CN108192000B

CN108192000B - Chitosan oligosaccharide graft copolymer G1.0 and preparation method and application thereof

Info

Publication number: CN108192000B
Application number: CN201810033904.2A
Authority: CN
Inventors: 熊春华; 杨雄; 阎亚利; 王小青; 陈青; 方嫱; 叶鑫妍
Original assignee: Zhejiang Gongshang University
Current assignee: Zhejiang Gongshang University
Priority date: 2018-01-14
Filing date: 2018-01-14
Publication date: 2021-06-01
Anticipated expiration: 2038-01-14
Also published as: CN108192000A

Abstract

The invention discloses a chitosan oligosaccharide graft copolymer G1.0 and a preparation method and application thereof. The invention adopts a free radical initiated grafting method, adopts a method of combining three action factors of physical, chemical and enzymolysis to degrade chitosan to prepare chitosan oligosaccharide, then takes the chitosan oligosaccharide as a raw material, firstly initiates an active group of chitosan oligosaccharide molecules by ammonium ceric nitrate to carry out free radical reaction to graft methyl acrylate for branching modification, and then carries out Michael addition reaction on the prepared chitosan oligosaccharide by using a dispersion method, grafts ethylenediamine, and prepares a graft copolymer G1.0. The invention provides a chitosan oligosaccharide graft copolymer with good water solubility and stronger bacteriostatic ability. The invention provides a preparation method of a chitosan oligosaccharide graft copolymer. The chitosan graft copolymer has the advantages of good water solubility and strong bacteriostatic ability, and can be used as a food preservative bacteriostatic agent.

Description

Chitosan oligosaccharide graft copolymer G1.0 and preparation method and application thereof

Technical Field

The invention belongs to the field of food chemistry, relates to a bacteriostatic agent in food chemistry, and particularly relates to a chitosan oligosaccharide graft copolymer, and a preparation method and application thereof.

Background

Chitosan is a polysaccharide product with the deacetylation degree of Chitin (Chitin) reaching more than 55 percent and is a natural alkaline polysaccharide composed of beta- (1, 4) -2-amino-2-deoxy-D-glucose and rarely existing in nature [1 ]. Chitin is the second largest natural polysaccharide resource for vitamins in natural world in annual biosynthetic quantities (up to 1010 tons/year) [ 2; 3] the chemical structural unit of which is N-acetyl-2-amino-2-deoxy-D-glucose dyadic linear macromolecular polymer connected by beta- (1, 4) glycosidic bond [4], mainly exists in the shells and inner cuticles of marine invertebrates such as shrimps, crabs and crustaceans, fungi (yeast, mould, mushroom, etc.), algae and the cell walls of higher plants [5 ]. Although the chitin source is widely used, because an oxygen atom on one sugar ring group and a hydrogen atom in the other sugar ring hydroxyl group are easy to combine with each other to form a hydrogen bond in an intramolecular or intermolecular adjacent two sugar unit structures, and a plurality of hydrogen bonds [6-8] closely connected with CO-NH exist in the molecule, the free rotation of the glycosidic bond is hindered by the action of a large number of hydrogen bonds, so that chitin molecules form a highly ordered crystal region. The chitin exists in alpha, beta and gamma crystal forms [9], wherein the beta form is the most common. The regular crystal structure increases the rigidity of chitin molecules, so that the chitin molecules have poor solubility, and are insoluble in water, olefine acid, dilute alkali and common organic reagents, thereby greatly limiting the application and development of the chitin molecules. Compared with chitin, chitosan has improved solubility, but still has more hydrogen bonds in the molecule, the pK value is about 6.5 to the power of 10, and the chitosan can only be dissolved in dilute acid solution and is insoluble in water and common organic solvents, and the application of the chitosan is limited to a certain extent due to the weakness. Although the solubility of chitosan is poor after deacetylation of chitin to form chitosan, the structural unit of chitosan contains active hydroxyl groups on C-3 and C-6 and active amino groups on C-2, when the chitosan is dispersed in an olefine acid solution with the weight of less than 10 percent, the-NH 2 on C-2 and H + undergo protonation reaction to form a positive-charged-NH 3+ cationic electrolyte, so that the chitosan has positive electricity [11 ]. This major change makes chitosan possess many unique chemical properties: good biocompatibility, degradability, no toxic side effect and biological activities of antisepsis, bacteriostasis, promoting wound healing, anticancer, etc. [3 ].

The research and application of chitosan and derivatives thereof in the aspect of bacteriostasis become a hot spot at present, but the application of chitosan in the actual production process is greatly limited due to poor water solubility and limited bacteriostasis capability.

Chitosan is the second largest natural polysaccharide resource second to cellulose at present, and is also a natural alkaline polysaccharide existing in nature. The microbial fuel has rich reserves, wide sources, low price, good biocompatibility, degradability, no toxic and side effects, bacteriostatic activity and other biochemical activities, and is widely applied to various fields of medicines, foods, environmental protection, agriculture and the like. Inhibition from chitosan

Disclosure of Invention

Aiming at the problems in the background art, the invention provides a chitosan oligosaccharide graft copolymer with good water solubility and stronger bacteriostatic ability.

Another object of the present invention is to provide a method for preparing the chitosan oligosaccharide graft copolymer of the present invention.

Still another object of the present invention is to use the chitosan oligosaccharide graft copolymer of the present invention as a food preservative and bacteriostatic agent.

The technical scheme adopted by the invention is as follows: a chitosan oligosaccharide graft copolymer G1.0 has the following molecular formula:

where n is any integer value.

A preparation method of a chitosan oligosaccharide graft copolymer G1.0 comprises the following specific steps:

1) weighing a certain amount of chitosan oligosaccharide, adding 1% (v/v) acetic acid as a reaction solvent, wherein the relationship between the adding amount of the acetic acid and the total amount of the chitosan oligosaccharide is as follows: adding 25ml of the acetic acid solution into every 1g of chitosan oligosaccharide; after the solution is fully dissolved, introducing nitrogen into the bottle to remove oxygen; adding a certain amount of ammonium ceric nitrate as an initiator under the protection of nitrogen, wherein the mass ratio of the addition amount of the initiator to the chitosan oligosaccharide is 0.5:1-2.5: 1; then slowly adding a proper amount of methyl acrylate dropwise; the reaction molar ratio of the chitosan oligosaccharide to the methyl acrylate is 1: 4-1: 10; setting the stirring speed at 200rpm/min, and continuously reacting for 2-10 h at the temperature of 40-55 ℃; after the reaction is finished, filtering out a reaction product G0.5, then carrying out suction filtration on the filtered reaction product G0.5 for 3 times by using a reaction solvent 1% (v/v) acetic acid, repeatedly washing the reaction product G for 3 times by using acetone, diethyl ether and absolute ethyl alcohol in sequence, removing unreacted methyl acrylate, and finally putting the reaction product G into a vacuum drying oven at 50 ℃ for drying for later use;

2) weighing G0.5 prepared in the steps, adding a chromatographic grade methanol solvent, soaking for 12 hours, controlling the reaction temperature to be 20 ℃, dropwise adding analytically pure ethylenediamine, and reacting for 8 hours under the protection of N2; the amount of methanol added and the total amount of G0.5 are related as follows: adding 0.5ml of methanol into every 1mg of G0.5; the amount of ethylenediamine added and the total amount of G0.5 are as follows: 0.08ml of ethylenediamine is added into every 1mg of G0.5; after the reaction is finished, filtering out a product G1.0 obtained by the reaction, washing the product for 3 times by using a reaction solvent methanol, repeatedly washing the product for 3 times by using absolute ethyl alcohol and distilled water in sequence, and placing the product at 50 ℃ for vacuum drying to obtain the catalyst.

