Marine bioactive polypeptide, preparation and application thereof

Abstract

This invention provides a marine bioactive polypeptide, its preparation, and its application, relating to the field of bioactive peptide preparation technology. The invention involves pulverizing abalone, adding a compound enzyme for preliminary enzymatic hydrolysis, and then adding a specific protease mixture containing prolyl endopeptidase, chymotrypsin, and proteinase K for a two-step enzymatic hydrolysis. The resulting polypeptide solution exhibits high ACE inhibition. Analysis and screening of the obtained polypeptide yielded polypeptides with amino acid sequences as shown in SEQ ID NO. 1-5. The polypeptide of this invention exhibits high ACE inhibition and an IC50 inhibition rate against ACE. 50 It is present in the range of 0.05 mg / mL to 0.40 mg / mL, and therefore can act as an angiotensin-converting enzyme inhibitor to lower blood pressure.

Description

[0001] This application is a divisional application of application number 202510153404.2, filed on February 12, 2025, entitled "A polypeptide with angiotensin-converting enzyme inhibition, a method for its preparation and application thereof". Technical Field

[0002] This invention relates to the field of bioactive peptide preparation technology, and in particular to a marine bioactive polypeptide and its preparation and application. Background Technology

[0003] Bioactive peptides (BAPs) are peptide compounds that are beneficial or physiologically active in the life activities of organisms. They are a class of polypeptides with a relative molecular mass of less than 6000 Da and possess a variety of biological functions. Their molecular structures vary in complexity, ranging from polymers composed of just two amino acids to dozens linked by peptide bonds. These polypeptides can be modified through phosphorylation, glycosylation, or acylation. In recent years, marine bioactive peptides have become a research hotspot in the food industry due to their diverse functions, wide availability, high specificity, and low toxicity. Among them, angiotensin-converting enzyme (ACE) inhibitory peptides have been extensively studied due to their blood pressure-lowering effects. ACE inhibitory peptides bind to the active pocket of ACE, thereby inhibiting ACE activity and preventing the conversion of angiotensin I (Ang I) to Ang II, thus preventing the inactivation of the vasodilator bradykinin and achieving the goal of lowering hypertension.

[0004] Abalone is a marine gastropod, a single-shelled mollusc, with tender meat and a rich flavor, making it popular with consumers both domestically and internationally. my country is a major abalone farming country. Abalone is not only highly nutritious, but numerous studies have also shown that it contains bioactive components such as polypeptides, polysaccharides, free amino acids, and fatty acids. Currently, the most common method for preparing bioactive peptides is enzymatic hydrolysis, where the main active enzymes are often complex enzymes such as alkaline protease, neutral protease, trypsin, and papain. This results in products with complex compositions and relatively low bioactivity. Research on how to extract peptides with high angiotensin-converting enzyme inhibitory activity from abalone is still lacking. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a highly active marine bioactive polypeptide in the prior art.

[0006] To address the aforementioned technical problems, this invention provides a marine bioactive polypeptide, its preparation, and its application. This invention involves pulverizing abalone, adding a compound enzyme for initial enzymatic hydrolysis, and then adding a specific protease mixture containing prolyl endopeptidase, chymotrypsin, and proteinase K for secondary enzymatic hydrolysis. The resulting polypeptide exhibits high ACE inhibitory activity. Analysis and screening of the obtained polypeptide yielded polypeptides with amino acid sequences as shown in SEQ ID NO1-5. The polypeptide of this invention exhibits high ACE inhibitory activity and high inhibition of angiotensin-converting enzyme IC50. 50 The concentration is between 0.05 mg / mL and 0.40 mg / mL, therefore it can act as an angiotensin-converting enzyme inhibitor to lower blood pressure.

[0007] The first objective of this invention is to provide a marine bioactive polypeptide, the amino acid sequence of which is shown in any one of SEQ ID NO. 1-5.

[0008] Furthermore, SEQ ID NO.1: FDRLF;

[0009] SEQ ID NO.2: SPPFFDGMTR;

[0010] SEQ ID NO.3: FDFRQF;

[0011] SEQ ID NO.4: GFDFRQF;

[0012] SEQ ID NO.5: PEHFPF.

[0013] A second objective of this invention is to provide a gene encoding the aforementioned bioactive polypeptides from marine organisms.

[0014] A third objective of this invention is to provide an application of the aforementioned marine bioactive polypeptide in the preparation of antihypertensive products.

