阅读说明：本技术 一种支化聚氨基酸抑菌剂及应用 (Branched polyamino acid bacteriostatic agent and application thereof ) 是由 季生象 刘骁 韩苗苗 刘亚栋 郭建伟 于 2018-01-31 设计创作，主要内容包括：本发明提供了一种支化聚氨基酸抑菌剂,包括支化聚氨基酸；所述支化聚氨基酸由两种或两种以上的氨基酸单元共聚得到。本发明所用的原料为各种氨基酸,均为天然的生物材料,无毒性,无副作用,是一种绿色环保型的新型抑菌剂,使用者易于接受。聚氨基酸的支化结构,使得该物质具有许多活性官能团,可以进一步进行修饰,具有良好的生物相容性,且该抑菌剂可撕裂细菌膜造成其死亡,长期使用不会产生耐药性。(The invention provides a branched polyamino acid bacteriostatic agent, which comprises branched polyamino acid; the branched polyamino acid is obtained by copolymerizing two or more than two amino acid units. The raw materials used by the invention are various amino acids, are all natural biological materials, have no toxicity and no side effect, are a novel environment-friendly bacteriostatic agent, and are easy to accept by users. The branched structure of the polyamino acid enables the substance to have a plurality of active functional groups, can be further modified, has good biocompatibility, can tear a bacterial film to cause death, and cannot generate drug resistance after long-term use.)

1. A branched polyamino acid bacteriostatic agent is characterized by comprising branched polyamino acid;

the branched polyamino acid is obtained by copolymerizing two or more than two amino acid units;

the amino acid unit has a general structural formula shown in formula I or a salt thereof:

wherein the content of the first and second substances,

a. b, c, d, e and f are independent integers of 0-6, and a + b + c + d + e + f is more than or equal to 1 and less than or equal to 20;

the T is₁、T₂、T₃、T₄、T₅And T₆Independently selected from any of the following structures:

and T of at least one amino acid₁、T₂、T₃、T₄、T₅、T₆At least one of which is

2. The bacteriostatic agent according to claim 1, wherein a + b + c + d + e + f is less than or equal to 10.

3. The bacteriostatic agent according to claim 1, wherein the branched polyamino acid is obtained by copolymerizing two or more branch-point amino acid units; or is copolymerized from one or more branch-point amino acid units and one or more copolymerized amino acid units;

the branch point amino acid unit is glutamic acid, lysine, ornithine, arginine, histidine, asparagine, glutamine, serine, tryptophan, aspartic acid, threonine, tyrosine or cysteine;

the copolymerized amino acid unit is glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline or methionine.

4. The bacteriostatic agent according to claim 1, wherein the two or more amino acid units comprise at least one basic amino acid unit.

5. The bacteriostatic agent according to claim 1 or 4, wherein the two or more amino acid units comprise at least one or more of lysine, ornithine, arginine and histidine.

6. The bacteriostatic agent according to claim 1, wherein the terminal amino groups in the branched polyamino acid are modified to the following groups:

wherein X, Y, Z, Q is independently selected from hydrogen, C1-C22 alkyl and its derivatives, C6-C30 aryl and its derivatives, C3-C18 cycloalkyl and its derivatives, C2-C18 alkene, alkyne and its derivatives, C1-C18 alkoxy and its derivatives, carboxylic acid and its derivatives, amine and its derivatives, nitrogen heterocycle and its derivatives, oxygen heterocycle and its derivatives or sulfur heterocycle and its derivatives.

7. The bacteriostatic agent according to claim 1, further comprising an adjuvant.

8. The bacteriostatic agent according to claim 1, wherein the bacteriostatic agent is a solid, a solution, a suspension, an emulsion, a hydrogel, an oleogel, an aerosol, grafted to a solid surface, and mixed with a polymer in one or more of the states.

9. Use of a bacteriostatic agent according to any one of claims 1 to 8 for inhibiting one or more of gram negative bacteria, gram positive bacteria and fungi.

Technical Field

The invention relates to the technical field of bacteriostats, in particular to a branched polyamino acid bacteriostat and application thereof.

