WO2017016022A1

WO2017016022A1 - Chito-oligosaccharide-o-kojic acid-mannich base derivative antibacterial agent and preparation method thereof

Info

Publication number: WO2017016022A1
Application number: PCT/CN2015/088398
Authority: WO
Inventors: 夏文水; 刘晓丽; 姜启兴; 许艳顺; 于沛沛
Original assignee: 江南大学
Priority date: 2015-07-24
Filing date: 2015-08-28
Publication date: 2017-02-02
Also published as: CN105218700A; AU2015403788A1; CN105218700B; AU2015403788B2

Abstract

A chito-oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent and preparing method thereof. The method comprises the following steps: (1) mixing N-Methylpiperazine with a chloro kojic acid and reacting the same to obtain a product of a chloro kojic acid-Mannich base; (2) mixing a chito-oligosaccharide Schiff base with the chloro kojic acid-Mannich base and reacting and processing the same to obtain a chito-oligosaccharide Schiff base-O-kojic acid-Mannich base derivative, and performing an amine deprotection reaction and passivation to obtain a chito-oligosaccharide-O-kojic acid-Mannich base derivative.

Description

Chitooligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent and preparation method thereof

This application claims the Chinese patent with the application date of July 24, 2015, application number 2015104438518, the invention name is "an chitosan oligosaccharide-O-kojic acid-mannich base derivative antibacterial agent and its preparation method" The priority of the application, the entire contents of which is incorporated herein by reference.

Technical field

The invention belongs to the technical field of food additives, and in particular relates to a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent and a preparation method thereof.

Background technique

In recent years, food safety has become one of the world's priorities for the global public. Food in the process of production, processing, transportation, storage and sales may be exposed to some harmful micro-organisms, these harmful microorganisms can contaminate food, so that food spoilage, not only will cause serious waste of food resources, but also wrong The eater's body causes serious damage, so the preservation of various foods is always an important issue to be solved. Natural antiseptic and bacteriostatic agents such as chitosan and kojic acid have been favored by people for their safety, non-toxicity and good thermal stability. They have been developed with chemical modification to have stronger antibacterial activity and broader antibacterial spectrum. Natural food preservatives have become a hot topic of research.

Chitooligosaccharide is a low molecular weight basic aminooligosaccharide obtained by cleavage of chitosan backbone by physical, chemical or enzymatic degradation. It is biodegradable, biocompatible, biologically non-toxic and chemically reactive. It inhibits a range of microorganisms including bacteria and fungi and is considered an ideal material for the development of new natural food preservatives.

However, since chitosan oligosaccharide is a natural macromolecular product, when used as a food antiseptic antibacterial agent, it has disadvantages such as low antibacterial activity compared with conventional common chemical preservatives, so the application in the food industry is not very current. universal. Therefore, the structure of chitosan oligosaccharide can be chemically modified. Due to the chemically reactive amino group and hydroxyl group in the molecular chain, these sites are ideal sites for chemical modification of chitooligosaccharides, which can be further improved. Its antibacterial activity, which is also an effective method for research at home and abroad.

In view of the above defects, the designer actively researches and innovates in order to create a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent and a preparation method thereof, so as to make it more industrially valuable. .

Summary of the invention

In order to solve the above technical problems, an object of the present invention is to provide a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent and a preparation method thereof, and an active antibacterial group in kojic acid-γ-pyran The keto group and N-methylpiperazine are introduced into the chitosan oligosaccharide molecular chain to synergize with the chitooligosaccharide molecule to enhance the antibacterial activity of chitooligosaccharide, and obtain a well-soluble and synergistic chitosan derivative. .

The chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent proposed by the present invention has the chemical structural formula shown below, wherein n is 6-20 and the degree of substitution is 1.21-1.78.

The invention also provides a preparation method of the chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent, comprising the following steps:

(1) Dissolving chitosan oligosaccharide Schiff base and chlorodeteric acid-Mannich base in an organic solvent, mixing and stirring, adding an organic solvent to precipitate the product after completion of the reaction, filtering, and precipitating the product with an organic solvent. Extracting, freeze-drying to obtain chitooligosaccharide Schiff base-O-kojic acid-Mannich base derivative;

(2) adding a mixture of 0.25 mol/L hydrochloric acid/ethanol (V/V = 1:4) to the chitooligosaccharide Schiff base-O-kojic acid-Mannich base derivative of the step (1). After mixing and stirring, the pH was adjusted to neutral after completion of the reaction, washing with an organic solvent, suction filtration, and freeze-drying to obtain a sample.

