CN106008746B - A kind of graft copolymer and preparation method thereof of Natta mycin and O, N- carboxymethyl chitosan oligosaccharide - Google Patents

Abstract

The present invention relates to a kind of medicinal and food antiseptic antibacterial agent Natta mycins and O, N- carboxymethyl chitosan oligosaccharide graft copolymer and preparation method thereof.By Natta mycin and O, N- carboxymethyl chitosan oligosaccharide graft copolymerization realizes the producing high-molecular of bacteriostatic agent Natta mycin.Meaning with following three aspect：First is that improving the stability of Natta mycin；Second is that improving the solubility in water；Third is that having widened scope of restraining fungi.

Description

A Graft Copolymer of Natamycin and O,N-Carboxymethyl Chitooligosaccharide and Its Preparation method

technical field

The invention belongs to the field of medicine and food additives, in particular to high-efficiency medicine and food antiseptic and antibacterial agent and its preparation method.

Background technique

Natamycin is a natural antifungal compound produced by Streptomyces fermentation. It belongs to the polyene macrolide antibacterial agent. Produced, it can be widely used in food preservation and antifungal treatment. Due to the continuous growth of the demand for biological preservatives in economically developed countries such as Europe and the United States and the steady and rapid development of the domestic food industry, the demand for natamycin at home and abroad has continued to grow in recent years. It is estimated that by 2015, the potential domestic demand The volume is expected to exceed 660t.

Natamycin relies on its lactone ring structure to interact with sterol compounds on the fungal cell membrane to form antibiotic-sterol compounds, thereby destroying the structure of the fungal cytoplasmic membrane. The hydrophilic part (polyol part) of the macrolide forms water holes on the membrane, which damages the permeability of the cell membrane, which in turn causes the leakage of amino acids, electrolytes and other substances in the bacteria, and the bacteria die. When there is no sterol compound on the cell membrane of certain microorganisms, natamycin has no effect on it, so natamycin only inhibits fungi, and has no antibacterial activity against bacteria and viruses. In addition, natamycin has the characteristics of low water solubility and poor chemical stability under high temperature and high pH conditions.

The macromolecularization of the bacteriostatic agent natamycin was achieved through the graft copolymerization of natamycin and O, N-carboxymethyl chitosan oligosaccharide. It has the following three meanings: one is to improve the stability; the other is to improve the solubility in water; the third is to broaden the scope of antibacterial.

Contents of the invention

The invention designs and prepares a graft polymer with natamycin as a side chain and chitosan oligosaccharide as a main chain. The present invention carries out the amidation reaction between the monosaccharide amino group of natamycin and the chitosan oligosaccharide acid chloride group, and grafts natamycin to O, N-carboxymethyl chitosan oligosaccharides to prepare natamycin and O , a graft copolymer of N-carboxymethyl chitosan oligosaccharides. The water solubility of this polymer is better than that of natamycin, and it also has the antibacterial characteristics of natamycin and chitooligosaccharide.

Detailed ways

Example 1

In the first step, disperse 24.5g oligochitosan in 70ml deionized water, stir and dissolve at room temperature; weigh 14.5g monochloroacetic acid and add 30ml deionized water to dissolve, stir at room temperature and add 25.2g sodium bicarbonate in batches to neutralize, Then, the neutralization solution was added to the above reaction solution at one time, stirred, and the temperature was gradually raised to 70° C., and stirred and reacted at this temperature for 3 hours. Cool to room temperature, neutralize to PH=6-6.5 with 5% hydrochloric acid, then add 100ml of absolute ethanol, a dark brown viscous solid precipitates out. Filter, wash with absolute ethanol 3 times, dry, weigh about 32.2g, and save for future use.

In the second step, 16.1 g of O,N-carboxymethyl chitosan oligosaccharide generated in step 1 was dispersed in 100 ml of NMP, and 10.0 g of thionyl chloride was added dropwise to the above reaction solution under stirring, and then heated to 75° C. Stir the reaction for 5 hours. Continue to heat up to 90°C, distill off the remaining thionyl chloride by fractional distillation, cool to room temperature, add 50ml of isopropanol to precipitate the product, filter with suction, wash with anhydrous ether for 3 times, dry and weigh about 24.6g .

