WO2022165940A1 - 一种壳聚糖衍生物及其制备方法和应用 - Google Patents

一种壳聚糖衍生物及其制备方法和应用 Download PDF

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WO2022165940A1
WO2022165940A1 PCT/CN2021/082319 CN2021082319W WO2022165940A1 WO 2022165940 A1 WO2022165940 A1 WO 2022165940A1 CN 2021082319 W CN2021082319 W CN 2021082319W WO 2022165940 A1 WO2022165940 A1 WO 2022165940A1
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chitosan
salt
preparation
acid
flavan
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PCT/CN2021/082319
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French (fr)
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巫世贵
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珠海市自然之旅生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof

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  • the invention belongs to the field of chitosan materials, and in particular relates to a chitosan derivative and a preparation method and application thereof.
  • Chitin is a naturally occurring polymer formed by connecting N-acetyl-D-glucosamine and D-glucosamine by beta-1,4 glycosidic bonds. It can be processed to partially or completely remove the acetyl groups and obtain a polymer called chitosan. Most commercial chitosan is obtained by first extracting chitin from fishery by-products such as shrimp or crab shells, followed by deacylation with alkali or acid.
  • Chitosan is known to possess various biological properties, including its antibacterial activity, which can lead to applications in the food or pharmaceutical industries. Chitosan is a food additive that has been approved in the national standard GB2760-2014, and its specific functions are thickener and coating agent.
  • problems in the application process of chitosan first, it is insoluble in water; second, as an antimicrobial agent, chitosan needs to be added at a higher concentration to have an antimicrobial effect, which is different from other antimicrobial agents. Compared with other agents, the application cost of chitosan is high and the benefit is low. Therefore, in the specific application process of chitosan, chitosan needs to be improved to meet the needs of actual production.
  • the preparation method of chitosan derivatives is introduced in the existing related art, and the chitosan-polyphenol conjugate is obtained.
  • the antibacterial, antioxidant and water solubility of the chitosan-polyphenol conjugate are relatively high. Chitosan has improved.
  • the chitosan and the polyphenol conjugate are connected by covalent bonds, and the preparation process needs to introduce catalytic enzymes (such as laccase), or introduce non-food-grade chemical reagents (such as hydrogen peroxide), and the preparation process is relatively complicated , the cost is high, and the antibacterial, antioxidant, or stability is not very good. This hinders the application of chitosan.
  • the present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art.
  • the present invention proposes a chitosan derivative, a preparation method and application thereof.
  • the chitosan derivative has good antibacterial properties and antioxidant properties, especially good high temperature stability. For example, after 120 After being treated at a temperature above °C, the chitosan derivative still has good antibacterial properties, and even the antibacterial properties are further improved.
  • the water solubility of the chitosan derivative is significantly improved compared to that of chitosan.
  • the inventive concept of the present invention is a salt formed by electrostatic interaction between the hydroxyl group on the flavan-3-ol and the amino group of the side chain of chitosan.
  • the preparation process of the chitosan derivative requires chitosan-acid salt as an important reaction intermediate, and then the anionic group formed by the acid in the chitosan-acid salt is replaced by a flavan-3-ol group, Chitosan-flavan-3-alkoxides are formed, and the hydroxyl groups in flavan-3-ols interact with amino groups in chitosan to form ionic bonds.
  • the reacting group is that the hydrogen ion in the organic acid reacts with the hydroxyl group of the chitosan side chain to form a covalently conjugated derivative.
  • One object of the present invention is to provide a chitosan derivative.
  • a chitosan derivative includes a flavan-3-ol structure and a chitosan structure; the flavan-3-ol structure and the chitosan structure are connected by ionic bonds.
  • the chitosan derivative of the present invention is a salt
  • the chitosan structure acts as a positive ion
  • the flavan-3-ol structure acts as an anion
  • the hydroxyl group in the flavan-3-ol and the amino group in the chitosan generate static electricity interact to form salts.
  • the flavan-3-ol structure is selected from catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, gallocatechin gallate, At least one of epigallocatechin gallate, anthocyanin or theaflavin.
  • the deacetylation degree of the chitosan structure is 50-100%; further preferably, the deacetylation degree of the chitosan structure is 80-99%.
  • the molecular weight of the chitosan derivative is 1 kDa to 5000 kDa; further preferably, the molecular weight of the chitosan derivative is 1 kDa to 2500 kDa; more preferably 120 kDa to 1000 kDa.
  • the chitosan derivative is selected from the group consisting of chitosan-catechin salt, chitosan-epicatechin salt, chitosan-epigallocatechin salt, chitosan-epicatechin salt At least one of gallate salt, chitosan-epigallocatechin gallate salt, chitosan-gallocatechin gallate salt, or chitosan-theaflavin salt.
  • the structural formula of the chitosan-epigallocatechin gallate salt is Among them, the value of n is 1-10000.
  • the connection between epigallocatechin gallate and chitosan is through ionic bonds (the chitosan-epigallocatechin gallate salt can also be any one of the structures of epigallocatechin gallate)
  • the hydroxyl groups interact electrostatically with the amino groups in the chitosan structure to form ionic bonds).
  • the second object of the present invention is to provide a preparation method of chitosan derivatives.