Preferably, the methyl acrylate added in step 1) of the preparation process of the chitosan oligosaccharide graft copolymer G1.0 contains p-methoxyphenol MEHQ, a polymerization inhibitor to prevent self-polymerization.

Preferably, the chitosan oligosaccharide graft copolymer G1.0 is prepared by the method of step 1) in which the reaction molar ratio of chitosan oligosaccharide and methyl acrylate is 1:8 to 1: 8.5.

Preferably, the preparation method of the chitosan oligosaccharide graft copolymer G1.0, step 1), is set to have a stirring speed of 200rpm/min and a continuous reaction time of 8 hours at a temperature of 40 ℃ to 55 ℃.

Preferably, the preparation method of the chitosan oligosaccharide graft copolymer G1.0 comprises the following steps: 1) aWeighing a certain amount of chitosan, fully dissolving the chitosan in a certain amount of 1% (v/v) acetic acid solution through high-speed stirring, transferring the solution into a quartz three-necked bottle, adding a certain amount of 30% (v/v) H2O2, and correctly installing the solution in a cavity of a microwave synthesizer to ensure that an ultrasonic probe is submerged below an interface of the solution; setting the microwave power at 300w, reacting for 30min, turning on the ultraviolet lamp for auxiliary degradation, after the experiment is finished, carrying out water bath rotary evaporation concentration on the solution at 40 ℃, and then carrying out freeze drying for 24h to obtain a chitooligosaccharide sample with the viscosity-average molecular weight M eta of about 25000; the relationship between the addition amount of the acetic acid and the total amount of the chitosan is as follows: every 3g of chitosan was added with 100mL of the above acetic acid solution, H₂O₂The relationship between the added amount and the total amount of chitosan is as follows: 0.1mL 30% (v/v) H per 1g chitosan was added₂O₂；

2) And the mass ratio of 1: 1: 1, weighing a certain amount of papain, pectinase and cellulase, dissolving in HAC-NaAC buffer solution with pH of 5.5, and preparing into 1mg/mL enzyme solution for later use; adjusting the pH value of the shell oligosaccharide solution after microwave degradation to 5.5 according to the concentration ratio of enzyme to substrate of 1: 10, adding the enzyme solution to be used, uniformly stirring, and placing in a constant-temperature water bath at 45 ℃ for enzymolysis for 4 hours; boiling and heating for 10min to inactivate enzyme, and concentrating at 40 deg.C under reduced pressure to obtain chitosan oligosaccharide sample solution.

Preferably, the preparation step of the chitosan oligosaccharide as the reaction raw material in the preparation method of the chitosan oligosaccharide graft copolymer G1.0 further comprises the steps of carrying out molecular weight classification on the prepared chitosan oligosaccharide sample solution, and selecting the chitosan oligosaccharide with the molecular weight of 3500-7000 as the raw material; the specific grading method is as follows: respectively selecting dialysis bags with the molecular weight cut-off of 7000 and 34mm and 3500 diameters for pretreatment, and removing metal ions; sleeving a dialysis bag with a smaller diameter into a dialysis bag with a larger diameter, fixing one end of the dialysis bag with a dialysis bag clamp, washing the dialysis bag with distilled water, then transferring the chitosan oligosaccharide sample solution prepared in claim 6 by using a 5mL liquid transfer gun, filling the chitosan oligosaccharide sample solution into an inner-layer dialysis bag, filling the gap between the double-layer dialysis bags with the distilled water, sealing the dialysis bag clamp, and then putting the dialysis bag into a large beaker filled with the distilled water for dialysis for 5 d; after the dialysis is finished, the liquid in the gap between the two layers of membranes is taken out, and freeze drying is carried out, thus obtaining the reaction raw material chitosan oligosaccharide with 3500-7000 molecular weight.

The chitosan oligosaccharide graft copolymer G1.0 is applied as a food preservative bacteriostatic agent.

The invention has the following advantages:

1. the chitosan oligosaccharide graft copolymer G1.0 provided by the invention has good water solubility and strong bacteriostatic ability, and can be used as a food preservative bacteriostatic agent.

2. The invention adopts a free radical initiated grafting method, degrades chitosan by a method combining three action factors of physics, chemistry and enzymolysis to prepare chitosan oligosaccharide, then carries out molecular weight grading on the chitosan oligosaccharide by a double-layer dialysis method, selects a specific amount of molecular weight as a raw material, firstly initiates an active group of chitosan oligosaccharide molecules by ammonium ceric nitrate to carry out free radical reaction to graft methyl acrylate, carries out branching modification, then carries out Michael addition reaction on the prepared chitosan oligosaccharide by a dispersion method, grafts ethylenediamine, and prepares a graft copolymer G1.0. Completely different from the quaternization reaction in the prior art.

3. The invention carries out microwave/ultrasonic wave/UV auxiliary H on chitosan₂O₂The method can be carried out under a lower concentration, effectively avoids the defect that the reduction of the activity of amino groups is caused by the side reaction of carbonyl ammonia condensation generated when the chitosan is degraded by hydrogen peroxide, and has mild experimental conditions, high degradation speed and high efficiency.

4. The chitosan oligosaccharide graft copolymer G1.0 provided by the invention has outstanding antibacterial performance: according to the invention, an MIC (minimal inhibitory concentration) determination method is adopted, and the MIC values of two gram-negative bacteria of escherichia coli and pseudomonas aeruginosa and two gram-positive bacteria of staphylococcus aureus and bacillus subtilis are determined, so that G1.0 has a remarkable bacteriostatic effect on the four tested bacteria.

Drawings

FIG. 1 is a schematic diagram of the separation of chitooligosaccharides of specific molecular weight by double membrane dialysis.

FIG. 2 is a graph showing the effect of the reaction molar ratio on the G0.5 graft ratio.

FIG. 3 is a graph showing the effect of reaction temperature on the G0.5 graft ratio.

FIG. 4 shows the effect of the amount of initiator added on the G0.5 graft ratio.

FIG. 5 is a graph showing the effect of reaction time on the G0.5 graft ratio.

FIG. 6 is a 3D plot and a contour plot of the AB, AC, BC cross-reacted response surface.

FIG. 7 is a bar graph of the percentage of carbon and nitrogen in different algebraic molecules.

FIG. 8 is a thermogravimetric plot of G0.5-3.0.

FIG. 9 is an infrared spectrum of Chitosan Oligosaccharide (COS), Methyl Acrylate (MA) and G0.5.

FIG. 10 is an infrared spectrum of G0.5, Ethylenediamine (EDA) and G1.0.

