Chinese forest frog polypeptide with anti-saccharification and antihypertensive functional activity as well as preparation method and application of Chinese forest frog polypeptide

Abstract

The invention discloses a Chinese forest frog source anti-glycated peptide and ACE inhibitory peptide and application thereof. Dried Chinese forest frog is used as a raw material, hydrolysis is carried out through an enzymolysis method, the enzymatic hydrolysate is decolorized and deodorized, and the Chinese forest frog polypeptide powder is obtained after filtration. The invention further provides a Chinese forest frog polypeptide separation and purification method, peptide fragments with AGEs inhibitory activity and ACE inhibitory activity are identified and screened from the Chinese forest frog polypeptide separation and purification method, and the peptide fragments can be used for preparing cosmetics or functional foods with anti-saccharification and / or antihypertensive effects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to Rana chensinensis polypeptides with anti-glycation and blood pressure lowering functional activities, their preparation methods and applications. Background Art

[0002] Rana chensinensis, also known as "snow frog", "Hasma", etc., has a good foundation for resource protection and industrial development. It is not only an important part of water and forest ecosystems, but also a precious frog species with multiple uses such as food and medicine. Appropriate processing will have a wide range of uses. The snow frog after oil extraction is called snow frog dry, which contains rich proteins, and its enzymatic hydrolysis products have activities such as antioxidant and antibacterial. The "NYT1516-2020 Green Food Frogs and Their Products" stipulates various indicators of snow frog dry, providing support for snow frog dry as a food with high nutritional value. So far, the development and utilization of snow frog dry mostly remain in the basic research stage, and the conversion rate of achievements is relatively low, and this resource has not been efficiently utilized. The present invention turns snow frog dry from "waste" into treasure, increases the product added value, and opens up a new path for the deep processing of snow frog and the development of series products.

[0003] Glycation, also known as glycosylation, refers to a series of non-enzymatic reactions such as condensation, rearrangement, cleavage and oxidative modification of the free amino groups of biological macromolecules such as proteins, amino acids, lipids, etc. and the carbonyl groups of reducing sugars (glucose, fructose, etc.). Advanced Glycation End products (AGEs) are a general term for a class of stable, irreversible, covalently polymerized substances with diverse structures formed after this series of reactions. AGEs can cause changes in protein structure, lead to protein dysfunction, and then affect the normal functions of body tissues and organs, accelerating the process of body aging. Angiotensin-converting enzyme (ACE) is an important zinc-containing metallopeptidase in the renin-angiotensin system (RAS), which can catalyze the hydrolysis of angiotensin I (Ang I) into angiotensin II (Ang II), thereby increasing blood pressure. In addition, Ang II may promote the formation of AGEs by increasing oxidative stress and abnormal glucose metabolism, and AGEs can activate RAS, forming a vicious cycle. ACE inhibitors can not only block the cycle and indirectly reduce the accumulation of AGEs by reducing the production of Ang II, but also directly inhibit the activity of ACE to achieve the purpose of lowering blood pressure. Anti-glycation and blood pressure lowering are long-term processes. Long-term use of chemically synthesized inhibitors will pose challenges to the health of the body. Therefore, the use of safe and efficient natural products to combat glycosylation and aging has gradually received wide attention.

[0004] In the prior art, there is no disclosure of the Rana chensinensis polypeptides with anti-glycation and blood pressure lowering functional activities, their preparation methods and applications as claimed in the present invention. <于<2000010>Summary of the Invention

[0005] Based on this, the present invention provides a method for preparing the polypeptide fraction of snow frog, the method comprising the following steps: (1) After soaking the snow frog raw material, soak it in sodium bicarbonate solution, adjust the pH value to 8~12, add hydrogen peroxide solution and stir, drain, and obtain pretreated snow frog raw material; (2) Place the pretreated snow frog raw material in a container, add water, adjust the pH value to 9-12, add animal protein hydrolysate to carry out enzymatic hydrolysis, filter, and obtain enzymatic hydrolysate; (3) Place the enzymatic hydrolysate in a container, add activated carbon powder, stir to adsorb, filter, and obtain filtrate; (4) Concentrate the filtrate to obtain a concentrated solution, filter the concentrated solution, and dry it to obtain snow frog polypeptide powder; (5) Take an appropriate amount of snow frog polypeptide powder, add water, filter, and obtain a snow frog polypeptide mixture solution; and (6) The mixture of snow frog peptides was injected into a pretreated rapid separation column and separated by preparative liquid chromatography. The peptide solutions corresponding to the second to sixth peptide peaks were collected and dried to obtain the purified second, third, fourth, fifth and sixth snow frog peptide fractions.

[0006] Further, in step (1), the snow frog raw material is dried snow frog remains after removing snow frog eggs.

[0007] Furthermore, in step (1), the evaporation is carried out in purified water.

[0008] Furthermore, in step (1), the soaking time is 1 to 12 hours, for example, 5 to 12 hours.

[0009] Further, in step (1), after the soaking, immersion and / or stirring, the sample is rinsed with purified water.

[0010] Furthermore, the rinsing is performed 2 to 5 times, for example, 3 times.

[0011] Furthermore, in step (1), the concentration of the sodium bicarbonate solution is 0.1% to 1%, for example, about 0.5%.

[0012] Furthermore, in step (1), the soaking time is 1 to 12 hours, for example, 5 to 12 hours.

[0013] Furthermore, in step (1), the concentration of the hydrogen peroxide solution is 0.5% to 5%, for example, about 1%.

[0014] Furthermore, in step (1), the stirring time is 0.5 to 3 hours, for example, about 1 hour.

[0015] Furthermore, in step (2), the water is purified water.

[0016] Furthermore, in step (2), the container is an enzymatic hydrolysis tank.