Background

The invention of antibiotics saves hundreds of millions of people's lives, and the people are free from the pain of bacterial infection, so the invention is a revolution in medicine. However, with the abuse of small molecule antibiotics, coupled with the short life cycle and gene level transfer characteristics of bacteria, various drug resistant bacteria have emerged. The spread of pathogenic bacteria is even more serious, endangering people's normal life. One recent study showed that by 2050 superbacteria, if not controlled, would result in approximately 1000 million people per year. Drug-resistant bacteria have become a hot spot of general concern in countries around the world, and are related to the health of human beings, the development of economy and the stability of society all over the world.

Compared with micromolecular antibiotics, the polymer bacteriostatic agent can be non-specifically combined with a negatively charged bacterial membrane and then inserted into the bacterial membrane to cause the bacterial membrane to be broken and further kill bacteria, so that the polymer bacteriostatic agent is not easy to generate drug resistance. Therefore, the development and use of the bacteriostatic polymer material which has high safety, good bacteriostatic effect, sustainable use and biodegradability is a significant topic with profound significance.

Amino acid is a renewable resource, is mainly synthesized by hydrolyzing and fermenting biomass (starch, cellulose and the like) raw materials, and the global annual yield reaches megaton level. At present, amino acid is mainly used as food and feed additives, and the additional value is low. How to develop a new high value-added product is a problem which needs to be solved urgently in the amino acid industry. The application of the branched polyamino acid in the bacteriostatic field is not reported so far.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a branched polyamino acid bacteriostatic agent and an application thereof, wherein the branched polyamino acid polymer prepared by using amino acid as a raw material has excellent bacteriostatic performance.

In order to solve the technical problems, the invention provides a branched polyamino acid bacteriostatic agent, which comprises branched polyamino acid;

the branched polyamino acid is obtained by copolymerizing two or more than two amino acid units;

the amino acid unit has a general structural formula shown in formula I or a salt thereof:

wherein the content of the first and second substances,

a. b, c, d, e and f are independent integers of 0-6, and a + b + c + d + e + f is more than or equal to 1 and less than or equal to 20;

the T is₁、T₂、T₃、T₄、T₅And T₆Independently selected from any of the following knotsStructure:

and T of at least one amino acid₁、T₂、T₃、T₄、T₅、T₆At least one of which is

The salt may be an amino acid salt well known to those skilled in the art, preferably a hydrochloride, sulfate, phosphate, carbonate or nitrate salt.

Preferably, a + b + c + d + e + f is less than or equal to 10.

Preferably, the branched polyamino acid is obtained by copolymerizing two or more branch point amino acid units; or is copolymerized from one or more branch-point amino acid units and one or more copolymerized amino acid units;

the branch point amino acid unit is glutamic acid, lysine, ornithine, arginine, histidine, asparagine, glutamine, serine, tryptophan, aspartic acid, threonine, tyrosine or cysteine;

the copolymerized amino acid unit is glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline or methionine.

Preferably, the two or more amino acid units comprise at least one basic amino acid unit.

More preferably, the two or more amino acid units include at least one or more of lysine, arginine, ornithine and histidine.

In certain embodiments of the invention, the branched polyamino acid is copolymerized from arginine and alanine, or from ornithine and leucine, or from lysine and alanine, or from histidine and phenylalanine, or from lysine and arginine.

The method for preparing the branched polyamino acid is not particularly limited in the present invention, and the branched polyamino acid can be prepared according to a method well known to those skilled in the art, and preferably according to the following method:

mixing amino acids, and reacting at 25-250 ℃ for 1 min-96 h in an inert gas atmosphere to obtain the branched polyamino acid.

The inert gas is preferably nitrogen.

The reaction temperature is preferably 150-200 ℃, and the reaction time is preferably 30 min-24 h, and more preferably 2 h-12 h.