Further, the method further comprises the step of purifying after the step (2), dialysis the dried product, adding an organic solvent to precipitate, suction filtration, and freeze-drying to finally obtain purified chitooligosaccharide-O-kojic acid. - Mannich base derivative.

Further, the ratio by mass of the chitooligosaccharide Schiff base to the chlorodeteric acid-Mannich base is 2:(1-5).

Further, the chitosan oligosaccharide has a molecular weight of 1000 to 5000 Da and a degree of deacetylation of 90 to 95%.

Further, in the step (1), the organic solvent for dissolving the chloroderivative-Mannich base is dimethyl sulfoxide or dimethylformamide, and the amount of the chloroderivative-Mannich base is 2 -4 times; the organic solvent for dissolving chitooligosaccharide Schiff base is dimethylformamide and pyridine, the amount of dimethylformamide is 2-4 times that of chitooligosaccharide Schiff base, and the amount of pyridine is chitosan oligosaccharide 2-6 times the Schiff base.

Further, in the step (1), the reaction temperature is -35 ° C to 40 ° C, and the reaction time is 2 to 6 h.

Further, in the step (2), the reaction temperature is room temperature, and the reaction time is 12-24 h.

Further, in the step (1), the preparation method of the chlorodeteric acid-Mannich base comprises the following steps:

Dissolve N-methylpiperazine and chlorodecanic acid in methanol and formalin solution, stir rapidly at room temperature to form a brown precipitate, collect, filter, wash several times with anhydrous methanol, and use anhydrous methanol Crystallization and drying give chlorogeric acid-Mannich base.

Further, the ratio by mass of the N-methylpiperazine and the chloroderivative acid is 1: (2-3), and the ratio of the volume of the methanol to the formalin solution is (10-20):1. .

By the above scheme, the present invention has at least the following advantages: the present invention is based on the idea of substructure linkage, and the active group - γ-pyranone and N-methyl piperazine in kojic acid are linked to chitooligosaccharide molecules. In the chain, the three have synergistic antibacterial activity, and developed a new type of food antiseptic and bacteriostatic agent.

The above description is only an overview of the technical solution of the present invention, in order to be able to understand the present issue more clearly. The technical means of the present invention can be implemented in accordance with the contents of the specification, and the following detailed description will be made with reference to the preferred embodiments of the present invention and the accompanying drawings.

DRAWINGS

Figure 1 is an infrared spectrum of chitosan oligosaccharide;

2 is an infrared spectrum diagram of a chitosan oligosaccharide derivative prepared in Example 1 of the present invention;

Figure 3 is a ¹ H-NMR chart of the chitosan oligosaccharide derivative prepared in Example 1 of the present invention;

Figure 4 is a ¹³ C-NMR chart of the chitosan oligosaccharide derivative prepared in Example 1 of the present invention;

Figure 5 is an infrared spectrum diagram of a chitosan oligosaccharide derivative prepared in Example 2 of the present invention;

Figure 6 is an infrared spectrum of a chitosan oligosaccharide derivative prepared in Example 3 of the present invention.

detailed description

The specific embodiments of the present invention are further described in detail below with reference to the drawings and embodiments. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The preparation method of chitosan oligosaccharide-O-kojic acid-Mannich base derivative having good water solubility and antibacterial activity is as follows: using N-methylpiperazine and chloroderivative acid as raw materials, firstly obtained by Mannich reaction Chlorothetrex-Mannich base is then alkylated with an amino-protected chitooligosaccharide Schiff base, and then subjected to amino deprotection and purification to obtain a chitosan oligosaccharide-O-koroic acid which retains the active amino group. Mannich base derivatives, the synthetic route is as follows:

Embodiment 1:

The preparation method of the chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent comprises the following steps:

1. Preparation of chlorinated kojic acid-Mannich base

N-methylpiperazine and chlorodeteric acid were dissolved in 100 mL of methanol and 10 mL of formalin at a mass ratio of 1:2, and rapidly stirred at 25 ° C to form a brown precipitate, collected, filtered, and dried. The mixture was washed with methanol for 5 times, recrystallized from anhydrous methanol, and dried to give chlorotrimetic acid-Mannich base.