[0017] Further, in step (2), the mass / volume (g / mL) ratio of the pretreated snow frog raw material to the water is 0.125 to 0.25, for example, about 0.2.

[0018] Further, in step (2), the mass ratio of the pretreated snow frog raw material to the animal protein hydrolysate is 0.001 to 0.01, for example, about 0.005.

[0019] Further, in step (2), the enzymatic hydrolysis reaction is carried out as follows: heating to 40~60℃, stirring for 1~8 h, heating to about 100℃ and holding for 5~40 min, and cooling to 50~70℃.

[0020] Furthermore, the enzymatic hydrolysis reaction proceeds as follows: heating to approximately 50°C, stirring for approximately 2 hours, heating to approximately 100°C and holding for approximately 10 minutes or approximately 30 minutes, and cooling to approximately 60°C.

[0021] Furthermore, in step (2), the filtration is a 0.45 μm plate and frame filter.

[0022] Furthermore, in step (3), the container is a mixing tank.

[0023] Further, in step (3), the amount of activated carbon powder added is 0.5% to 10% of the total weight of the enzymatic hydrolysate, for example, 2% to 10%, for example, about 5%.

[0024] Furthermore, in step (3), the temperature of the stirring adsorption is 40~70°C, for example about 50°C or about 60°C.

[0025] Furthermore, in step (3), the stirring adsorption time is 0.5 to 2 hours, for example, about 1 hour.

[0026] Furthermore, in step (3), the filtration is a 0.22 μm plate and frame filter.

[0027] Furthermore, in step (4), the concentration is carried out in a vacuum concentration tank.

[0028] Furthermore, in step (4), the concentration is a vacuum-reduced concentration.

[0029] Furthermore, in step (4), the solid content of the concentrate is 1% to 40%, for example, 4% to 40%.

[0030] Furthermore, in step (4), the filtration is a 0.22 μm plate and frame filter.

[0031] Furthermore, in step (4), the drying is carried out in a spray tower.

[0032] Furthermore, in step (4), the spray rate of the dryer is 3 to 10 L / h, for example, about 5 L / h.

[0033] Furthermore, the pH value is adjusted to approximately 10 using a sodium hydroxide solution.

[0034] Furthermore, the concentration of the sodium hydroxide solution is 0.5~2 mol / L, for example, about 1 mol / L.

[0035] Furthermore, in step (5), the filtration is membrane filtration.

[0036] Furthermore, the pore size of the filter membrane is 0.22 μm.

[0037] Further, in step (5), the concentration of the snow frog polypeptide mixture solution is 0.1~2 g / mL, for example, about 1 g / mL.

[0038] Further, in step (5), the mass of the snow frog polypeptide mixture is 1 to 3 g, for example, about 2 g.

[0039] Furthermore, in step (5), the water is ultrapure water.

[0040] Furthermore, the volume of the water is 1 to 3 mL, for example, about 2 mL.

[0041] Furthermore, in step (6), the specification of the rapid separation column is approximately 330 g.

[0042] Furthermore, the packing material for this rapid separation column is UniPMM 40-500.

[0043] Furthermore, in step (6), the pretreatment involves rinsing the column with 2 to 6 times the volume of pure water, for example, using approximately 4 times the volume of pure water.

[0044] Further, in step (6), the conditions for preparing the liquid phase are as follows: mobile phase A is water, mobile phase B is methanol, the gradient elution program is: 0~15 min, 0%B; 15.1~25 min, 20%B; 25.1~35 min, 40%B; 35.1~50 min, 60%B; the flow rate is 5~15 mL / min, the column temperature is room temperature, and the ultraviolet detection wavelength is 180~300 nm.

[0045] Furthermore, the flow rate is approximately 10 mL / min.

[0046] Furthermore, the ultraviolet detection wavelength is approximately 220 nm.

[0047] Furthermore, in step (6), the drying is freeze drying.

[0048] Furthermore, the freeze-drying time is 12 to 36 hours, for example, about 24 hours.

[0049] According to another aspect of the present invention, a purified second, third, fourth, fifth, and / or sixth snow frog polypeptide fraction prepared by the above preparation method is provided.

[0050] According to another aspect of the present invention, a snow frog peptide or a salt thereof is provided, wherein the amino acid sequence of the snow frog peptide is SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10.

[0051] Furthermore, this snow frog peptide is derived from the aforementioned sixth snow frog polypeptide fraction.

[0052] Furthermore, this snow frog peptide is synthesized using a solid-phase synthesis method.

[0053] Furthermore, the solid-phase synthesis method includes the steps of (a) swelling the resin, (b) deprotecting the resin, (c) washing the deprotected resin, (d) detecting the deprotection, (e) condensing the amino acid, (f) deprotection, (g) washing the deprotected resin, (h) detecting the deprotection, (i) condensation, and (j) detecting the reaction.

[0054] According to another aspect of the present invention, the use of the above-mentioned snow frog polypeptide powder, or the above-mentioned purified second, third, fourth, fifth and / or sixth snow frog polypeptide fractions, or the above-mentioned snow frog peptides or their salts, in the preparation of cosmetics, functional foods, pharmaceuticals and / or health products with anti-glycation functional activity is provided.

[0055] According to another aspect of the present invention, the above-mentioned snow frog peptide or its salt is provided for use in the preparation of food, pharmaceutical and / or health products having antihypertensive functional activity.

[0056] Furthermore, this anti-glycation functional activity includes inhibition of advanced glycation end products (AGEs).

[0057] Furthermore, this antihypertensive activity includes angiotensin-converting enzyme inhibitory activity.