In certain embodiments of the invention, the terminal amino group in the branched polyamino acid may be modified to the following group:

wherein X, Y, Z, Q is independently selected from hydrogen, C1-C22 alkyl and its derivatives, C6-C30 aryl and its derivatives, C3-C18 cycloalkyl and its derivatives, C2-C18 alkene, alkyne and its derivatives, C1-C18 alkoxy and its derivatives, carboxylic acid and its derivatives, amine and its derivatives, nitrogen heterocycle and its derivatives, oxygen heterocycle and its derivatives or sulfur heterocycle and its derivatives.

The derivative is preferably C1-C5 alkyl substituent, C1-C5 alkoxy substituent, halogen, hydroxyl, sulfydryl, nitro, cyano, C5-C8 aryl, C5-C8 heteroaryl, C3-C5 cycloalkyl, carboxyl, amino, amido substituent, or any one or more C atoms are replaced by O or S.

More preferably, X, Y, Z, Q is independently selected from hydrogen, C1-C4 alkyl or amino.

The method of modification in the present invention is not particularly limited, and a method of amino acid modification known to those skilled in the art may be used.

The terminal amino group refers to a branched polyamino acidOf (2) is-NH₂A group.

According to the invention, the bacteriostatic agent can further comprise an auxiliary agent, and the content of the auxiliary agent is preferably 0-99 wt%.

The kind of the adjuvant is not particularly limited in the present invention, and may be one suitable for bacteriostatic agents well known to those skilled in the art.

The bacteriostatic adjuvant preferably comprises: [i] inorganic bacteriostatic agents such as metals, metal ions, metal salts and oxides thereof; [ ii ] organic bacteriostatic agents such as organic metals, organic halides, guanidines, organic nitro compounds, organic phosphorus and organic arsenic compounds, furan and derivatives thereof, pyrrole, imidazole, acylaniline, thiazole and derivatives thereof, quaternary ammonium salts and the like; (iii) one or more of natural bacteriostatic agents such as natural bacteriostatic peptides, macromolecular saccharides and the like; [ iv ] non-toxic additives or carriers such as glycerin, PEG, high molecular saccharides, polypeptides, plastics, ceramics, glass, apatite, resins, fibers, rubbers, and the like.

More preferably, the bacteriostatic auxiliary agent is metal Ti, Ag⁺，Cu²⁺，Fe³⁺,Zn²⁺One or more of quaternary ammonium salt, halamine, polybiguanides, halogenated phenol, chitosan, protamine, natural antibacterial peptide and the like.

In certain embodiments of the invention, the adjuvant is polyhexamethylene biguanide.

The formulation of the bacteriostatic agent is not particularly limited in the invention, and the bacteriostatic agent can be solid, solution, suspension, emulsion, hydrogel, oleogel, aerosol, grafted on the surface of the solid, and mixed with polymer in one or more states for use.

The preparation method of the bacteriostatic agent is not particularly limited, and the branched polyamino acid and the auxiliary agent are mixed.

The mixing process may or may not use a solvent.

The solvent may be water or an organic solvent.

The organic solvent is preferably one or more of methanol, ethanol, ethyl acetate, n-heptane, dimethylformamide, dimethylacetamide, tetrahydrofuran, chloroform, dichloromethane, carbon tetrachloride, acetonitrile, petroleum ether, n-hexane, cyclohexane, dioxane, dimethyl sulfoxide, xylene, toluene, benzene, chlorobenzene, bromobenzene, acetone and ionic liquid.

The bacteriostatic agent provided by the invention has the advantages of simple preparation process, low equipment requirement, easiness in operation, easiness in obtaining materials, low cost, industrial application prospect and broad-spectrum bacteriostatic property.

The invention also provides application of the bacteriostatic agent in inhibiting one or more of gram-negative bacteria, gram-positive bacteria and fungi.

The bacteriostatic agent can be applied to the fields of food, cosmetics, hygienic products, medical products and the like.

Such as food preservative, cosmetic additive, mouthwash, disinfectant, multifunctional care solution, preservative in eye drops, swimming pool disinfectant, and can also be used for toothpaste, facial cleanser, hand sanitizer, disinfectant soap, disinfectant preservative for fruit and vegetable storage, etc.