In the third step, take 8.8 g of the powder product in step 2 and disperse it in 50 ml of deionized water, adjust the pH value to between 8.5 and 9.0 with 5% aqueous sodium hydroxide solution, and add 0.68 g (0.001 mol) of Natta at one time under stirring Mycin, under nitrogen protection, stirred at 50°C, the solid gradually dissolved, reacted for 5 hours, and cooled to room temperature. 5% hydrochloric acid was neutralized to pH=6.5-7.0, and then 50ml of absolute ethanol was added, and a brown solid powder was precipitated. After filtering, washing with ether three times, vacuum drying, and weighing, about 2.2 g of the product was obtained, and the grafting rate was 1.5%. Store in vacuum and away from light.

Example 2

In the first step, disperse 24.5g oligochitosan in 70ml deionized water, stir and dissolve at room temperature; weigh 14.5g monochloroacetic acid and add 30ml deionized water to dissolve, stir at room temperature and add 25.2g sodium bicarbonate in batches to neutralize, Then, the neutralization solution was added to the above reaction solution at one time, stirred, and the temperature was gradually raised to 70° C., and stirred and reacted at this temperature for 3 hours. Cool to room temperature, neutralize to PH=6-6.5 with 5% hydrochloric acid, then add 100ml of absolute ethanol, a dark brown viscous solid precipitates out. Filter, wash with absolute ethanol 3 times, dry, weigh about 32.2g, and save for future use.

In the second step, 16.1 g of O,N-carboxymethyl chitosan oligosaccharide generated in step 1 was dispersed in 100 ml of NMP, and 10.0 g of thionyl chloride was added dropwise to the above reaction solution under stirring, and then heated to 75° C. Stir the reaction for 5 hours. Continue to heat up to 90°C, distill off the remaining thionyl chloride by fractional distillation, cool to room temperature, add 50ml of isopropanol to precipitate the product, filter with suction, wash with anhydrous ether for 3 times, dry and weigh about 24.6g .

In the third step, take 8.8 g of the powder product in step two and disperse it in 50 ml of deionized water, adjust the pH value to between 8.5 and 9.0 with 5% aqueous sodium hydroxide solution, and add 2.05 g (0.003 mol) of Nata at one time under stirring Mycin, under nitrogen protection, stirred at 50°C, the solid gradually dissolved, reacted for 5 hours, and cooled to room temperature. 5% hydrochloric acid was neutralized to pH=6.5-7.0, and then 50ml of absolute ethanol was added, and a brown solid powder was precipitated. After filtering, washing with ether three times, vacuum drying, and weighing, about 6.5 g of the product was obtained, and the grafting rate was 2.8%. Store in vacuum and away from light.

Example 3

In the first step, disperse 24.5g chitosan oligosaccharide in 70ml deionized water, stir and dissolve at room temperature; weigh 14.5g monochloroacetic acid and add 30ml deionized water to dissolve, stir at room temperature and add 25.2g sodium bicarbonate in batches to neutralize, Then, the neutralization solution was added to the above reaction solution at one time, stirred, and the temperature was gradually raised to 70° C., and stirred and reacted at this temperature for 3 hours. Cool to room temperature, neutralize to PH=6-6.5 with 5% hydrochloric acid, then add 100ml of absolute ethanol, a dark brown viscous solid precipitates out. Filter, wash with absolute ethanol 3 times, dry, weigh about 32.2g, and save for future use.

In the second step, 16.1 g of O,N-carboxymethyl chitosan oligosaccharide generated in step 1 was dispersed in 100 ml of NMP, and 10.0 g of thionyl chloride was added dropwise to the above reaction solution under stirring, and then heated to 75° C. Stir the reaction for 5 hours. Continue to heat up to 90°C, distill off the remaining thionyl chloride by fractional distillation, cool to room temperature, add 50ml of isopropanol to precipitate the product, filter with suction, wash with anhydrous ether for 3 times, dry and weigh about 24.6g .

In the third step, take 8.8 g of the powder product in step 2 and disperse it in 50 ml of deionized water, adjust the pH value to between 8.5 and 9.0 with 5% aqueous sodium hydroxide solution, and add 3.4 g (0.005 mol) of Nata at one time under stirring Mycin, under nitrogen protection, stirred at 50°C, the solid gradually dissolved, reacted for 5 hours, and cooled to room temperature. 5% hydrochloric acid was neutralized to pH=6.5-7.0, and then 50ml of absolute ethanol was added, and a brown solid powder was precipitated. After filtering, washing with ether three times, vacuum drying, and weighing, the obtained product was about 10.7 g, and the grafting rate was 4.9%. Store in vacuum and away from light.