  • a preparation method of a chitosan derivative comprising the following steps:
  • Chitosan is mixed with acid solution, heated to obtain chitosan-acid salt, and then flavan-3-ol is added, stirred and reacted to prepare the chitosan derivative.
  • the molecular weight of the chitosan is 1 kDa to 4000 kDa; further preferably, the molecular weight of the chitosan is 1 kDa to 1500 kDa; more preferably, the molecular weight of the chitosan is 120 kDa to 840 kDa.
  • the degree of deacetylation of the chitosan is 50-100%; further preferably, the degree of deacetylation of the chitosan is 80-99%.
  • the acid solution is selected from solutions of hydrochloric acid, formic acid, lactic acid, citric acid or acetic acid.
  • the volume concentration of the acid solution is 0.5-10%; further preferably, the volume concentration of the acid solution is 2-8% (the volume concentration of the acid solution here refers to the acid solution after mixing with chitosan , the resulting mixture, the volume concentration of acid in the mixture).
  • the mass-volume ratio of the chitosan to the acid solution is (5-20) g: (5-30) mL; further preferably, the mass-volume ratio of the chitosan to the acid solution is (8- 15) g: (10-25) mL.
  • the pH of the mixture formed by mixing the chitosan and the acid solution is 1-6; further preferably, the pH is 2-5.
  • the heating temperature is 30-300°C; further preferably, the heating temperature is 50-100°C.
  • the heating time is 10-500 minutes; further preferably, the heating time is 20-200 minutes.
  • the mass ratio of the added amount of the flavan-3-ol to the chitosan-acid salt is (0.1-5): 1; further preferably, the added amount of the flavan-3-ol
  • the mass ratio with the chitosan-acid salt is (0.5-4):1.
  • the temperature of the reaction is 30-300°C; further preferably, the temperature of the reaction is 50-100°C.
  • the reaction time is 10-500 minutes; further preferably, the reaction time is 20-200 minutes.
  • the added amount of the flavan-3-ol and the appropriate reaction temperature and reaction time of the chitosan-acid salt make the prepared chitosan derivative have better antibacterial properties, especially It is still able to maintain good antibacterial properties after high temperature (eg 120 °C) treatment.
  • a preparation method of a chitosan derivative comprises the following steps:
  • the specific process of the first impurity removal is: drying the mixture obtained after heating, and then dialysis and purification (commercially available cellulose filter membrane, the pore size is adjusted according to the molecular weight requirements, and the ultra-low temperature is used at room temperature. Purified by dialysis of pure water for 3-7 days) to remove excess unreacted acid. Helps reduce impurities in chitosan-acid salts.
  • the specific process of the second impurity removal is as follows: drying the mixture obtained after the reaction, and then performing dialysis purification to remove excess unreacted flavan-3-ol. Helps reduce impurities in chitosan derivatives, thereby improving the antibacterial and antioxidant properties of chitosan derivatives.
  • a kind of preparation method of chitosan derivative comprises the following steps:
  • drying and grinding are performed after the second impurity removal to obtain powdery chitosan derivatives.
  • the specific method of the above drying can be air-drying, freeze-drying, spray-drying or heat-drying.
  • the third object of the present invention is to provide an application of chitosan derivatives.
  • the coating is an antibacterial coating.
  • the food or cosmetic is a food or cosmetic with good antibacterial and antiseptic effect.
  • the chitosan derivative of the present invention is a salt formed by electrostatic interaction between the hydroxyl group on flavan-3-ol and the amino group of the side chain of chitosan.
  • the preparation process of the chitosan derivative requires chitosan-acid salt as an important reaction intermediate, and then the anionic group formed by the acid in the chitosan-acid salt is replaced by a flavan-3-ol group, Chitosan-flavan-3-alkoxides are formed, and the hydroxyl groups in flavan-3-ols interact with amino groups in chitosan to form ionic bonds.
  • the reacting group is that the hydrogen ion in the organic acid reacts with the hydroxyl group of the chitosan side chain to form a covalently conjugated derivative. Therefore, the chitosan derivative of the present invention has better water solubility (at 25°C, the solubility is not less than 1g/100g water, such as 1g/100g water to 12g/100g water), and has good antibacterial and antioxidant properties. In particular, it has good high temperature stability. For example, after being treated at a temperature above 120 ° C, the chitosan derivative still has good antibacterial properties, and even the mold properties are further improved.
  • the chitosan derivatives prepared by the present invention have good high temperature stability, good antibacterial properties and antioxidative properties, they can be widely used in medicines, foods and cosmetics.
  • FIG. 1 is a Fourier transform infrared spectrogram of the chitosan-epigallocatechin gallate salt prepared in Example 1.
  • FIG. 1 is a Fourier transform infrared spectrogram of the chitosan-epigallocatechin gallate salt prepared in Example 1.
  • the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels unless otherwise specified, or can be obtained by existing known methods.
  • a preparation method of a chitosan derivative comprising the following steps:
  • Fig. 1 is the Fourier transform infrared spectrogram of the chitosan-epigallocatechin gallate salt prepared in this embodiment 1 (the ordinate in Fig. 1 is " Transmittance " represents “ transmittance ", abscissa is ""Wavenumber” means "wave number”, and the unit of abscissa is “cm -1 "; CHIT means chitosan, CHIT-AC means chitosan-acetate, CHIT-FLAV means chitosan-epigallocatechin gallate salt, FLAV stands for epigallocatechin gallate).
