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及应用,包括支化聚氨基酸；所述支化聚氨基酸由一种氨基酸单元均聚得到；所述氨基酸单元具有式Ⅰ所示结构或其盐。本发明所用的原料为氨基酸,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 and application thereof, wherein the bacteriostatic agent comprises branched polyamino acid; the branched polyamino acid is obtained by homopolymerization of an amino acid unit; the amino acid unit has a structure represented by formula I or a salt thereof. The raw materials used in the invention are amino acids, are 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, comprising branched polyamino acid;

the branched polyamino acid is obtained by homopolymerization of an amino acid unit;

the amino acid unit has a structure represented by formula I:

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;

T₁、T₂、T₃、T₄、T₅and T₆Independently selected from any of the following structures:

and T₁、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 amino acid unit is glutamic acid, lysine, arginine, ornithine, histidine, aspartic acid, tryptophan, serine, tyrosine, cysteine, asparagine, glutamine or threonine.

4. The bacteriostatic agent according to claim 1, wherein the branched polyamino acid is a homopolymer of a basic amino acid.

5. The bacteriostatic agent according to claim 1 or 4, wherein the branched polyamino acid is branched polylysine, branched polyarginine, branched polyornithine or branched polyhistidine.

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 homopolymerization of an amino acid unit;

the amino acid unit has a structure represented by formula I:

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;

T₁、T₂、T₃、T₄、T₅and T₆Independently selected from hydrogen, C1-C8 alkyl and derivatives thereof, C6-C30 aryl and derivatives thereof, C1-C8 alkoxy and derivatives thereof, carboxylic acid and derivatives thereof, amine and derivatives thereof, nitrogen heterocycle and derivatives thereof, oxygen heterocycle and derivatives thereof or sulfur heterocycle and derivatives thereof; and T₁、T₂、T₃、T₄、T₅、T₆At least one of them is selected from alkoxy of C1-C8 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; and T₁、T₂、T₃、T₄、T₅And T₆Not H at the same time;

the group of the derivative is hydroxyl, sulfydryl, guanidyl, nitryl, cyano, C5-C8 aromatic heterocycle, acylamino, or any one or more C atoms are replaced by O or S;

the branched polyamino acid is obtained by homopolymerization of an amino acid unit shown in a formula I.

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.

In the formula I, 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.

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

[]T in (1)₁、T₂、T₃、T₄、T₅、T₆Represents a random combination of functional groups.

T₁、T₂、T₃、T₄、T₅And T₆Independently selected from hydrogen, C1-C8 alkyl and derivatives thereof, C6-C30 aryl and derivatives thereof, and C1-C8 alkoxyC1-C8 carboxylic acid and derivatives thereof, C1-C8 amine and derivatives thereof, C2-C8 azacyclic ring and derivatives thereof, C2-C8 oxacyclic ring and derivatives thereof or C2-C8 thiacyclic ring and derivatives thereof; and T₁、T₂、T₃、T₄、T₅、T₆At least one of the compounds is selected from alkoxy of C1-C8 and derivatives thereof, carboxylic acid of C1-C8 and derivatives thereof, amine of C1-C8 and derivatives thereof, nitrogen heterocycle of C2-C8 and derivatives thereof, oxygen heterocycle of C2-C8 and derivatives thereof or sulfur heterocycle and derivatives thereof; and T₁、T₂、T₃、T₄、T₅And T₆Not H at the same time.

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.

Preferably, said T is₁、T₂、T₃、T₄、T₅And T₆Independently selected from H, C1-C5 alkyl, or C1-C5 substituted alkyl; the substituted alkyl group is preferably a hydroxyl-containing substituent, a mercapto-containing substituent, an aryl substituent, a heteroaryl substituent, a carboxyl substituent, a heterocyclic substituent, an amido substituent, an amino substituent, or a C atom substituted with O or S.

The number of carbon atoms in the aryl substituent, the heteroaryl substituent and the heterocyclic substituent is preferably 5 to 12, and more preferably 5 to 8.

The number of carbon atoms in the carboxyl substituent, the amido substituent and the amino substituent is preferably 1 to 8, and more preferably 1 to 5.

More preferably, said T₁、T₂、T₃、T₄、T₅And T₆Independently selected from any of the following structures:

in the present invention, said T is₁、T₂、T₃、T₄、T₅And T₆And H is not simultaneously obtained, and the obtained branched polyamino acid is of a branched structure.

The branched polyamino acid preferably has the following structure:

wherein r, u and q are integers, r is more than or equal to 1 and less than or equal to 9, u is more than or equal to 1 and less than or equal to 6, and q is more than or equal to 1 and less than or equal to 6. G is preferably one of C2-C8 alkene, alkyne and derivatives thereof, carboxylic acid and derivatives thereof, alkyl alcohol and derivatives thereof, amine and derivatives thereof, nitrogen heterocycle and derivatives thereof, oxygen heterocycle and derivatives thereof, sulfur heterocycle and derivatives thereof; t and L are independently preferably one of hydrogen, C1-C8 alkyl and derivatives thereof, C6-C30 aryl and derivatives thereof, C3-C8 cycloalkyl and derivatives thereof, C2-C8 alkene, alkyne and derivatives thereof, C1-C8 alkoxy, carboxylic acid and derivatives thereof, amine and derivatives thereof, nitrogen heterocycle and derivatives thereof, oxygen heterocycle and derivatives thereof, sulfur heterocycle and derivatives thereof.

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.

In the present invention, the amino acid unit is preferably glutamic acid, lysine, arginine, ornithine, histidine, aspartic acid, tryptophan, serine, tyrosine, cysteine, asparagine, glutamine or threonine.

More preferably, the branched polyamino acid is a homopolymer of a basic amino acid.