FIG. 11 is a G0.5 and G1.0 infrared spectra.

FIG. 12 shows Zeta potential values of COS/PAMAM of different generations in aqueous solution.

Detailed Description

The present invention will be further described with reference to specific examples, but the present invention is not limited to the following examples.

1 preparation of Chitosan oligosaccharide

1.1.1 Experimental materials and reagents

TABLE 2-1 Main materials and reagents

2.1.2 laboratory instruments and apparatus

TABLE 2-2 Main instruments and Equipment

1.2.1 preparation of Chitosan oligosaccharide by stepwise degradation of Chitosan

1.2.1.1 microwave/ultrasonic/UV-assisted rapid viscosity reduction of H2O2

Accurately weighing 3g of chitosan, stirring at high speed to fully dissolve in 100mL of acetic acid solution with specific concentration (v/v), transferring the solution into a 250mL quartz three-neck bottle, and adding a certain amount of 30% H₂O₂The ultrasonic probe is correctly arranged in the cavity of the microwave synthesizer, and the ultrasonic probe is required to be ensured to be submerged below the interface of the solution. Fixing the power of ultrasonic wave at 200W, setting the microwave power and reaction time, and turning on the ultraviolet lamp (70 uw/cm)²) And (3) opening and closing to perform auxiliary degradation, after the experiment is finished, performing water bath rotary evaporation concentration on the solution at the temperature of 40 ℃, and then performing freeze drying for 24 hours to obtain a chitooligosaccharide sample. The molecular weight of the chitosan is reduced to tens of thousands of orders of magnitude by the treatment.

In this experiment will pass L₉(3⁴) Orthogonal test versus reaction time (min), H₂O₂Four important conditions of the addition amount (mL), the microwave power (W) and the acetic acid concentration (V/V,%) are optimized and screened, and finally the optimal rapid viscosity reduction condition is obtained. The factor levels for the experimental design are shown in tables 2-3:

tables 2 to 3L₉(3⁴) Orthogonal experiment factor level meter

1.2.1.2 nonspecific enzymolysis

According to the mass ratio of 1: 1: 1, weighing a certain amount of papain, pectinase and cellulase, dissolving in HAc-NaAc buffer solution with pH of 5.5, and preparing into 1mg/mL enzyme solution for later use. Adjusting the pH value of the chitosan oligosaccharide solution subjected to microwave degradation to 5.5 according to the concentration ratio of enzyme to substrate of 1: 10, adding 3mg of enzyme solution, stirring uniformly, and placing in a constant-temperature water bath at 45 ℃ for enzymolysis for 4 hours. Boiling and heating for 10min to inactivate enzyme, and vacuum distilling and concentrating at 40 deg.C to obtain chitosan oligosaccharide sample solution.

1.2.1.3 product of the double Membrane dialysis isolation

In order to solve the problem of uneven molecular weight distribution of the product in the preparation process of the chitosan oligosaccharide, the product obtained by the method of double-layer membrane dialysis is creatively used for separating. The method comprises the following specific operations: respectively selecting dialysis bags with the molecular weight cut-off of 7000 and 3500 diameters of 25mm and 34mm for pretreatment, and removing metal ions. And sleeving a dialysis bag with a smaller diameter into a dialysis bag with a larger diameter, fixing one end of the dialysis bag with a dialysis bag clamp, washing the dialysis bag with distilled water, then transferring a proper amount of the prepared chitosan oligosaccharide sample solution into an inner-layer dialysis bag by using a 5mL liquid transfer gun, filling a gap between the double-layer dialysis bags with the distilled water, sealing the dialysis bag clamp, and dialyzing the dialysis bag in the distilled water for 5 days at room temperature. And after the dialysis is finished, taking out the liquid in the gap between the double layers of membranes, and freezing and drying to obtain a light yellow powdery chitosan oligosaccharide sample with a specific molecular weight. The principle of double-membrane dialysis is shown in FIG. 1.

1.2.1.4 microwave/ultrasonic/UV-assisted H₂O₂Fast viscosity reduction orthogonal test result

Tables 2-4 results of orthogonal experiments

The results of the above orthogonal experiments can be combined to obtain: the experimental factors influencing the degradation of chitosan are as follows in sequence: time of microwave>H₂O₂Adding amount of>Microwave power>Acetic acid concentration; the optimal conditions for the experiment were: h₂O₂The addition amount is 0.3mL, the microwave power is 300W, the reaction time is 30min, and the acetic acid concentration is 1%. The tests are repeated under the optimal experimental conditions, and a chitooligosaccharide sample with the viscosity-average molecular weight M eta of about 25000 can be obtained.

2 preparation of G1.0

2.1.1 Experimental materials and reagents

TABLE 3-1 Main Agents

2.1.2 laboratory instruments and apparatus

TABLE 3-2 Main instrumentation

2.2.1 preparation of G0.5

1g of the chitosan oligosaccharide sample prepared in the previous chapter is accurately weighed and placed in a 100mL three-necked flask, 25mL 1% (v/v) acetic acid is added as a reaction solvent, the mixture is fully dissolved, and nitrogen is introduced into the flask for 30min to remove oxygen. Adding a certain amount of cerous ammonium nitrate as an initiator under the protection of nitrogen, and slowly dropwise adding a proper amount of methyl acrylate (containing p-methoxyphenol MEHQ which prevents self-polymerization), wherein the p-methoxyphenol MEHQ is from an avastin reagent net, and the product number is M100033, > 99.0% (GC), and contains MEHQ stabilizer of less than or equal to 100 ppm. The stirring speed is set to be 200rpm/min, and the reaction is continuously carried out for 8 hours at a certain temperature. After the reaction is finished, filtering out G0.5, filtering the reaction solvent for 3 times, repeatedly washing the reaction solvent for 3 times by using acetone, diethyl ether and absolute ethyl alcohol in sequence, removing unreacted methyl acrylate, and finally putting the reaction solvent into a vacuum drying oven at 50 ℃ for drying for later use.

Four important factors for the above experiments: the amount of the initiator (g), the molar ratio of methyl acrylate to chitosan oligosaccharide (mL/g), the reaction temperature (. degree. C.), and the reaction time were investigated for the conditions of the single-factor experiment. Fixing the reaction time, discussing the cross influence between every two other three main factors by a response surface method according to the principle of Box-Benhnken center Design, carrying out factor level analysis by using Design-expert8.0.4, and screening the optimal synthesis condition of G0.5. The factor level table for the response surface design is shown in tables 3-3:

TABLE 3-3 factor horizon of response surface method experimental design

The principle of the free radical grafting reaction between the ceric ammonium nitrate-initiated ligand molecule and the chitosan oligosaccharide is explained as follows^[83]：Ce⁴⁺C of sugar residue first₂-NH₂And C₃-OH, forming a complex. Then C₂And C₃Disproportionation occurs to form two radicals, NH and-CH (oh). The free radical may be in C₂And C₃The formation of atomic center and the formation of-NH are possible, so that ammonium cerium nitrate may initiate the chitosan oligosaccharide molecule to generate three different free radicals, and then the grafting reaction is carried out, and finally the chitosan oligosaccharide derivative is generated.