2. Preparation of chitooligosaccharide-O-kojic acid-Mannich base derivative

Under rapid stirring, 0.5 g of chlorodeco-Mannich base was dissolved in 2 mL of dimethyl sulfoxide, and 1 g of chitooligosaccharide Schiff base (molecular weight 1000 Da, deacetylation degree 90%) was dissolved in 2 mL of two In a mixture of methyl formamide and 2 mL of pyridine, chitosan sulphur Schiff base dissolved in a mixture of dimethylformamide and pyridine is added dropwise to chloroderivative-Mani in dimethylformamide. In the base, the reaction mixture was magnetically stirred in a -35 ° C water bath for 2 hours, poured into excess acetone to precipitate the product, and the precipitate was filtered. The precipitate was subjected to Soxhlet extraction with ethanol and acetone to remove dimethylformamide and pyridine. After lyophilization, 1 g of chitooligosaccharide Schiff base-O-kojic acid-Mannich base derivative was obtained.

To the chitosan oligosaccharide Schiff base-O-koroic acid-Mannich base derivative prepared above, 20 mL of a mixture of 0.25 mol/L hydrochloric acid (V/V = 1:4) was added, and the mixture was stirred at room temperature for 12 hours. The pH was adjusted to neutral with 10% Na ₂ CO ₃ , washed repeatedly with acetone 5 times, suction filtered, and lyophilized.

1 gram of crude product was suspended in 5 mL of ultrapure water, placed in a dialysis bag, dialyzed for 24 hours, concentrated, and a sufficient amount of acetone was added for precipitation, suction filtration, and lyophilization to finally obtain purified brown chitosan oligosaccharide-O-curved Acid-Mannich base derivative.

The yield of the chitosan oligosaccharide derivative was 62.1%, which was characterized by infrared spectroscopy and nuclear magnetic resonance.

Fig. 1 is an infrared spectrum of chitosan oligosaccharide, wherein 3422.36 cm ^-1 is the stretching vibration absorption peak of OH and NH, 2923.83 cm ^-1 is the absorption peak of CH stretching vibration, and 1628.76 cm ^-1 is the bending vibration absorption of NH ₂ . The peaks, 1155.97 cm ^-1 and 1071.52 cm ^-1 are absorption peaks of CO stretching vibration, and 893.24 cm ^-1 is a ring stretching vibration absorption peak.

Figure 2 is the infrared spectrum of chitosan oligosaccharide derivative according to the present embodiment, wherein wherein β- glycosidic bonds pyran corresponding to an absorption peak at 890.44cm ^-1, 1519.37cm ^-1 stretching C = C derivative that The vibration absorption peak, 1223.02 cm ^-1 is the COC stretching vibration absorption peak of the derivative, and 970.39 cm ^-1 is the absorption peak of the covalently bonded bond of chlorodetergentic acid-Mannich base with chitosan oligosaccharide, 1223.02 cm ^-1 and The presence of 970.39 cm ^-1 demonstrates the formation of the target product.

Figure 3 is a ¹ H-NMR chart of the chitosan oligosaccharide derivative in the present example, the peak at which the chemical shift occurs at 1.968 ppm corresponds to the proton peak of -CH ₃ on the acetamido residue; at 2.47-2.796 ppm The peak corresponds to the proton peak of H ₂ on the glucosamine residue and the acetamido residue; the peak at 3.089-3.837 ppm corresponds to the corresponding proton peak at H3, H4, H5, H6 on glucosamine and acetylglucosamine The peak at 4.7 ppm is the solvent peak; two new chemical shifts appear at 4.26 and 6.82 ppm, which can be assigned to the -CH2 (H-7') and H-3, respectively, on the 5-hydroxypyrone skeleton of kojic acid. 'The chemical shift of each proton; and the chemical shift of H ₂ on the glucosamine residue at δ = 2.98 ppm is still present. This result is consistent with the results of the infrared spectrum, so these new chemical shifts demonstrate 5-hydroxypyridyl in kojic acid. The ketone and the amino group of the chitosan oligosaccharide are alkylated; at 2.88, 3.07 and 3.12 ppm, the chemical shift on the N-methylpiperazine ring; therefore, by the assignment of the above chemical shifts, it can be proved that the chloroform Acid-Mannich base and O-alkylation reaction of chitooligosaccharide Schiff base, chitooligosaccharide-O-kojic acid-Mannich base Preparation of biological success.