Example 4

In the first step, disperse 24.5g chitosan oligosaccharide in 70ml deionized water, stir and dissolve at room temperature; weigh 14.5g monochloroacetic acid and add 30ml deionized water to dissolve, stir at room temperature and add 25.2g sodium bicarbonate in batches to neutralize, Then, the neutralization solution was added to the above reaction solution at one time, stirred, and the temperature was gradually raised to 70° C., and stirred and reacted at this temperature for 3 hours. Cool to room temperature, neutralize to PH=6-6.5 with 5% hydrochloric acid, then add 100ml of absolute ethanol, a dark brown viscous solid precipitates out. Filter, wash with absolute ethanol 3 times, dry, weigh about 32.2g, and save for future use.

In the second step, 16.1 g of O,N-carboxymethyl chitosan oligosaccharide generated in step 1 was dispersed in 100 ml of NMP, and 10.0 g of thionyl chloride was added dropwise to the above reaction solution under stirring, and then heated to 75° C. Stir the reaction for 5 hours. Continue to heat up to 90°C, distill off the remaining thionyl chloride by fractional distillation, cool to room temperature, add 50ml of isopropanol to precipitate the product, filter with suction, wash with anhydrous ether for 3 times, dry and weigh about 24.6g .

In the third step, take 8.8 g of the powder product in step 2 and disperse it in 50 ml of deionized water, adjust the pH value to between 8.5 and 9.0 with 5% aqueous sodium hydroxide solution, and add 4.75 g (0.007 mol) of Nata at one time under stirring Mycin, under nitrogen protection, stirred at 50°C, the solid gradually dissolved, reacted for 5 hours, and cooled to room temperature. 5% hydrochloric acid was neutralized to pH=6.5-7.0, and then 50ml of absolute ethanol was added, and a brown solid powder was precipitated. After filtering, washing with ether three times, vacuum drying, and weighing, about 14.9 g of the product was obtained, and the grafting rate was 6.7%. Store in vacuum and away from light.

Example 5

Accurately weigh 1.0 g of the O,N-carboxymethyl chitosan oligosaccharide solid powder prepared in the first step of Examples 1, 2, 3 and 4 and dissolve it in 50 ml of deionized water. Then titrate the pH value to 7.0 with 0.1mol/L NaOH aqueous solution at room temperature, and calculate the number of NaOH moles required to neutralize 1.0gO, N-carboxymethyl chitosan, thereby calculating 1.0gO, N-carboxymethyl The total molar number of carboxyl groups in chitosan oligosaccharides is counted as A; meanwhile, the natamycin and O, N-carboxymethyl shells prepared in the third step of 1.0g embodiment 1, 2, 3 and 4 were accurately weighed respectively The solid powder of the graft copolymer of oligosaccharides was dissolved in 50 ml of deionized water. Then titrate the pH value to 7.0 with 0.1mol/L NaOH aqueous solution at room temperature, respectively calculate and neutralize Natamycin and O in 1.0g embodiment 1,2,3 and 4, N-carboxymethyl chitosan oligosaccharide The required NaOH moles of the graft copolymer, thus calculated 1.0g natamycin and O, the total moles of carboxyl and acid chlorides that do not participate in the reaction in the N-carboxymethyl chitosan graft copolymer, calculated for B. Wherein O, the average molecular weight of N-carboxymethyl chitosan oligosaccharide is 2770 (the average degree of polymerization is 6), and the molecular weight of natamycin is 665.7, suppose every mole of O, there is X in the N-carboxymethyl chitosan molecule Moles of Natamycin are grafted by carboxyl or acid chloride, then Natamycin and O, the average molecular weight of the graft copolymer of N-carboxymethyl chitosan oligosaccharide is:

2270+(665.7-17)X

Then X:

2270A-(2270+648.7X)B=X

X＝2270×(A-B)/(1+648.7B)

The grafting rate of Natamycin can be calculated:

X/20

The results are shown in Table 1

Table 1. The Natamycin grafting rate of embodiment 1～4

Example A,mol B,mol X,mol Grafting rate, % 1 18.60 17.37 0.30 1.5 2 18.42 16.28 0.56 2.8 3 18.45 15.01 0.98 4.9 4 18.56 14.13 1.34 6.7

Example 5

The O,N-carboxymethyl chitosan oligosaccharides prepared in Examples 1, 2, 3 and 4 with different grafting ratios of natamycin were dissolved in deionized water, and the viscosities of their aqueous solutions with different concentrations were measured at 25°C. The results are shown in Table 2.