2.2.2 preparation of G1.0

Weighing G0.550mg synthesized under the optimal condition, placing the G0.550mg in a 100mL three-necked bottle, adding 25mL of methanol solvent, soaking for 12h, controlling the reaction temperature to be 20 ℃, and dropwise adding 4mL of ethylenediamine and N₂And reacting for 8 hours under protection. After the reaction is finished, filtering out G1.0, washing for 3 times by using a reaction solvent, repeatedly washing for a plurality of times by using absolute ethyl alcohol and distilled water in sequence, and placing at 50 ℃ for vacuum drying.

3.2.5.1 element analysis to determine half-generation grafting rate

1.000mg to 2.000mg of chitosan oligosaccharide and each generation of grafting product are accurately weighed by a one-millionth balance, wrapped by a tin bag, sequentially placed in a sample tray, and subjected to C, N, H element content measurement by an element analyzer, and the grafting rate (percent) of the half generation product is calculated by the following formula 3-1. And (3) carrying out single-factor experimental exploration and response surface condition optimization by taking the grafting rate as a dependent variable.

Wherein: x- -product grafting (%);

F_o-G1.5 and G2.5 are the amino content in the upper half-generation, G0.5 is the degree of deacetylation (%) of the chitosan oligosaccharide;

mc, the relative atomic mass of the Mn- -C, N atom;

nc- -the number of C atoms contained in the grafted molecule;

r- -C, N percent of the product.

3.2.5.2 measurement of amino content of the entire generation by double-leap potential

In the synthesis process, the whole generation of molecules generated after grafting the ethylenediamine is an open-end compound with amino as a terminal group, the amino content of the whole generation of molecules can be measured by utilizing double-jump potentiometric titration, and the grafting efficiency can be evaluated. Accurately weighing 0.1G of G1.0, G2.0 and G3.0 samples, dissolving in 0.1mol/L hydrochloric acid standard solution, and titrating with a calibrated 0.1mol/L NaOH solution under a constant pH/mV automatic potentiometric titrator. The amino content of the sample was calculated by equation 3-2:

wherein, V₁- -the volume of NaOH consumed in the first jump (mL);

V₂-the volume of naoh (ml) consumed in the second burst;

c- -concentration of NaOH standard solution (mol/L);

0.016- -amount of amine (g) corresponding to 1mL of a 1mol/L NaOH solution;

m- -sample mass (g).

3.2.5.3 Infrared structural characterization

The samples were mixed with KBr (spectral purity) at a ratio of 1: 50-1: 100, fully grinding to prepare a transparent sheet, and carrying out FTIR spectrum on the structure of the transparent sheet by a Nicolet-380 Fourier infrared spectrometerScanning with a scanning range of 4000cm^-1-400cm^-1Resolution of 4cm^-1The number of scans was 32.

3.2.5.4 thermogravimetric analysis (TGA)

Thermogravimetric analysis is an important characterization means for determining the thermal stability of a substance, along with the trend of temperature rise set by a program, a substance to be detected is degraded at a corresponding temperature to cause mass change, the sensitivity degree of the substance to heat can be obtained according to a change curve of the weight loss rate along with the temperature, and the structure of the substance is further qualitatively analyzed. The specific operation is as follows: a sample of about 20.000mg, dried to constant weight, is placed in an alumina crucible and slowly placed over the sample cell in the furnace cavity of the thermogravimetric instrument, the chamber door is closed, and the program automatically displays the exact mass of the sample. The measurement conditions were set as follows: the flow rate of the nitrogen carrier gas is 20mL/min, the heating rate is 10 ℃/min, and the starting temperature and the stopping temperature are 25-800 ℃.

3.2.5.5 Zeta potential measurement

The termini of the entire generation of COS/PAMAM dendrimer derivatives are all active-NH₂At the beginning, each generation of grafting has the change of amino, the amino is an active group with positive charge, and the change of the potential can be observed by utilizing the Zeta potential, so that the defect that the structural characteristics of the infrared characteristic absorption peak cannot be judged due to no obvious change after repeated grafting is carried out for many times is overcome. The sample is prepared into 0.05mg/mL aqueous solution, and then the Zeta potential value is measured by using a nanometer particle size analyzer to continuously scan for three times at 25 ℃.

3.3.1 Single factor conditional exploration of G0.5 Synthesis

3.3.1.1 Effect of reaction molar ratio on G0.5 graft ratio

In general, the reaction is only carried out to the maximum extent when the reaction sites and the graft functional groups are present in the appropriate ratio. As can be seen from FIG. 2, the grafting ratio increases with the increase of the content of the reactive monomer within a certain range, mainly because the diffusion rate of the reactive functional group increases with the increase of the content of the grafting monomer, so that the number of effective collisions with the active sites of the grafted material increases, and the reaction proceeds in the forward direction. When the reaction saturation ratio is reached, namely the ratio of the chitosan oligosaccharide to the methyl acrylate in the figure reaches 1: when 8, the system viscosity is increased due to the excessive monomer content, the diffusion speed of the functional groups is reduced, the reactive monomers compete with each other for active sites to form an inhibiting effect, and the side reaction is increased due to the easy self-polymerization of methyl acrylate, so that the grafting rate is reduced finally.

3.3.1.2 Effect of reaction temperature on G0.5 graft ratio

Most of the synthesis reactions are endothermic reactions, and proper temperature can give energy necessary for the reactions, so that Gibbs free energy change is generated in the reactions, and then the reactions are generated. As can be seen from fig. 3: in the initial stage of the reaction, the grafting rate is increased along with the increase of the reaction temperature, the activity of a reaction site can be furthest excited by increasing the temperature, and an initiator, namely ammonium cerium nitrate, used in the experiment can meet the bond dissociation energy required by the reaction only under the condition that the temperature is higher than 40 ℃, so that active groups in chitosan oligosaccharide molecules are initiated to generate free radicals. When the temperature reaches 45 ℃, the primary free radical concentration of the system reaches the maximum, and the grafting rate is the highest. When the temperature is continuously increased, side reactions in the reaction system are increased, the number of reaction functional groups is reduced, the main reaction is inhibited, and the grafting rate is reduced.

3.3.1.3 Effect of initiator addition on G0.5 graft ratio

In the experiment, the addition amount of the initiator ammonium cerium nitrate is one of the main factors influencing the grafting rate of the reaction. Under the action of Ce + of ammonium ceric nitrate, a large number of free radicals are generated in the chitosan oligosaccharide unit molecules to enhance the electron attraction effect on C ═ C in methyl acrylate, and under the attack of the free radicals, double bonds are opened to form chemical bonds with the chitosan oligosaccharide molecules, so that a grafting reaction is generated. According to the principle that ammonium ceric nitrate initiates chitosan oligosaccharide to generate free radical reaction, a certain amount of chitosan oligosaccharide molecules need a corresponding amount of cerium ions to initiate, as can be seen from fig. 4, as the addition amount of ammonium ceric nitrate increases, the reaction grafting rate gradually increases, and when 1.5g of ammonium ceric nitrate is added, the reaction grafting rate reaches the maximum. However, after the reaction has proceeded to half, the difficulty of the reaction increases due to the increase in side reactions, and further increase in the amount of the initiator added does not play a positive role.