  • the peak at 1556 cm -1 is the carboxylate anion COO -
  • the peak at 1408 cm -1 is the stretching mode of the carboxylate anion
  • the peak at 1230cm -1 in FLAV and CHIT-FLAV which represents the hydroxyl group of epigallocatechin gallate CO bond in .
  • chitosan The structural formula of chitosan is: (Since the chosen molecular weight of chitosan is 280kDa, n here is also determined).
  • chitosan-acetate (Since the chosen molecular weight of chitosan is 280kDa, n here is also determined).
  • chitosan-epigallocatechin gallate salt (Since the chosen molecular weight of chitosan is 280kDa, n here is also determined).
  • a preparation method of a chitosan derivative comprising the following steps:
  • chitosan-hydrochloride powder weigh 10g of chitosan-hydrochloride powder and dissolve it in 1L of water, then add 15g of catechin, under closed conditions, at 75°C, stir and react for 2 hours, then carry out lyophilization and grinding for 24 hours, and then carry out dialysis Purification to remove unreacted catechins, and then freeze-drying and grinding to prepare chitosan derivatives (chitosan-catechin salts).
  • a preparation method of a chitosan derivative comprising the following steps:
  • chitosan-acetate powder weigh 10g of chitosan-acetate powder and dissolve it in 1L of water, then add 11g of epicatechin, under closed conditions, at 70 ° C, stir and react for 2 hours, then carry out lyophilization and grinding for 24 hours, and then carry out
  • the chitosan derivative (chitosan-epicatechin salt) is prepared by dialysis and purification to remove the unreacted epicatechin, and then freeze-drying and grinding.
  • a preparation method of a chitosan derivative comprising the following steps:
  • chitosan-acetate powder weigh 10g of chitosan-acetate powder and dissolve it in 1L of water, then add 18g of epigallocatechin, under closed conditions, at 80 ° C, stir and react for 1 hour, then carry out lyophilization and grinding for 24 hours, and then Dialysis and purification are performed to remove unreacted epigallocatechin, and then freeze-drying and grinding are performed to prepare a chitosan derivative (chitosan-epigallocatechin salt).
  • a preparation method of a chitosan derivative comprising the following steps:
  • a preparation method of a chitosan derivative comprising the following steps:
  • chitosan-lactate powder weigh 10g of chitosan-lactate powder and dissolve it in 1L of water, then add 22g of gallocatechin gallate, under closed conditions, at 90 °C, stir and react for 2 hours, and then carry out lyophilization and grinding for 24 hours , followed by dialysis purification to remove unreacted gallocatechin gallate, and then freeze-drying and grinding to prepare chitosan derivatives (chitosan-gallocatechin gallate salt).
  • a preparation method of a chitosan derivative comprising the following steps:
  • chitosan-citrate powder weigh 10g of chitosan-citrate powder and dissolve it in 1L of water, then add 13g of theaflavins, under closed conditions, at 90°C, stir and react for 2 hours, then lyophilize and grind for 24 hours, and then dialyze Purify, remove unreacted theaflavins, and then perform freeze-drying and grinding to prepare chitosan derivatives (chitosan-theaflavins).
  • a preparation method of a chitosan derivative comprising the steps of: selecting chitosan with a molecular weight of 180kDa and a degree of deacetylation of 88% (through hydrogen nuclear magnetic resonance) Spectrum verification), weigh 10g of the chitosan, dissolve it in 1L of water, add acetic acid (the volume concentration of acetic acid is 2.5%) and mix to form a mixture, then heat it at 80°C for 1 hour, and freeze it for 24 hours after the heating is completed. Dry treatment, grinding to obtain powder, the powder is subjected to dialysis purification treatment to remove excess unreacted acetic acid, and then lyophilized and ground again to obtain chitosan-acetate powder;
  • chitosan-acetate powder weigh 10g of chitosan-acetate powder and dissolve it in 1L of water, then add 6g of epigallocatechin gallate, under closed conditions, at 70 ° C, stir and react for 1.5 hours, and then carry out lyophilization and grinding for 24 hours Treatment, followed by dialysis purification, and then freeze-drying and grinding, to prepare a chitosan derivative (chitosan-epigallocatechin gallate salt).
  • a preparation method of a chitosan derivative comprising the following steps:
  • chitosan-lactate powder weigh 10g of chitosan-lactate powder and dissolve it in 1L of water, then add 15g of anthocyanins, and under closed conditions, at 70°C, stir and react for 2 hours, then lyophilize and grind for 24 hours, followed by dialysis After purification, lyophilization and grinding, the chitosan derivative (chitosan-anthocyanin salt) is prepared.
  • Example 10 Compared with Example 1, the only difference of Example 10 is that in Example 10, the reaction temperature of chitosan-acetate and epigallocatechin gallate is 110 ° C, and the reaction time is 1.5 hours, The rest of the procedure is the same as in Example 1.
  • Comparative example 1 (preparation method of chitosan-epigallocatechin gallate conjugated product in prior art)
  • Example 10 Take the chitosan derivatives prepared in Examples 1-7, Example 10, and Comparative Example 1, and test them against bacteria (including Gram-positive bacteria and Gram-negative bacteria, wherein Gram-positive bacteria include gold Staphylococcus aureus, Bacillus cereus, Lactobacillus plantarum, Gram-negative bacteria including Escherichia coli, Pseudomonas aeruginosa), yeast (Malassezia furfur, Candida albicans), molds (Aspergillus niger, Penicillium italia) Minimum inhibitory concentration (MIC) (sample inoculation concentration unit is ppm, concentration gradient is 4000ppm, 2000ppm, 1000ppm, 500ppm).