Compared with the prior art, the invention provides a branched polyamino acid bacteriostatic agent, which consists of branched polyamino acid and an auxiliary agent; the branched polyamino acid is obtained by copolymerizing two or more than two amino acid units; the amino acid unit is arginine, alanine, ornithine, leucine, lysine, histidine or phenylalanine. The raw materials used by the invention are various amino acids, are all natural biological materials, have no toxicity and no side effect, are a novel environment-friendly bacteriostatic agent, and are easy to accept by users. The branched structure of the polyamino acid enables the substance to have a plurality of active functional groups, can be further modified, has good biocompatibility, can tear a bacterial film to cause death, and cannot generate drug resistance after long-term use.

Drawings

FIG. 1 shows the nuclear magnetic hydrogen spectrum of a branched polyamino acid prepared in example 3 of the present invention.

Detailed Description

In order to further illustrate the present invention, the following examples are provided to describe the branched polyamino acid bacteriostatic agent, its preparation method and application in detail.

In the present invention, the reaction materials used in the following examples are all commercially available products.

Example 1

Adding 80 g of arginine and 20g of alanine into a 500mL round-bottom flask, connecting a water distribution device, pumping nitrogen for three times, wherein each time is longer than 10 minutes, finally keeping the nitrogen atmosphere, stirring and heating at 180 ℃ for reaction for 4 hours, stopping heating, then cooling the reaction system to room temperature, and dissolving and precipitating a polymer into ether by using methanol to obtain 78.7 g of hyperbranched polyamino acid.

Example 2

Adding 50 g of ornithine, 50 g of leucine and 10mg of scandium trifluoromethanesulfonate into a 500mL round-bottom flask, connecting a water distribution device, changing nitrogen for three times, wherein each time is longer than 10 minutes, finally keeping the nitrogen atmosphere, stirring and heating at 180 ℃ for reaction for 4 hours, stopping heating, and dissolving and precipitating a polymer into ether by using methanol to obtain 81.5 g of hyperbranched polyamino acid.

Example 3

91.32 g of lysine hydrochloride, 28.05 g of KOH and 20g of alanine were added to a 500mL round-bottomed flask, a water-dividing apparatus was connected, nitrogen was purged three times for more than 10 minutes each, finally, a nitrogen atmosphere was maintained, the reaction was heated with stirring at 180 ℃ for 4 hours, heating was stopped, then the reaction system was cooled to room temperature, and the polymer was dissolved in methanol and precipitated into ether to give 75.2 g of a branched polyamino acid.

The nuclear magnetic hydrogen spectrum of the synthesized branched polyamino acid is shown in figure 1.

Example 4

Adding 90 g of histidine, 10 g of phenylalanine and 10mg of ferric trichloride into a 500mL round-bottom flask, connecting a water diversion device, pumping nitrogen for three times, wherein each time is more than 10 minutes, finally keeping the nitrogen atmosphere, stirring and heating at 180 ℃ for reaction for 4 hours, stopping heating, cooling the reaction system to room temperature, and dissolving and precipitating a polymer into diethyl ether by using methanol to obtain 79.5 g of hyperbranched polyamino acid.

Example 5

Adding 80 g of ornithine and 20g of 6-aminocaproic acid into a 500mL round-bottom flask, connecting a water distribution device, pumping nitrogen for three times, wherein each time is more than 10 minutes, finally keeping the nitrogen atmosphere, stirring and heating at 180 ℃ for reaction for 4 hours, stopping heating, cooling the reaction system to room temperature, dissolving the polymer with methanol, and precipitating the polymer into ether to obtain 82.3 g of hyperbranched polyamino acid.

Example 6

Adding 91.32 g of lysine hydrochloride, 20g of NaOH and 20g of alanine into a 500mL round-bottom flask, connecting a water distribution device, pumping nitrogen for three times, wherein each time is more than 10 minutes, finally keeping the nitrogen atmosphere, stirring and heating at 180 ℃ for reaction for 36 hours, stopping heating, cooling the reaction system to room temperature, dissolving the polymer by using methanol, and precipitating the polymer into ether to obtain 74.8 g of hyperbranched polyamino acid.

