CN102153673A - N-parabenzyloxycarboxymethyl chitosan quaternary ammonium salt and preparation method thereof - Google Patents

Abstract

其中，X在0.65-0.95之间，分子量为45-50KD。其物化指标是：浅黄色粉末状固体，常温下可溶于乙醇和异丙醇，微溶于水和二氯甲烷，对金黄色葡萄球菌和黑曲霉的最低抑菌浓度均达到0.5%。本发明通过改性增强了壳聚糖的抗菌活性，扩大了壳聚糖的抗菌范围，对金黄色葡萄球菌和黑曲霉具有良好的抗菌活性，可用于食品抗菌防腐添加剂。The invention discloses a kind of N-p-benzyloxymethyl chitosan quaternary ammonium salt, its structure is:

Wherein, X is between 0.65-0.95, and the molecular weight is 45-50KD. Its physical and chemical indicators are: light yellow powdery solid, soluble in ethanol and isopropanol at room temperature, slightly soluble in water and methylene chloride, and the minimum inhibitory concentration against Staphylococcus aureus and Aspergillus niger reaches 0.5%. The invention enhances the antibacterial activity of the chitosan through modification, expands the antibacterial range of the chitosan, has good antibacterial activity against Staphylococcus aureus and Aspergillus niger, and can be used as an antibacterial and antiseptic additive for food.

Description

N-p-benzyloxymethyl chitosan quaternary ammonium salt and preparation method thereof

technical field

The invention relates to a chemically modified functional derivative of chitosan, which is applied in the technical field of food industry and belongs to the technical field of polymer compounds.

Background technique

Chitosan is the deacetylation product of chitin, which is the only alkaline polysaccharide that exists in large quantities in the biological world. Due to intramolecular and intermolecular hydrogen bonds, chitosan is only soluble in acids and acidic aqueous solutions. The chitosan molecular chain contains a large number of modifiable groups such as hydroxyl and amino groups, and various derivatives with special functions can be obtained through chemical modification, so its application range can be expanded. For example, chitosan has excellent biodegradability, compatibility, film-forming properties and strong antibacterial, anti-corrosion and fresh-keeping ability, and has been widely used in the food industry and other fields. Chitosan can only be dissolved in acidic medium, which limits its application range. Usually, people introduce quaternary ammonium salts, alkyl groups, inorganic acids, etc. into chitosan molecules to improve the solubility or flocculation of chitosan.

Contents of the invention

The purpose of the present invention is to overcome the water-insoluble shortcoming of chitosan, provide a kind of chitosan derivative with good antibacterial effect---N-p-benzyloxymethyl chitosan quaternary ammonium salt, it can be used As an antibacterial and antiseptic additive in food.

Another object of the present invention is to provide the preparation method of above-mentioned N-p-benzyloxymethyl chitosan quaternary ammonium salt.

The object of the present invention is achieved by the following technical scheme: N-p-phenylmethoxymethyl chitosan quaternary ammonium salt is characterized in that: its structure is as shown in the following formula:

Wherein, X is between 0.65-0.95, and the molecular weight is 45-50KD. The physical and chemical indicators of the compound are: light yellow powdery solid, soluble in ethanol and isopropanol at normal temperature, slightly soluble in water and dichloromethane; the minimum inhibitory concentration of the compound to Staphylococcus aureus and Aspergillus niger can reach 0.5%.

In the present invention, chitosan and p-methoxybenzaldehyde (anisaldehyde) are stirred and reacted in a three-necked flask to generate chitosan Schiff base, and then sodium borohydride is added to react to obtain alkylated chitosan; After adjusting the pH to weak alkaline with sodium hydroxide, add methyl iodide and sodium iodide with a weight ratio of 1:1 to the above solution, control the reaction temperature and keep stirring. After the reaction is completed, add acetone, let stand overnight, and suction filter. Wash with a mixture of ethanol and ether, and dry under vacuum at constant temperature to obtain a solid. The solid product was placed in a Soxhlet extractor and extracted with absolute ethanol under reflux, and the solid was vacuum-dried to constant weight to obtain the target product N-p-benzyloxymethyl chitosan quaternary ammonium salt.

