radical scavenging rate of the cornus seed antioxidant peptides of Examples 1 to 5 and Comparative Example 3 was determined as follows: 2 mL of samples of different concentrations were accurately weighed and placed in a test tube, and then 2 mL of 2×10 -4 mol / LDPPH solution, shake well, and let it stand at room temperature for 30 minutes. Measure its absorbance at a wavelength of 517 nm to obtain the absorbance of the sample group, which is recorded as A1. Add 2 mL of DPPH solution to 2 mL of ethanol and mix well. Measure its absorbance at 517 nm to obtain the absorbance of the blank group, which is recorded as A0. Calculate the DPPH free radical scavenging rate according to the following formula:

[0098] DPPH free radical scavenging rate % = (A0-A1) / A0×100%

[0099] Where: A1 is the absorbance of the sample group, and A0 is the absorbance of the blank group.

[0100] The DPPH free radical scavenging rates of the cornus officinalis seed antioxidant peptides of Examples 1 to 5 of the present invention and Comparative Example 3 are shown in Table 3.

[0101] Table 3 Scavenging rate of DPPH free radicals by cornus seed antioxidant peptides

[0102]

[0103] As shown in Table 3, the cornus officinalis seed antioxidant peptide extracted by the present invention has a significant scavenging effect on DPPH free radicals. The linear relationship diagram of the DPPH scavenging rate of cornus officinalis seed antioxidant peptides at different concentrations was drawn using Origin2019b software, as shown in Figure 2 As shown, the IC of the clearance corresponding to Examples 1 to 5 was calculated using the DoseRsep function nonlinear fitting. 50 The distribution range of the values ​​is (3.43~3.51) mg / mL. The IC value of comparative example 3 for DPPH free radical scavenging rate is 50 It is 7.80 mg / mL, indicating that the cornus seed antioxidant peptide obtained by the present invention has better scavenging ability for DPPH free radicals than comparative example 3.

[0104] (2) ABTS+ free radical scavenging rate determination

[0105] The ABTS+ free radical scavenging rate of the cornus seed antioxidant peptides prepared in Examples 1 to 5 and Comparative Example 3 was determined as follows: a Trolox standard solution (0–1000 μM) was prepared, and different concentrations of Trolox solution (e.g., 20 μL) were mixed with ABTS + The working solution (180 μL) was mixed and reacted at room temperature for 6 min. The absorbance (Abs) at 734 nm was measured and the clearance-concentration curve was plotted.

[0106] Take 20 μL of sample solution (needs gradient dilution) and 180 μL ABTS + Mix the working solution and react at room temperature in the dark for 6 minutes. Measure the absorbance at 734 nm (A1). For a blank control, replace the sample with ethanol and measure A0. Calculate the ABTS+ free radical scavenging rate according to the following formula:

[0107] ABTS+ free radical scavenging rate % = (A0-A1) / A0×100%

[0108] Where: A1 is the absorbance of the sample group, and A0 is the absorbance of the blank control group.

[0109] The ABTS+ free radical scavenging rates of cornus officinalis seed antioxidant peptides of Examples 1 to 5 and Comparative Example 3 are shown in Table 4.

[0110] Table 4 Scavenging rate of ABTS+ free radicals by cornus seed antioxidant peptides

[0111]

[0112] As shown in Table 4, the cornus officinalis seed antioxidant peptide extracted by the present invention has a significant scavenging effect on ABTS+ free radicals. The linear relationship diagram of the ABTS+ scavenging rate of cornus officinalis seed antioxidant peptides at different concentrations was drawn using Origin2019b software, as shown in Figure 3 As shown, the IC of the clearance corresponding to Examples 1 to 5 was calculated using the DoseRsep function nonlinear fitting. 50 The distribution range of values ​​is (5.59~6.42) mg / mL. The IC value of comparative example 3 for ABTS+ free radical scavenging rate is 50 14.22 mg / mL, IC 50 The lower the value, the stronger the antioxidant ability, which indicates that the cornus seed antioxidant peptide obtained in the present invention has better scavenging ability against ABTS+ free radicals than comparative example 3.

[0113] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the present invention. Those skilled in the art can modify or replace the technical solutions of the present invention according to the concept of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by coupling supercritical CO2 extraction with a deep eutectic solvent (DES), characterized in that: The following steps are involved: S1. Microwave-treating cornus officinalis seeds, drying, crushing, and sieving to obtain cornus officinalis seed powder; S2, adding the cornus officinalis seed powder prepared in step S1 to a supercritical CO2 extraction reactor, using a deep eutectic solvent as an entrainer, and simultaneously introducing the deep eutectic solvent and CO2 into the reactor for extraction, and obtaining residue A and cornus officinalis seed oil after the extraction is completed; S3, mixing the residue A with the deep eutectic solvent for secondary extraction, and filtering after the extraction to obtain a residue B and a filtrate; S4, adding water to the filtrate obtained in step S3, mixing and centrifuging, collecting the aqueous phase and the oil phase respectively, recovering the deep eutectic solvent in the aqueous phase for recycling, and combining the oil phase with the cornus seed oil obtained in step S2 after vacuum distillation to complete the extraction of cornus seed oil; S5. Add the residue B obtained in step S3 into water and mix well to obtain a mixture, add a mixed enzyme to the mixture for enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis reaction, filter to obtain a filtrate, and concentrate and dry the filtrate to obtain the cornus seed antioxidant peptide.

2. The method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with deep eutectic solvent DES according to claim 1, characterized in that: In step S2 and step S3, the deep eutectic solvent is prepared by mixing choline chloride, glycerol and citric acid.

3. The method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with deep eutectic solvent DES according to claim 2, characterized in that: The molar ratio of choline chloride, glycerol and citric acid in the deep eutectic solvent is 1: (1.5-2.5): (0.3-0.7).

4. The method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with deep eutectic solvent DES according to claim 1, characterized in that: In step S2, the flow rate of the deep eutectic solvent is 2 to 5 mL / min; in step S3, the amount of the deep eutectic solvent used is 3% to 7% of the molar flow rate of CO2.

5. The method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with deep eutectic solvent DES according to claim 1, characterized in that: In step S2, the CO2 flow rate is 15-20 L / h, the extraction pressure is 350-450 MPa, the extraction temperature is 35-45°C, and the extraction time is 3-5 h.

6. The method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with deep eutectic solvent DES according to claim 1, characterized in that: In step S5, the mixed enzyme is prepared by mixing alkaline protease and flavor protease.

7. The method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with deep eutectic solvent DES according to claim 6, characterized in that: In step S5, the mass ratio of alkaline protease to flavor protease in the enzyme mixture is 1:1 to 1:

3.

8. The method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with deep eutectic solvent DES according to claim 1, characterized in that: In step S5, the enzymatic hydrolysis reaction temperature is 30-50° C., the enzymatic hydrolysis reaction pH is 5-7, and the enzymatic hydrolysis reaction time is 1-2 h.

9. The method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with deep eutectic solvent DES according to claim 1, characterized in that: In step S5, the amount of the mixed enzyme is 0.02% to 0.04% by weight of the residue B.

10. The method for synergistically extracting cornus officinalis seed oil and antioxidant peptides by supercritical CO2 extraction coupled with deep eutectic solvent DES according to claim 1, characterized in that: In step S1, the microwave power is 500-1000 W, the microwave treatment time is 30-60 s, and the microwave treatment temperature is 40-60° C.

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