[0058] This invention provides a method for preparing snow frog oligopeptide powder, the method comprising the following steps: (1) Pre-processing Soak the dried snow frog limbs (after removing the snow frog eggs) in purified water for 5-12 hours. After draining the water, rinse with purified water 2-5 times, then soak in a 0.1%-1% sodium bicarbonate solution for 5-12 hours. Remove the sodium bicarbonate solution, rinse the limbs with purified water until neutral, then soak the limbs in 3-5 times the amount of purified water. Adjust the pH to 8-12 with a 1 mol / L sodium hydroxide solution, add 1%-5% hydrogen peroxide, stir for 1-3 hours, discard the solution, rinse the limbs with purified water 3 times, drain the water, and set aside.

[0059] (2) Enzymatic hydrolysis Place the processed limbs in an enzymatic hydrolysis vessel, add 4-8 times the amount of purified water, adjust the pH to 9-12 with 1 mol / L sodium hydroxide solution, then add 0.1%-1% of animal protein hydrolysing enzyme by weight of the limbs, raise the temperature to 50 ℃-60 ℃, stir and react for 2-8 h, raise the temperature to 100 ℃ to inactivate the enzyme for 30 min, stop heating, and wait for the enzymatic hydrolysate to cool to 60 ℃. Filter the solution through a 0.45 μm plate and frame filter, and set the filtrate aside for use.

[0060] (3) Decolorization and deodorization Place the filtrate in a mixing tank, add 2%-10% activated carbon powder, stir and adsorb at 50 ℃ for 0.5-2 h, filter through a 0.22 μm plate and frame filter, and set the filtrate aside for use.

[0061] (4) Vacuum decompression concentration The decolorized and deodorized enzymatic hydrolysate was placed in a vacuum concentration tank and concentrated under vacuum until the solid content was 4%-40%. It was then filtered again using a 0.22 μm plate and frame filter, and the filtrate was set aside for use.

[0062] (5) Obtaining snow frog oligopeptide powder The concentrated enzymatic hydrolysate was passed into a spray tower to obtain snow frog oligopeptide powder.

[0063] The second technical solution provided by this invention is to obtain the snow frog polypeptide fraction using the method described in the first technical solution, and to separate and purify the snow frog polypeptide mixture. The method includes the following steps: (1) Prepare a solution of snow frog polypeptide mixture with a concentration of 0.1 g / mL-1 g / mL, and filter the dissolved polypeptide mixture solution through a 0.22 μm filter membrane.

[0064] (2) Rinse the column with 2 to 4 times the volume of pure water, and inject the polypeptide mixture solution after passing through the 0.22 μm membrane into the top with a syringe. After the separation column is installed, start the separation and purification.

[0065] (3) Preparation liquid phase conditions: column temperature: room temperature; mobile phase A: water; mobile phase B: methanol; UV detection wavelength: 220 nm. Peptide peaks were collected manually and freeze-dried for 24 h.

[0066] The third technical solution provided by this invention is to identify and screen the purified peptides to obtain peptide sequences with anti-glycation and blood pressure-lowering potential. This method includes the following steps: (1) Peptide identification: CE-MS analysis was performed using a PS2 neutral-coated capillary. Peptide sequencing was performed on the acquired data using the de novo sequencing module of Peaks Studio software (version: 12.0).

[0067] (2) Peptide screening: Peptides are screened based on activity score and affinity for receptor, and the peptides are subjected to virtual enzymatic digestion.

[0068] The beneficial effects of this invention are: This invention utilizes alkaline protease to enzymatically hydrolyze dried snow frog to obtain snow frog polypeptides. This method is simple, rapid, safe, efficient, environmentally friendly, and low-cost.

[0069] The snow frog polypeptide powder obtained by this invention has anti-glycation effects and can significantly reduce the formation of AGEs. The identified peptides have anti-glycation effects and ACE inhibitory activity, and can be used in the fields of ordinary food, health food, special medical purpose formula food and cosmetics, providing a new solution for people with needs such as anti-glycation and blood pressure reduction.

[0070] This invention screened and synthesized peptides with AGEs-inhibiting and ACE-inhibiting activities. Compared with small molecule inhibitors, these peptides exhibit no cytotoxicity and are suitable for long-term use. By inhibiting AGEs formation and ACE activity, protecting the function of biological macromolecules, and synergistically providing antioxidant effects, these peptides demonstrate highly efficient and safe properties in preventing glycation, hypertension-related diseases, and aging. They possess both academic innovation and industrialization value, and are particularly suitable for the cosmetics, functional foods, and pharmaceutical fields. Attached Figure Description

[0071] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0072] Figure 1 The image shows the separation and purification chromatograms of snow frog polypeptide powder. F1-F6 represent six different fractions obtained from the purification of the snow frog polypeptide powder, corresponding to the first, second, third, fourth, fifth, and sixth purified snow frog polypeptide fractions, respectively.

[0073] Figure 2 This is a schematic diagram showing the AGEs inhibitory activity results of snow frog polypeptide powder and its F1-F6 fractions. NC: negative control; PC: positive control (guanidine salt of amino acids, 10 mg / mL); * indicates the difference compared to NC. p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001; A represents the AGEs inhibition activity results of snow frog polypeptide powder; B represents the AGEs inhibition activity results of F1-F6 sites.