Table 2. Solution viscosity of products with different grafting ratios (mPa.S, 25°C)

Example 6

Antibacterial experiments were carried out on several typical molds, yeasts, bacteria, etc. with the grafted rate of 2.8%, 4.9% and 6.7% respectively. The test method is as follows: Divide the culture medium into two groups, A and B. No bacteriostatic agent was added to plate A as a control, 50 mg/kg bacteriostatic agent was added to plate B, and then strains were inserted into plates A and B respectively. After culturing in an incubator, the results were observed after 3 days, see Table 3. The above results show that the product not only has an inhibitory effect on mold and yeast, but also has antibacterial activity on bacteria.

Table 3. Bacterial inhibition spectrum of products with different grafting ratios (25°C)

Example 7

The products with grafting rate of 4.9% and 6.7% were used to do antibacterial experiments on several typical molds, yeasts, bacteria, etc., and their effective antibacterial concentrations were determined. The test method is as follows: Divide the culture medium into multiple parts, add different amounts of bacteriostatic agents respectively, then inoculate the strains respectively, put them into the incubator for cultivation, and observe the results after 3 days. The results are shown in Tables 4 and 5.

Table 4. The effective bacteriostasis concentration (25 ℃) of graft rate 4.9% product

Table 5. The effective bacteriostatic concentration (25 ℃) of graft rate 6.7% product

Example 8

The product aqueous solution with a concentration of 0.1% and a grafting rate of 4.9% was placed in a sealed tank, and aged at 90° C., 120° C., and 150° C. for 48 hours. Measure its antibacterial activity by the same method of embodiment 7. The results are shown in Tables 6, 7, and 8. The antibacterial ability of the product with a concentration of 0.1% and a grafting rate of 4.9% did not decrease after aging at 90°C for 48 hours; after aging at 120°C for 48 hours, its antibacterial ability still did not decrease; at 150°C After aging for 48 hours, its antibacterial ability decreased, but when the concentration was greater than 30mg/kg, it still had antibacterial ability against six strains.

Table 6. Effective bacteriostatic concentration (25°C) of 4.9% grafting rate product aged at 90°C for 48h

Table 7. The effective inhibitory concentration (25°C) of the product with a grafting rate of 4.9% aged at 120°C for 48h

Table 8. The effective inhibitory concentration (25°C) of the product with a grafting rate of 4.9% aged at 150°C for 48h

Claims (2)

1. a kind of Natamycin and O, the graft copolymer of N-carboxymethyl chitosan oligosaccharide is characterized in that structure is represented by following general formula: In the formula, m+n=2～20. 2. a kind of natamycin described in claim 1 and O, the preparation method of N-carboxymethyl chitosan graft copolymer is characterized in that implementing as follows: In the first step, disperse 24.5g chitosan oligosaccharide in 70mL deionized water, stir and dissolve at room temperature; weigh 14.5g monochloroacetic acid and add 30mL deionized water to dissolve, stir at room temperature and add 25.2g sodium bicarbonate in batches to neutralize, Then add the neutralization solution to the above reaction solution at one time, stir, gradually raise the temperature to 70°C, stir and react at this temperature for 3 hours, cool to room temperature, neutralize with 5% hydrochloric acid to pH=6-6.5, and then add 100mL Anhydrous ethanol, dark brown viscous solid precipitated, filtered, washed 3 times with anhydrous ethanol, dried, weighed to 32.2g, and stored for future use; In the second step, 16.1 g of O, N-carboxymethyl chitosan oligosaccharide generated in the first step was dispersed in 100 mL of NMP, and 10.0 g of thionyl chloride was added dropwise to the above reaction solution under stirring, and then heated to 75°C , stirred and reacted for 5 hours, continued to heat up to 90°C, distilled off the remaining thionyl chloride, cooled to room temperature, added 50mL isopropanol to precipitate the product, filtered it with suction, washed three times with anhydrous ether, dried and weighed 24.6 g; In the third step, take 8.8 g of the powder product in the second step and disperse it in 50 mL of deionized water, adjust the pH value to 8.5-9.0 with 5% aqueous sodium hydroxide solution, and add 0.68 to 4.75 g of Nata at one time while stirring Mycin, under nitrogen protection, stirred at 50°C, the solid gradually dissolved, reacted for 5 hours, cooled to room temperature, neutralized with 5% hydrochloric acid to pH=6.5-7.0, then added 50mL of absolute ethanol, brown solid powder precipitated, filtered , washed 3 times with ether, dried in vacuo, and weighed to obtain 2.2-14.9 g of the product, which was stored in a vacuum and protected from light.