3.3.1.4 Effect of reaction time on G0.5 grafting

The radical reaction includes three processes of chain initiation, chain growth and chain termination, and sufficient reaction time is necessary for the reaction. FIG. 5 is a graph showing the effect of reaction time on the G0.5 graft ratio. As can be seen from the figure, at the initial stage of the reaction, the grafting rate rapidly increased within 6 hours due to the large concentration of the reaction product present in the system, and the grafting reaction proceeded sequentially with the passage of time, and the grafting amount gradually increased. However, when the reaction is carried out for 8 hours, side reactions in the reaction system are increased, which has a certain influence on the product and leads to serious reduction of the grafting rate.

3.3.2G0.5 synthetic response surface optimization analysis

3.3.2.1 response surface design scheme and test results

On the basis of a single-factor test result, three main factors of reaction molar ratio, initiator addition amount and reaction temperature are selected for response surface analysis and optimization. The following 17 sets of experiments are designed by utilizing the center design principle of Box-Benhnken, the grafting ratio is taken as a response value, the synthetic conditions are further optimized by a three-factor three-level Response Surface Method (RSM), and the response degree of each factor and the result of pairwise crossing influence are examined. The experimental design and results are shown in tables 3-4 below:

tables 3-4 response surface design and Experimental results

3.3.2.2 multivariate regression fitting analysis and mathematical model establishment

And (3) performing multiple regression fitting analysis on the experimental data in the table 3-4 by using DesignExpert8.0.4 software to obtain a multiple quadratic linear equation which is in accordance with the addition amount, the reaction molar ratio and the reaction temperature of the grafting rate and the influencing factors, wherein the mathematical model is shown in a formula 3-3:

Y＝+52.62+9.70*A+1.96*B+3.08*C-1.03*A*B-0.030*A*C-1.30*B*C-13.74*A²-2.05*B²-2.89*C² (3-3)

wherein, Y-grafting rate; a-initiator addition amount; b-reaction molar ratio; c-reaction temperature.

TABLE 3-5 response surface quadratic analysis of variance

Analysis of variance is to determine whether the mathematical model makes sense by determining whether process parameters have a significant effect on conversion. The results of the response surface quadratic analysis of variance are shown in tables 3-5, and the mathematical model for the development of this response is very significant as indicated by the F-value 106.58 of the model, and the F-value has only 0.01% of the chance of being due to noise. The F value of the mismatching term is 2.55, and only 19.42% of the chance is due to noise, which is insignificant relative to pure error, and these meet the requirement of a multiple linear fit. Correlation coefficient R of equation actual value²0.9928 with predicted R²0.9200 are close and both are close to 1, indicating that there is a good fit between the actual and predicted values of G0.5 grafting. Correction decision coefficient AdjR²A variation distribution illustrating a response value of 98.34% can be explained by this equation, which is 0.9834. In addition, the coefficient of variation (C.V ═ 3.09%) and the signal-to-noise ratio (Adeq Precision 28.356 > 4) of the model were within the allowable ranges of the model. In conclusion, the mathematical model obtained by the multivariate linear fitting has effective significance for the analysis and prediction of experimental results.

In A, B, C, AB, AC, BC, A²、B²、C²Among the nine model terms, A, B, C, A²、B²、C²Pr > F < 0.0500, indicating that these model terms are significantThe remaining three terms, Pr > F > 0.1000, are not significant. Simplifications may be made for unimportant model items (excluding model items that support the model hierarchy) to improve the model. Meanwhile, comparing the F value of A, B, C, it can be found that the influence of the addition amount of the initiator, the reaction molar ratio and the reaction temperature on the grafting ratio of G0.5 is as follows: the addition amount of the initiator is more than the reaction temperature and more than the reaction molar ratio, and the F value of A is very obvious (Pr)>F value<0.0001), factor A²It is also very remarkable, suggesting that the initiator is the main driving factor for the grafting of methyl acrylate onto chitosan oligosaccharide. This is in agreement with the results of prior art studies which show that methyl acrylate is difficult to react with chitosan in the absence of an initiator, and that methyl acrylate is generally methylated and then grafted onto chitosan by alkylation or induced by physical factors such as plasma, gamma radiation, etc.

3.3.2.3 Cross-acting influencing factors

One of the conditions of the addition amount of the initiator, the reaction molar ratio and the reaction temperature is kept at the optimal level, and a 3D curved surface graph and an contour line graph are drawn to obtain the cross-influence effect of the other two factors on the grafting rate. The center point of the contour line is the highest point in the three-dimensional surface map, i.e., the maximum value of the grafting rate achieved under the interaction of the two independent variables. The shape of the contour line can reflect the strength of the cross action of the two variables, the more elliptical the contour line is, the stronger the cross action is, and the more circular the contour line is, the weaker the cross action is. AB. Fig. 6 shows a 3D diagram and a contour diagram of the response surface of the cross action of AC and BC, and the strength of the cross action is sequentially as follows from the shape of the contour: BC > AB > AC, consistent with the results of the analysis of variance. Since the independence of the factor a is particularly strong, its interaction with the other two variables becomes weaker, and instead the interaction between the factors BC is the strongest. It is demonstrated that under the condition of ensuring the proper amount of the initiator, the grafting rate shows a trend of decreasing after increasing along with the increase of the reaction molar ratio and the increase of the reaction temperature, because the proper reaction molar ratio and temperature can increase the effective collision between the reaction monomer and the chitosan oligosaccharide molecules and improve the reaction grafting rate, and when the reaction molar ratio is too large or the reaction temperature is too high, the methyl acrylate is easy to self-polymerize, so that the side reaction of the reaction system is increased, and the grafting rate is reduced.

3.3.2.4 determination and verification of optimal synthesis conditions

The optimal synthesis conditions for G0.5 by response surface optimization analysis were: the amount of the initiator added was 1.67G, the reaction molar ratio COS: MA was I:8.48, the reaction temperature was 47.39 ℃, and the predicted graft ratio of G0.5 synthesized under these conditions was 55.2522%. In actual operation, experimental conditions are difficult to achieve as accurate as the above optimal conditions, so the following adjustments are made: the addition amount of the initiator is 1.65g, and the reaction molar ratio is 1:8.5, the reaction temperature is 47.5 ℃. Repeated tests are carried out according to the adjusted experimental scheme, the measured average grafting rate is 52.38%, and the average grafting rate is basically consistent with the predicted value, so that the quadratic regression equation established in the experiment can effectively predict the grafting rate of G0.5 under certain synthesis conditions.

And (4) conclusion: by single factor experiments on G0.5 synthesis conditions: the reaction molar ratio of COS to MA (g/mL) was 1:8, the reaction temperature was 45 ℃, the amount of initiator added was 1.5g, and the reaction time was 8 h. Fixing the reaction time to be 8h on the basis of single factor, and selecting three main factors of reaction molar ratio, initiator addition amount and reaction temperature to optimize the response surface to obtain the optimal synthesis conditions as follows: the amount of the initiator added was 1.65g, the reaction molar ratio of COS to MA (g/mL) was 1:8.5, and the reaction temperature was 47.5 ℃. And by square difference analysis yields: the influence of three factors on the grafting rate is in turn: the initiator addition amount > reaction molar ratio > reaction temperature, wherein the results show that the influence of the initiator addition amount is most significant.

