Dark tea antihypertensive peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioactive peptides, and discloses a dark tea antihypertensive peptide as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) adding water and a compound enzyme into a dark tea raw material for enzymolysis; (2) adjusting the pH value of the enzymatic hydrolysate to 5.0-12.0, adding alkaline protease for enzymolysis, centrifuging, taking supernatant, and freeze-drying the supernatant to obtain dark green tea protein powder; (3) adding the dark tea protein powder into water, and carrying out ultrasonic-pulsed electric field cooperative treatment; (4) carrying out enzymolysis treatment by adopting subtilisin and flavourzyme in sequence; and (5) carrying out multi-stage membrane separation and purification, sequentially removing unhydrolyzed protein, small molecule peptide fragments and salt in the supernatant to obtain a purified solution, and carrying out vacuum freeze drying treatment on the purified solution to obtain the dark tea antihypertensive peptide. The ultrasonic-pulsed electric field is adopted to fully stretch the protein, the enzymolysis efficiency is improved, the compound enzyme synergistic hydrolysis technology is adopted to extract the dark tea antihypertensive peptide, the preparation method is simple, the cost is low, the enzyme dosage is reduced, and the safety is good.

Description

Technical Field

[0001] The invention belongs to the technical field of active peptides, and specifically relates to a dark tea antihypertensive peptide and a preparation method and application thereof. Background Art

[0002] Hypertension is the most common cardiovascular disease, with more than 15 million deaths worldwide each year. Hypertension has become one of the "number one killers" threatening human health. The treatment of hypertension is not only about lowering blood pressure, but also about reducing the risk of coronary heart disease, stroke, kidney damage and other diseases. ACE inhibitors, as effective drugs for blood pressure regulation, are the first-line drugs for the treatment of hypertension. Synthetic ACE inhibitors (ACEI) have a series of side effects during their use. To solve these difficulties, it is urgent to develop innovative drugs with low side effects and strengthen basic research to explore the pathological mechanisms and new therapies of hypertension.

[0003] Active peptides are small molecule protein fragments composed of amino acids, which are easily absorbed by the human body and have strong targeting properties. They can be obtained through food and synthesis by modern biotechnology. Currently, active peptides have been widely used in the fields of drug development, health food and cosmetics, and play an important role in enhancing immunity and regulating blood pressure.

[0004] Traditional methods for extracting antihypertensive peptides have problems such as low efficiency, poor purity, and complex processes. For example, single enzymatic hydrolysis has poor selectivity for protein substrates, insufficient release rate of active peptides, resulting in uneven molecular weight distribution of peptides, or harmful residues introduced by solvent extraction. The separation process is cumbersome, and high temperature or extreme pH conditions destroy the peptide structure and seriously reduce biological activity. Therefore, it is urgent to develop an efficient, low-cost, and industrially suitable antihypertensive peptide extraction technology. Summary of the invention

[0005] In view of the problems and shortcomings in the prior art, the present invention aims to provide a dark tea antihypertensive 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 a dark tea antihypertensive peptide, comprising the following steps:

[0008] (1) crushing the dried dark tea, adding water and complex enzyme to the dark tea, and performing enzymolysis at 25-60° C. to obtain an enzymolysis solution;

[0009] (2) adjusting the pH of the enzymatic hydrolyzate prepared in step (1) to 5.0-12.0, adding alkaline protease to the enzymatic hydrolyzate, performing enzymatic hydrolysis at 30-75° C., centrifuging to obtain a supernatant after the enzymatic hydrolysis is completed, and freeze-drying the supernatant to obtain dark tea protein powder;

[0010] (3) Add water to the dark tea protein powder obtained in step (2), mix well to obtain a mixed solution, and then treat the mixed solution with ultrasonic waves and pulsed electric fields simultaneously. After the treatment, a stock solution is obtained.

[0011] (4) Sequentially use subtilisin and flavor protease to carry out enzymatic hydrolysis treatment on the stock solution prepared in step (3). After the enzymatic hydrolysis is completed, carry out enzyme inactivation treatment, and then centrifuge. The supernatant is reserved for use.