The preparation method of described N-p-phenylmethoxymethyl chitosan quaternary ammonium salt, its preparation steps are as follows:

(1) A certain amount of chitosan is added to a certain volume mass percentage of 3% glacial acetic acid at a ratio of 20ml per gram to swell for 30min, transferred to a three-necked flask, and 1/2 times the volume of the solution is added to the flask. Stir at -50°C;

(2) Add anise aldehyde test solution with the same weight as chitosan into the constant pressure dropping funnel, add a certain volume of absolute ethanol at a volume ratio of 1:20 and shake to dissolve, slowly drop anise aldehyde into the above chitosan solution Aldehyde dehydrated ethanol solution, under stirring reaction 3h obtains chitosan Schiff base;

(3) Add sodium borohydride in the same molar amount as chitosan into the beaker, add a certain volume of distilled water at a ratio of 30ml per gram to dissolve, and slowly drop the sodium borohydride solution into the chitosan Schiff base with a constant pressure dropping funnel And keep stirring until a large amount of white flocs appear, continue stirring for 2-3h to obtain alkylated chitosan;

(4) Slowly add an appropriate amount of sodium hydroxide to adjust pH=8-9 in step (3) alkylation chitosan reaction solution, respectively place the methyl iodide reagent and the sodium iodide solid of 2 times of chitosan molar weight in the beaker, and Add a certain volume of absolute ethanol and stir to dissolve at a ratio of 10ml per gram of sodium iodide, and slowly drop the mixed solution into the reaction solution (3) with a constant pressure dropping funnel and react for 3 hours under constant stirring;

(5) Add 3 times the volume of acetone to the reaction solution in step (4), let stand overnight, filter with suction, wash with ethanol and ether mixture of 1/5 times the volume of the filtrate, and dry at a constant temperature in vacuum to obtain a solid;

(6) Place the product of step (5) in a Soxhlet extractor and extract it with absolute ethanol under reflux, and dry the solid in vacuum to constant weight to obtain the product N-p-benzyloxymethyl chitosan quaternary ammonium salt.

Described ⑴, ⑵, ⑶ and the process condition of stirring in step ⑷ are: use constant temperature stirrer, adjust to reaction temperature, and adjust to higher rotating speed, make the magnetron rotate at a constant speed at the bottom of the there-necked flask; The technical conditions are as follows: use a filter bottle and a Buchner funnel to bake in an oven at 50-65° C. for 1-3 hours, then filter the reaction liquid with a water pump at room temperature, and wash and collect the solid obtained in the Buchner funnel after the suction filtration is completed.

The weight ratio of chitosan: anisaldehyde: methyl iodide is 1: (1-2): (2-3).

In the step (4), methyl iodide: the weight ratio of sodium iodide is 1:1.

The mixing volume ratio of absolute ethanol and absolute ether in step (5) is 1: (1-10).

The process conditions of the Soxhlet extraction in step (6) are: heating in a water bath at 65-75°C, and extracting for 24-30 hours.

The process conditions of vacuum drying in steps (5) and (6) are: 30-50°C, vacuum degree of 8×10 ⁴ Pa, drying for 12-24 hours.

The application of the N-p-benzyloxymethyl chitosan quaternary ammonium salt as a food antiseptic additive.

The present invention synthesizes a novel chitosan quaternary ammonium salt, the chitosan quaternary ammonium salt has good antibacterial activity to Staphylococcus aureus and Aspergillus niger, strengthens the antibacterial activity of chitosan, and expands the antibacterial activity of chitosan Antibacterial range, can be used for food antiseptic additives.

Description of drawings

Fig. 1 is the infrared spectrogram of chitosan.

Fig. 2 is the infrared spectrogram of the prepared N-p-benzyloxymethyl chitosan quaternary ammonium salt.

Fig. 3 is the ¹ H NMR spectrum of chitosan.