[0074] Figure 3 This is a schematic diagram showing the AGEs inhibitory activity results of the synthetic peptides from snow frog. NC: negative control; PC: positive control (guanidine salt of amino acids, 10 mg / mL); * indicates the difference compared to NC. p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

[0075] Figure 4 This is a schematic diagram showing the ACE inhibitory activity results of the synthetic peptides from *Cephalotaxus fortunei*. In the diagram, NC represents the negative control, and PC represents the positive control (captopril, 10 nmol / L). * indicates a difference compared to NC. p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. Detailed Implementation

[0076] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0077] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0078] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0079] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0080] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0081] As described in the background section, anti-glycation and blood pressure reduction are long-term processes, and long-term use of chemically synthesized inhibitors will pose challenges to the body's health. To address the above problems, this invention provides a method for preparing snow frog polypeptide fractions, which includes the following steps: (1) After soaking the snow frog raw material, soak it in sodium bicarbonate solution, adjust the pH value to 8~12, add hydrogen peroxide solution and stir, drain, and obtain pretreated snow frog raw material; (2) Place the pretreated snow frog raw material in a container, add water, adjust the pH value to 9-12, add animal protein hydrolysate to carry out enzymatic hydrolysis, filter, and obtain enzymatic hydrolysate; (3) Place the enzymatic hydrolysate in a container, add activated carbon powder, stir to adsorb, filter, and obtain filtrate; (4) Concentrate the filtrate to obtain a concentrated solution, filter the concentrated solution, and dry it to obtain snow frog polypeptide powder; (5) Take an appropriate amount of snow frog polypeptide powder, add water, filter, and obtain a snow frog polypeptide mixture solution; and (6) The mixture of snow frog peptides was injected into a pretreated rapid separation column and separated by preparative liquid chromatography. The peptide solutions corresponding to the second to sixth peptide peaks were collected and dried to obtain the purified second, third, fourth, fifth and sixth snow frog peptide fractions.

[0082] In this invention, when pH, time, frequency, concentration, ratio, final concentration, temperature, amount added, solid content, spray speed, mass, volume, multiple, flow rate, detection wavelength, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “8~12” is disclosed, the described range should be interpreted as including ranges “8~12”, “8~11”, “8~10”, “8~9”, “9~12”, “9~11”, “9~10”, “10~12”, “10~11”, “11~12”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0083] In a preferred embodiment, in step (1), the snow frog raw material is dried snow frog remains after removing snow frog eggs.

[0084] In a preferred embodiment, in step (1), the soaking is carried out in purified water.

[0085] In a preferred embodiment, in step (1), the soaking time is 1 to 12 hours, for example, 5 to 12 hours.

[0086] In a preferred embodiment, in step (1), after the soaking, immersion and / or stirring, the sample is rinsed with purified water.

[0087] In a preferred embodiment, the rinsing is performed 2 to 5 times, for example, 3 times.

[0088] In a preferred embodiment, in step (1), the concentration of the sodium bicarbonate solution is 0.1% to 1%, for example, about 0.5%.

[0089] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5%" includes 0.5% ± 5%, or from 0.475% to 0.525%.

[0090] In a preferred embodiment, in step (1), the soaking time is 1 to 12 hours, for example, 5 to 12 hours.

[0091] In a preferred embodiment, in step (1), the concentration of the hydrogen peroxide solution is 0.5% to 5%, for example, about 1%.

[0092] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1%" includes 1% ± 5%, or from 0.95% to 1.05%.

[0093] In a preferred embodiment, the stirring time in step (1) is 0.5 to 3 hours, for example, about 1 hour.

[0094] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0095] In a preferred embodiment, in step (2), the water is purified water.

[0096] In a preferred embodiment, in step (2), the container is an enzymatic hydrolysis tank.

[0097] In a preferred embodiment, in step (2), the mass / volume (g / mL) ratio of the pretreated snow frog raw material to the water is 0.125 to 0.25, for example, about 0.2.

[0098] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21.

[0099] In a preferred embodiment, in step (2), the mass ratio of the pretreated snow frog raw material to the animal protein hydrolysate is 0.001 to 0.01, for example, about 0.005.

[0100] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.005" includes ±5% of 0.005, or from 0.00475 to 0.00525.

[0101] In a preferred embodiment, in step (2), the enzymatic hydrolysis reaction is performed as follows: heating to 40-60°C, stirring for 1-8 h, heating to about 100°C and holding for 5-40 min, and cooling to 50-70°C.

[0102] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0103] In a preferred embodiment, the enzymatic hydrolysis reaction is performed by heating to about 50°C, stirring for about 2 hours, heating to about 100°C and holding for about 10 minutes or about 30 minutes, and cooling to about 60°C.

[0104] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 100" includes ±5% of 100, or from 95 to 105; "about 10" includes ±5% of 10, or from 9.5 to 10.5; "about 30" includes ±5% of 30, or from 28.5 to 31.5; and "about 60" includes ±5% of 60, or from 57 to 63.

[0105] In a preferred embodiment, in step (2), the filtration is a 0.45 μm plate and frame filter.

[0106] In a preferred embodiment, in step (3), the container is a mixing tank.

[0107] In a preferred embodiment, in step (3), the amount of activated carbon powder added is 0.5% to 10% of the total weight of the enzymatic hydrolysate, for example, 2% to 10%, for example, about 5%.

[0108] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5%" includes 5% ± 5%, or from 4.75% to 5.25%.

[0109] In a preferred embodiment, in step (3), the temperature of the stirring adsorption is 40~70°C, for example about 50°C or about 60°C.

[0110] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5; "about 60" includes ±5% of 60, or from 57 to 63.

[0111] In a preferred embodiment, in step (3), the stirring and adsorption time is 0.5 to 2 hours, for example, about 1 hour.

[0112] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0113] In a preferred embodiment, in step (3), the filtration is a 0.22 μm plate and frame filter.

[0114] In a preferred embodiment, in step (4), the concentration is carried out in a vacuum concentration tank.

[0115] In a preferred embodiment, in step (4), the concentration is a vacuum concentration.

[0116] In a preferred embodiment, in step (4), the solid content of the concentrate is 1% to 40%, for example, 4% to 40%.

[0117] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3%" includes 3% ± 5%, or from 2.85% to 3.15%.

[0118] In a preferred embodiment, in step (4), the filtration is a 0.22 μm plate and frame filter.

[0119] In a preferred embodiment, the drying in step (4) is carried out in a spray tower.

[0120] In a preferred embodiment, in step (4), the spray rate for drying is 3 to 10 L / h, for example, about 5 L / h.