[0012] (5) Carry out multi-stage membrane separation and purification on the supernatant obtained in step (4), sequentially remove the unhydrolyzed protein, small molecular peptide segments and salts in the supernatant to obtain a purified solution. The purified solution is subjected to vacuum freeze-drying treatment to obtain dark tea antihypertensive peptide.

[0013] Preferably, the hydrolysis conditions of subtilisin in step (4) are: pH is 2.0 - 9.0, and the temperature is 10 - 60 °C; the hydrolysis conditions of flavor protease are: pH is 3.5 - 8.5, and the temperature is 20 - 65 °C.

[0014] Preferably, the addition amount of subtilisin in step (4) is 0.8% - 3.0% of the total mass of the stock solution, and the enzymatic hydrolysis time is 2 - 5 h; the addition amount of flavor protease is 0.8% - 3.2% of the total mass of the stock solution, and the enzymatic hydrolysis time is 1 - 7 h.

[0015] Preferably, the composite enzyme in step (1) is composed of cellulase and pectinase according to a mass ratio of 2:1, and the addition amount of the composite enzyme is 0.1% - 0.5% of the mass of the dark tea.

[0016] Preferably, the ultrasonic power in step (3) is 100 - 500 W, the pulsed electric field intensity is 5 - 40 kV / CM, and the treatment time of ultrasonic waves and pulsed electric fields is 30 - 120 min.

[0017] Preferably, the addition amount of alkaline protease in step (2) is 0.1% - 1.3% of the mass of the dark tea.

[0018] Preferably, in step (5), an ultrafiltration membrane is used to remove unhydrolyzed macromolecular proteins; a nanofiltration membrane is used to retain small molecular peptide segments; a reverse osmosis membrane is used for desalination.

[0019] More preferably, the reverse osmosis membrane in step (5) is a polyethersulfone composite membrane.

[0020] Preferably, the cut-off molecular weight of the ultrafiltration membrane in step (5) is 100 kDa, and the cut-off molecular weight of the nanofiltration membrane is 1 - 5 kDa.

[0021] Preferably, the enzymatic hydrolysis time in step (1) is 1 - 5 h, and the enzymatic hydrolysis time in step (2) is 0.5 - 4 h.

[0022] Preferably, after the enzymatic hydrolysis in steps (1) and (2) is completed, the temperature is lowered to 4 °C to terminate the reaction.

[0023] Preferably, the amount of water added in steps (1) and (3) is calculated based on a solid-to-liquid ratio of 1:(5 - 30).

[0024] Preferably, the dark tea in step (1) is pulverized to 50 - 100 mesh.

[0025] In the second aspect of the present invention, a dark tea antihypertensive peptide prepared by any of the methods in the first aspect is provided.

[0026] In the third aspect of the present invention, an application of the dark tea antihypertensive peptide described in the second aspect in the preparation of antihypertensive drugs is provided.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) First, the present invention enzymatically hydrolyzes dark tea with a composite enzyme to disrupt the cell wall. The enzymatic hydrolysate is subjected to targeted hydrolysis of the protein-polyphenol complex by alkaline protease to obtain dark tea protein powder. Ultrasonic-pulsed electric field is applied to the solid-to-liquid of the dark tea protein powder to fully unfold the protein, improving the enzymatic hydrolysis efficiency in the next step. Then, a composite enzyme synergistic hydrolysis technology is used to extract dark tea antihypertensive peptides. The enzymatic hydrolysis step adopts sequential enzymatic hydrolysis with subtilisin and flavor protease. The former cuts off hydrophobic amino acid sites, and the latter further degrades large molecular peptide segments to release highly active small molecular antihypertensive peptides.

[0029] (2) The small molecular antihypertensive peptides pass through a multi-stage membrane separation and purification system and are vacuum freeze-dried to obtain antihypertensive peptides with a smaller molecular weight, easier to be absorbed by the human body, and higher ACE inhibition rate. The yield of the dark tea polypeptide prepared by the method of the present invention can reach 32%, the purity reaches 90%, and the ACE inhibition rate reaches 87%.