  • bacteria including Gram-positive bacteria and Gram-negative bacteria, wherein Gram-positive bacteria include gold Staphylococcus aureus, Bacillus cereus, Lactobacillus plantarum, Gram-negative bacteria including Escherichia coli, Pseudomonas aeruginosa),
  • the cultured system is a commercially available nutrient broth (provided by Guangdong Huankai Microorganism Technology Co., Ltd., model is 022010), the pH of the cultured system is 6, and the bacterial culture condition is that it is cultured for 7 days at a temperature of 36°C, The culture conditions of yeast and mold were cultured at a temperature of 28°C for 7 days. Table 1 shows the results.
  • the chitosan derivatives prepared in Examples 1-7 and Example 10 of the present invention have better antibacterial effects than Comparative Example 1.
  • the chitosan-epigallocatechin gallate conjugated product prepared in Comparative Example 1 had no antibacterial effect on mold. It can be seen from the data of Example 1 and Example 10 that the reaction temperature of chitosan-acetate and epigallocatechin gallate has a certain influence on the antibacterial effect of the prepared chitosan derivatives .
  • the antibacterial effects of the chitosan derivatives prepared in other examples were similar to those of Example 1.
  • the chitosan derivative prepared in Example 1 was taken, and its antibacterial effect after being treated with different temperature conditions was tested, and the stability of the chitosan derivative prepared in Example 1 was further tested.
  • the culture system and culture conditions were the same as above (culture conditions for bacteria were cultured at a temperature of 36°C for 7 days, and culture conditions for yeast and mold were cultured at a temperature of 28°C for 7 days). Different temperature conditions are specifically divided into: normal temperature 25 °C for 1 hour, high pressure moist heat treatment (101KPa, 121 °C, 15 minutes), normal pressure water bath 121 °C for 1 hour, normal pressure oil bath for 1 hour at 180 °C.
  • the antibacterial results are shown in Table 2.
  • the chitosan derivative prepared in Example 1 has a good effect on leather after being treated by high pressure wet heat treatment for 1 hour, normal pressure water bath at 121°C for 1 hour, and normal pressure oil bath at 180°C for 1 hour.
  • the antibacterial effects of blue-positive bacteria, gram-negative bacteria and yeast are consistent with the room temperature of 25°C, indicating that the chitosan derivatives prepared in the examples of the present invention have good high temperature stability.
  • the inventor also unexpectedly found that the chitosan derivative prepared in Example 1 was subjected to high-pressure wet heat treatment for 1 hour, normal pressure water bath treatment at 121 °C for 1 hour, and normal pressure oil bath treatment at 180 °C for 1 hour.
  • the antibacterial effect of the chitosan derivative prepared in Example 1 on mold was unexpectedly enhanced, and an unexpected technical effect was achieved.
  • the chitosan derivatives prepared in the remaining examples have similar high temperature stability to those of the chitosan derivatives prepared in Example 1 above.
  • Each group was prepared with 2% saline solution by mass, and added chitosan (the concentration of chitosan in saline was 3000 ppm), epigallocatechin gallate (epigallocatechin gallate in saline), respectively.
  • the concentration of the chitosan derivative prepared in Example 1 is 3000 ppm), the chitosan derivative prepared in Example 1 (the concentration of the chitosan derivative prepared in Example 1 in the saline solution is 3000 ppm), potassium sorbate (the concentration of potassium sorbate in the saline solution is 75 ppm) ), (in the potassium sorbate group, record the original pH value of the salt water, add citric acid to adjust the pH to 5.0, then add potassium sorbate and stir evenly, which is beneficial to exert the antibacterial effect of potassium sorbate), heat it in a pot, put it Add 2 large pieces of beef, after the fire is boiled, turn to low heat and simmer for about 1 hour. The specific heat is when the beef is soft and rotten but not scattered;
  • the chitosan derivative group prepared in Example 1 can also be maintained at 1.02log cfu/mL. It can be seen that the chitosan derivative prepared in Example 1 has a good anti-corrosion and fresh-keeping effect. The chitosan derivatives prepared in other examples also have similar preservative and fresh-keeping effects.
  • Chitosan and epigallocatechin gallate alone do not have good antibacterial properties when added to the above foods.
  • the chitosan derivatives prepared by the present invention have good high temperature stability, good antibacterial properties and antioxidative properties, they can be widely used in medicines, cosmetics or coatings.
  • the solubility of the chitosan derivative prepared in Example 1 of the present invention in water is 10g/100g water, the solubility of chitosan is less than 0.01g/100g water, and the chitosan derivative prepared in Comparative Example 1 -
  • the solubility of the epigallocatechin gallate conjugated product is less than 0.01 g/100 g water.