Amino content of 3.3.3.1G 1.0

TABLE 3-7 amino content of the metathesis products determined by the double-jump potential

Tables 3-7 show: the decrease in the amino group content with the increase in the number of grafting generations is due primarily to the fact that the reaction is primarily at an increase in the overall molecular weight, which is greater than the increase in the amino groups in the product, and therefore shows a tendency toward a decrease in the amino group content. However, according to the reaction scheme, the number of terminal amino groups is increasing as the reaction proceeds.

3.3.3.2G0.5-G3.0 carbon to nitrogen ratio

The grafted methyl acrylate and ethylenediamine in the experiment are mainly related to the change of the contents of carbon and nitrogen elements, so that the carbon-nitrogen percentage can be used for estimating the reaction progress. FIG. 7 is a bar graph of the percentage of carbon and nitrogen in molecules of different generations of products, and it can be seen from the graph that the continuous change of the carbon and nitrogen ratios of adjacent generations indicates that the chemical reaction is performed. The carbon-nitrogen ratio of the whole generation molecule G1.0 increases with the number of generations of grafting, mainly because the amount of carbon element increases more than that of nitrogen element at the reaction. Half-generation molecular grafting is methyl acrylate, and only the content of carbon element is increased.

3.3.3.2 thermal stability analysis

From FIG. 8, it can be concluded that the modified COS/PAMAM derivatives of various generations have different degrees of changes in thermal stability compared with chitosan oligosaccharide. They both undergo mainly two weight loss phases: the weight lost from the initial temperature to around 100 ℃ is caused by evaporation of water in the sample and initial degradation of the crystal structure; the sharp weight reduction at the temperature of 150-400 ℃ is mainly caused by the thermal decomposition of the main skeleton of the sugar unit molecules of the chitosan oligosaccharide and the derivatives thereof, the opening of the chemical bond in the pyranose ring and the complete destruction of the crystal structure; the slow weight reduction after 400 ℃ may be caused by the decomposition of the ash residue remaining after the degradation. Wherein the thermal stability of G0.5 and G1.0 is improved compared with that of chitosan oligosaccharide, and the quality of the final ash residue is more than that of the chitosan oligosaccharide, which indicates that the previous two steps are successful in the branching modification of the chitosan oligosaccharide. However, as the number of grafting generations increases, the grafting rate gradually decreases and side reactions increase, resulting in little change in thermal stability, which is almost close to chitosan oligosaccharide.

4 Infrared structural analysis

4.1G 0.5 generation infrared atlas analysis

Shown in FIG. 9Shown as a comparison of the IR spectra of chitooligosaccharide, methyl acrylate and G0.5. In the synthesized G0.5 spectrum, 3404.7cm in the primary chitosan oligosaccharide band^-1The overlapping peak of the O-H and N-H stretching vibration absorption at the position is obviously reduced and has a certain deviation from the original 3404cm_- ¹Has a broad peak direction and a low wave number of 3385cm_- ¹And (4) moving. Illustrating C in oligosaccharide under the initiation of ammonium ceric nitrate₂At position-NH₂Or C₃the-OH at position undergoes a radical reaction. Chitosan oligosaccharide 1590cm^-1The deformation vibration absorption peak of N-H also has a trend of obviously reducing at the corresponding position of G0.5, and further shows that the reaction is likely to occur in the chitosan oligosaccharide C₂On the active amino group. Compared with the spectrum of methyl acrylate, 1654cm of the spectrum^-1The middle-strength stretching vibration absorption peak of C ═ C completely disappears in the spectrum of G0.5, and is 1384cm_- ¹The new strong characteristic absorption peak is probably-CH in methyl acrylate₃The characteristic absorption peak of (a) may also be caused by a change in the C — C skeleton of methyl acrylate due to a graft reaction between C ═ C and chitosan oligosaccharide. In addition, 1075cm_- ¹The enhancement of the C-O-C shock absorption peak is probably the result of the ester group effect after the methyl acrylate is grafted on the chitosan oligosaccharide, which indicates that the chitosan oligosaccharide and the methyl acrylate have a grafting reaction.

4.2G 1.0 generation Infrared Spectroscopy

A comparison of the IR spectra for G0.5, ethylenediamine, and G1.0 is shown in FIG. 10, from which it can be seen that: the most significant change in the G1.0 band compared to G0.5 was 1629cm in the original absorption band_- ¹The C ═ O stretching vibration absorption peak of the amide I band is increased, and-CH₃At 1384cm^-1The characteristic absorption peak at (A) becomes significantly smaller, indicating that the ester group on G0.5 undergoes Michael addition reaction with ethylenediamine. 2923cm_- ¹And 2878cm_- ¹Are respectively-CH₂3385cm for asymmetric and symmetric stretching vibration enhancement_- ¹The stretching vibration absorption peak at N-H became deep and shifted with respect to that in G0.5, also demonstrating that G1.0 was synthesized.

In this experiment, the michael addition reaction mainly involves amidation of an ester group in the original methyl acrylate and an amino group in ethylenediamine, and in this process, in order to prevent the formation of a bridge structure in the molecule, sufficient ethylenediamine is added, but the excess ethylenediamine and the reactant are liable to undergo a competitive reaction, so that the high-generation dendrimer contains low-generation molecules, and the monodispersity of the product is reduced, so that a large amount of absolute ethanol and distilled water is required to remove unreacted ethylenediamine after each reaction is completed, thereby improving the purity of the product.

4.3G 0.5-G1.0 comparative infrared spectra

FIG. 11 is a comparison of the IR spectra of G0.5 and G1.0, from which it can be seen that: 3400cm with increasing grafting generation number^-1The size of the stretching vibration peak of N-H in the amino group is alternated, 1390cm_- ¹Is in the form of-CH₃The characteristic absorption peaks all change as a result of alternating grafting of methyl acrylate and ethylenediamine. 2880cm^-1Gradual enhancement of absorption peak at methylene, 1640cm^-1The increase of the C ═ O stretching vibration peak is due to the action effect of group accumulation during the synthesis process. While the change in the characteristic absorption peak at 1070cm-1 is caused by C-O-C grafting in methyl acrylate. Because only the group alternation in methyl acrylate and ethylenediamine exists in the whole reaction process and no other characteristic functional group is introduced, the shapes and the trends of the spectral lines of the infrared characteristic absorption peaks of G0.5 and G1.0 are basically consistent, and the spectral lines only have the difference of the peak shapes and the peak sizes. The result of the element analysis can be combined to obtain the grafting reaction of the chitosan oligosaccharide and the branched molecules.