[0121] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0122] In a preferred embodiment, the pH adjustment is performed by adjusting the pH value to approximately 10 using a sodium hydroxide solution.

[0123] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0124] In a preferred embodiment, the concentration of the sodium hydroxide solution is 0.5 to 2 mol / L, for example, about 1 mol / L.

[0125] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0126] In a preferred embodiment, in step (5), the filtration is membrane filtration.

[0127] In a preferred embodiment, the filter membrane has a pore size of 0.22 μm.

[0128] In a preferred embodiment, in step (5), the concentration of the snow frog polypeptide mixture solution is 0.1~2 g / mL, for example, about 1 g / mL.

[0129] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0130] In a preferred embodiment, in step (5), the mass of the snow frog polypeptide mixture is 1 to 3 g, for example, about 2 g.

[0131] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0132] In a preferred embodiment, in step (5), the water is ultrapure water.

[0133] In a preferred embodiment, the volume of the water is 1 to 3 mL, for example, about 2 mL.

[0134] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0135] In a preferred embodiment, in step (6), the rapid separation column has a specification of about 330 g.

[0136] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 330" includes ±5% of 330, or from 313.5 to 346.5.

[0137] In a preferred embodiment, the packing material for the rapid separation column is UniPMM 40-500.

[0138] In a preferred embodiment, in step (6), the pretreatment is to use 2 to 6 times the volume of pure water to wet the column, for example, to use about 4 times the volume of pure water to wet the column.

[0139] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0140] In a preferred embodiment, in step (6), the conditions for preparing the liquid phase are as follows: mobile phase A is water, mobile phase B is methanol, the gradient elution program is: 0~15 min, 0%B; 15.1~25 min, 20%B; 25.1~35 min, 40%B; 35.1~50 min, 60%B; the flow rate is 5~15 mL / min, the column temperature is room temperature, and the ultraviolet detection wavelength is 180~300 nm.

[0141] In a preferred embodiment, the flow rate is approximately 10 mL / min.

[0142] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0143] In a preferred embodiment, the ultraviolet detection wavelength is approximately 220 nm.

[0144] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 220" includes ±5% of 220, or from 209 to 231.

[0145] In a preferred embodiment, in step (6), the drying is freeze drying.

[0146] In a preferred embodiment, the freeze-drying time is 12 to 36 hours, for example, about 24 hours.

[0147] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 24" includes ±5% of 24, or from 22.8 to 25.2.

[0148] According to another aspect of the present invention, a purified second, third, fourth, fifth, and / or sixth snow frog polypeptide fraction prepared by the above preparation method is provided.

[0149] According to another aspect of the present invention, a snow frog peptide or a salt thereof is provided, wherein the amino acid sequence of the snow frog peptide is SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10.

[0150] In a preferred embodiment, the snow frog peptide is derived from the sixth snow frog polypeptide fraction mentioned above.

[0151] In a preferred embodiment, the snow frog peptide is a snow frog peptide synthesized using a solid-phase synthesis method.

[0152] In a preferred embodiment, the solid-phase synthesis method includes the steps of (a) swelling the resin, (b) deprotecting the resin, (c) washing the deprotected resin, (d) detecting the deprotected resin, (e) condensing the amino acid, (f) deprotecting the resin, (g) washing the deprotected resin, (h) detecting the deprotected resin, (i) condensing the resin, and (j) detecting the reaction.

[0153] According to another aspect of the present invention, the use of the above-mentioned snow frog polypeptide powder, or the above-mentioned purified second, third, fourth, fifth and / or sixth snow frog polypeptide fractions, or the above-mentioned snow frog peptides or their salts, in the preparation of cosmetics, functional foods, pharmaceuticals and / or health products with anti-glycation functional activity is provided.

[0154] According to another aspect of the present invention, the above-mentioned snow frog peptide or its salt is provided for use in the preparation of food, pharmaceutical and / or health products having antihypertensive functional activity.

[0155] In a preferred embodiment, the anti-glycation functional activity includes advanced glycation end product (AGE) inhibition activity.

[0156] In a preferred embodiment, the antihypertensive activity includes angiotensin-converting enzyme inhibitory activity.

[0157] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0158] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0159] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0160] Experimental instruments and materials Multifunctional extraction and concentration system (Shanghai Yuyan Machinery Equipment Co., Ltd.); YC-019 spray dryer (Shanghai Yacheng Instrument Equipment Co., Ltd.); Rapid preparative liquid chromatograph (Changzhou Santai Technology Co., Ltd.); SpectraMax ID3 microplate reader (Meigu Molecular Instruments (Shanghai) Co., Ltd.); Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific); Sodium bicarbonate (Sinopharm Chemical Reagent Co., Ltd.); Sodium hydroxide (Sinopharm Chemical Reagent Co., Ltd.); 1M dilute hydrochloric acid solution (Shenzhen Bolinda Technology Co., Ltd.); UniPMM 40-500 packing material (Changzhou Santai Technology Co., Ltd.).

[0161] The snow frog comes from Changbai Mountain and the Greater and Lesser Khingan Mountains, and has been identified as genuine.

[0162] Example 1: Preparation of Snow Frog Polypeptides (1) Pre-processing Soak the dried snow frog limbs (after removing the snow frog eggs) in purified water for 5 hours. After draining the water, rinse the limbs three times with purified water, then soak them in a 0.5% sodium bicarbonate solution for 5 hours. Remove the sodium bicarbonate solution, rinse the limbs with purified water until neutral, then soak the limbs in 5 times the amount of purified water. Adjust the pH to 10 with a 1 mol / L sodium hydroxide solution, add 1% hydrogen peroxide, stir for 1 hour, discard the solution, rinse the limbs three times with purified water, drain the water, and set aside.

