Functional marine biological polypeptide and preparation method thereof

Abstract

The invention relates to the technical field of peptides, and discloses a functionalized marine biological polypeptide and a preparation method thereof.Marine bioactive peptide, quaternary ammonium salt modified imidazolyl chitosan and epoxy cellulose are added into a stirrer and stirred, and the functionalized marine biological polypeptide is obtained after stirring is finished. In the stirring process, an epoxy group in epoxy cellulose and a hydroxyl group in quaternary ammonium salt modified imidazolyl chitosan are subjected to a ring-opening reaction and are cross-linked with each other to form a net structure, and the epoxy cellulose, the quaternary ammonium salt modified imidazolyl chitosan and the marine bioactive peptide are intertwined to form a cross-linked network; the specific surface area and the pore structure of the marine bioactive peptide are increased, so that the marine bioactive peptide has better water absorption. Imidazole groups and quaternary ammonium salt groups in the epoxy cellulose and quaternary ammonium salt modified imidazolyl chitosan disclosed by the invention have a relatively good antibacterial effect.

Description

Technical Field

[0001] The invention relates to the field of peptide technology, in particular to a functionalized marine biological polypeptide and a preparation method thereof. Background Art

[0002] Marine bioactive peptides are a class of polypeptides with multiple biological functions, which are widely used in food, cosmetics, medical and other fields. If their antibacterial effect is not good, food, cosmetics and medical products will be invaded by bacteria, thus affecting their normal use. For example, patent CN200510000032.2 provides a method for preparing a marine antioxidant active peptide. The marine antioxidant active peptide provided by the invention has the advantages of strong antioxidant activity and good water solubility, but its antibacterial and water absorption effects are not improved, which greatly limits its application range. Summary of the invention

[0003] 1. Technical issues to be solved

[0004] In view of the deficiencies of the prior art, the present invention provides a functionalized marine biological polypeptide and a preparation method thereof, which has good antibacterial and water absorption effects.

[0005] (II) Technical solution

[0006] To achieve the above objectives, the present invention provides the following technical solutions: a functionalized marine biological polypeptide and a preparation method thereof, comprising the following components by weight: 15-25 parts by weight of marine biological active peptides, 5-10 parts by weight of quaternary ammonium salt modified imidazole chitosan, and 4-8 parts by weight of epoxy cellulose.

[0007] Preferably, the preparation method of the quaternary ammonium salt modified imidazole chitosan is:

[0008] (1) Under a nitrogen atmosphere, 12-16 parts by weight of 1-vinylimidazole and 8-15 parts by weight of chloroacetic acid are sequentially poured into a reactor filled with N,N-dimethylformamide solvent, mixed evenly, reacted at 45-60° C., cooled to room temperature after completion, distilled under reduced pressure, washed, and dried to obtain intermediate 1;

[0009] (2) adding intermediate 1 and nanocrystalline cellulose to a dimethyl sulfoxide solvent, stirring, and then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, reacting at 30-45° C. for 12-20 hours, filtering, and washing with ethanol to obtain olefinated cellulose;

[0010] (3) adding 4-9 parts by weight of 1-(3-aminopropyl)imidazole and 6-12 parts by weight of olefinated cellulose to a dimethylformamide solvent, performing Michael addition reaction, reacting at 55-80° C. for 20-25 hours, rotary distilling after the reaction, filtering, and vacuum drying to obtain an intermediate 2;

[0011] (4) adding chitosan and chloroacetyl chloride to N,N-dimethylformamide, stirring and mixing, and continuously adding triethylamine catalyst thereto, heating to 70-90° C. and stirring for reaction for 10-14 hours, adding distilled water after the reaction to precipitate, filtering the solvent, and washing with acetone to obtain chloro-based chitosan;

[0012] (5) Add chlorinated chitosan and intermediate 2 into a reactor, stir for 5-8 min, heat to 40-50° C., react for 2-4 h, and wash after reaction to obtain quaternary ammonium salt-modified imidazole chitosan.

[0013] Preferably, the reaction time in (1) is 20-24 hours.

[0014] Preferably, in said (2), the mass ratio of the intermediate 1, nanocrystalline cellulose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 2.4-3.5:1:0.01-0.03:0.02-0.03.

[0015] Preferably, the mass ratio of chitosan, chloroacetyl chloride and triethylamine catalyst in (4) is 1:1.2-1.5:0.01-0.04.

[0016] Preferably, the mass ratio of chlorinated chitosan to intermediate 2 in (5) is 0.8-1.3:1.