[0030] (3) The method for preparing the dark tea antihypertensive peptide of the present invention is simple, low in cost, less in enzyme dosage, and good in safety. Specific Embodiments

[0031] To make the purpose, technical solutions and advantages of the present invention clearer and more definite, the present invention is further described in detail below through examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] Example 1

[0033] A method for preparing a dark tea antihypertensive peptide, the specific steps are as follows:

[0034] (1) Dry the dark tea raw materials and then crush them to 80 mesh. Add deionized water at a material-liquid ratio of 1:15. Add a composite enzyme accounting for 0.3% of the mass of the dark tea for pretreatment, and carry out enzymatic hydrolysis at 45 °C for 3 h. Immediately cool down to 4 °C to terminate the enzymatic hydrolysis, obtaining an enzymatic hydrolysate. The composite enzyme is composed of cellulase and pectinase at a mass ratio of 2:1;

[0035] (2) Adjust the pH of the enzymatic hydrolysate in step (1) to 9.0, add an alkaline protease accounting for 0.8% of the mass of the dark tea, and carry out enzymatic hydrolysis at 50 °C for 2 h to release the bound proteins. Immediately cool down to 4 °C to terminate the enzymatic hydrolysis, centrifuge at 8000 rpm for 15 min, and take the supernatant. Freeze-dry the supernatant to obtain dark tea protein powder;

[0036] (3) Add deionized water to the dark tea protein powder obtained in step (2) at a material-liquid ratio of 1:15, and carry out ultrasonic-pulsed electric field synergistic treatment for 80 min. After the treatment, obtain the stock solution, where the ultrasonic power is 300 W and the pulsed electric field strength is 25 kV / cm;

[0037] (4) Enzymatically hydrolyze the stock solution obtained in step (3) successively with subtilisin and flavor protease: First, under the conditions of the optimal pH 7.0 and temperature 45 °C, add subtilisin accounting for 1.5% of the total mass of the stock solution for enzymatic hydrolysis for 2 h. Adjust the pH of the enzymatic hydrolysate after subtilisin enzymatic hydrolysis to 6.5, and then continue to add flavor protease accounting for 1.5% of the total mass of the stock solution for enzymatic hydrolysis for 2 h at a temperature of 45 °C. Then carry out enzyme inactivation treatment, heat at 85 °C for 10 min, cool down, centrifuge at 4000 r / min for 15 min, and reserve the supernatant;

[0038] (5) Purify the supernatant in step (4) by multi-stage membrane separation: First, use a 100 kDa ultrafiltration membrane to remove the unhydrolyzed macromolecular proteins from the above enzymatic hydrolysate. Then use a series of 5 kDa and 1 kDa nanofiltration membranes to retain the peptide segments with a target molecular weight of 1 - 5 kDa. Finally, use a reverse osmosis membrane polyethersulfone (PES) composite membrane to desalt and concentrate to a solid content of ≥20%, and make it into a powder by vacuum freeze-drying (-40 °C, 0.1 Pa), which is the dark tea antihypertensive peptide.

[0039] Example 2

[0040] A preparation method of dark tea antihypertensive peptide, the specific steps are as follows:

[0041] (1) Dry the dark tea raw materials and then crush them to 50 mesh. Add deionized water at a material-liquid ratio of 1:5. Add a composite enzyme accounting for 0.1% of the mass of the dark tea for pretreatment, and carry out enzymatic hydrolysis at 25 °C for 5 h. Immediately cool down to 4 °C to terminate the enzymatic hydrolysis, obtaining an enzymatic hydrolysate. The composite enzyme is composed of cellulase and pectinase at a mass ratio of 2:1;

[0042] (2) Adjust the pH of the enzymatic hydrolysate in step (1) to 5.0, add alkaline protease at 0.1% of the mass of the dark tea, and carry out enzymatic hydrolysis at 35 °C for 0.5 h to target the hydrolysis of protein-polyphenol complexes and release bound proteins; immediately cool down to 4 °C to terminate the enzymatic hydrolysis, centrifuge at 8000 rpm for 15 min to obtain the supernatant, and freeze-dry the supernatant to obtain dark tea protein powder;

[0043] (3) Add water to the dark tea protein powder obtained in step (2) at a solid-liquid ratio of 1:15, and perform ultrasonic-pulsed electric field synergistic treatment for 30 min to obtain the stock solution, where the ultrasonic power is 100 W and the pulsed electric field strength is 5 kV / cm;