Fig. 4 is the ¹ H NMR spectrum of N-p-benzyloxymethyl chitosan quaternary ammonium salt.

Fig. 5 is the inhibitory rate of different concentrations of chitosan and N-p-benzyloxymethyl chitosan quaternary ammonium salt to Staphylococcus aureus.

Fig. 6 is the inhibitory rate of different concentrations of chitosan and N-p-benzyloxymethyl chitosan quaternary ammonium salt to Aspergillus niger.

Figure 7 is a diagram of the main reaction steps involved in the present invention. in, .

Analysis of the two infrared spectra in Figure 1 and Figure 2 shows that in the infrared spectrum of chitosan, the 1649 cm ^-1 is the amide Ι band. Due to the higher degree of deacetylation, the peak is smaller, and the 1597 cm ^-1 is the strong amino- _NH2 absorption band. 1373 cm ^-1 is OH bending vibration, 1159 cm ^-1 and 893 cm ^-1 are chitosan sugar-based absorption bands. In the infrared spectrum of quaternary ammonium salts, the amino deformation vibration absorption band at 1597 cm ^-1 is obviously weakened, which proves that the substitution reaction occurs on -NH ₂ , and the appearance of new peaks at 1511-1573 cm ^-1 is the characteristic absorption band of quaternary ammonium salts, 1415 The sharp peak at cm ^-1 is the CH bending vibration in the newly introduced -CH ₃ and -CH ₂ -, and the appearance of a new peak at 835 cm ^-1 is the out-of-plane bending vibration of =CH in the p-substituted benzene ring, 1107 cm ^-1 The enhancement of the peak is due to the newly introduced characteristic absorption peak of -OCH ₃ , which proves the successful synthesis of chitosan quaternary ammonium salt.

In Figure 3 and Figure 4, chitosan was dissolved in the mixture of CD ₃ COOD and D ₂ O, and N-p-benzyloxymethyl chitosan quaternary ammonium salt was dissolved in D ₂ O for determination. The small peak at 2.02ppm in Figure 3 is caused by the presence of _-CH in the acetamide group of chitosan, and the peak is smaller due to the higher degree of deacetylation; while the independent peak at 2.96ppm is attributed to the presence of _H Existence; various peaks from 3.50-3.72ppm are completely consistent with H ₃ , H ₄ , H ₅ , and H ₆ in chitosan; the peak at 4.67ppm is the function of H ₁ . In Figure 4, the peak at 1.82ppm is the characteristic peak of -CH ₃ in the acetamide group, the weak peak at around 2.70ppm represents the resonance absorption peak of the proton in -N ⁺ (CH ₃ ) ₂ I ^- , and the peak at 4.66ppm is the peak of H ₁ Absorption peak, the multiplet at 3.50-3.68ppm represents the signal peak of protons in H ₃ , H ₄ , H ₅ , H ₆ and -OCH ₃ , the signal peak at 2.95ppm is H ₂ , and the multiplet at 6.74-7.68ppm is The proton absorption peak of the para-disubstituted benzene ring and the proton absorption peak of -CH ₂ - appeared at 4.32ppm, which proved that the chitosan quaternary ammonium salt was successfully synthesized.

Different concentrations of chitosan and N-benzyloxymethyl chitosan quaternary ammonium salt in Figure 5 all produce inhibitory effect on Staphylococcus aureus, and N-p-benzyloxymethyl chitosan quaternary ammonium salt The antibacterial ability of chitosan is obviously greater than that of chitosan, and its minimum inhibitory concentration is 0.5%.

Fig. 6 is the inhibition curve of different concentrations of chitosan and N-p-benzyloxymethyl chitosan quaternary ammonium salt to Aspergillus niger. When the concentration of chitosan is lower than 0.5%, it not only has no inhibitory effect on Aspergillus niger, but helps its growth. The reason may be that Aspergillus niger belongs to fungi, and the cell wall of fungi contains chitosan, and there is a high content of chitosan in Aspergillus niger, which makes Aspergillus niger have certain resistance to the antibacterial properties of chitosan, which determines the Glycans have no inhibitory ability to Aspergillus niger, but may be used to promote cell growth and reproduction. After adding N-p-benzyloxymethyl chitosan quaternary ammonium salt, the dry weight of Aspergillus niger was smaller, indicating that the product had a stronger inhibitory ability to Aspergillus niger, and the minimum inhibitory concentration was 0.5%.