[0017] Preferably, the preparation method of the epoxy cellulose is: at 25-40° C., 15-20 parts by weight of olefinic cellulose is added to an N,N-dimethylformamide solvent, stirred evenly, and then 12-18 parts by weight of an aqueous hydrogen peroxide solution is added thereto, stirred for reaction for 3-6 hours, and after the reaction is completed, the solvent is removed by reduced pressure distillation, washed and dried to obtain epoxy cellulose.

[0018] Preferably, the method for preparing the functionalized marine biological polypeptide is as follows: adding the marine biological active peptide, quaternary ammonium salt-modified imidazole chitosan, and epoxy cellulose into a stirrer, stirring for 10-14 minutes, and obtaining the functionalized marine biological polypeptide after stirring.

[0019] (III) Beneficial technical effects

[0020] The invention adds marine biological active peptides, quaternary ammonium salt modified imidazole chitosan and epoxy cellulose into a stirrer, and stirs to obtain functionalized marine biological polypeptides.

[0021] The tertiary amine group in 1-vinylimidazole reacts with the chlorine in chloroacetic acid to generate a quaternary ammonium salt group, and an olefinic group and a carboxyl group are introduced to obtain intermediate 1, which is further esterified with the hydroxyl group in nanocrystalline cellulose to obtain olefinic cellulose. The amino group in 1-(3-aminopropyl)imidazole and the olefinic group in the olefinic cellulose are subjected to Michael addition to obtain intermediate 2. Chitosan is reacted with chloroacetyl chloride to obtain chlorinated chitosan. The chlorine in the chlorinated chitosan is reacted with the tertiary amine group in intermediate 2 to obtain quaternary ammonium salt-modified imidazole chitosan; the olefinic group in the olefinic cellulose is epoxidized to obtain epoxy cellulose.

[0022] During the stirring process, the epoxy groups in the epoxy cellulose react with the hydroxyl groups in the quaternary ammonium salt modified imidazole chitosan to form a cross-linked network structure, and the epoxy cellulose, the quaternary ammonium salt modified imidazole chitosan and the marine bioactive peptide are entangled to form a cross-linked network, which increases the specific surface area and pore structure of the marine bioactive peptide and makes it absorb water better. The imidazole group and the quaternary ammonium salt group in the epoxy cellulose and the quaternary ammonium salt modified imidazole chitosan of the present invention have good antibacterial effect, and the grafting sites on the cellulose are more, which further improves the substitution degree of the antibacterial group, so that the antibacterial effect is better, and the chitosan itself has a good antibacterial effect. DETAILED DESCRIPTION

[0023] Marine bioactive peptides: composed of 7 kinds of amino acids, namely aspartic acid, glycine, threonine, alanine, proline, valine and lysine, and the molar ratio of them is 1:1:1:1:1:1:1:1.

[0024] Example 1

[0025] (1) Under a nitrogen atmosphere, 12 parts by weight of 1-vinylimidazole and 8 parts by weight of chloroacetic acid are sequentially poured into a reactor filled with N,N-dimethylformamide solvent, mixed evenly, and reacted at 45° C. for 20 hours. After the reaction, the mixture is cooled to room temperature, distilled under reduced pressure, washed, and dried to obtain an intermediate 1;

[0026] (2) adding intermediate 1 and nanocrystalline cellulose to a dimethyl sulfoxide solvent, stirring, and then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, wherein the mass ratio of intermediate 1, nanocrystalline cellulose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 2.4:1:0.01:0.02, reacting at 30° C. for 12 h, filtering, and washing with ethanol to obtain olefinated cellulose;

[0027] (3) adding 4 parts by weight of 1-(3-aminopropyl)imidazole and 6 parts by weight of ethylenated cellulose to a dimethylformamide solvent, performing Michael addition reaction, reacting at 55° C. for 20 h, and after the reaction, performing rotary distillation, filtering, and vacuum drying to obtain an intermediate 2;

[0028] (4) adding chitosan and chloroacetyl chloride to N,N-dimethylformamide, stirring and mixing, and continuing to add triethylamine catalyst, wherein the mass ratio of chitosan, chloroacetyl chloride and triethylamine catalyst is 1:1.2:0.01, heating to 70° C. and stirring for 10 hours, adding distilled water after the reaction to precipitate, filtering the solvent, washing with acetone, and obtaining chlorinated chitosan;

[0029] (5) adding chlorinated chitosan and intermediate 2 into a reactor, wherein the mass ratio of chlorinated chitosan to intermediate 2 is 0.8:1, stirring for 5 minutes, heating to 40° C. for reaction for 2 hours, and washing after completion to obtain quaternary ammonium salt-modified imidazole chitosan;

[0030] (6) adding 15 parts by weight of olefinated cellulose to N,N-dimethylformamide solvent at 25° C., stirring evenly, adding 12 parts by weight of hydrogen peroxide aqueous solution thereto, stirring and reacting for 3 hours, and after the reaction is completed, removing the solvent by distillation under reduced pressure, washing and drying to obtain epoxy cellulose;

[0031] (7) 15 parts by weight of marine bioactive peptides, 5 parts by weight of quaternary ammonium salt-modified imidazole chitosan, and 4 parts by weight of epoxy cellulose are added into a stirrer and stirred for 10 minutes to obtain functionalized marine bioactive peptides.