[0163] (2) Enzymatic hydrolysis The processed limb was placed in an enzymatic hydrolysis vessel, 5 times the volume of purified water was added, the pH was adjusted to 10 with 1 mol / L sodium hydroxide solution, and then 0.5% of the limb mass of animal protein hydrolase was added. The temperature was raised to 50 °C and stirred for 2 h. The temperature was then raised to 100 °C to inactivate the enzyme for 10 min. Heating was stopped, and the hydrolysate was allowed to cool to 60 °C. It was then filtered through a 0.45 μm plate and frame filter, and the filtrate was set aside for use.

[0164] (3) Decolorization and deodorization Place the filtrate in a mixing tank, add 5% activated carbon powder, stir and adsorb at 50 ℃ for 1 h, filter through a 0.22 μm plate and frame filter, and set the filtrate aside for use.

[0165] (4) Vacuum decompression concentration The decolorized and deodorized enzymatic hydrolysate was placed in a vacuum concentration tank and concentrated under vacuum until the solid content was 4%. It was then filtered again using a 0.22 μm plate and frame filter, and the filtrate was set aside for use.

[0166] (5) Obtaining snow frog oligopeptide powder The concentrated enzymatic hydrolysate was passed into a spray tower and treated at a spray rate of 5 L / h to obtain snow frog oligopeptide powder.

[0167] Example 2: Isolation and purification of snow frog polypeptides (1) Take 2 g of frozen snow frog polypeptide powder sample, add 2 mL of ultrapure water, prepare a snow frog polypeptide solution with a concentration of 1 g / mL, and filter the dissolved polypeptide solution through a 0.22 μm filter membrane.

[0168] (2) Load UniPMM 40-500 packing into a standard rapid separation column (330 g), rinse the column with four times the volume of pure water, and inject the polypeptide solution after passing through a 0.22 μm membrane into the top of the column using a syringe. After the separation column is installed, start the separation and purification process.

[0169] (3) Preparative liquid phase conditions: column temperature: room temperature; mobile phase A: water; mobile phase B: methanol; elution gradient as shown in Table 1; flow rate: 10 mL / min; UV detection wavelength: 220 nm. The peptide peaks were manually collected and divided into six parts, F1-F6, and then freeze-dried for 24 h.

[0170] Table 1. Elution gradient of mobile phase

[0171] Example 3 Assay of the inhibitory activity of snow frog peptide on AGEs A mixed solution containing 80 mg / mL bovine serum albumin and 240 mg / mL glucose was prepared using PBS and filtered through a 0.22 μm filter to serve as the 2× glycation reaction solution. The reaction systems for each group were prepared according to the table. The final concentration of bovine serum albumin in each reaction system was 40 mg / mL, and the final concentration of glucose was 120 mg / mL. The final concentrations of the samples or positive controls are shown in Table 2.

[0172] Table 2 AGEs Removal Test Reaction System

[0173] After thorough mixing, the mixture was incubated at 55 °C for 4 days. PBS was used as a negative control instead of the sample, 10% aminoguanidine hydrochloride as a positive control, and PBS was used instead of the glycosylation reaction solution as a control system. After the reaction, the incubated solution was cooled to room temperature, centrifuged at 2000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm filter. 200 μL of the reaction solution was then added to each well of a 96-well plate. The AGEs inhibition rate was measured using a fluorescence microplate reader at an excitation wavelength of 320 nm and an emission wavelength of 460 nm, and calculated using the following formula: AGEs inhibition rate (%) = [1 - (AB) / (CD)] × 100 Where: A - fluorescence intensity of the saccharification system with added test substance; B - Fluorescence intensity of the PBS solution containing the test substance; C - Fluorescence intensity of the saccharified system without the addition of the test substance; D - Fluorescence intensity of PBS solution without test substance.

[0174] The results are as follows Figure 2 As shown in Figure A, compared with the BC group, the AGEs inhibition rate in the PC group was significantly increased ( p < 0.001 indicates that the experimental system is effective; compared with group BC, the inhibition rate of AGEs increased significantly with increasing concentration ( p The inhibition rate was < 0.05%, but decreased to some extent at 500 mg / mL. Experimental results indicate that the sample possesses anti-glycation ability.

[0175] The results are as follows Figure 2 As shown in B, compared with the BC group, the AGEs inhibition rate in the PC group was significantly increased ( p < 0.001 indicates that the experimental system is effective; compared with group BC, F1 did not show AGEs inhibitory activity at any concentration; compared with group BC, F2 showed significantly increased AGEs inhibition rate at concentrations of 0.625 mg / mL and 1.25 mg / mL ( p < 0.05); Compared with the BC group, the AGEs inhibition rates of F3, F4, F5, and F6 were significantly increased at concentrations of 0.625 mg / mL, 1.25 mg / mL, and 2.5 mg / mL, respectively. p < 0.05) indicates that the sample has anti-glycation ability; F6 has the strongest AGEs inhibitory activity, and the activity increases with the increase of sample concentration. F6 was selected for subsequent peptide identification.

[0176] Example 4: Identification and Screening of Active Polypeptides from Snow Frog CE-MS analysis was performed using a PS2 neutral-coated capillary (BGE: 50% HAC; SL: 50 mmol / L ammonium acetate + 2.5% acetic acid). Sample injection was 500 mbar, 10 s; mass spectrometry data acquisition range was 1000-3200 m / z; separation voltage was 30 kV. Peptide sequencing was performed on the acquired data using Peaks Studio software (version 12.0) with the de novo sequencing module. The primary precursor ion error was set to ±10 ppm, considering variable modifications: N, Q deamidation, M oxidation.

[0177] Peptide activity was scored using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ). Virtual enzymatic digestion of peptides was performed using the ENZYME(S) ACTION section of the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php). Molecular docking was used to predict the binding degree of peptides to the BSA receptor, and molecular docking with the ACE receptor was also performed. All databases are open access, with searchable text and structures, and are continuously updated.