[0015] Furthermore, the blood pressure-lowering product works by inhibiting the activity of angiotensin-converting enzyme.

[0016] The fourth objective of this invention is to provide a method for preparing the above-mentioned marine bioactive polypeptides, comprising the following steps:

[0017] S1. Abalone is heated and crushed to obtain crude abalone product. A compound enzyme is added to the crude abalone product for preliminary enzymatic hydrolysis to obtain a preliminary enzymatic hydrolysis product. The compound enzyme includes papain, neutral protease, alkaline protease, trypsin and lipase.

[0018] S2. Add a specific protease mixture to the initial enzymatic hydrolysis product for secondary enzymatic hydrolysis to obtain the polypeptide;

[0019] The specific protease mixture includes prolyl endopeptidase, chymotrypsin, and proteinase K.

[0020] Furthermore, the mass ratio of papain: neutral protease: alkaline protease: trypsin: lipase is 1:2:2:2:1.

[0021] Furthermore, the heating temperature in step S1 is 100-150°C, and the heating time is 3-5 hours.

[0022] Further, the mass ratio of the protease to the crude abalone product in step S1 is (2-4):100.

[0023] Furthermore, the pH of the initial enzymatic hydrolysis in step S1 is 6.5-7.5.

[0024] Furthermore, the initial enzymatic hydrolysis temperature in step S1 is 50-60℃.

[0025] Further, in step S2, the mass ratio of the specific protease mixture to the crude enzymatic hydrolysis product is (0.5-2):100.

[0026] Furthermore, the mass ratio of prolyl endopeptidase: chymotrypsin: proteinase K is (1-2): (1-2): (1-2).

[0027] Furthermore, the temperature for the secondary enzymatic hydrolysis in step S2 is 30-40℃.

[0028] The beneficial effects of this invention are:

[0029] This invention provides a marine bioactive polypeptide, its preparation, and its application. The amino acid sequence of the polypeptide obtained by this invention is shown in SEQ ID NO. 1-5. The polypeptide of this invention has high ACE inhibitory activity and inhibits the IC50 of angiotensin-converting enzyme. 50 The concentration is between 0.05 mg / mL and 0.40 mg / mL, therefore it can act as an angiotensin-converting enzyme inhibitor to lower blood pressure. Attached Figure Description

[0030] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0031] Figure 1 This is a flowchart of the method for preparing and screening polypeptides according to the present invention;

[0032] Figure 2 This is a graph showing the ACEi activity assay results of the enzymatic hydrolysis product obtained in Example 2 of the present invention;

[0033] Figure 3 This is a schematic diagram of the Sephadex G-15 gel filtration chromatography chromatogram and fraction collection of the present invention;

[0034] Figure 4 This is a graph showing the ACEi activity determination results after gel filtration sequence distillation according to the present invention. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0036] The compound enzymes were: papain (>200,000 u / g) provided by Shenzhen Xiameng Biotechnology Co., Ltd., China; neutral protease (>2.4 AU-A / g) and alkaline protease (>6.5 AU-N / g) from Novozymes (China) Biotechnology Co., Ltd.; trypsin (>250,000 u / g) from Shanghai Maclean Biotechnology Co., Ltd., China; and lipase (>30,000 u / g) from Shanghai Yuanye Biotechnology Co., Ltd., China. The mass ratio of papain:neutral protease:alkaline protease:trypsin:lipase was 1:2:2:2:1.

[0037] Methods for detecting ACE inhibition:

[0038] Using FAPGG as a substrate (FAPGG: N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine, FAPGG is a commonly used substrate for angiotensin-converting enzyme (ACE) and is often used for the in vitro quantitative determination of ACE activity), the reactants were added to the microplate according to the table. The absorbance A1 before the reaction was measured at 340 nm using a microplate reader. After incubation at 37°C for 30 min, the absorbance A2 was measured. ΔA was calculated as ΔA = A1 - A2. The change in absorbance per unit time represents the ACE enzyme activity. The ACE inhibition rate was calculated using the following formula:

[0039] ACE inhibition rate (%) = 1 - ΔA sample / ΔA blank × 100%

[0040] In the formula: ΔA blank represents the change in absorbance within 30 minutes when buffer solution is added; ΔA sample represents the change in absorbance within 30 minutes when inhibitor is added.

[0041] Example 1

[0042] (1) After steaming abalone at 110℃ for 4 hours, add water and mix evenly. After crushing, obtain abalone meat paste. Freeze the abalone meat paste for more than 12 hours, and then place it in a low-temperature freeze dryer for more than 4 days to obtain abalone meat powder.