Example 7

Adding 91.32 g of lysine hydrochloride, 20g of NaOH and 20g of alanine into a 500mL round-bottom flask, connecting a water distribution device, pumping nitrogen for three times, wherein each time is more than 10 minutes, finally keeping the nitrogen atmosphere, stirring and heating at 100 ℃ for reaction for 96 hours, stopping heating, cooling the reaction system to room temperature, and dissolving and precipitating a polymer into ether by using methanol to obtain 72.8 g of hyperbranched polyamino acid.

Example 8

Adding 91.32 g of lysine hydrochloride, 20g of NaOH and 20g of alanine into a 500mL round-bottom flask, connecting a water distribution device, pumping nitrogen for three times, wherein each time is more than 10 minutes, finally keeping the nitrogen atmosphere, stirring and heating at 250 ℃ for reaction for 1 hour, stopping heating, cooling the reaction system to room temperature, dissolving the polymer by using methanol, and precipitating the polymer into ether to obtain 73.1 g of hyperbranched polyamino acid.

Example 9

Adding 50 g of lysine and 50 g of arginine into a 500mL round-bottom flask, connecting a water distribution device, pumping nitrogen for three times, wherein each time is longer than 10 minutes, finally keeping the nitrogen atmosphere, stirring and heating at 180 ℃ for reaction for 4 hours, stopping heating, then cooling the reaction system to room temperature, dissolving the polymer with methanol, and precipitating the polymer into ether to obtain 80.1 g of branched polyamino acid.

Example 10

2g of the hyperbranched polyamino acid prepared in example 2 was dissolved in 5mL of methanol, 4g of 1H-pyrazole-1-carboxamidine hydrochloride and 4mL of triethylamine were added, the reaction was carried out at 65 ℃ for 12 hours, then the heating was stopped, the reaction solution was cooled to room temperature, and the reaction solution was precipitated in ether to obtain 2.1g of a guanidino-modified hyperbranched polyamino acid.

Example 11

2g of the hyperbranched polyamino acid prepared in example 8 was dissolved in 5mL of methanol, 4g of 1H-pyrazole-1-carboxamidine hydrochloride and 4mL of triethylamine were added, the reaction was carried out at 65 ℃ for 12 hours, then the heating was stopped, the reaction solution was cooled to room temperature, and the reaction solution was precipitated in ether to obtain 2.2g of a guanidino-modified hyperbranched polyamino acid.

Example 12

Adding 20g of phenylalanine, 10 g of alanine, 70 g of lysine hydrochloride and 20g of KOH into a 500mL round-bottom flask, connecting a water distribution device, pumping nitrogen for three times, wherein each time is more than 10 minutes, finally keeping the nitrogen atmosphere, stirring and heating at 100 ℃ for reaction for 96 hours, stopping heating, cooling the reaction system to room temperature, and dissolving and precipitating a polymer into ether by using methanol to obtain 68.5 g of hyperbranched polyamino acid.

Example 13

Heating and dissolving 2g of the hyperbranched polyamino acid obtained in the example 3 in 20mL of DMF, adding 5g of bromobutane, stirring and reacting at 80 ℃ for 24 hours, stopping heating, cooling to room temperature, and settling in ethyl acetate to obtain 2.4g of the quaternary ammonium salt modified hyperbranched polyamino acid.

Example 14

2g of the hyperbranched polyamino acid prepared in example 6 and 2g of polyhexamethylene biguanide were dissolved in 5mL of water, uniformly dispersed with stirring, and then lyophilized to obtain 4g of a mixture of the hyperbranched polyamino acid and polyhexamethylene biguanide.

Example 15

2g of the hyperbranched polyamino acid prepared in example 2 and 2g of polyhexamethylene biguanide were dissolved in 5mL of water, uniformly dispersed with stirring, and then lyophilized to obtain 4g of a mixture of the hyperbranched polyamino acid and polyhexamethylene biguanide.