[0178] Table 3. Evaluation of the activity and prediction of the properties of snow frog polypeptides

[0179] The screening results are shown in Table 3. Peptides with high biological activity (PeptideRanker score > 0.6) and high binding affinity (Molecular docking score < -7) were selected, resulting in 10 peptides. Among them, 8 peptides were resistant to pepsin and 9 peptides were resistant to trypsin, indicating that they may have stability in vivo.

[0180] The above-mentioned polypeptide was synthesized by Nanjing GenScript using a solid-phase synthesis method. The basic process includes: (1) Swelling resin: Add resin to the reactor, add DCM and stir to swell and activate.

[0181] (2) Resin deprotection: Vacuum dry the swelling reagent, add Pip / DMF and stir for several minutes.

[0182] (3) Deprotection washing: Vacuum dry the deprotection reagent, wash with DMF, and dry.

[0183] (4) Deprotection test: Put the resin into the test tube, add the ninhydrin test reagent, put the test tube into the water bath, take it out and observe the color of the resin. If the resin color becomes darker, it is positive, indicating that the deprotection is successful.

[0184] (5) Condensation of the first amino acid: Dissolve Fmoc-Pro-OH and HOBT in an appropriate amount of DMF, then add DIC to activate them and pour them into the reaction vessel for stirring reaction.

[0185] (6) Deprotection: Add Pip / DMF and stir.

[0186] (7) Deprotection washing: Vacuum dry the deprotection reagent, then wash with DMF. (8) Deprotection test: Put the resin into the test tube, add the ninhydrin test reagent, heat in a water bath, take it out and observe the color of the resin. If the resin color becomes darker, it is positive, indicating that the deprotection is successful.

[0187] (9) Condensation: Dissolve Fmoc-Pro-OH and HOBT in an appropriate amount of DMF, then add DIC to activate them and pour them into the reactor.

[0188] (10) Reaction detection: Take out and observe the color of the resin. If there is no obvious change in the color of the resin, it indicates that the reaction condensation is successful.

[0189] (11) Repeat steps 7 to 12 to condense the amino acids in the sequence from right to left.

[0190] (12) After the last amino acid condensation is completed, the deprotection is performed in sequence. After the deprotection washing step, the resin is washed with methanol and then vacuum dried.

[0191] Example 4: Inhibitory activity of synthetic peptides against AGEs The experimental method was the same as in Example 3. The concentration of the synthesized peptide was 0.5 mg / mL, and the concentration of the positive standard was 10 mg / mL.

[0192] The results are as follows Figure 3 As shown, SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 exhibit AGEs inhibitory activity. Among them, SEQ ID NO.1 and SEQ ID NO.3 show strong AGEs inhibition rates, which is consistent with the results of molecular docking. Both SEQ ID NO.1 and SEQ ID NO.3 have strong binding affinity to BSA (score < -9).

[0193] Example 5: ACE inhibitory activity of synthetic peptides ACE inhibitory activity assay In a 96-well plate, 40 μL of sample (2.5 mg / mL) was added to each well, followed by 50 μL of FAPGG solution and 10 μL of ACE enzyme solution (0.1 U / mL). After mixing, the absorbance was immediately measured at 340 nm. The plate was then incubated at 37 °C in the dark for 30 min, and the absorbance at 340 nm was measured again. The HEPES buffer concentration was 80 mmol / L (pH = 8.30, NaCl concentration = 300 mmol / L). A 1.0 mmol / L FAPGG solution was prepared using HEPES buffer and stored in the dark.

[0194] The formula for calculating the ACE inhibition rate is: .

[0195] In the formula: A is the decrease in absorbance of the blank well; B is the decrease in absorbance of the sample well.

[0196] The results are as follows Figure 4 As shown, 7 peptides exhibited some ACE inhibitory activity, while SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 showed no activity. Among them, SEQ ID NO.1, SEQ ID NO.8, and SEQ ID NO.9 showed strong ACE inhibitory activity, with SEQ ID NO.3 exhibiting the strongest activity. p < 0.05), reaching a maximum of 71.32%.

[0197] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing a polypeptide fraction of Concha Haliotidis, characterized in that, The preparation method includes the following steps: (1) After soaking the snow frog raw material, soak it in sodium bicarbonate solution, adjust the pH value to 8~12, add hydrogen peroxide solution and stir, drain, and obtain pretreated snow frog raw material; (2) Place the pretreated snow frog raw material in a container, add water, adjust the pH value to 9-12, add animal protein hydrolysate to carry out enzymatic hydrolysis, filter, and obtain enzymatic hydrolysate; (3) Place the enzymatic hydrolysate in a container, add activated carbon powder, stir to adsorb, filter, and obtain filtrate; (4) Concentrate the filtrate to obtain a concentrated solution, filter the concentrated solution, and dry it to obtain snow frog polypeptide powder; (5) Take an appropriate amount of snow frog polypeptide powder, add water, filter, and obtain a snow frog polypeptide mixture solution; as well as (6) The mixture of snow frog peptides was injected into a pretreated rapid separation column and separated by preparative liquid chromatography. The peptide solutions corresponding to the second to sixth peptide peaks were collected and dried to obtain the purified second, third, fourth, fifth and sixth snow frog peptide fractions.

