Soybean polypeptide with antibacterial function and preparation method thereof

Abstract

The invention relates to the technical field of polypeptides, and discloses a soybean polypeptide with an antibacterial function and a preparation method thereof.The soybean polypeptide with the antibacterial function is obtained by adding soybean polypeptide, quaternized cholic acid and modified chitosan into N, N-dimethylformamide and stirring for reaction. A chitosan group, a quaternary ammonium salt group, an imidazole group and a cholic acid group contained in the soybean polypeptide with the antibacterial function jointly form an antibacterial system, and a good antibacterial effect is achieved; wherein the chitosan group can destroy bacterial cell walls to death bacteria, so that the antibacterial effect is achieved, and the cholic acid group also has a relatively good antibacterial effect; the imidazole group rapidly kills bacteria by destroying bacterial cell membranes, affecting bacterial metabolism and inhibiting formation of biological membranes, a good antibacterial effect is achieved, quaternary ammonium salt is adsorbed to the negatively charged bacterial cell membranes through the cationic characteristics of quaternary ammonium salt, the cell membranes are broken, and therefore the bacteria are rapidly killed.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, in particular to a soybean polypeptide with antibacterial function and a preparation method thereof. Background Art

[0002] In the pharmaceutical and animal husbandry industries, the extensive use of antibiotics has led to the emergence of a large number of drug-resistant strains and drug residues, which has caused great harm to health. Therefore, it is necessary to develop a substance that is not easy to resist and has antibacterial effects. Soybean polypeptide is a biologically active polypeptide mixture with certain antibacterial properties. Therefore, the present invention blends it with quaternized bile acid and modified chitosan to prepare soybean polypeptide with antibacterial function. For example, patent CN112661814 discloses an antibacterial peptide compound and an antibacterial peptide extract and its preparation method and use. The antibacterial peptide compound and extract of the invention have good antibacterial effects, but its antibacterial substance is relatively single and the antibacterial effect needs to be improved. Summary of the invention

[0003] 1. Technical issues to be resolved

[0004] In view of the deficiencies in the prior art, the present invention provides a soybean polypeptide with antibacterial function and a preparation method thereof, which has good antibacterial effect.

[0005] (II) Technical solution

[0006] To achieve the above object, the present invention provides the following technical solution: a soybean polypeptide with antibacterial function, comprising the following weight components: 15-30 weight parts of soybean polypeptide, 5-10 weight parts of quaternized bile acid, and 6-12 weight parts of modified chitosan.

[0007] Preferably, the preparation method of the quaternized bile acid is:

[0008] (1) adding bile acid to a reactor containing a dimethylformamide solvent, adding tri(2-aminoethyl)amine to the reactor after dissolution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the reactor under an ice bath, stirring to dissolve and activating for 8-12 minutes, adding 1-hydroxybenzotriazole, stirring and reacting at room temperature for 20-24 hours, precipitating in a sodium bicarbonate aqueous solution after the reaction, and washing the precipitate 2-4 times to obtain a bile acid trimer;

[0009] (2) adding 7-12 parts by weight of bile acid trimer and 5-10 parts by weight of 4-vinylbenzyl chloride to dimethyl sulfoxide solvent, stirring to dissolve, stirring to react at 60-80° C., precipitating in ether after completion, washing with ether 2-4 times, and drying to obtain intermediate 1;

[0010] (3) adding intermediate 1 and 1-methyl-1H-imidazole-2-carbonyl chloride to N,N-dimethylformamide solvent, stirring and mixing, and continuously adding pyridine catalyst thereto, reacting at 75-90° C. for 6-10 hours, and then distilling under reduced pressure, washing and drying to obtain intermediate 2;

[0011] (4) Under a nitrogen atmosphere, 9-14 parts by weight of hexamethyldisilazane and 8-16 parts by weight of intermediate 2 are added to n-butanol solvent, stirred and dispersed, and 0.8-1 parts by weight of chloroplatinic acid catalyst is added thereto, reacted at 65-75° C. for 40-60 min, cooled to room temperature after reaction, filtered and washed to obtain quaternized bile acid.

[0012] Preferably, the mass ratio of cholic acid, tris(2-aminoethyl)amine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole in (1) is 1.1-1.3:1:0.01-0.03:0.01-0.02.

[0013] Preferably, the reaction time in (2) is 20-24 hours.

[0014] Preferably, the mass ratio of the intermediate 1, 1-methyl-1H-imidazole-2-carbonyl chloride and pyridine catalyst in (3) is 1:2.2-3.6:0.02-0.03.