Figure 4 is a ¹³ C-NMR chart of the chitosan oligosaccharide derivative in the present example, δ = 22.74, 56.33, 60.46, 71.03, 75.11, 76.95, 98.63, 174.18 ppm respectively assigned to chitosan oligosaccharide molecules -CH ₃ , C 2 , chemical shifts of C6, C3, C5, C4, C1 and -C=O; δ = 45.83, 47.08, 56.38 ppm, respectively assigned to -CH2 and -CH ₃ on the N-methylpiperazine ring in the derivative C. The chemical shifts at δ=60.44, 114.64, 139.82, 145.87, 159.35 and 176.99 ppm are assigned to the 5-hydroxypyranone group in the derivative, C-7', C-3', C-6', C-5 The chemical shifts of ', C-2' and C-4' indicate that the chitooligosaccharide-O-kojic acid-Mannich base derivative was successfully prepared.

Embodiment 2:

1. Preparation of chlorinated kojic acid-Mannich base

N-methylpiperazine and chlorodeteric acid were dissolved in 150 mL of methanol and 10 mL of formalin at a mass ratio of 1:2.5, and rapidly stirred at 25 ° C to form a brown precipitate, which was collected, filtered, and dried. The mixture was washed with methanol for 5 times, recrystallized from anhydrous methanol, and dried to give chlorotrimetic acid-Mannich base.

2. Preparation of chitooligosaccharide-O-kojic acid-Mannich base derivative

1.5 g of chlorodeco-Mannich base was dissolved in 3 mL of dimethylformamide with rapid stirring, and 1 g of chitooligosaccharide Schiff base (molecular weight 3000 Da, deacetylation degree 93%) was dissolved in 3 mL. In a mixture of dimethylformamide and 4 mL of pyridine, the chitooligosaccharide Schiff base dissolved in a mixture of dimethylformamide and pyridine is added dropwise to the chloroderivative-manate dissolved in dimethylformamide. In the Nich base, the reaction mixture was magnetically stirred in a water bath at -5 ° C for 4 hours, poured into excess acetone to precipitate the product, and the precipitate was filtered, and the precipitate was subjected to Soxhlet extraction with ethanol and acetone to remove dimethylformamide and Pyridine, and finally 1.8 g of the product after vacuum freeze-drying was obtained.

To the chitosan oligosaccharide Schiff base-O-kojic acid-Mannich base derivative prepared above, 36 mL of a mixture of 0.25 mol/L hydrochloric acid (V/V = 1:4) was added, and the mixture was stirred at room temperature for 12 hours. The pH was adjusted to neutral with 10% Na ₂ CO ₃ , washed repeatedly with acetone 5 times, suction filtered, and lyophilized.

1 gram of crude product was suspended in 5 mL of ultrapure water, placed in a dialysis bag, dialyzed for 24 hours, concentrated, and a sufficient amount of acetone was added for precipitation, suction filtration, and lyophilization to obtain a purified brown. Chitosan oligosaccharide-O-kojic acid-Mannich base derivative.

The yield of the chitooligosaccharide derivative was 65.2%. IR spectra for samples of FIG. 5, wherein, wherein β- glycosidic bonds pyran corresponding to an absorption peak at 892.24cm ^-1, 1518.32cm ^-1 derivative for C = C stretching vibration absorption peaks, 1226.23cm ^-1 is the COC stretching vibration absorption peak of the derivative, and 971.91 cm ^-1 is the absorption peak of the covalently bonded bond of chloric acid-Mannich base and chitooligosaccharide, and the existence of 1226.23 cm ^-1 and 971.91 cm ^-1 Prove the formation of the target product.

Embodiment 3:

1. Preparation of chlorinated kojic acid-Mannich base

N-methylpiperazine and chlorodeteric acid were dissolved in 200 mL of methanol and 10 mL of formalin at a mass ratio of 1:3, and rapidly stirred at 25 ° C to form a brown precipitate, which was collected, filtered, and dried. The mixture was washed with methanol for 5 times, recrystallized from anhydrous methanol, and dried to give chlorotrimetic acid-Mannich base.