[0043] (2) Take 10g of abalone meat powder from (1), add 300mL of water and mix evenly. Place it in a 55℃ constant temperature water bath and a stirrer at 800rpm. Add a compound enzyme with a mass concentration of 3% (based on the mass of abalone meat powder) for 4h of enzymatic hydrolysis. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme at 90-95℃ for 10-15min. Then centrifuge the system at 10000rpm for 10min, take the supernatant, freeze the supernatant for more than 12h, and then place it in a low temperature freeze dryer for more than 4d to obtain abalone crude enzymatic hydrolysis product powder.

[0044] (3) Take 10g of the abalone crude enzymatic hydrolysis product dry powder from (2), dissolve it in ultrapure water and make up to 200mL. Place it in a constant temperature water bath at 37℃ and stir at 800rpm. While mixing, add a specific protease with a mass concentration of 0.5% (based on the mass of the abalone crude enzymatic hydrolysis product) for enzymatic hydrolysis for 4h to obtain abalone enzymatic hydrolysate. In the specific protease, the mass concentration of prolyl endopeptidase is 0.125%, the mass concentration of chymotrypsin is 0.25%, and the mass concentration of proteinase K is 0.125%.

[0045] (4) Inactivate the enzyme in the abalone enzymatic hydrolysate in (3) at 90-95℃ for 10-15 min, then centrifuge the system at 10000 rpm for 10 min, and take the supernatant to obtain the abalone polypeptide solution of the present invention.

[0046] Example 2

[0047] The preparation method of the abalone polypeptide solution in this embodiment is the same as that in Example 1, except that the mass concentration of the specific protease added in this embodiment is 1%, wherein the mass concentration of prolyl endopeptidase is 0.25%, the mass concentration of chymotrypsin is 0.5%, and the mass concentration of proteinase K is 0.25%. The specific steps are as follows:

[0048] (1) After steaming abalone at 110℃ for 4 hours, add water and mix evenly. After crushing, obtain abalone meat paste. Freeze the abalone meat paste for more than 12 hours, and then place it in a low-temperature freeze dryer for more than 4 days to obtain abalone meat powder.

[0049] (2) Take 10g of abalone meat powder from (1), add 300mL of water and mix evenly. Place it in a 55℃ constant temperature water bath and a stirrer at 800rpm. Add a compound enzyme with a mass concentration of 3% (based on the mass of abalone meat powder) for 4h of enzymatic hydrolysis. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme at 90-95℃ for 10-15min. Then centrifuge the system at 10000rpm for 10min, take the supernatant, freeze the supernatant for more than 12h, and then place it in a low temperature freeze dryer for more than 4d to obtain abalone crude enzymatic hydrolysis product powder.

[0050] (3) Take 10g of the abalone crude enzymatic hydrolysis product dry powder from (2), dissolve it in ultrapure water and make up to 200mL. Place it in a constant temperature water bath at 37℃ and stir at 800rpm. While mixing, add a specific protease with a mass concentration of 1% (based on the mass of the abalone crude enzymatic hydrolysis product) for 4h to obtain abalone enzymatic hydrolysate. In the specific protease, the mass concentration of prolyl endopeptidase is 0.25%, the mass concentration of chymotrypsin is 0.5%, and the mass concentration of proteinase K is 0.25%.

[0051] (4) Inactivate the enzyme in the abalone enzymatic hydrolysate in (3) at 90-95℃ for 10-15 min, then centrifuge the system at 10000 rpm for 10 min, and take the supernatant to obtain the abalone polypeptide solution of the present invention.

[0052] Example 3

[0053] The preparation method of abalone polypeptide solution in this embodiment is the same as that in Example 1, except that the mass concentration of the specific protease added in this embodiment is 2%, wherein the mass concentration of prolyl endopeptidase is 0.5%, the mass concentration of chymotrypsin is 1%, and the mass concentration of proteinase K is 0.5%. The specific steps are as follows:

[0054] (1) After steaming abalone at 110℃ for 4 hours, add water and mix evenly. After crushing, obtain abalone meat paste. Freeze the abalone meat paste for more than 12 hours, and then place it in a low-temperature freeze dryer for more than 4 days to obtain abalone meat powder.