5 thermal stability analysis

5.1 Zeta potential measurement results

The free amino group, when dissolved in water, will react with H in water⁺Protonation occurs to generate excessive OH^-Ions, resulting in a decrease in the pH of the solution and a concomitant decrease in the potential value. FIG. 12 shows the Zeta potential change diagram of COS/PAMAM derivatives of different generations in aqueous solution, when synthesizing half-generation molecule, because methyl acrylate reacts with amino group to consume amino group and reduce its content, generate protonReduced action, H in solution⁺The concentration is increased, resulting in the increase of the Zeta potential value; when the whole generation of molecules is synthesized, the grafted ethylene diamine can generate a large amount of amino groups, so that the protonation in the solution is enhanced, and OH^-The concentration is increased, and the overall potential value of the solution is reduced and even reaches a negative value.

6.1G 1.0 bacteriostatic agent bactericidal performance test material and equipment

6.1.1 Experimental materials and reagents

TABLE 4-1 test strains

TABLE 4-2 major reagents

6.1.2 Experimental instruments and apparatus

TABLE 4-3 Main instrumentation

6.2.1G 1.0 bacteriostatic agent bactericidal performance test

6.2.1.1 Strain activation and preparation of suspensions

Transferring 50 μ L of strain preserved in Glycine max (L.) Linne, inoculating into 50ml nutrient broth (LB culture medium), placing in 37 deg.C constant temperature oscillator, and shake culturing for 16-18h to activate strain to logarithmic phase. Taking a proper amount of activated bacteria liquid in a 10mL centrifuge tube, centrifuging for 10min at 4000rpm/min, pouring out supernatant, washing white bacterial plaque for 1-2 times by using 0.85% physiological saline under the centrifugation condition, and removing residual glycerol during preservation. Transferring 1ml of the treated bacterial liquid into a test tubeDiluting with 0.85% physiological saline by ten-fold dilution method to obtain bacterial suspension with different concentration gradients, and diluting 0.2mL to 10^-4CFU·mL^-1And plate counting of the bacteria solutions at the following concentrations. Selecting concentration gradient with colony number of 4-5 to determine the optimal growth concentration of the strain, wherein the concentration is 10^-8cfu·mL^-1. Bacterial suspension with the concentration is prepared according to the method for later use.

6.2.1.2 MIC assay

Referring to the determination method of the MIC of the bactericide in Zhoudqing second edition microbiology experiment course, the liquid dilution method is utilized to test the bactericidal performance of the COS/PAMAM bactericide prepared by the experiment. The method comprises the following specific operations: firstly, a sample is dissolved in 0.1mol/L CH₃COONa-0.2mol/L CH₃COOH buffer solution was prepared into 1000. mu.g/mL solution, solutions with different drug concentrations were prepared in test tubes according to the method shown in Table 4-4, and then 0.2mL of 10 prepared in 6.2.1.1 was added to each tube^-8cfu·mL^-1The test inoculum was run using inoculum only and culture broth containing sample only as blank controls. The above tubes were incubated at 37 ℃ for 12 hours in an incubator at 200rpm, and the turbidity of each tube was visually observed, and if the sample group became clear and transparent as in the control group from a certain tube, it was indicated as a Minimum Inhibitory Concentration (MIC).

TABLE 4-4 preparation of test tubes of different sample contents

6.2.1.3 MBC determination

The determination of the Minimum Bactericidal Concentration (MBC) was based on the MIC determination of 4.2.2.2. All clarified tube solutions in the MIC test were inoculated into solid medium, the growth of colonies was observed, and finally the minimum sample concentration at which no significant colony growth occurred in the plate was defined as the Minimum Bactericidal Concentration (MBC) of the sample against the test bacteria.

6.2.1.4 bactericidal rate curve

Inoculating 2mL of test bacteria into 50mL of liquid culture medium, subpackaging the test tubes into small test tubes, dividing the test tubes into an experimental group and a control group, adding a sample solution with the MIC concentration into the experimental group, adding physiological saline with the same volume into the control group, and shaking up to ensure that the bacteriostatic agent is fully contacted with the test bacteria. According to the method provided by GB/T4789.3-2010, the solutions of the groups are respectively diluted to appropriate concentration step by step, then 1mL of the solution is transferred by a liquid transfer gun and placed on an agar culture medium, after the solution is uniformly coated by a glass coating rod, a flat plate is inverted, the flat plate is placed in a constant temperature incubator at 37 ℃ for culturing for different time, and viable bacteria count of the flat plate is carried out at set time points. Is calculated by the following formula^[95]The bactericidal rate of the microspheres is:

wherein: b% represents the sterilization rate; n is a radical of₀The number of viable bacteria in the control group; n is a radical of₁The number of viable bacteria in the experimental group.

6.2.2 preparation of COS/PAMAM dendrimer adsorbed Ag + and Cu2+ composite bacteriostatic agent

Accurately weighing 15.0mg of the prepared COS/PAMAM sample into a 100mL iodine vial, and adding 25.0mL HAc-NaAc buffer solutions with different pH values to dissolve them sufficiently. Then 5.0mL of 3.0 mg/mL solution was added to the iodophor^-1Ag of (A)⁺、 Cu²⁺The solution was subjected to a blank test at 25 ℃ at 100rpm min without adding a sample group^-1The oven oscillates for 24 h. And after the adsorption is balanced, taking supernatant to determine the content of the residual ions in the solution. The adsorption quantity Q was estimated according to the following formula^[96]。

Wherein: c_oConcentration of metal ions before adsorption (mg. mL)^-1)；C_eConcentration of metal ions after adsorption equilibration (mg. mL)^-1) (ii) a Static saturation adsorption capacity (mg. g) of Q-COS/PAMAM molecules^-1) (ii) a V-volume of solution (mL); m isDry weight (g) of COS/PAMAM samples.

6.2.3 COS/PAMAM/Cu2+ bacteriostat bactericidal Performance test

The experimental procedure was as in 6.2.1.

6.2.4 COS/PAMAM and COS/PAMAM/Cu2+ sterilization mechanism

6.2.4.1 extracellular DNA and RNA determination

The action mechanism of the bactericide is mainly as follows: one is that the bactericide can break the hydrophobic bond and the metal bridge of the subunit connection point on the mycoderm, so that the surface of the mycoderm has cracks or gaps and loses physiological function; the second is that the positive charge of the bactericide can generate electrostatic attraction with the negative charge on the surface of the bacteria to form an electrovalence bond, so as to cause bacteriolysis, destroy the cell wall and cell membrane structure of the bacteria, further cause the leakage of cell contents such as DNA, RNA and the like, and finally cause cell death. Therefore, the rupture degree of the cells is judged by measuring the content of DNA and RNA, and indirectly, the rupture degree of the test bacteria cells can be judged by colorimetric measurement of an absorbance value at 260nm by using an ultraviolet spectrophotometer.

6.2.4.2 TTC dehydrogenase assay

TTC (red tetrazolium) can receive H (H) generated by dehydrogenase in the process of cell respiration⁺E), a red trityl methyl beam (TF) is formed, the reaction formula is as follows:

TTC, under the action of living bacteria, passes through cell walls and membranes from the environment into the interior of cells. Therefore, TTC can be used to determine the viability of the cells of the test bacteria, i.e., the more viable the number of cells, the stronger the metabolism, and the more TTC taken up from the environment, the more TF is generated. On the other hand, if the bactericide acts on the living bacteria to cause reduction in the amount or death, the amount of TF produced becomes small. Therefore, the antibacterial effect of the bactericide can be examined by measuring the amount of TF produced, and the sterilization mechanism can be further studied.