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Abstract

本发明属于壳聚糖类材料领域，公开了一种壳聚糖衍生物及其制备方法和应用。该壳聚糖衍生物，包括黄烷-3-醇结构和壳聚糖结构；黄烷-3-醇结构与壳聚糖结构之间通过离子键连接；黄烷-3-醇结构选自儿茶素、表儿茶素、没食子儿茶素、表没食子儿茶素、表儿茶素没食子酸酯、没食子儿茶素没食子酸酯、表没食子儿茶素没食子酸酯、花青素或茶黄素中的至少一种。该壳聚糖衍生物具有良好的抗菌性和抗氧化性，特别是具有良好的耐高温稳定性，例如经过120℃以上的温度处理后，壳聚糖衍生物依然具有良好的抗菌性，甚至抗霉菌性有进一步提升。另外，壳聚糖衍生物在25℃下，溶解度不小于1g/100g水。

Description

一种壳聚糖衍生物及其制备方法和应用 技术领域
本发明属于壳聚糖类材料领域，特别涉及一种壳聚糖衍生物及其制备方法和应用。
背景技术
几丁质是天然存在的聚合物，是由β-1,4糖苷键连接N-乙酰基-D-葡萄糖胺和D-葡萄糖胺形成的。可以对其进行加工，以实现部分或完全除去乙酰基，并获得称为壳聚糖的聚合物。大多数商用壳聚糖，是先通过从渔业副产品(例如虾或蟹壳)中提取几丁质，然后再用碱或酸进行脱酰作用，得到壳聚糖。
已知壳聚糖具有多种生物特性，包括其抗菌活性，从而可应用于食品或制药行业。壳聚糖是已经在国家标准GB2760-2014中得到批准的食品添加剂，具体功能为增稠剂和涂层剂。但是，壳聚糖的应用过程中存在以下问题：第一，难溶于水；第二，壳聚糖作为抗微生物剂，需要添加较高的浓度，才具有抗微生物的效果，与其他抗微生物剂相比，壳聚糖的应用成本高，效益较低。因此，在壳聚糖的具体应用过程中，需要对壳聚糖进行改进，以满足实际生产的需求。
现有的相关技术中介绍了壳聚糖衍生物的制备方法，制得壳聚糖-多酚共轭物，该壳聚糖-多酚共轭物的抗菌性，抗氧化性和水溶性相对壳聚糖有提升。但该壳聚糖与多酚共轭物之间通过共价键连接，制备过程需要引入催化酶(例如漆酶)，或引入非食品级的化学试剂(例如过氧化氢)，制备过程相对复杂，成本较高，且抗菌性、抗氧化性或稳定性并不是很好。这对壳聚糖的应用起到了一定的阻碍作用。
因此，亟需提供一种新的壳聚糖衍生物，该壳聚糖衍生物不仅良好的抗菌性和抗氧化性，而且具有良好的稳定性，有助于该壳聚糖衍生物的应用。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出一种壳聚糖衍生物及其制备方法和应用，所述壳聚糖衍生物具有良好的抗菌性和抗氧化性，特别是具有良好的耐高温稳定性，例如经过120℃以上的温度处理后，所述壳聚糖衍生物依然具有良好的抗菌性，甚至抗菌性有进一步提升。另外，所述壳聚糖衍生物的水溶性相对壳聚糖而言，有显著的提升。
本发明的发明构思：本发明所述壳聚糖衍生物是通过黄烷-3-醇上的羟基与壳聚糖侧链氨基之间，发生静电交互作用，从而形成的盐。所述壳聚糖衍生物的制备过程需要壳聚糖-酸盐作为重要的反应中间产物，然后通过黄烷-3-醇基团取代了壳聚糖-酸盐中酸形成的阴离子基团，生成了壳聚糖-黄烷-3-醇盐，黄烷-3-醇中的羟基与壳聚糖中的氨基发生作用，形成离子键。而现有技术中的 壳聚糖酸盐衍生物，发生反应的基团是有机酸中的氢离子与壳聚糖侧链的羟基反应而生成共价键共轭衍生物。
本发明目的一，提供一种壳聚糖衍生物。
具体的，一种壳聚糖衍生物，包括黄烷-3-醇结构和壳聚糖结构；所述黄烷-3-醇结构与壳聚糖结构之间通过离子键连接。
本发明所述壳聚糖衍生物是一种盐，壳聚糖结构充当正离子，黄烷-3-醇结构充当阴离子，黄烷-3-醇中的羟基与壳聚糖中的氨基发生静电交互作用，从而形成盐。
优选的，所述黄烷-3-醇结构选自儿茶素、表儿茶素、没食子儿茶素、表没食子儿茶素、表儿茶素没食子酸酯、没食子儿茶素没食子酸酯、表没食子儿茶素没食子酸酯、花青素或茶黄素中的至少一种。
优选的，所述壳聚糖结构的脱乙酰度50-100％；进一步优选的，所述壳聚糖结构的脱乙酰度为80-99％。
优选的，所述壳聚糖衍生物的分子量为1kDa到5000kDa；进一步优选的，所述壳聚糖衍生物的分子量为1kDa到2500kDa；更优选120kDa到1000kDa。
优选的，所述壳聚糖衍生物选自壳聚糖-儿茶素盐、壳聚糖-表儿茶素盐、壳聚糖-表没食子儿茶素盐、壳聚糖-表儿茶素没食子酸酯盐、壳聚糖-表没食子儿茶素没食子酸酯盐、壳聚糖-没食子儿茶素没食子酸酯盐或壳聚糖-茶黄素盐中的至少一种。