[0015] Preferably, the preparation method of the modified chitosan is: add chitosan and chloromethylbenzoyl chloride to N,N-dimethylformamide, stir to dissolve, continue to add triethylamine catalyst, heat to 55-70°C and stir to react for 10-15h, add deionized water after the reaction to precipitate, filter the solvent, wash with acetone to obtain modified chitosan.

[0016] Preferably, the mass ratio of chitosan, chloromethylbenzoyl chloride and triethylamine catalyst is 1:1.4-1.6:0.02-0.03.

[0017] Preferably, the preparation method of the soybean polypeptide with antibacterial function is: add soybean polypeptide, quaternized bile acid, and modified chitosan to N,N-dimethylformamide, stir and react at 60-75°C for 10-12h, add distilled water after the reaction to precipitate, filter, and wash to obtain the soybean polypeptide with antibacterial function.

[0018] (III) Beneficial technical effects

[0019] The method comprises adding soybean polypeptide, quaternized bile acid and modified chitosan into N,N-dimethylformamide, stirring and reacting the mixture, and obtaining the soybean polypeptide with antibacterial function after the reaction.

[0020] The amine group in the carboxytris(2-aminoethyl)amine in the bile acid is subjected to an amidation reaction to generate an amide group, and a hydroxyl group and a tertiary amine group are introduced to obtain a bile acid trimer. The tertiary amine group in the bile acid trimer and the chlorine in 4-vinylbenzyl chloride are subjected to a quaternization reaction to generate a quaternary ammonium salt, and an alkenyl group is introduced to obtain an intermediate 1. The hydroxyl group in the intermediate 1 and the acyl chloride group in 1-methyl-1H-imidazole-2-carbonyl chloride are reacted to introduce an imidazole group and increase the substitution degree of imidazole to obtain an intermediate 2. The silicon-hydrogen bond in hexamethyldisilazane and the alkenyl group in the intermediate 2 are subjected to an addition reaction to obtain quaternized bile acid. Chitosan and chloromethylbenzoyl chloride are reacted to obtain modified chitosan.

[0021] The chitosan group, quaternary ammonium salt group, imidazole group and bile acid group contained in the soybean polypeptide with antibacterial function of the present invention jointly constitute an antibacterial system, which has a good antibacterial effect; the chitosan group can destroy the bacterial cell wall, kill the bacteria, and achieve the antibacterial effect, and the bile acid group itself also has a good antibacterial effect; the imidazole group destroys the bacterial cell membrane, affects the bacterial metabolism and inhibits the formation of the biofilm, thereby quickly killing the bacteria and having a good antibacterial effect; after the 1-methyl-1H-imidazole-2-carbonyl chloride reacts with the intermediate 1, the substitution degree of the imidazole group is increased, and the antibacterial effect is further improved; the quaternary ammonium salt is adsorbed onto the negatively charged bacterial cell membrane through its cationic characteristics, resulting in the rupture of the cell membrane and the leakage of the contents, thereby quickly killing the bacteria.

[0022] During the stirring reaction, the chlorine in the modified chitosan will react with the tertiary amine on the imidazole group in the quaternized bile acid to generate a quaternary ammonium salt group, which has a good antibacterial effect. DETAILED DESCRIPTION

[0023] Example 1

[0024] (1) adding cholic acid to a reactor containing a dimethylformamide solvent, adding tri(2-aminoethyl)amine to the reaction mixture after dissolution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the reaction mixture under an ice bath, stirring and dissolving the mixture, activating the mixture for 8 minutes, and adding 1-hydroxybenzotriazole, wherein the mass ratio of cholic acid, tri(2-aminoethyl)amine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole is 1.1:1:0.01:0.01, reacting the mixture under stirring at room temperature for 20 hours, precipitating the mixture in a sodium bicarbonate aqueous solution after the reaction, and washing the precipitate twice to obtain a cholic acid trimer;

[0025] (2) Add 7 parts by weight of bile acid trimer and 5 parts by weight of 4-vinylbenzyl chloride to dimethyl sulfoxide solvent, stir to dissolve, stir and react at 60° C. for 20 h, precipitate in ether, wash twice with ether, and dry to obtain intermediate 1;

[0026] (3) adding intermediate 1 and 1-methyl-1H-imidazole-2-carbonyl chloride to N,N-dimethylformamide solvent, stirring and mixing, and continuously adding pyridine catalyst thereto, wherein the mass ratio of intermediate 1, 1-methyl-1H-imidazole-2-carbonyl chloride, and pyridine catalyst is 1:2.2:0.02, reacting at 75° C. for 6 h, and then distilling under reduced pressure, washing, and drying to obtain intermediate 2;