6.2.4.3 scanning electron microscope morphology observation

The action mechanism of the currently accepted cationic bactericide is mainly that positive charges carried by the bactericide and negative charges carried by the surface of bacteria generate electrostatic attraction, the charge distribution on the surface of a cell membrane is disturbed, the physiological activity of the bactericide is damaged, and the bactericide penetrates into cells to generate bacteriolysis, so that cell contents such as DNA (deoxyribonucleic acid), RNA (ribonucleic acid) and the like flow out, and finally the cells die. After the cell death, the cell fragments can be adsorbed on the surface of the bactericide, so that the bactericidal sample can be observed by surface topography to intuitively reflect the action effect of the bactericide.

6.3.1 Bactericidal Effect of COS/PAMAM derivatives

6.3.1.1 MIC measurement results

Tables 4-5 MIC values of G1.0 action against four test bacteria

By testing the MIC values of G1.0 against two gram-negative bacteria of Escherichia coli and Pseudomonas aeruginosa and two gram-positive bacteria of Staphylococcus aureus and Bacillus subtilis, it can be concluded that: g1.0 has obvious bacteriostatic effect on 4 tested bacteria. From the perspective of the tested bacteria, the three bactericides have better antibacterial performance on escherichia coli than other strains, namely bacillus subtilis, staphylococcus aureus is in the third place, and the three bactericides have the worst antibacterial performance on pseudomonas aeruginosa, mainly because the tested bacteria have strong drug resistance.

Claims

1. A chitosan oligosaccharide graft copolymer G1.0 is characterized in that the molecular formula is as follows:

where n is any integer value.

2. A preparation method of the chitosan oligosaccharide graft copolymer G1.0 as claimed in claim 1, which is characterized by comprising the following specific steps:

1) weighing a certain amount of chitosan oligosaccharide, adding 1% (v/v) acetic acid as a reaction solvent, wherein the relationship between the adding amount of the acetic acid and the total amount of the chitosan oligosaccharide is as follows: adding 25ml of the acetic acid solution into every 1g of chitosan oligosaccharide; after the solution is fully dissolved, introducing nitrogen into the bottle to remove oxygen; adding a certain amount of ammonium ceric nitrate as an initiator under the protection of nitrogen, wherein the mass ratio of the addition amount of the initiator to the chitosan oligosaccharide is 1.6:1-2.5: 1; then slowly adding a proper amount of methyl acrylate dropwise; after the reaction is finished, filtering out a reaction product G0.5, then carrying out suction filtration on the filtered reaction product G0.5 for 3 times by using a reaction solvent 1% (v/v) acetic acid, repeatedly washing the reaction product with acetone, diethyl ether and absolute ethyl alcohol for 3 times in sequence, removing unreacted methyl acrylate, and finally putting the reaction product into a vacuum drying box at 50 ℃ for drying for later use, wherein the added methyl acrylate contains a polymerization inhibitor p-methoxyphenol MEHQ for preventing self polymerization, and the reaction molar ratio of the chitosan oligosaccharide to the methyl acrylate is 1: 8-1: 8.5; setting the stirring speed to be 200rpm and the continuous reaction time to be 8h at the temperature of 40-55 ℃;

2) weighing G0.5 prepared by the above steps, adding a chromatographic grade methanol solvent, soaking for 12h, controlling the reaction temperature to be 20 ℃, and dropwise adding analytically pure ethylenediamine and N₂Reacting for 8 hours under protection; the amount of methanol added and the total amount of G0.5 are related as follows: adding 0.5ml of methanol into every 1mg of G0.5; the amount of ethylenediamine added and the total amount of G0.5 are as follows: 0.08ml of ethylenediamine is added into every 1mg of G0.5; after the reaction is finished, filtering out a product G1.0 obtained by the reaction, washing for 3 times by using a reaction solvent methanol, repeatedly washing for 3 times by using absolute ethyl alcohol and distilled water in sequence, and placing at 50 ℃ for vacuum drying to obtain the catalyst;

the preparation steps of the reaction raw material chitosan oligosaccharide in the step 1) are as follows:

a) weighing a certain amount of chitosan, fully dissolving the chitosan in a certain amount of 1% (v/v) acetic acid solution by high-speed stirring, transferring the solution into a quartz three-neck bottle, and adding a certain amount of 30% (v/v) H₂O₂The ultrasonic probe is correctly arranged in a cavity of the microwave synthesizer, so that the ultrasonic probe is submerged below the interface of the solution; setting the microwave power at 300w, reacting for 30min, turning on the ultraviolet lamp for auxiliary degradation,after the experiment is finished, carrying out water bath rotary steaming concentration on the solution at 40 ℃, and then carrying out freeze drying for 24 hours to obtain a chitooligosaccharide sample with the viscosity average molecular weight M eta of 25000; the relationship between the addition amount of the acetic acid and the total amount of the chitosan is as follows: for every 3g of chitosan, 100mL of the above acetic acid solution was added, and the amount of H2O2 added was related to the total amount of chitosan as follows: 0.1mL 30% (v/v) H per 1g chitosan was added₂O₂；

b) And the mass ratio of 1: 1: 1, weighing a certain amount of papain, pectinase and cellulase, dissolving in HAC-NaAC buffer solution with pH of 5.5, and preparing into 1mg/mL enzyme solution for later use; adjusting the pH value of the chitosan oligosaccharide solution subjected to microwave degradation to 5.5 according to the concentration ratio of enzyme to substrate of 1: 10, adding the enzyme solution to be used, uniformly stirring, and placing in a constant-temperature water bath at 45 ℃ for enzymolysis for 4 hours; boiling and heating for 10min to inactivate enzyme, and vacuum distilling and concentrating at 40 deg.C to obtain chitosan oligosaccharide sample solution;

the preparation method further comprises the steps of carrying out molecular weight grading on the prepared chitosan oligosaccharide sample solution, and selecting chitosan oligosaccharide with molecular weight of 3500-7000 as a raw material; the specific grading method is as follows: respectively selecting dialysis bags with the molecular weight cut-off of 7000 and 34mm and 3500 diameters for pretreatment, and removing metal ions; sleeving a dialysis bag with a smaller diameter into a dialysis bag with a larger diameter, fixing one end of the dialysis bag with a dialysis bag clamp, washing the dialysis bag with distilled water, transferring the chitosan oligosaccharide sample solution prepared in the step b) by using a 5mL liquid transfer gun, filling the chitosan oligosaccharide sample solution into an inner-layer dialysis bag, filling the gap between the double-layer dialysis bags with the distilled water, sealing the dialysis bag clamp, and then putting the dialysis bag into a large beaker filled with the distilled water for dialysis for 5 d; after the dialysis is finished, the liquid in the gap between the two layers of membranes is taken out, and freeze drying is carried out, thus obtaining the reaction raw material chitosan oligosaccharide with 3500-7000 molecular weight.

3. Use of the chitosan oligosaccharide graft copolymer G1.0 as defined in claim 1 as a food preservative and bacteriostatic agent.