Example 16

36mg of the hyperbranched polyamino acid prepared in examples 1-15 are respectively dissolved by 3mL of sterile PBS to obtain 12mg/mL mother liquor, and the antibacterial activity of the hyperbranched polyamino acid-based antibacterial agent is tested according to the following method, and the experimental results are shown in tables 1 and 2.

The various strains used in the following examples were purchased from the china biologicals institute.

The antibacterial activity of the hyperbranched polyamino acid-based antibacterial agent is detected by a 96-well plate method, epsilon-polylysine synthesized by a fermentation method is used as a reference to evaluate the antibacterial capability of the obtained hyperbranched polyamino acid-based antibacterial agent, and the Minimum Inhibitory Concentration (MIC) is defined as the lowest polymer concentration which inhibits 90% of bacterial growth compared with a control group.

Selecting a small amount of strains from an agar slant culture medium by using an inoculating loop, culturing at 37 ℃ overnight to recover the strains and achieve exponential growth, diluting the bacteria liquid to make the concentration of the bacteria liquid be 106CFU/mL, adding 175 mu L of bacteria liquid and 25 mu L of polymer solution with different concentrations into each hole, culturing a 96-hole plate at 37 ℃ for 20H, and detecting the OD600 value by using a microplate reader.

TABLE 1

TABLE 2

Example 17

36mg of the hyperbranched polyamino acids prepared in examples 1 to 15 were respectively dissolved in 3mL of sterile PBS to obtain a mother solution of 12mg/mL, and the antibody extravascular hemolytic activity of the hyperbranched polyamino acid-based antibacterial agent was tested according to the following method, and the experimental results are shown in tables 3 and 4.

And (3) detecting the in vitro hemolytic activity of the hyperbranched polyamino acid-based antibacterial agent by adopting a 96-well plate method, and evaluating the in vitro hemolytic activity of the obtained hyperbranched polyamino acid-based antibacterial agent by taking epsilon-polylysine synthesized by a fermentation method as a reference.

Preparing 2% (v/v) erythrocyte suspension, taking 2mL of fresh healthy human blood, diluting with 10mL of PBS buffer solution without endotoxin, putting the diluted blood into a triangular flask containing glass beads, shaking for 10 minutes, or stirring the blood with a glass rod, removing fibrous protein to enable the blood to become defibrinated, centrifuging for 10-15 minutes at 1000-1500 r/min at 20 ℃, removing supernatant, and washing the precipitated erythrocytes for 4 times by using the PBS buffer solution according to the method until the supernatant does not show red. The obtained red blood cells are prepared into 2 percent suspension by using normal saline for subsequent tests.

The polymer was diluted with PBS buffer solution to prepare solutions of different concentrations, added to a 96-well plate, PBS buffer solution alone was used as a negative control, 0.2% Triton-X-100 was dissolved in water as a positive control for in vitro hemolytic activity detection, and washed erythrocytes (2% v/v, 50. mu.L) were added to the 96-well plate, mixed well and cultured. The absorbance at 540nm was measured with a microplate reader.

TABLE 3

TABLE 4

Example 18

This example was used to test the acute toxicity of hyperbranched polyamino acid based antibacterial agents in animals and the control was epsilon-polylysine synthesized by fermentation to evaluate the resulting hyperbranched polyamino acid based antibacterial agents for acute toxicity in animals.

100 mice were treated with 31. + -.3 g of each of the male and female halves, and the hyperbranched polyamino acid-based antibacterial agents prepared in examples 1 to 15 were administered to the mice at a dose of 1mg/mL once a day for 15 consecutive days to observe the toxic reaction of the mice. The experimental result shows that after the continuous intramuscular injection for 15 days, except the activity of individual mice is reduced, the rest mice have no obvious abnormal reaction, and all mice survive, which proves that the obtained hyperbranched polyamino acid based antibacterial agent has smaller in vivo toxicity.

As can be seen from the examples and comparative examples, the hyperbranched polyamino acid based antibacterial agent prepared by the invention has excellent antibacterial performance, no toxicity and no side effect.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.