[0055] (2) Take 10g of abalone meat powder from (1), add 300mL of water and mix evenly. Place it in a 55℃ constant temperature water bath and a stirrer at 800rpm. Add a compound enzyme with a mass concentration of 3% (based on the mass of abalone meat powder) for 4h of enzymatic hydrolysis. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme at 90-95℃ for 10-15min. Then centrifuge the system at 10000rpm for 10min, take the supernatant, freeze the supernatant for more than 12h, and then place it in a low temperature freeze dryer for more than 4d to obtain abalone crude enzymatic hydrolysis product powder.

[0056] (3) Take 10g of the abalone crude enzymatic hydrolysis product dry powder from (2), dissolve it in ultrapure water and make up to 200mL. Place it in a constant temperature water bath at 37℃ and stir at 800rpm. While mixing, add a specific protease with a mass concentration of 3% (based on the mass of the abalone crude enzymatic hydrolysis product) for 4h to obtain abalone enzymatic hydrolysate. In the specific protease, the mass concentration of prolyl endopeptidase is 0.5%, the mass concentration of chymotrypsin is 1%, and the mass concentration of proteinase K is 0.5%.

[0057] (4) Inactivate the enzyme in the abalone enzymatic hydrolysate in (3) at 90-95℃ for 10-15 min, then centrifuge the system at 10000 rpm for 10 min, and take the supernatant to obtain the abalone polypeptide solution of the present invention.

[0058] Example 4

[0059] The preparation method of the abalone polypeptide solution in this embodiment is the same as that in Example 2. The mass concentration of the specific protease added in this embodiment is also 1%, the difference being that the mass concentration of prolyl endopeptidase is 0.17%, the mass concentration of chymotrypsin is 0.33%, and the mass concentration of proteinase K is 0.5%. The specific steps are as follows:

[0060] (1) After steaming abalone at 110℃ for 4 hours, add water and mix evenly. After crushing, obtain abalone meat paste. Freeze the abalone meat paste for more than 12 hours, and then place it in a low-temperature freeze dryer for more than 4 days to obtain abalone meat powder.

[0061] (2) Take 10g of abalone meat powder from (1), add 300mL of water and mix evenly. Place it in a 55℃ constant temperature water bath and a stirrer at 800rpm. Add a compound enzyme with a mass concentration of 3% (based on the mass of abalone meat powder) for 4h of enzymatic hydrolysis. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme at 90-95℃ for 10-15min. Then centrifuge the system at 10000rpm for 10min, take the supernatant, freeze the supernatant for more than 12h, and then place it in a low temperature freeze dryer for more than 4d to obtain abalone crude enzymatic hydrolysis product powder.

[0062] (3) Take 10g of the abalone crude enzymatic hydrolysis product dry powder from (2), dissolve it in ultrapure water and make up to 200mL. Place it in a constant temperature water bath at 37℃ and stir at 800rpm. While mixing, add a specific protease with a mass concentration of 1% (based on the mass of the abalone crude enzymatic hydrolysis product) for 4h to obtain abalone enzymatic hydrolysate. In the specific protease, the mass concentration of prolyl endopeptidase is 0.17%, the mass concentration of chymotrypsin is 0.33%, and the mass concentration of proteinase K is 0.5%.

[0063] (4) Inactivate the enzyme in the abalone enzymatic hydrolysate in (3) at 90-95℃ for 10-15 min, then centrifuge the system at 10000 rpm for 10 min, and take the supernatant to obtain the abalone polypeptide solution of the present invention.

[0064] The abalone polypeptide solutions obtained in Examples 1-4 and Comparative Examples 1-2 were subjected to ACEi activity assay at 1 mg / mL, and the results are shown in Table 1.

[0065] Table 1. ACEi activity assay of abalone polypeptide solutions in Examples 1-4 and Comparative Examples 1-2

[0066]

[0067] As can be seen from Table 1, the technical solution of the present invention significantly improves the ACEi activity of abalone polypeptide liquid. Specifically, compared with Comparative Example 1, the ACEi activity of the abalone polypeptide liquid obtained in Example 2 of the present invention after 3 hours can be increased by up to 228%, which fully proves the feasibility of the preparation method of the present invention.

[0068] Example 5: Screening of ACE inhibitory peptides from abalone

[0069] (1) The abalone polypeptide solution obtained in Example 2 was filtered using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and the components with molecular weights in the range of 0-3 kDa were collected.