[0044] (4) Enzymatically hydrolyze the stock solution obtained in step (3) successively with subtilisin and flavor protease: First, under the conditions of the optimal pH of 7.0 and temperature of 10 °C, add subtilisin at 0.8% of the total mass of the stock solution and carry out enzymatic hydrolysis for 1 h; adjust the pH of the enzymatic hydrolysate after subtilisin enzymatic hydrolysis to 6.5, then raise the temperature to 20 °C, add flavor protease at 1.2% of the total mass of the stock solution and carry out enzymatic hydrolysis for 4 h; then perform enzyme inactivation treatment, heat at 85 °C for 10 min, cool down and centrifuge at 4000 r / min for 15 min, and reserve the supernatant;

[0045] (5) Purify the supernatant in step (4) by multi-stage membrane separation: First, use a 100 kDa ultrafiltration membrane to remove unhydrolyzed macromolecular proteins from the above enzymatic hydrolysate; then use a series of 5 kDa and 1 kDa nanofiltration membranes to retain peptide segments with a target molecular weight of 1-5 kDa; finally, use a reverse osmosis membrane polyethersulfone (PES) composite membrane to desalt and concentrate to a solid content of ≥20%, and make it into powder by vacuum freeze-drying (-40 °C, 0.1 Pa), which is the dark tea antihypertensive peptide.

[0046] Example 3

[0047] A preparation method of dark tea antihypertensive peptide, the specific steps are as follows:

[0048] (1) Dry and crush the dark tea raw material to 50 meshes, and add deionized water at a solid-liquid ratio of 1:30; add 0.5% of the composite enzyme of the mass of the dark tea for pretreatment, and carry out enzymatic hydrolysis at 60 °C for 5 h, immediately cool down to 4 °C to terminate the enzymatic hydrolysis, and obtain the enzymatic hydrolysate, where the composite enzyme is composed of cellulase and pectinase in a mass ratio of 2:1;

[0049] (2) Adjust the pH of the enzymatic hydrolysate in step (1) to 12.0, add alkaline protease at 1.3% of the mass of the dark tea, and carry out enzymatic hydrolysis at 75 °C for 2 h to release bound proteins; immediately cool down to 4 °C to terminate the enzymatic hydrolysis, centrifuge at 8000 rpm for 15 min to obtain the supernatant, and freeze-dry the supernatant to obtain dark tea protein powder;

[0050] (3) Add water to the dark tea protein powder obtained in step (2) at a material-liquid ratio of 1:15, and perform ultrasonic-pulsed electric field co-treatment for 120 min to obtain the stock solution, where the ultrasonic power is 500 W and the pulsed electric field intensity is 40 kV / cm;

[0051] (4) Enzymatically hydrolyze the stock solution obtained in step (3) successively with subtilisin and flavor protease: First, under the conditions of the optimal pH of 7.0 and temperature of 60 °C, add 3.0% of the total mass of the stock solution of subtilisin and enzymatically hydrolyze for 5 h; adjust the pH of the enzymatic hydrolysate after subtilisin hydrolysis to 6.5, then raise the temperature to 65 °C, add 3.2% of the total mass of the stock solution of flavor protease and enzymatically hydrolyze for 7 h; then perform enzyme inactivation treatment, heat at 85 °C for 10 min, cool and centrifuge at 4000 r / min for 15 min, and reserve the supernatant;

[0052] (5) Perform multi-stage membrane separation and purification on the supernatant in step (4): First, use a 100 kDa ultrafiltration membrane to remove the unhydrolyzed macromolecular proteins from the above enzymatic hydrolysate; then use a series of 5 kDa and 1 kDa nanofiltration membranes to retain the peptide segments with a target molecular weight of 1-5 kDa; finally, use a reverse osmosis membrane polyethersulfone (PES) composite membrane to desalt and concentrate to a solid content of ≥20%, and make it into a powder by vacuum freeze-drying (-40 °C, 0.1 Pa), which is the dark tea antihypertensive peptide.

[0053] Determination of the indicators of the dark tea antihypertensive peptide:

[0054] (1) Determination of the yield

[0055] Determine the yield of the dark tea antihypertensive peptide prepared in the example, and the calculation formula is as follows:

[0056]

[0057] Among them, the mass of the antihypertensive peptide is the mass of the finished product of the dark tea antihypertensive peptide in step (5), and the mass of the raw material is the mass of the dark tea raw material in step (1).