优选的，所述壳聚糖-表没食子儿茶素没食子酸酯盐的结构式为
Figure PCTCN2021082319-appb-000001
其中，n的取值为1-10000。表没食子儿茶素没食子酸酯与壳聚糖之间通过离子键连接(所述壳聚糖-表没食子儿茶素没食子酸酯盐还可以是表没食子儿茶素没食子酸酯结构上的任一羟基与壳聚糖结构中的氨基发生静电交互作用，形成离子键)。
本发明目的二，提供一种壳聚糖衍生物的制备方法。
具体的，一种壳聚糖衍生物的制备方法，包括以下步骤：
将壳聚糖与酸液混合，加热，获得壳聚糖-酸盐，然后加入黄烷-3-醇，搅拌，反应，制得所述壳聚糖衍生物。
优选的，所述壳聚糖的分子量为1kDa到4000kDa；进一步优选的，所述壳聚糖的分子量为1kDa到1500kDa；更优选的，所述壳聚糖的分子量为120kDa到840kDa。
优选的，所述壳聚糖的脱乙酰度为50-100％；进一步优选的，所述壳聚糖的脱乙酰度为80-99％。
优选的，所述酸液选自盐酸、甲酸、乳酸、柠檬酸或乙酸的溶液。
优选的，所述酸液的体积浓度为0.5-10％；进一步优选的，所述酸液的体积浓度为2-8％(此处酸液的体积浓度是指酸液与壳聚糖混合后，形成的混合物中，酸在混合物中的体积浓度)。
优选的，所述壳聚糖与酸液的质量体积比为(5-20)g:(5-30)mL；进一步优选的，所述壳聚糖与酸液的质量体积比为(8-15)g:(10-25)mL。
优选的，所述壳聚糖与酸液混合后形成的混合物的pH为1-6；进一步优选的，pH为2-5。
优选的，所述加热的温度为30-300℃；进一步优选的，所述加热的温度为50-100℃。
优选的，所述加热的时间为10-500分钟；进一步优选的，所述加热的时间为20-200分钟。
优选的，所述黄烷-3-醇的加入量与所述壳聚糖-酸盐的质量比为(0.1-5):1；进一步优选的，所述黄烷-3-醇的加入量与所述壳聚糖-酸盐的质量比为(0.5-4):1。
优选的，所述反应的温度为30-300℃；进一步优选的，所述反应的温度为50-100℃。
优选的，所述反应的时间为10-500分钟；进一步优选的，所述反应的时间为20-200分钟。
优选的，所述黄烷-3-醇的加入量与所述壳聚糖-酸盐合适的反应温度以及合适的反应时间，使得制得的壳聚糖衍生物具有更好的抗菌性，特别是经过高温(例如120℃)处理后，依然能保持良好的抗菌性。
优选的，一种壳聚糖衍生物的制备方法，包括以下步骤：
将壳聚糖与酸液混合，加热，然后第一次除杂，获得壳聚糖-酸盐，然后加入黄烷-3-醇，搅拌，反应，第二次除杂，制得所述壳聚糖衍生物。
优选的，所述第一次除杂的具体过程为：将加热后得到的混合物进行干燥，然后进行透析纯化(市售的纤维素滤膜，孔径大小跟根据分子量需求进行调节，室温下利用超纯水透析纯化3-7天)，以除去多余的未反应的酸。有助于减少壳聚糖-酸盐中的杂质。
优选的，所述第二次除杂的具体过程为：将反应后得到的混合物进行干燥，然后进行透析纯化，以除去多余的未反应的黄烷-3-醇。有助于减少壳聚糖衍生物中的杂质，从而提高壳聚糖衍生物的抗菌性及抗氧化性。
更优选的，一种壳聚糖衍生物的制备方法，包括以下步骤：
将壳聚糖与酸液混合，加热，干燥，然后第一次除杂，获得壳聚糖-酸盐，将所述壳聚糖-酸盐与溶剂混合，然后加入黄烷-3-醇，搅拌，反应，干燥，第二次除杂，制得所述壳聚糖衍生物。
优选的，所述第二次除杂后还进行干燥、研磨处理，获得粉末状的壳聚糖衍生物。有助于壳聚糖衍生物的使用。
上述干燥的具体方式可为风干、冻干、喷干或热干。
本发明目的三，提供一种壳聚糖衍生物的应用。
本发明所述壳聚糖衍生物在制备药品、食品、化妆品或涂料中的应用。
优选的，所述涂料为抗菌涂料。
优选的，所述食品或化妆品是具有良好的抗菌防腐功效的食品或化妆品。
相对于现有技术，本发明的有益效果如下：
(1)本发明所述壳聚糖衍生物是通过黄烷-3-醇上的羟基与壳聚糖侧链氨基之间，发生静电交互作用，从而形成的盐。所述壳聚糖衍生物的制备过程需要壳聚糖-酸盐作为重要的反应中间产物，然后通过黄烷-3-醇基团取代了壳聚糖-酸盐中酸形成的阴离子基团，生成了壳聚糖-黄烷-3-醇盐，黄烷-3-醇中的羟基与壳聚糖中的氨基发生作用，形成离子键。而现有技术中的壳聚糖酸盐衍生物，发生反应的基团是有机酸中的氢离子与壳聚糖侧链的羟基反应而生成共价键共轭衍生物。故，本发明所述壳聚糖衍生物水溶性更好(25℃下，溶解度不小于1g/100g水，例如为1g/100g水至12g/100g水)，具有良好的抗菌性和抗氧化性，特别是具有良好的耐高温稳定性，例如经过120℃以上的温度处理后，所述壳聚糖衍生物依然具有良好的抗菌性，甚至霉菌性有进一步提升。