In certain embodiments of the invention, the branched polyamino acid is a branched polylysine, a branched polyarginine, a branched polyornithine, or a branched polyhistidine. Preferably branched polylysine or branched polyornithine.

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:

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

The inert gas is preferably nitrogen or argon.

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-NH in the branched polyamino acid₂A 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.

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 in the invention.

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 and application thereof, wherein the branched polyamino acid bacteriostatic agent comprises branched polyamino acid; the branched polyamino acid is obtained by homopolymerization of an amino acid unit; the amino acid unit has a structure represented by formula I or a salt thereof. The raw materials used in the invention are amino acids, are 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 is a nuclear magnetic hydrogen spectrum of a branched polylysine prepared in example 2;

FIG. 2 is a nuclear magnetic hydrogen spectrum of branched polyarginine prepared in example 10.

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 100 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, 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 82.7 g of hyperbranched poly-arginine.

Example 2

Adding 91.32 g of lysine hydrochloride and 28.05 g 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 250 ℃ for 1 minute, stopping heating, dissolving the polymer by using methanol, and precipitating the polymer into diethyl ether to obtain 84 g of hyperbranched polylysine.

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

Example 3

Adding 50 g of serine and 50mL of n-hexanol into a 500mL round-bottom flask, connecting a water distribution device, stirring and heating at 190 ℃ for 10h under the nitrogen atmosphere, stopping heating, cooling the reaction system to room temperature, and dissolving and precipitating a polymer into diethyl ether by using methanol to obtain 28.5 g of hyperbranched polyserine.

Example 4

Adding 91.32 g of lysine hydrochloride, 28.05 g of KOH and 10mg of antimony trioxide 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 100 ℃ for reaction for 96 hours, stopping heating, cooling the reaction system to room temperature, and dissolving and precipitating a polymer into diethyl ether by using methanol to obtain 55.5 g of hyperbranched polylysine.

Example 5

Adding 80 g of lysine, 20 g of lysine hydrochloride and 6.14g 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, dissolving the polymer with methanol, and precipitating the polymer into ether to obtain 68.5 g of hyperbranched polylysine.

Example 6

Adding 50 g of cysteine and 100mL of DMF (dimethyl formamide) into a 500mL round-bottom flask, connecting a water distribution device, stirring and heating at 180 ℃ for 10h under the nitrogen atmosphere, stopping heating, cooling the reaction system to room temperature, and dissolving and precipitating the polymer into diethyl ether by using methanol to obtain 33.5 g of hyperbranched poly-cysteine.

Example 7

Adding 50 g of glutamic acid and 100mL of ethylene glycol into a 500mL round-bottom flask, connecting a water distribution device, stirring and heating at 200 ℃ for 1 minute under the nitrogen atmosphere, stopping heating, cooling the reaction system to room temperature, and dissolving and precipitating the polymer into diethyl ether by using methanol to obtain 31.5 g of hyperbranched polyglutamic acid.

Example 8

Adding 50 g of arginine and 100mL of ethylene glycol into a 500mL round-bottom flask, connecting a water distribution device, stirring and heating at 150 ℃ under the nitrogen atmosphere for 96h for reaction, stopping heating, then cooling the reaction system to room temperature, and dissolving and precipitating the polymer into diethyl ether by using methanol to obtain 34.5 g of hyperbranched poly-arginine.

Example 9

Adding 100 g of lysine and 0.1 g of phosphoric 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 100 ℃ for reaction for 96 hours, stopping heating, cooling the reaction system to room temperature, dissolving a polymer by using methanol, and precipitating the polymer into ether to obtain 78.5 g of hyperbranched polylysine.

Example 10

Adding 100 g of arginine and 200 g of ethylene glycol into a 500mL round-bottom flask, connecting a water distribution device, bubbling N2 for 30min, pumping nitrogen for three times, wherein each time is more than 10 min, finally keeping the nitrogen atmosphere, stirring and heating at 180 ℃ for reaction for 8h, stopping heating, cooling the reaction system to room temperature, separating the ethylene glycol, and precipitating the polymer into ether to obtain 71.2 g of hyperbranched poly-arginine.

The nuclear magnetic hydrogen spectrum of the synthesized branched polyamino acid is shown in figure 2.

Example 11

Adding 100 g of histidine and 200 g of ethylene glycol into a 500mL round-bottom flask, connecting a water distribution device, bubbling N2 for 30min, pumping nitrogen for three times, wherein each time is more than 10 min, finally keeping the nitrogen atmosphere, stirring and heating at 180 ℃ for reaction for 8h, stopping heating, cooling the reaction system to room temperature, separating the ethylene glycol, and washing the polymer with diethyl ether for 5 times to obtain 71.2 g of hyperbranched polyhistidine.

Example 12

Dissolving 2g of hyperbranched polyornithine in 5mL of methanol, adding 4.8g of methylisothiourea hemisulfate and 5mL of triethylamine, reacting at 65 ℃ for 12 hours, stopping heating, cooling to room temperature, and settling in diethyl ether to obtain 2.1g of guanidino-modified hyperbranched polyornithine.

Example 13

2g of the hyperbranched polyarginine from example 1 and 0.2g of silver nitrate were dissolved in 5mL of water, uniformly dispersed with stirring and then lyophilized to give 2.18g of a mixture of hyperbranched polyarginine and silver ions.

Example 14

2g of the hyperbranched polylysine of example 2 and 0.2g of chitosan were dissolved in 5mL of water, uniformly dispersed with stirring, and then lyophilized to obtain 2.18g of a mixture of hyperbranched polylysine and chitosan.

Example 15

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 16

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

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

Example 17

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

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 2

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 foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.