[0058] (2) Determination of the purity

[0059] Determine the purity of the dark tea antihypertensive peptide prepared in the example, and the method is as follows:

[0060] Filler pretreatment: Weigh 25 g of Sephadex G-10 dry gel powder in a beaker and add an appropriate amount of ultrapure water. Weigh in the beaker, add an appropriate amount of ultrapure water and stir, then soak it at room temperature for 24 h to completely dissolve it. Column packing: Select a chromatographic column of 16 mm × 60 cm, pour the dissolved filler into the chromatographic column, and pay attention not to have air bubbles. Sample: Prepare the freeze-dried black tea antihypertensive peptide prepared in the example into 100 mg / mL, and then pass it through a 0.22 μm aqueous filter membrane, and take 10 mL of the sample. Collection: The eluent is ultrapure water; the flow rate is: 0.2 mL / min; the detection wavelength: 220 nm; the collection time: A: 210 - 290 min, B: 370 - 460 min, C: 370 - 460 min. Collect the peptide peak, freeze-dry it to obtain high-purity antihypertensive peptide.

[0061]

[0062] (3) Determination of ACE inhibitory rate

[0063] Perform ACE inhibitory rate determination on the black tea antihypertensive peptide prepared in the example. The determination method of angiotensin-converting enzyme (ACE) inhibitory rate is as follows:

[0064] Dissolve 1 mg of black tea antihypertensive peptide powder in 1 mL of distilled water, and adjust the pH of each solution to the same value (pH = 7) with 0.5 mol / L NaOH or 1 mol / L HCl solution to prepare a black tea antihypertensive peptide solution for standby. Dissolve ACE and HHL (benzoyl-L-histidyl-L-leucine) separately in a borate buffer solution with pH 8.3 and 0.1 mol / L (containing 0.3 mol / L of NaCl). The enzyme activity of the ACE solution is 0.1 U / mL, and the concentration of the HHL solution is 0.005 mol / L to prepare an ACE solution and an HHL solution for standby. Take 20 μL of the ACE solution and 80 μL of the black tea antihypertensive peptide solution and mix them evenly in a 4 mL centrifuge tube as the experimental group. The control group is 20 μL of the ACE solution added with 80 μL of an equal amount of borate buffer solution and mixed evenly in a 4 mL centrifuge tube.

[0065] After incubation at 37 °C for 5 min, 170 μL of HHL solution was added to each centrifuge tube. After mixing evenly, the mixture was incubated at 37 °C for 30 min. Then, 150 μL of 1 mol / L HCl solution was added to each centrifuge tube, and the mixture was shaken to mix evenly to terminate the reaction. Finally, 1 mL of ethyl acetate was added to each centrifuge tube, vortexed for 30 s, and centrifuged at 4000 r / min for 15 min. After standing for 5 min, when the organic layer and the aqueous layer were completely separated, 0.8 mL of the organic layer was transferred to a 5 mL centrifuge tube. The centrifuge tube was placed in an 80 °C water bath to evaporate the ethyl acetate. Finally, 3.2 mL of deionized water was added to the residue to dissolve it completely, and the absorbance value (A228) of the solution at 228 nm was measured.

[0066]

[0067] Where: I is the ACE inhibition rate, A0 is the absorbance value of the control group, and A1 is the absorbance value of the test sample.

[0068] The yields, purities, and ACE inhibition rates of the dark tea antihypertensive peptides in Examples 1 to 3 of the present invention are shown in Table 1.

[0069] Table 1 Determination results of the indexes of the dark tea antihypertensive peptides in Examples 1 to 3

[0070] serial number Peptide yield% purity% ACE inhibition rate% Example 1 32 90 87 Example 2 21.2 82 73 Example 3 20.5 78 70