Detailed ways

The present invention will be further illustrated below by specific examples.

Example 1:

Add 1g chitosan to 20ml mass percentage 3% glacial acetic acid to swell for 30min, transfer it to a 250ml three-neck flask, add 10ml absolute ethanol, stir, slowly add 1ml dissolved in 20ml absolute ethanol to the constant pressure dropping funnel anisaldehyde, stirred for 3 hours, slowly added 0.3 g of sodium borohydride solution dissolved in 10 ml of distilled water into a constant pressure dropping funnel and stirred until a large amount of white flocs appeared, continued to stir for 2 hours, and added sodium hydroxide to the reaction solution to adjust pH=8-9, dissolve 0.4ml methyl iodide and 2g sodium iodide in 20ml ethanol solution and stir evenly, and slowly drop the mixture into the above reaction solution, control the reaction temperature to 50°C and continue stirring for 3h, stop To react, add 250ml of acetone to the reaction solution, let stand overnight, filter with suction, wash with 50ml of ethanol and ether with a volume ratio of 1:1, and dry at constant temperature to obtain a solid, put the solid into a Soxhlet extractor and extract it with absolute ethanol for 24 hours , the product was dried to constant weight to obtain the target product N-p-benzyloxymethyl chitosan quaternary ammonium salt.

Example 2:

The antibacterial rate (%) of N-p-benzyloxymethyl chitosan quaternary ammonium salt and chitosan of the present invention to Staphylococcus aureus

Example 3:

The antibacterial rate (%) of N-p-benzyloxymethyl chitosan quaternary ammonium salt and chitosan of the present invention to Aspergillus niger

Determination of the above-mentioned bacteriostatic rate: prepare different mass fractions of chitosan solution and chitosan quaternary ammonium salt solution (using 0.5% acetic acid solution as solvent). 0.1 mL of bacterial suspension and 0.1 mL of sample solutions of different concentrations were evenly spread on the medium plate, and the blank was the bacterial suspension without sample solution. All plates were cultured in a constant temperature incubator at 37 °C for 24 hours, and the growth of bacteria on each plate was observed, and the antibacterial rate was calculated:

I = (n ₁ -n ₂ )/ n ₁ ×100%

Among them, _n1 and _n2 are the number of colonies on blank and sample-coated plates, respectively.

For Aspergillus niger, the antibacterial efficiency of chitosan and its derivatives was determined by dry weight method. Chitosan solutions and chitosan quaternary ammonium salt solutions (with 0.5% acetic acid solution as solvent) were prepared with different mass fractions. Add 1 mL of Aspergillus niger suspension and 2 mL of sample solutions of different concentrations to Sabouraud medium, the blank is the bacterial suspension without sample solution, cultivate in a constant temperature shaking incubator at 28 ℃ for 48 h, and filter with suction. , dry and weigh the dry weight of the thalli, and calculate the bacteriostatic rate:

I = (m ₁ -m ₂ )/ m ₁ ×100%

Among them, m ₁ and m ₂ are the dry weight of mycelium in culture solution without sample and in culture solution with sample, respectively.

Those skilled in the art will understand that within the protection scope of the present invention, modifications, additions and substitutions are all possible to the above embodiments, and none of them exceed the protection scope of the present invention.