[0032] Example 2

[0033] (1) Under a nitrogen atmosphere, 16 parts by weight of 1-vinylimidazole and 15 parts by weight of chloroacetic acid were sequentially poured into a reactor filled with N,N-dimethylformamide solvent, mixed evenly, and reacted at 60° C. for 24 hours. After the reaction, the mixture was cooled to room temperature, distilled under reduced pressure, washed, and dried to obtain an intermediate 1;

[0034] (2) adding intermediate 1 and nanocrystalline cellulose to a dimethyl sulfoxide solvent, stirring, and then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, wherein the mass ratio of intermediate 1, nanocrystalline cellulose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 3.5:1:0.03:0.03, reacting at 45° C. for 20 hours, filtering, and washing with ethanol to obtain olefinated cellulose;

[0035] (3) adding 9 parts by weight of 1-(3-aminopropyl)imidazole and 12 parts by weight of olefinated cellulose to a dimethylformamide solvent, performing Michael addition reaction, reacting at 80° C. for 25 h, and after the reaction, performing rotary distillation, filtering, and vacuum drying to obtain an intermediate 2;

[0036] (4) adding chitosan and chloroacetyl chloride to N,N-dimethylformamide, stirring and mixing, and continuing to add triethylamine catalyst, wherein the mass ratio of chitosan, chloroacetyl chloride and triethylamine catalyst is 1:1.5:0.04, heating to 90° C. and stirring for 14 h, adding distilled water after the reaction to precipitate, filtering the solvent, washing with acetone, and obtaining chlorinated chitosan;

[0037] (5) adding chlorinated chitosan and intermediate 2 into a reactor, wherein the mass ratio of chlorinated chitosan to intermediate 2 is 1.3:1, stirring for 8 minutes, heating to 50° C. for reaction for 4 hours, and washing after completion to obtain quaternary ammonium salt-modified imidazole chitosan;

[0038] (6) adding 20 parts by weight of olefinic cellulose to N,N-dimethylformamide solvent at 40° C., stirring evenly, adding 18 parts by weight of hydrogen peroxide aqueous solution thereto, stirring and reacting for 6 hours, and after the reaction is completed, removing the solvent by reduced pressure distillation, washing and drying to obtain epoxy cellulose;

[0039] (7) 25 parts by weight of marine bioactive peptides, 10 parts by weight of quaternary ammonium salt-modified imidazole chitosan, and 8 parts by weight of epoxy cellulose are added into a stirrer and stirred for 14 minutes to obtain functionalized marine bioactive peptides.

[0040] Example 3

[0041] (1) Under a nitrogen atmosphere, 14 parts by weight of 1-vinylimidazole and 11 parts by weight of chloroacetic acid were sequentially poured into a reactor filled with N,N-dimethylformamide solvent, mixed evenly, and reacted at 52° C. for 22 hours. After the reaction, the mixture was cooled to room temperature, distilled under reduced pressure, washed, and dried to obtain an intermediate 1;

[0042] (2) adding intermediate 1 and nanocrystalline cellulose to a dimethyl sulfoxide solvent, stirring, and then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, wherein the mass ratio of intermediate 1, nanocrystalline cellulose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 2.9:1:0.02:0.025, reacting at 37° C. for 16 h, filtering, and washing with ethanol to obtain olefinated cellulose;

[0043] (3) adding 6 parts by weight of 1-(3-aminopropyl)imidazole and 9 parts by weight of olefinated cellulose to dimethylformamide solvent, performing Michael addition reaction, reacting at 67° C. for 22 h, and after the reaction, performing rotary distillation, filtering, and vacuum drying to obtain intermediate 2;

[0044] (4) adding chitosan and chloroacetyl chloride to N,N-dimethylformamide, stirring and mixing, and continuing to add triethylamine catalyst, wherein the mass ratio of chitosan, chloroacetyl chloride and triethylamine catalyst is 1:1.3:0.02, heating to 80° C. and stirring for 12 h, adding distilled water after the reaction to precipitate, filtering the solvent, washing with acetone, and obtaining chlorinated chitosan;