[0027] (4) under a nitrogen atmosphere, 9 parts by weight of hexamethyldisilazane and 8 parts by weight of intermediate 2 were added to n-butanol solvent, stirred and dispersed, and 0.8 parts by weight of chloroplatinic acid catalyst was added thereto, reacted at 65° C. for 40 min, cooled to room temperature after completion, filtered and washed to obtain quaternized bile acid;

[0028] (5) Add chitosan and chloromethylbenzoyl chloride to N,N-dimethylformamide, stir to dissolve, continue to add triethylamine catalyst, wherein the mass ratio of chitosan, chloromethylbenzoyl chloride and triethylamine catalyst is 1:1.4:0.02, heat to 55°C and stir to react for 10 hours, add deionized water after the reaction to precipitate, filter the solvent, wash with acetone, and obtain modified chitosan;

[0029] (6) Add 15 parts by weight of soybean polypeptide, 5 parts by weight of quaternized bile acid, and 6 parts by weight of modified chitosan to N,N-dimethylformamide, stir and react at 60°C for 10 hours, add distilled water after the reaction to precipitate, filter, and wash to obtain a soybean polypeptide with antibacterial function.

[0030] Example 2

[0031] (1) adding cholic acid to a reactor containing a dimethylformamide solvent, adding tri(2-aminoethyl)amine to the reaction mixture after dissolution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the reaction mixture under an ice bath, stirring and dissolving the mixture, activating the mixture for 12 minutes, and adding 1-hydroxybenzotriazole, wherein the mass ratio of cholic acid, tri(2-aminoethyl)amine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole is 1.3:1:0.03:0.02, reacting the mixture under stirring at room temperature for 24 hours, precipitating the mixture in a sodium bicarbonate aqueous solution after the reaction, and washing the precipitate 4 times to obtain a cholic acid trimer;

[0032] (2) Add 12 parts by weight of bile acid trimer and 10 parts by weight of 4-vinylbenzyl chloride to dimethyl sulfoxide solvent, stir to dissolve, stir and react at 80° C. for 24 hours, precipitate in ether, wash with ether 4 times, and dry to obtain intermediate 1;

[0033] (3) adding intermediate 1 and 1-methyl-1H-imidazole-2-carbonyl chloride to N,N-dimethylformamide solvent, stirring and mixing, and continuously adding pyridine catalyst thereto, wherein the mass ratio of intermediate 1, 1-methyl-1H-imidazole-2-carbonyl chloride, and pyridine catalyst is 1:3.6:0.03, reacting at 90° C. for 10 h, and then distilling under reduced pressure, washing, and drying to obtain intermediate 2;

[0034] (4) under a nitrogen atmosphere, 14 parts by weight of hexamethyldisilazane and 16 parts by weight of intermediate 2 were added to n-butanol solvent, stirred and dispersed, and 1 part by weight of chloroplatinic acid catalyst was added thereto, reacted at 75° C. for 60 min, cooled to room temperature after completion, filtered and washed to obtain quaternized bile acid;

[0035] (5) Add chitosan and chloromethylbenzoyl chloride to N,N-dimethylformamide, stir to dissolve, continue to add triethylamine catalyst, wherein the mass ratio of chitosan, chloromethylbenzoyl chloride and triethylamine catalyst is 1:1.6:0.03, heat to 70°C and stir to react for 15 hours, add deionized water after the reaction to precipitate, filter the solvent, wash with acetone, and obtain modified chitosan;

[0036] (6) Add 30 parts by weight of soybean peptide, 10 parts by weight of quaternized bile acid, and 12 parts by weight of modified chitosan to N,N-dimethylformamide, stir and react at 75°C for 12 hours, add distilled water after the reaction to precipitate, filter, and wash to obtain a soybean peptide with antibacterial function.

[0037] Example 3

[0038] (1) adding cholic acid to a reactor containing a dimethylformamide solvent, adding tri(2-aminoethyl)amine to the reactor after dissolution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the reactor under an ice bath, stirring and dissolving the mixture, activating the mixture for 10 minutes, and adding 1-hydroxybenzotriazole, wherein the mass ratio of cholic acid, tri(2-aminoethyl)amine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole is 1.2:1:0.02:0.015, reacting the mixture under stirring at room temperature for 22 hours, precipitating the mixture in a sodium bicarbonate aqueous solution after the reaction, and washing the precipitate three times to obtain a cholic acid trimer;

[0039] (2) adding 9.5 parts by weight of bile acid trimer and 7.5 parts by weight of 4-vinylbenzyl chloride to dimethyl sulfoxide solvent, stirring to dissolve, stirring and reacting at 70° C. for 22 hours, precipitating in ether, washing with ether three times, and drying to obtain intermediate 1;