2. Preparation of chitooligosaccharide-O-kojic acid-Mannich base derivative

2.5 g of chlorodeco-Mannich base was dissolved in 4 mL of dimethyl sulfoxide with rapid stirring, and 1 g of chitooligosaccharide Schiff base (molecular weight 5000 Da, degree of deacetylation 95%) was dissolved in 4 mL of two In a mixture of methyl formamide and 6 mL of pyridine, the chitooligosaccharide Schiff base dissolved in a mixture of dimethylformamide and pyridine was added dropwise to chloroderivative-Muni in dimethylformamide. In the base, the reaction mixture was magnetically stirred in a 40 ° C water bath for 6 hours, poured into excess acetone to precipitate the product, and the precipitate was filtered, and the precipitate was subjected to Soxhlet extraction with ethanol and acetone to remove dimethylformamide and pyridine, respectively. Finally, 3 g of the product after vacuum freeze-drying was obtained.

Adding 60 mL of a mixture of 0.25 mol/L hydrochloric acid (V/V = 1:4) to the chitosan oligosaccharide Schiff base-O-kojic acid-Mannich base derivative prepared above, and stirring at room temperature for 12 hours, The pH was adjusted to neutral with 10% Na ₂ CO ₃ , washed repeatedly with acetone 5 times, suction filtered, and lyophilized.

The yield of the chitooligosaccharide derivative was 63.5%. IR spectra for samples of FIG. 6, wherein the glycosidic bond wherein β- pyranose corresponding to an absorption peak at 893.34cm ^-1, 1514.18cm ^-1 derivative for C = C stretching vibration absorption peaks, 1223.73cm ^-1 is the COC stretching vibration absorption peak of the derivative, and 970.39 cm ^-1 is the absorption peak of the covalent bond of chloroderivative with chitosan oligosaccharide, and the presence of 1223.13 cm ^-1 and 970.39 cm ^-1 proves the formation of the target product. .

Antibacterial test

1. Preparation of antibacterial agent solution

The samples in the first embodiment, the second embodiment and the third embodiment are respectively named as derivative 1, derivative 2, and derivative 3, and chitosan oligosaccharide, kojic acid, derivative 1, derivative 2 and The derivative 3 was dissolved and formulated into a solution having a concentration gradient of 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 mg/mL, and filtered through a 0.22 μm microporous membrane. ,spare.

2, antibacterial activity determination

1 mL of the initial bacterial solution was mixed with 98 mL of the sterilized nutrient broth, and 1 mL of the above-mentioned antibacterial agent with different concentration gradients was added thereto, and 1 mL of deionized water was used instead of the antibacterial agent solution as a blank, and shake culture was carried out at 37 ° C. . During the culture, 1 mL of the culture solution was taken out after 8 hours, and the number of colonies was counted by the plate method after dilution, and each sample was repeated 3 times. The antibacterial rate (I) is calculated according to the following formula:

I(%)=N ₁ -N ₂ /N ₁ ×100

Wherein, N1: the total number of colonies in the initial culture solution;

N2: Total number of colonies in the culture medium containing the antibacterial agent.

Taking the concentration of the antibacterial agent as the abscissa and the inhibition rate as the ordinate, the IC ₅₀ value is obtained.

Table 1 Detection of antibacterial properties

As can be seen from Table 1, the antibacterial agent prepared by the present invention has good antibacterial activity against Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Salmonella typhimurium, Shigella dysenteriae and Escherichia coli. Moreover, the antibacterial property is much better than that of chitosan oligosaccharide and kojic acid, and has potential application value in food preservation.

In summary, the principle of the present invention is as follows: there are three chemically modified active sites in the molecular structure of chitosan oligosaccharide. If an alkylation reaction occurs, the amino acid at the C-2 position first occurs according to the magnitude of the activity at three positions. The upper one is followed by the primary hydroxyl group at the C-6 position, and the second is the secondary hydroxyl group at the C-3 position. Since the C-2 amino group in the chitosan oligosaccharide molecule is an active group which acts as an antibacterial agent in an acidic solution, the active amino group at the C-2 position is first protected with benzaldehyde, and an alkane is formed. After the base reaction, it is deprotected in an acid alcohol solution to release the active amino group. The hydroxyl group at the C-3 position does not participate in the reaction due to the steric hindrance effect. Thus, the hydroxyl group at the C-6 position undergoes an O-alkylation reaction with the chloro- tretric acid-Mannich base.