[0045] (5) adding chlorinated chitosan and intermediate 2 into a reactor, wherein the mass ratio of chlorinated chitosan to intermediate 2 is 1.05:1, stirring for 6 minutes, heating to 45° C. for reaction for 3 hours, and washing after completion to obtain quaternary ammonium salt-modified imidazole chitosan;

[0046] (6) adding 17 parts by weight of olefinic cellulose to N,N-dimethylformamide solvent at 32° C., stirring evenly, adding 15 parts by weight of hydrogen peroxide aqueous solution thereto, stirring and reacting for 4 hours, and after the reaction is completed, removing the solvent by distillation under reduced pressure, washing and drying to obtain epoxy cellulose;

[0047] (7) 20 parts by weight of marine bioactive peptides, 7 parts by weight of quaternary ammonium salt-modified imidazole chitosan, and 6 parts by weight of epoxy cellulose are added into a stirrer and stirred for 12 minutes to obtain functionalized marine bioactive peptides.

[0048] Example 4

[0049] (1) Under a nitrogen atmosphere, 12 parts by weight of 1-vinylimidazole and 8 parts by weight of chloroacetic acid are sequentially poured into a reactor filled with N,N-dimethylformamide solvent, mixed evenly, and reacted at 45° C. for 20 hours. After the reaction, the mixture is cooled to room temperature, distilled under reduced pressure, washed, and dried to obtain an intermediate 1;

[0050] (2) adding intermediate 1 and nanocrystalline cellulose to a dimethyl sulfoxide solvent, stirring, and then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, wherein the mass ratio of intermediate 1, nanocrystalline cellulose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 2.4:1:0.01:0.02, reacting at 30° C. for 12 h, filtering, and washing with ethanol to obtain olefinated cellulose;

[0051] (3) adding 9 parts by weight of 1-(3-aminopropyl)imidazole and 12 parts by weight of olefinated cellulose to a dimethylformamide solvent, performing Michael addition reaction, reacting at 80° C. for 25 h, and after the reaction, performing rotary distillation, filtering, and vacuum drying to obtain an intermediate 2;

[0052] (4) adding chitosan and chloroacetyl chloride to N,N-dimethylformamide, stirring and mixing, and continuing to add triethylamine catalyst, wherein the mass ratio of chitosan, chloroacetyl chloride and triethylamine catalyst is 1:1.5:0.04, heating to 90° C. and stirring for 14 h, adding distilled water after the reaction to precipitate, filtering the solvent, washing with acetone, and obtaining chlorinated chitosan;

[0053] (5) adding chlorinated chitosan and intermediate 2 into a reactor, wherein the mass ratio of chlorinated chitosan to intermediate 2 is 1.05:1, stirring for 6 minutes, heating to 45° C. for reaction for 3 hours, and washing after completion to obtain quaternary ammonium salt-modified imidazole chitosan;

[0054] (6) adding 17 parts by weight of olefinic cellulose to N,N-dimethylformamide solvent at 32° C., stirring evenly, adding 15 parts by weight of hydrogen peroxide aqueous solution thereto, stirring and reacting for 4 hours, and after the reaction is completed, removing the solvent by distillation under reduced pressure, washing and drying to obtain epoxy cellulose;

[0055] (7) 20 parts by weight of marine bioactive peptides, 7 parts by weight of quaternary ammonium salt-modified imidazole chitosan, and 6 parts by weight of epoxy cellulose are added into a stirrer and stirred for 12 minutes to obtain functionalized marine bioactive peptides.

[0056] Comparative Example 1

[0057] 20 parts by weight of marine bioactive peptides and 6 parts by weight of epoxy cellulose were added into a stirrer and stirred for 12 minutes to obtain functionalized marine bioactive peptides.

[0058] Comparative Example 2

[0059] 20 parts by weight of marine biological active peptides and 7 parts by weight of quaternary ammonium salt-modified imidazole chitosan were added into a stirrer and stirred for 12 minutes to obtain functionalized marine biological peptides.

[0060] Using Staphylococcus aureus, Escherichia coli or Candida albicans as the test strains, 0.5 mL of the bacterial suspension of the test strain (concentration of 1×106 CFU / mL) was added to a sterile culture dish containing beef extract peptone medium, and then the functionalized marine biological polypeptide sample was attached to the surface of the culture medium, and cultured in a constant temperature incubator at 37°C for 24 hours. After culture, the diameter of the inhibition zone was measured. The test results are shown in Table 1.