[0071] As can be seen from Table 1, the ACE inhibition rate of the dark tea antihypertensive peptide prepared by the preparation method of the present invention is above 70%, the yield of the antihypertensive peptide is above 20%, and the purity is above 78%. Among them, the ACE inhibition rate of the dark tea antihypertensive peptide in Example 1 is the highest, which is 87%, the yield of the antihypertensive peptide is 32%, and the purity is 90%. The reason is that: in Example 1, the composite enzyme cellulase: pectinase was added at the optimal ratio of 2:1 and the addition amount of 0.3% to enzymatically hydrolyze dark tea, which can destroy the cell wall to the greatest extent. The enzymatic hydrolysate was subjected to targeted hydrolysis of the protein-polyphenol complex by alkaline protease to obtain more dark tea protein powder. The ultrasonic-pulsed electric field was used to synergistically treat the material liquid of the dark tea protein powder to fully stretch the protein, expose more hydrophobic groups and enzymatic cleavage sites, and improve the enzymatic hydrolysis efficiency in the next step. Then, the composite enzyme synergistic hydrolysis technology was used to extract the dark tea antihypertensive peptide. The enzymatic hydrolysis step adopted sequential enzymatic hydrolysis with subtilisin (optimal pH 7.0) and flavor protease (optimal pH 6.5). The former cuts off the hydrophobic amino acid sites, and the latter further degrades the macromolecular peptide segments. When the ratio of the two is appropriate and the conditions are moderate, more highly active small molecule antihypertensive peptides can be released, thereby increasing the yield of the antihypertensive peptide and improving the ACE inhibition rate. Appropriate enzyme ratio can reduce the residue of undigested protein, thereby improving the peptide purity of the antihypertensive peptide; under the synergistic action of ultrasonic-pulse at an appropriate power, the mechanical shear force can be enhanced, the penetration of the protein hydrolase can be accelerated, the enzymatic hydrolysis time can be shortened, and the purity of the antihypertensive peptide can be indirectly improved.

[0072] In Example 2, the enzymolysis time was too short, hydrophobic amino acids (such as Leu, Phe, Val) were not sufficiently exposed, the hydrophilicity of the peptide segments was too strong, the protein was not sufficiently hydrolyzed, the proportion of large molecular peptide segments was relatively high, and due to steric hindrance effects, long-chain peptides were difficult to bind to the ACE active site, resulting in a low inhibition rate; in Example 3, the enzymolysis time was too long, and over-hydrolysis led to further cleavage of the bioactive peptides, destroying key functional groups (such as Pro-Phe sequence or Trp-His-Lys domain), weakening the binding stability with ACE, and reducing the inhibitory efficacy. When the ultrasonic power in Example 3 was too high, the thermal effect (temperature increase) might accelerate chemical reactions, leading to protein denaturation or excessive destruction of cell structures, reducing functionality. Excessively high pulsed electric field power might trigger irreversible electroporation, destroying the target molecular structure; in Example 2, when the ultrasonic power and pulsed electric field were too low, the protein unfolding was insufficient, affecting the release of subsequent small molecule antihypertensive peptides. Both too high and too low would affect the yield of antihypertensive peptides, reducing the ACE inhibition rate.

[0073] In Example 2, the enzyme ratio was too low, and insufficient activity of the key enzyme would lead to incomplete hydrolysis, leaving residual macromolecular impurities (such as undegraded protein fragments), which would affect the purity of the peptide; in Example 3, the excessive enzyme ratio might over-hydrolyze the target peptide segments, producing short-chain inactive peptides or free amino acids, also reducing the purity of the peptide. In Example 2, too low ultrasonic power could not effectively break protein aggregates or cell walls, resulting in incomplete release of antihypertensive peptides, leaving residual undegraded protein fragments and reduced purity; in Example 3, too high ultrasonic power would cause excessive cleavage of proteins or peptide chains, generating short-chain inactive peptide segments or free amino acids, increasing the proportion of impurities and reducing the purity of the final product. High-power ultrasound was prone to cause local high temperatures (such as >60 °C), leading to inactivation of some enzymes or denaturation of the target peptides, reducing the subsequent enzymolysis efficiency, leaving residual macromolecular impurities that were not completely hydrolyzed, affecting the activity and purity.

[0074] Comparative Example 1

[0075] It was basically the same as Example 1, except that in step (4), only flavor protease was used for enzymolysis, and the enzymolysis was carried out at a temperature of 45 °C, a substrate concentration of 8%, and an enzyme addition amount of 1.5% for 4 h.