Claims (9)

1. a kind of N-p-benzyloxymethyl chitosan quaternary ammonium salt, it is characterized in that: the compound structure is as shown in the following formula:

Wherein, X is between 0.65-0.95, and the molecular weight is 45-50KD. 2. a preparation method of N-p-phenylmethoxymethyl chitosan quaternary ammonium salt as claimed in claim 1, is characterized in that: described preparation step is as follows: (1) A certain amount of chitosan is added to a certain volume mass percentage of 3% glacial acetic acid at a ratio of 20ml per gram to swell for 30min, transferred to a three-necked flask, and 1/2 times the volume of the solution is added to the flask. Stir at -50°C; (2) Add anise aldehyde test solution with the same weight as chitosan into the constant pressure dropping funnel, add a certain volume of absolute ethanol at a volume ratio of 1:20 and shake to dissolve, slowly drop anise aldehyde into the above chitosan solution Aldehyde absolute ethanol solution, stirred for 3h to obtain chitosan Schiff base; (3) Add sodium borohydride in the same molar amount as chitosan into the beaker, add a certain volume of distilled water at a ratio of 30ml per gram to dissolve, and slowly drop the sodium borohydride solution into the chitosan Schiff base with a constant pressure dropping funnel And keep stirring until a large amount of white flocs appear, continue stirring for 2-3h to obtain alkylated chitosan; (4) Slowly add an appropriate amount of sodium hydroxide to adjust pH=8-9 in step (3) alkylation chitosan reaction solution, respectively place the methyl iodide reagent and the sodium iodide solid of 2 times of chitosan molar weight in the beaker, and Add a certain volume of absolute ethanol at a rate of 10ml per gram of sodium iodide and stir to dissolve. The mixed solution is slowly dropped into the above reaction solution with a constant pressure dropping funnel and reacted for 3 hours under constant stirring; (5) Add 3 times the volume of acetone to the reaction solution in step (4), let stand overnight, filter with suction, wash with ethanol and ether mixture of 1/5 times the volume of the filtrate, and dry at a constant temperature in vacuum to obtain a solid; (6) Place the product of step (5) in a Soxhlet extractor and extract it with absolute ethanol under reflux, and dry the solid in vacuum to constant weight to obtain the product N-p-benzyloxymethyl chitosan quaternary ammonium salt. 3. according to the preparation method of the described N-p-benzyloxymethyl chitosan quaternary ammonium salt of claim 2, it is characterized in that: described (1), (2), the technological condition of stirring in (3) and (4) step is: use Constant temperature stirrer, adjusted to the reaction temperature, and adjusted to a higher speed, so that the magnet rotates at a constant speed at the bottom of the three-necked flask; (5) The process conditions of the suction filtration in the step are: use a filter bottle and a Buchner funnel to bake in an oven at 50-65°C for 1-3 hours, and then filter the reaction solution with a water pump at room temperature. After the suction filtration, wash and collect the Buchner The resulting solid in the funnel. 4. according to the preparation method of the described N-p-benzyloxymethyl chitosan quaternary ammonium salt of claim 2, it is characterized in that: described chitosan: anisaldehyde: the feeding weight ratio of methyl iodide is 1 :(1-2):(2-3). 5. according to the preparation method of the described N-p-benzyloxymethyl chitosan quaternary ammonium salt of claim 2, it is characterized in that: in the described (4) step, methyl iodide: the weight ratio of feeding intake of sodium iodide is 1:1 . 6. according to the preparation method of the described N-p-phenylmethoxymethyl chitosan quaternary ammonium salt of claim 2, it is characterized in that: the dehydrated alcohol of described (5) step and the mixing volume ratio of dehydrated ether are 1 : (1-10). 7. according to the preparation method of the described N-p-benzyloxymethyl chitosan quaternary ammonium salt of claim 2, it is characterized in that: the processing condition of the Soxhlet extraction of described (6) step is: 65-75 ℃ water bath Heat and extract for 24-30 hours. 8. according to the preparation method of the described N-p-benzyloxymethyl chitosan quaternary ammonium salt of claim 2, it is characterized in that: the process condition of the vacuum drying of described (5) and (6) step is: 30-50 DEG C , with a vacuum degree of 8×10 ⁴ Pa, and dry for 12-24 hours. 9. according to the application of the described N-p-benzyloxymethyl chitosan quaternary ammonium salt of claim 1 as preparing food antiseptic additive.

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