[0040] (3) adding intermediate 1 and 1-methyl-1H-imidazole-2-carbonyl chloride to N,N-dimethylformamide solvent, stirring and mixing, and continuously adding pyridine catalyst thereto, wherein the mass ratio of intermediate 1, 1-methyl-1H-imidazole-2-carbonyl chloride, and pyridine catalyst is 1:2.9:0.025, reacting at 82° C. for 8 h, and then distilling under reduced pressure, washing, and drying to obtain intermediate 2;

[0041] (4) Under a nitrogen atmosphere, 11.5 parts by weight of hexamethyldisilazane and 12 parts by weight of intermediate 2 were added to n-butanol solvent, stirred and dispersed, and 0.9 parts by weight of chloroplatinic acid catalyst was added thereto, reacted at 70° C. for 50 min, cooled to room temperature after completion, filtered and washed to obtain quaternized bile acid;

[0042] (5) Add chitosan and chloromethylbenzoyl chloride to N,N-dimethylformamide, stir to dissolve, continue to add triethylamine catalyst, wherein the mass ratio of chitosan, chloromethylbenzoyl chloride and triethylamine catalyst is 1:1.5:0.025, heat to 62.5°C and stir to react for 12.5h, add deionized water after the reaction to precipitate, filter the solvent, wash with acetone, and obtain modified chitosan;

[0043] (6) Add 22.5 parts by weight of soybean polypeptide, 7.5 parts by weight of quaternized bile acid, and 9 parts by weight of modified chitosan to N,N-dimethylformamide, stir and react at 67°C for 11 hours, add distilled water after the reaction to precipitate, filter, and wash to obtain a soybean polypeptide with antibacterial function.

[0044] Example 4

[0045] (1) adding cholic acid to a reactor containing a dimethylformamide solvent, adding tri(2-aminoethyl)amine to the reaction mixture after dissolution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the reaction mixture under an ice bath, stirring and dissolving the mixture, activating the mixture for 8 minutes, and adding 1-hydroxybenzotriazole, wherein the mass ratio of cholic acid, tri(2-aminoethyl)amine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole is 1.1:1:0.01:0.01, reacting the mixture under stirring at room temperature for 20 hours, precipitating the mixture in a sodium bicarbonate aqueous solution after the reaction, and washing the precipitate twice to obtain a cholic acid trimer;

[0046] (2) Add 7 parts by weight of bile acid trimer and 5 parts by weight of 4-vinylbenzyl chloride to dimethyl sulfoxide solvent, stir to dissolve, stir and react at 60° C. for 20 h, precipitate in ether, wash twice with ether, and dry to obtain intermediate 1;

[0047] (3) adding intermediate 1 and 1-methyl-1H-imidazole-2-carbonyl chloride to N,N-dimethylformamide solvent, stirring and mixing, and continuously adding pyridine catalyst thereto, wherein the mass ratio of intermediate 1, 1-methyl-1H-imidazole-2-carbonyl chloride, and pyridine catalyst is 1:3.6:0.03, reacting at 90° C. for 10 h, and then distilling under reduced pressure, washing, and drying to obtain intermediate 2;

[0048] (4) under a nitrogen atmosphere, 14 parts by weight of hexamethyldisilazane and 16 parts by weight of intermediate 2 were added to n-butanol solvent, stirred and dispersed, and 1 part by weight of chloroplatinic acid catalyst was added thereto, reacted at 75° C. for 60 min, cooled to room temperature after completion, filtered and washed to obtain quaternized bile acid;

[0049] (5) Add chitosan and chloromethylbenzoyl chloride to N,N-dimethylformamide, stir to dissolve, continue to add triethylamine catalyst, wherein the mass ratio of chitosan, chloromethylbenzoyl chloride and triethylamine catalyst is 1:1.5:0.025, heat to 62.5°C and stir to react for 12.5h, add deionized water after the reaction to precipitate, filter the solvent, wash with acetone, and obtain modified chitosan;

[0050] (6) Add 22.5 parts by weight of soybean polypeptide, 7.5 parts by weight of quaternized bile acid, and 9 parts by weight of modified chitosan to N,N-dimethylformamide, stir and react at 67°C for 11 hours, add distilled water after the reaction to precipitate, filter, and wash to obtain a soybean polypeptide with antibacterial function.

[0051] Comparative Example 1

[0052] This comparative example is different from Example 4 in that no quaternized bile acid is added.

[0053] Comparative Example 2

[0054] The difference between this comparative example and Example 4 is that no modified chitosan is added.