[0076] Comparative Example 2

[0077] It was basically the same as Example 1, except that in step (4), only subtilisin was used for enzymolysis, and the enzymolysis was carried out at a temperature of 45 °C, a substrate concentration of 8%, and an enzyme addition amount of 1.5% for 4 h.

[0078] The yields, purities, and ACE inhibition rates of the dark tea antihypertensive peptides prepared in Comparative Example 1 and Comparative Example 2 were measured according to the method described in the examples, and the results are shown in Table 2 below.

[0079] Table 2 Index determination results of dark tea antihypertensive peptides in Example 1 and Comparative Examples 1 - 2

[0080] serial number Enzyme used in step (4) Peptide yield% purity% ACE inhibition rate% Example 1 Subtilisin and flavor protease 32 90 87 Comparative Example 1 Flavor protease 18.7 74 59 Comparative Example 2 Subtilisin 17.5 72 61

[0081] As can be seen from Table 2, compared with Comparative Examples 1 - 2, the yield, purity, and ACE inhibitory rate of the antihypertensive peptide in Example 1 were significantly improved. The reason is that in step (4) of Example 1, subtilisin and flavourzyme were used for stepwise enzymatic hydrolysis, which improved the enzymatic hydrolysis efficiency. In Comparative Example 2, using subtilisin alone mainly acted on the carboxyl terminus of hydrophobic amino acid residues (such as phenylalanine and tyrosine). Due to possible limitations in the cleavage sites, some potential bioactive peptide segments may not have been released. In Comparative Example 1, using flavourzyme alone preferred to cleave the amino terminus of hydrophilic amino acids (such as glutamic acid and aspartic acid). Due to the low degree of hydrolysis, the release efficiency of bioactive peptides was limited. In Example 1, the cleavage sites of the two enzymes were complementary, which could fully hydrolyze the protein substrate and release more bioactive peptide segments with diverse structures. The combination of subtilisin and flavourzyme could further hydrolyze the intermediate products that were not completely degraded, such as cleaving large peptide segments into smaller bioactive units, achieving "secondary enzymatic hydrolysis synergistic effect". Therefore, the combined use of subtilisin and flavourzyme could significantly enhance the yield and ACE inhibitory rate of antihypertensive peptides through complementary cleavage sites, optimized product molecular weight, and multi - mechanism inhibition. At the same time, in Comparative Examples 1 and 2, using a single enzyme to hydrolyze specific peptide bonds may only release some antihypertensive peptide fragments, resulting in the mixture of incompletely hydrolyzed proteins or polypeptide chains in the product, leading to a decrease in purity.

[0082] Comparative Example 3

[0083] It was basically the same as Example 1, except that in step (3), only ultrasonic treatment was used, and the ultrasonic power was 300 W.

[0084] Comparative Example 4

[0085] It was basically the same as Example 1, except that in step (3), only pulsed electric field treatment was used, and the pulsed electric field intensity was 40 kV / CM.

[0086] The yield, purity, and ACE inhibitory rate of the dark tea antihypertensive peptides prepared in Comparative Examples 3 - 4 were determined according to the method described in the example, and the results are shown in Table 3 below.

[0087] Table 3 Index determination results of dark tea antihypertensive peptides in Example 1 and Comparative Examples 3 - 4

[0088] serial number Processing of step (3) Peptide yield% purity% ACE inhibition rate% Example 1 Ultrasound and pulsed electric field synergy 32 90 87 Comparative Example 3 ultrasound 17.6 73 60 Comparative Example 4 Pulsed electric field 16.7 69 55

[0089] As can be seen from Table 3, compared with Comparative Examples 3 to 4, the ACE inhibition rate of Example 1 was significantly improved. The reason is that in step (3) of Example 1, the liquid material was treated by the synergistic action of ultrasonic wave and pulsed electric field. Compared with the use of a single ultrasonic wave or pulsed electric field, the enzymolysis efficiency was improved. When using ultrasonic wave alone, the protein structure was destroyed through the cavitation effect, exposing more enzyme cleavage sites and promoting the release of bioactive peptides, which could improve the yield and ACE inhibition rate; when using electric field treatment alone, the secondary structure of the antihypertensive peptide could be changed, exposing more ACE inhibitory active sites, thus improving the yield and ACE inhibition rate; the cavitation effect of the ultrasonic wave in their synergistic action promoted the penetration of the electric field into the protein, accelerated the unfolding of the molecular structure, and the synergistic treatment exposed more hydrophobic groups and enzyme cleavage sites, thus releasing antihypertensive peptide fragments with higher activity and further improving the yield and ACE inhibition rate of the dark tea antihypertensive peptide.