[0061] Table 1: Antimicrobial testing.

[0062]

[0063] It can be seen from Table 1 that Examples 1-4 of the present invention have better antibacterial effects than Comparative Examples 1-2.

[0064] The functionalized marine biological polypeptide was dried, weighed, immersed in distilled water for 12 hours, taken out, and the surface water was absorbed with filter paper, weighed, and the water absorption rate was calculated; water absorption rate Q = (mm 0 ) / m 0 ; m is the mass of marine biological polypeptide after absorbing water, m 0 It is the mass of marine biological polypeptide before absorbing water, and the test is shown in Table 2.

[0065] Table 2: Water absorption test.

[0066] project Water absorption (g / g) Example 1 257.9 Example 2 264.7 Example 3 253.6 Example 4 250.8 Comparative Example 1 110.6 Comparative Example 2 105.9

[0067] It can be seen from Table 2 that Examples 1-4 of the present invention have better water absorption than Comparative Examples 1-2.

[0068] 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 functionalized marine biological polypeptide, characterized in that: The invention comprises the following components by weight: 15-25 parts by weight of marine biological active peptides, 5-10 parts by weight of quaternary ammonium salt modified imidazole chitosan, and 4-8 parts by weight of epoxy cellulose.

2. The functionalized marine biological polypeptide according to claim 1, characterized in that: The preparation method of the quaternary ammonium salt modified imidazole chitosan is: (1) Under a nitrogen atmosphere, 12-16 parts by weight of 1-vinylimidazole and 8-15 parts by weight of chloroacetic acid are sequentially poured into a reactor filled with N,N-dimethylformamide solvent, mixed evenly, reacted at 45-60° C., cooled to room temperature after completion, distilled under reduced pressure, washed, and dried to obtain intermediate 1; (2) adding intermediate 1 and nanocrystalline cellulose to a dimethyl sulfoxide solvent, stirring, and then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, reacting at 30-45° C. for 12-20 hours, filtering, and washing with ethanol to obtain olefinated cellulose; (3) adding 4-9 parts by weight of 1-(3-aminopropyl)imidazole and 6-12 parts by weight of olefinated cellulose to a dimethylformamide solvent, performing Michael addition reaction, reacting at 55-80° C. for 20-25 hours, rotary distilling after the reaction, filtering, and vacuum drying to obtain an intermediate 2; (4) adding chitosan and chloroacetyl chloride to N,N-dimethylformamide, stirring and mixing, and continuously adding triethylamine catalyst thereto, heating to 70-90° C. and stirring for reaction for 10-14 hours, adding distilled water after the reaction to precipitate, filtering the solvent, and washing with acetone to obtain chloro-based chitosan; (5) Add chlorinated chitosan and intermediate 2 into a reactor, stir for 5-8 min, heat to 40-50° C., react for 2-4 h, and wash after reaction to obtain quaternary ammonium salt-modified imidazole chitosan.

3. The functionalized marine biological polypeptide according to claim 2, characterized in that: The reaction time in (1) is 20-24h.

4. The functionalized marine biological polypeptide according to claim 2, characterized in that: The mass ratio of the intermediate 1, nanocrystalline cellulose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in (2) is 2.4-3.5:1:0.01-0.03:0.02-0.

03.

5. The functionalized marine biological polypeptide according to claim 2, characterized in that: The mass ratio of chitosan, chloroacetyl chloride and triethylamine catalyst in (4) is 1:1.2-1.5:0.01-0.

04.

6. The functionalized marine biological polypeptide according to claim 2, characterized in that: The mass ratio of chlorinated chitosan to intermediate 2 in (5) is 0.8-1.3:

1.

7. The functionalized marine biological polypeptide according to claim 1, characterized in that: The preparation method of the epoxy cellulose is as follows: at 25-40° C., 15-20 parts by weight of olefinic cellulose is added to an N,N-dimethylformamide solvent, stirred evenly, and then 12-18 parts by weight of an aqueous hydrogen peroxide solution is added thereto, stirred for reaction for 3-6 hours, and after the reaction is completed, the solvent is removed by reduced pressure distillation, washed and dried to obtain the epoxy cellulose.

8. A method for preparing a functionalized marine biological polypeptide according to any one of claims 1 to 7, characterized in that: The method for preparing the functionalized marine biological polypeptide is as follows: adding the marine biological active peptide, quaternary ammonium salt-modified imidazole chitosan, and epoxy cellulose into a stirrer, stirring for 10-14 minutes, and obtaining the functionalized marine biological polypeptide after stirring.

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