[0055] The soybean peptide with antibacterial function was dissolved and cast into a film to make a thin sheet with a radius of 1 cm; then the bacteria were transferred to a sterile culture medium using an inoculation loop to culture the bacteria at a concentration of 10 6CFU / mL of bacterial suspension, then transfer 0.5mL of the bacterial suspension to a culture dish containing beef extract peptone medium, shake evenly and add the slice, and culture in a constant temperature incubator. After 48h of culture, measure the diameter of the inhibition zone.

[0056] Table 1: Inhibition zones of different bacteria.

[0057] sample Escherichia coli (mm) Staphylococcus aureus (mm) Example 1 17.6 16.8 Example 2 18.9 16.5 Example 3 18.5 17.4 Example 4 17.4 15.9 Comparative Example 1 6.5 6.2 Comparative Example 2 7.8 8.6

[0058] It can be seen from Table 1 that Examples 1-4 have better antibacterial effects than Comparative Examples 1-2.

[0059] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A soybean polypeptide with antibacterial function, characterized in that: The invention comprises the following components by weight: 15-30 parts by weight of soybean polypeptide, 5-10 parts by weight of quaternized bile acid and 6-12 parts by weight of modified chitosan.

2. The soybean polypeptide with antibacterial function according to claim 1, characterized in that: The preparation method of the quaternized bile acid is: (1) adding bile acid to a reactor containing a dimethylformamide solvent, adding tri(2-aminoethyl)amine to the reactor after dissolution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the reactor under an ice bath, stirring to dissolve and activating for 8-12 minutes, adding 1-hydroxybenzotriazole, stirring and reacting at room temperature for 20-24 hours, precipitating in a sodium bicarbonate aqueous solution after the reaction, and washing the precipitate 2-4 times to obtain a bile acid trimer; (2) adding 7-12 parts by weight of bile acid trimer and 5-10 parts by weight of 4-vinylbenzyl chloride to dimethyl sulfoxide solvent, stirring to dissolve, stirring to react at 60-80° C., precipitating in ether after completion, washing with ether 2-4 times, and drying to obtain intermediate 1; (3) adding intermediate 1 and 1-methyl-1H-imidazole-2-carbonyl chloride to N,N-dimethylformamide solvent, stirring and mixing, and continuously adding pyridine catalyst thereto, reacting at 75-90° C. for 6-10 hours, and then distilling under reduced pressure, washing and drying to obtain intermediate 2; (4) Under a nitrogen atmosphere, 9-14 parts by weight of hexamethyldisilazane and 8-16 parts by weight of intermediate 2 are added to n-butanol solvent, stirred and dispersed, and 0.8-1 parts by weight of chloroplatinic acid catalyst is added thereto, reacted at 65-75° C. for 40-60 min, cooled to room temperature after reaction, filtered and washed to obtain quaternized bile acid.

3. The soybean polypeptide with antibacterial function according to claim 2, characterized in that: The mass ratio of cholic acid, tris(2-aminoethyl)amine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole in (1) is 1.1-1.3:1:0.01-0.03:0.01-0.

02.

4. The soybean polypeptide with antibacterial function according to claim 2, characterized in that: The reaction time in (2) is 20-24h.

5. The soybean polypeptide with antibacterial function according to claim 2, characterized in that: The mass ratio of the intermediate 1, 1-methyl-1H-imidazole-2-carbonyl chloride and pyridine catalyst in (3) is 1:2.2-3.6:0.02-0.

03.

6. The soybean polypeptide with antibacterial function according to claim 1, characterized in that: The preparation method of the modified chitosan comprises: adding chitosan and chloromethylbenzoyl chloride to N,N-dimethylformamide, stirring and dissolving, continuously adding triethylamine catalyst thereto, heating to 55-70° C., stirring and reacting for 10-15 hours, adding deionized water after the reaction to precipitate, filtering the solvent, washing with acetone, and obtaining the modified chitosan.

7. The soybean polypeptide with antibacterial function according to claim 6, characterized in that: The mass ratio of the chitosan, chloromethylbenzoyl chloride and triethylamine catalyst is 1:1.4-1.6:0.02-0.

03.

8. A method for preparing a soybean polypeptide with antibacterial function according to any one of claims 1 to 7, characterized in that: The method for preparing the soybean polypeptide with antibacterial function comprises: adding soybean polypeptide, quaternized bile acid and modified chitosan to N,N-dimethylformamide, stirring and reacting at 60-75° C. for 10-12 hours, adding distilled water to precipitate after the reaction, filtering and washing to obtain the soybean polypeptide with antibacterial function.