The beneficial effects of the present invention are as follows: (1) O-alkylation reaction of a hydroxy group at the C-6 position of a chitooligosaccharide with a chloroderivative-Mannich base, and an active antibacterial group-γ in kojic acid -pyridyl The ketone group and N-methylpiperazine are introduced into the chitooligosaccharide molecular chain to synergize with the chitooligosaccharide molecule, which significantly enhances its antibacterial activity. (2) These new derivatives are low in toxicity, have good water solubility, can be dissolved in various inorganic and organic solvents, avoid the use of organic solvents, are more environmentally friendly, and expand their application fields. It has a wide range of applications in the fields of medicine, food, cosmetics and agriculture.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be noted that those skilled in the art can make some improvements without departing from the technical principles of the present invention. And modifications and variations are also considered to be within the scope of the invention.

Claims

A chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent, which has the chemical structural formula shown below, wherein n is 6-20 and the degree of substitution is 1.21-1.78.
The method for preparing a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent according to claim 1, comprising the steps of:

(1) Dissolving chitosan oligosaccharide Schiff base and chlorodeteric acid-Mannich base in an organic solvent, mixing and stirring, adding an organic solvent to precipitate the product after completion of the reaction, filtering, and precipitating the product with an organic solvent. Extracting, freeze-drying to obtain chitooligosaccharide Schiff base-O-kojic acid-Mannich base derivative;

(2) adding a mixture of 0.25 mol/L hydrochloric acid/ethanol (V/V = 1:4) to the chitooligosaccharide Schiff base-O-kojic acid-Mannich base derivative of the step (1). After mixing and stirring, the pH was adjusted to neutral after completion of the reaction, washing with an organic solvent, suction filtration, and freeze-drying to obtain a sample.
The method for preparing a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent according to claim 2, wherein the method further comprises the purifying step after the step (2), The dried product is dialyzed, precipitated with an organic solvent, suction filtered, and lyophilized to finally obtain a purified chitooligosaccharide-O-kojic acid-Mannich base derivative.
The method for preparing a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent according to claim 2, wherein the chitooligosaccharide Schiff base and chlorodeteric acid-Mannich base The mass ratio is 2: (1-5).
The method for preparing a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent according to claim 4, wherein the chitosan oligosaccharide has a molecular weight of 1000 to 5000 Da and a degree of deacetylation of 90- 95%.
The method for preparing a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent according to claim 2, wherein in the step (1), the chloroderivative-Mannich base is dissolved The organic solvent is dimethyl sulfoxide or dimethylformamide in an amount of from 2 to 4 times the mass of the chloric acid-Mannich base; the organic solvent for dissolving the chitooligosaccharide Schiff base is dimethylformamide and The amount of pyridine and dimethylformamide is 2-4 times that of chitooligosaccharide Schiff base, and the amount of pyridine is 2-6 times that of chitooligosaccharide Schiff base.
The method for preparing a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent according to claim 2, wherein in the step (1), the reaction temperature is -35 ° C to 40 ° C, The reaction time is 2 to 6 hours.
The method for preparing a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent according to claim 2, wherein in the step (2), the reaction temperature is room temperature, and the reaction time is 12- 24h.
The method for preparing a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent according to claim 2, wherein in the step (1), the chloroderivative-Mannich The preparation method of the base includes the following steps:

Dissolve N-methylpiperazine and chlorodecanic acid in methanol and formalin solution, stir rapidly at room temperature to form a brown precipitate, collect, filter, wash several times with anhydrous methanol, and use anhydrous methanol Crystallization and drying give chlorogeric acid-Mannich base.
The chitosan oligosaccharide-O-kojic acid-Mannich base derivative according to claim 9 The preparation method of the agent is characterized in that the ratio by mass of the N-methylpiperazine and the chloroderivic acid is 1: (2-3), and the ratio of the volume of the methanol to the formalin solution is ( 10-20): 1.