[0070] (2) The components collected in step 1 were further separated using a Sephadex G-15 gel column (2.6 × 40 cm), eluted with ultrapure water at a flow rate of 0.6 mL / min, and fractions with different absorption peaks at 280 nm were collected. The ACE inhibitory activity was then determined. Chromatograms and fraction collection details are shown below. Figure 3 The ACE inhibitory activities of different fractions are shown in the figure. Figure 4 Fraction C showed the highest ACE inhibitory activity, while fraction D had the highest peptide abundance.

[0071] (3) Use peptidomics technology to analyze the dominant fractions (i.e. fractions C and D) in step (2) to obtain the amino acid sequences of all contained polypeptides;

[0072] (4) Peptides with amino acid sequence scores > 0.9 in step (3) were screened using the PeptideRanker tool. After molecular docking prediction, the peptides were chemically synthesized and their biological activity was verified.

[0073] Specifically, peptidomics analysis revealed that components C and D contained a total of 1937 different peptide sequences. The potential biological activities of these 1937 peptide sequences were scored and ranked using PeptideRanker. The amino acid sequences of the five peptides with scores >0.9 are shown in Table 2 (SEQ ID NO. 1-5). After molecular docking, all five peptides were able to stably exist in the active pocket of ACE enzymes, with binding energies ranging from -6.2 to -9.7 kcal / mol. After chemical synthesis of the five peptides, their ACEi activity was measured, and the IC50 values ​​were... 50 The values ​​ranged from 0.05 to 0.40 mg / mL. Among them, peptide FDRLF (0.05 mg / mL) showed the best ACEi activity. No matching was found between these five peptide sequences in BIOPEP-UWM and EROP-Moscow, indicating that all five peptides are novel, previously unreported sequences.

[0074] The specific molecular docking method includes: molecular docking using AutoDock Vina 1.1.2 software. The crystal structure of the human ACE-lisinopril complex (1O86) was obtained from the RCSB protein database (https: / / www.rcsb.org / ). The peptide molecule was constructed using PyMol 2.6 with energy minimization. PyMol 2.6 was used to process the protein, removing water molecules and small ligands, and adding hydrogen, etc. AutoDock Tools was used to convert the receptor protein and peptide into PDBQT format. The center of the docking box was defined according to the position of the crystal ligands, and the side length of the box was [missing information]. The highest score from the Vina docking was selected as the final result, and a visualization analysis was performed in PyMol 2.6.

[0075] Table 2. Bioactivity assay of the peptides screened in Example 5.

[0076]

[0077] Comparative Example 1

[0078] (1) After steaming abalone at 110℃ for 4 hours, add water and mix evenly. After crushing, obtain abalone meat paste. Freeze the abalone meat paste for more than 12 hours, and then place it in a low-temperature freeze dryer for more than 4 days to obtain abalone meat powder.

[0079] (2) Take 10g of abalone meat powder from (1), add 300mL of water to mix the abalone meat and water evenly, place it in a 55℃ constant temperature water bath and stir at 800rpm, add a compound enzyme with a mass concentration of 3% (based on the mass of abalone meat powder) for 4h of enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide during the enzymatic hydrolysis, and inactivate the enzyme at 90-95℃ for 10-15min after the enzymatic hydrolysis is completed; then centrifuge the system at 10000rpm for 10min, take the supernatant to obtain crude enzymatic hydrolysate of abalone meat.

[0080] Comparative Example 2

[0081] (1) After steaming abalone at 110℃ for 4 hours, add water and mix evenly. After crushing, abalone meat paste is obtained. The abalone meat paste is frozen for more than 12 hours and then placed in a low-temperature freeze dryer for more than 4 days to obtain abalone meat powder.

[0082] (2) Take 10g of abalone meat powder from (1), add 300mL of water and mix well to form a mixture of abalone meat and water. Place it in a constant temperature water bath at 55℃ and stir at 800rpm. At the same time, add 0.25% prolyl endopeptidase, 0.5% chymotrypsin and 0.25% proteinase K by mass concentration (based on the mass of abalone meat powder) for 4h of enzymatic hydrolysis. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme at 90-95℃ for 10-15min. Then centrifuge the system at 10000rpm for 10min and take the supernatant to obtain the enzymatic hydrolysate.

[0083] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A marine bioactive polypeptide, characterized in that, The amino acid sequence of the marine bioactive polypeptide is shown in SEQ ID NO.

4.

2. A gene encoding the marine bioactive polypeptide of claim 1.

3. The application of the marine bioactive polypeptide of claim 1 in the preparation of antihypertensive products.

4. The application according to claim 3, characterized in that, The blood pressure-lowering product works by inhibiting the activity of angiotensin-converting enzyme.

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