[0090] In Comparative Examples 3 and 4, the single ultrasonic treatment may cause local overheating or excessive structural damage due to uneven energy distribution, leaving residual macromolecular fragments that are not completely hydrolyzed, affecting the purity of the antihypertensive peptide; the structural change of the single pulsed electric field may reduce the solubility of the peptide, increase the difficulty of solid-phase synthesis or chromatographic separation, and further affect the impurity separation efficiency in the purification process, reducing the purity of the antihypertensive peptide.

Claims

1. A preparation method of a dark tea antihypertensive peptide, characterized in that, It includes the following steps: (1) Crush the dried dark tea, then add water and a composite enzyme to the dark tea, and carry out enzymatic hydrolysis at 25-60°C to obtain an enzymatic hydrolysate; (2) Adjust the pH of the enzymatic hydrolysate prepared in step (1) to 5.0-12.0, add alkaline protease to the enzymatic hydrolysate, and carry out enzymatic hydrolysis at 30-75°C. After the enzymatic hydrolysis is completed, centrifuge to obtain the supernatant, and freeze-dry the supernatant to obtain dark tea protein powder; (3) Add water to the dark tea protein powder obtained in step (2), mix well to obtain a mixed solution, and then simultaneously treat the mixed solution with ultrasonic waves and pulsed electric fields. After the treatment is completed, obtain a stock solution; (4) Sequentially carry out enzymatic hydrolysis treatment on the stock solution prepared in step (3) with subtilisin and flavor protease. After the enzymatic hydrolysis is completed, carry out enzyme inactivation treatment, centrifuge, and reserve the supernatant; (5) Carry out multi-stage membrane separation and purification on the supernatant obtained in step (4), sequentially remove the unhydrolyzed protein, small molecular peptide segments and salts in the supernatant to obtain a purified solution, and the purified solution is subjected to vacuum freeze-drying treatment to obtain dark tea antihypertensive peptide.

2. The preparation method according to claim 1, characterized in that, In step (4), the hydrolysis conditions of subtilisin are: pH is 2.0-9.0, and the temperature is 10-60°C; the hydrolysis conditions of flavor protease are: pH is 3.5-8.5, and the temperature is 20-65°C.

3. The preparation method according to claim 2, wherein, In step (4), the addition amount of subtilisin is 0.8%-3.0% of the total mass of the stock solution, and the enzymatic hydrolysis time is 2-5h; the addition amount of flavor protease is 0.8%-3.2% of the total mass of the stock solution, and the enzymatic hydrolysis time is 1-7h.

4. The preparation method of the dark tea hypotensive peptide according to claim 1, wherein In step (1), the composite enzyme is composed of cellulase and pectinase according to a mass ratio of 2:1, and the addition amount of the composite enzyme is 0.1%-0.5% of the mass of the dark tea.

5. The preparation method according to claim 4, wherein In step (3), the ultrasonic power is 100-500W, and the pulsed electric field intensity is 5-40kV / CM.

6. The preparation method of the dark tea hypotensive peptide according to claim 4, characterized in that In step (2), the addition amount of alkaline protease is 0.1%-1.3% of the mass of the dark tea.

7. The preparation method of the dark tea hypotensive peptide according to claim 6, characterized in that, In step (5), an ultrafiltration membrane is used to remove unhydrolyzed macromolecular proteins; a nanofiltration membrane is used to retain small molecular peptide segments; a reverse osmosis membrane is used for desalination.

8. The preparation method according to claim 7, characterized in that, In step (5), the cut-off molecular weight of the ultrafiltration membrane is 100kDa, and the cut-off molecular weight of the nanofiltration membrane is 1-5kDa.

9. A dark tea antihypertensive peptide prepared by the method according to any one of claims 1-8.

10. Use of the dark tea antihypertensive peptide according to claim 9 in the preparation of antihypertensive drugs.

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