2. The production method according to claim 1, characterized by, In step (1), the snow frog raw material is dried snow frog remains after removing snow frog eggs; Preferably, in step (1), the soaking is carried out in purified water; Preferably, in step (1), the soaking time is 1 to 12 hours, for example, 5 to 12 hours; Preferably, in step (1), after the soaking, immersion and / or stirring, the sample is rinsed with purified water; More preferably, the rinsing is performed 2 to 5 times, for example, 3 times; More preferably, in step (1), the concentration of the sodium bicarbonate solution is 0.1% to 1%, for example, about 0.5%; More preferably, in step (1), the soaking time is 1 to 12 hours, for example, 5 to 12 hours; More preferably, in step (1), the concentration of the hydrogen peroxide solution is 0.5% to 5%, for example, about 1%; More preferably, in step (1), the stirring time is 0.5 to 3 hours, for example, about 1 hour; Preferably, in step (2), the water is purified water; Preferably, in step (2), the container is an enzymatic hydrolysis tank; Preferably, in step (2), the mass / volume (g / mL) ratio of the pretreated snow frog raw material to the water is 0.125~0.25, for example, about 0.2; Preferably, in step (2), the mass ratio of the pretreated snow frog raw material to the animal protein hydrolyzing enzyme is 0.001 to 0.01, for example, about 0.005; Preferably, in step (2), the enzymatic hydrolysis reaction is performed as follows: heating to 40-60°C, stirring for 1-8 hours, heating to approximately 100°C and holding for 5-40 minutes, and cooling to 50-70°C. More preferably, the enzymatic hydrolysis reaction steps are: heating to about 50°C, stirring for about 2 hours, heating to about 100°C and holding for about 10 minutes or holding for about 30 minutes, and cooling to about 60°C. More preferably, in step (2), the filtration is a 0.45 μm plate and frame filter; Particularly preferably, in step (3), the container is a mixing tank; Particularly preferably, in step (3), the amount of activated carbon powder added is 0.5% to 10% of the total weight of the enzymatic hydrolysate, for example, 2% to 10%, for example, about 5%; Particularly preferably, in step (3), the temperature of the stirring adsorption is 40~70°C, for example about 50°C or about 60°C; Particularly preferably, in step (3), the stirring and adsorption time is 0.5 to 2 hours, for example, about 1 hour; Particularly preferably, in step (3), the filtration is a 0.22 μm plate and frame filter; Particularly preferably, in step (4), the concentration is carried out in a vacuum concentration tank; Particularly preferably, in step (4), the concentration is vacuum concentration; Particularly preferably, in step (4), the solid content of the concentrate is 1% to 40%, for example, 4% to 40%; Particularly preferably, in step (4), the filtration is a 0.22 μm plate and frame filter; Particularly preferably, in step (4), the drying is carried out in a spray tower; Particularly preferably, in step (4), the spray rate for drying is 3 to 10 L / h, for example, about 5 L / h; Particularly preferred is that the pH value is adjusted to approximately 10 using a sodium hydroxide solution; Most preferably, the concentration of the sodium hydroxide solution is 0.5~2 mol / L, for example, about 1 mol / L.

3. The preparation method according to claim 1, characterized in that, In step (5), the filtration is membrane filtration; Preferably, the pore size of the filter membrane is 0.22 μm; Preferably, in step (5), the concentration of the snow frog polypeptide mixture solution is 0.1~2 g / mL, for example, about 1 g / mL; Preferably, in step (5), the mass of the snow frog polypeptide mixture is 1 to 3 g, for example, about 2 g; Preferably, in step (5), the water is ultrapure water; Preferably, the volume of the water is 1 to 3 mL, for example, about 2 mL; More preferably, in step (6), the rapid separation column has a specification of approximately 330 g; More preferably, the packing material of the rapid separation column is UniPMM 40-500; More preferably, in step (6), the pretreatment is to use 2 to 6 times the volume of pure water to wet the column, for example, to use about 4 times the volume of pure water to wet the column.

4. The production method according to claim 1, characterized by, In step (6), the conditions for preparing the liquid phase are: mobile phase A is water, mobile phase B is methanol, and the gradient elution program is: 0~15 min, 0% B; 15.1~25 min, 20% B; 25.1~35 min, 40%B; 35.1~50 min, 60%B; flow rate 5~15 mL / min, column temperature room temperature, UV detection wavelength 180~300 nm; Preferably, the flow rate is about 10 mL / min; Preferably, the ultraviolet detection wavelength is approximately 220 nm; More preferably, in step (6), the drying is freeze-drying; More preferably, the freeze-drying time is 12 to 36 hours, for example, about 24 hours.

5. A purified second, third, fourth, fifth, and / or sixth snow frog polypeptide fraction prepared by the preparation method according to any one of claims 1 to 4.

6. A conchiolin peptide or a salt thereof, characterized by, The amino acid sequence of the snow frog peptide is SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.

10.

7. The conch peptide or salt thereof according to claim 6, characterized in that, The snow frog peptide is derived from the sixth snow frog polypeptide fraction as described in any one of claims 1 to 5; Preferably, the snow frog peptide is a snow frog peptide synthesized using a solid-phase synthesis method; More preferably, the solid-phase synthesis method includes the steps of (a) swelling resin, (b) resin deprotection, (c) deprotection washing, (d) deprotection detection, (e) amino acid condensation, (f) deprotection, (g) deprotection washing, (h) deprotection detection, (i) condensation, and (j) reaction detection.

8. The use of the snow frog polypeptide powder according to any one of claims 1 to 4, or the purified second, third, fourth, fifth and / or sixth snow frog polypeptide fractions according to claim 5, or the snow frog peptide or its salt according to claim 6 or 7, in the preparation of cosmetics, functional foods, pharmaceuticals and / or health products with anti-glycation functional activity.

9. Use of the snow frog peptide or its salt as described in claim 6 or 7 in the preparation of food, pharmaceutical and / or health products with antihypertensive activity.

10. Use according to claim 8 or 9, characterized in that, The anti-glycation functional activity includes inhibition of advanced glycation end products (AGEs); Preferably, the antihypertensive activity includes angiotensin-converting enzyme inhibitory activity.

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