(2)由于本发明制得的壳聚糖衍生物具有良好的耐高温稳定性，以及良好的抗菌性和抗氧化性，因此，可广泛应用在药品、食品以及化妆品中。
附图说明
图1为本实施例1中制得的壳聚糖-表没食子儿茶素没食子酸酯盐的傅立叶红外光谱图。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案，现列举以下实施例进行说明。需要指出的是，以下实施例对本发明要求的保护范围不构成限制作用。
以下实施例中所用的原料、试剂或装置如无特殊说明，均可从常规商业途径得到，或者可以通过现有已知方法得到。
实施例1：壳聚糖衍生物(壳聚糖-表没食子儿茶素没食子酸酯盐)的制备
一种壳聚糖衍生物(壳聚糖-表没食子儿茶素没食子酸酯盐)的制备方法，包括以下步骤：
选择分子量为280kDa，脱乙酰度为88％的壳聚糖(经过核磁共振氢谱核验)，称量10g该壳聚糖溶解于1L水中并加入乙酸(乙酸的体积浓度为2％，即乙酸在壳聚糖、水以及乙酸形成的混合物中的体积浓度为2％)混合，形成混合物，该混合物的pH为3.5，然后在50℃下，加热1小 时，加热完成后，进行24小时冻干处理，研磨得到粉末，将该粉末进行透析纯化处理，去除多余未反应的乙酸，然后再次进行冻干、研磨处理，得到壳聚糖-乙酸盐粉末；
称量10g壳聚糖-乙酸盐粉末溶解于1L水中，然后加入10g表没食子儿茶素没食子酸酯，在封闭条件下(封闭条件下进行，有助于避免乙酸挥发)，70℃下，搅拌反应1.5小时，然后进行24小时冻干、研磨处理，随后进行透析纯化，除去未反应完全的表没食子儿茶素没食子酸酯，然后进行冻干、研磨处理，即制备得到壳聚糖衍生物(壳聚糖-表没食子儿茶素没食子酸酯盐)。
图1为本实施例1中制得的壳聚糖-表没食子儿茶素没食子酸酯盐的傅立叶红外光谱图(图1中的纵坐标为“Transmittance”表示“透射比”，横坐标为“Wave number”表示“波数”，横坐标的单位为“cm -1”；CHIT表示壳聚糖，CHIT-AC表示壳聚糖-乙酸盐，CHIT-FLAV表示壳聚糖-表没食子儿茶素没食子酸酯盐，FLAV表示表没食子儿茶素没食子酸酯)。从图1可以看出，CHIT-AC中，1556cm -1处峰，为羧酸根阴离子COO -，1408cm -1处峰，为羧酸根阴离子的拉伸模式，FLAV和CHIT-FLAV中，1693cm -1和1606cm -1处峰，表示表没食子儿茶素没食子酸酯苯环中的C＝C双键，FLAV和CHIT-FLAV中，1230cm -1处峰，表示表没食子儿茶素没食子酸酯的羟基中的C-O键。
通过傅立叶红外光谱，对本实施例1中获得的中间产物壳聚糖-乙酸盐以及产物壳聚糖-表没食子儿茶素没食子酸酯盐，进行结构分析，其图谱见图1。从图1中可以看出壳聚糖-乙酸盐为合成步骤中的重要中间产物，随后，乙酸基团，被表没食子儿茶素没食子酸酯基团替换，形成了壳聚糖-表没食子儿茶素没食子酸酯盐，具有和壳聚糖、表没食子儿茶素没食子酸酯盐、壳聚糖乙酸盐都不同的结构特征。
壳聚糖的结构式为：
Figure PCTCN2021082319-appb-000002
(由于选择的壳聚糖分子量为280kDa，因此，此处的n也是确定的)。
壳聚糖-乙酸盐的结构式为：
Figure PCTCN2021082319-appb-000003
(由于选择的壳聚糖分子量为280kDa，因此，此处的n也是确定的)。
壳聚糖-表没食子儿茶素没食子酸酯盐的的结构式为：
Figure PCTCN2021082319-appb-000004
(由于选择的壳聚糖分子量为280kDa，因此，此处的n也是确定的)。
实施例2：壳聚糖衍生物(壳聚糖-儿茶素盐)的制备
一种壳聚糖衍生物(壳聚糖-儿茶素盐)的制备方法，包括以下步骤：
选择分子量为100kDa，脱乙酰度为80％的壳聚糖(经过核磁共振氢谱核验)，称量8g该壳聚糖溶解于1L水中并加入盐酸(盐酸的体积浓度为3％)混合，形成混合物，该混合物的pH为3.0，然后在40℃下，加热40分钟，加热完成后，进行28小时冻干处理，研磨得到粉末，将该粉末进行透析纯化处理，去除多余未反应的盐酸，然后再次进行冻干、研磨处理，得到壳聚糖-盐酸盐粉末；
称量10g壳聚糖-盐酸盐粉末溶解于1L水中，然后加入15g儿茶素，在封闭条件下，75℃下，搅拌反应2小时，然后进行24小时冻干、研磨处理，随后进行透析纯化，除去未反应完全的儿茶素，然后进行冻干、研磨处理，即制备得到壳聚糖衍生物(壳聚糖-儿茶素盐)。
实施例3：壳聚糖衍生物(壳聚糖-表儿茶素盐)的制备
一种壳聚糖衍生物(壳聚糖-表儿茶素盐)的制备方法，包括以下步骤：
选择分子量为1000kDa，脱乙酰度为70％的壳聚糖(经过核磁共振氢谱核验)，称量12g该壳聚糖溶解于1L水中并加入乙酸(乙酸的体积浓度为4％)混合，形成混合物，然后在60℃下，加热50分钟，加热完成后，进行24小时冻干处理，研磨得到粉末，将该粉末进行透析纯化处理，去除多余未反应的乙酸，然后再次进行冻干、研磨处理，得到壳聚糖-乙酸盐粉末；
称量10g壳聚糖-乙酸盐粉末溶解于1L水中，然后加入11g表儿茶素，在封闭条件下，70℃下，搅拌反应2小时，然后进行24小时冻干、研磨处理，随后进行透析纯化，除去未反应完全的表儿茶素，然后进行冻干、研磨处理，即制备得到壳聚糖衍生物(壳聚糖-表儿茶素盐)。
实施例4：壳聚糖衍生物(壳聚糖-表没食子儿茶素盐)的制备
一种壳聚糖衍生物(壳聚糖-表没食子儿茶素盐)的制备方法，包括以下步骤：
选择分子量为1100kDa，脱乙酰度为75％的壳聚糖(经过核磁共振氢谱核验)，称量10g该壳聚糖溶解于1L水中并加入乙酸(乙酸的体积浓度为2.5％)混合，形成混合物，然后在70℃下， 加热70分钟，加热完成后，进行24小时冻干处理，研磨得到粉末，将该粉末进行透析纯化处理，去除多余未反应的乙酸，然后再次进行冻干、研磨处理，得到壳聚糖-乙酸盐粉末；
称量10g壳聚糖-乙酸盐粉末溶解于1L水中，然后加入18g表没食子儿茶素，在封闭条件下，80℃下，搅拌反应1小时，然后进行24小时冻干、研磨处理，随后进行透析纯化，除去未反应完全的表没食子儿茶素，然后进行冻干、研磨处理，即制备得到壳聚糖衍生物(壳聚糖-表没食子儿茶素盐)。
实施例5：壳聚糖衍生物(壳聚糖-表儿茶素没食子酸酯盐)的制备
一种壳聚糖衍生物(壳聚糖-表儿茶素没食子酸酯盐)的制备方法，包括以下步骤：
选择分子量为1200kDa，脱乙酰度为85％的壳聚糖(经过核磁共振氢谱核验)，称量10g该壳聚糖溶解于1L水中并加入甲酸(甲酸的体积浓度为2％)混合，形成混合物，然后在75℃下，加热75分钟，加热完成后，进行24小时冻干处理，研磨得到粉末，将该粉末进行透析纯化处理，去除多余未反应的甲酸，然后再次进行冻干、研磨处理，得到壳聚糖-甲酸盐粉末；
称量10g壳聚糖-甲酸盐粉末溶解于1L水中，然后加入20g表儿茶素没食子酸酯，在封闭条件下，85℃下，搅拌反应1.5小时，然后进行24小时冻干、研磨处理，随后进行透析纯化，除去未反应完全的表儿茶素没食子酸酯，然后进行冻干、研磨处理，即制备得到壳聚糖衍生物(壳聚糖-表儿茶素没食子酸酯盐)。
实施例6：壳聚糖衍生物(壳聚糖-没食子儿茶素没食子酸酯盐)的制备
一种壳聚糖衍生物(壳聚糖-没食子儿茶素没食子酸酯盐)的制备方法，包括以下步骤：
选择分子量为1500kDa，脱乙酰度为88％的壳聚糖(经过核磁共振氢谱核验)，称量10g该壳聚糖溶解于1L水中并加入乳酸(乳酸的体积浓度为2％)混合，形成混合物，然后在75℃下，加热75分钟，加热完成后，进行24小时冻干处理，研磨得到粉末，将该粉末进行透析纯化处理，去除多余未反应的乳酸，然后再次进行冻干、研磨处理，得到壳聚糖-乳酸盐粉末；
称量10g壳聚糖-乳酸盐粉末溶解于1L水中，然后加入22g没食子儿茶素没食子酸酯，在封闭条件下，90℃下，搅拌反应2小时，然后进行24小时冻干、研磨处理，随后进行透析纯化，除去未反应完全的没食子儿茶素没食子酸酯，然后进行冻干、研磨处理，即制备得到壳聚糖衍生物(壳聚糖-没食子儿茶素没食子酸酯盐)。
实施例7：壳聚糖衍生物(壳聚糖-茶黄素盐)的制备
一种壳聚糖衍生物(壳聚糖-茶黄素盐)的制备方法，包括以下步骤：
选择分子量为100kDa，脱乙酰度为88％的壳聚糖(经过核磁共振氢谱核验)，称量10g该壳聚糖溶解于1L水中并加入柠檬酸(乳酸的体积浓度为2.5％)混合，形成混合物，然后在80℃下， 加热70分钟，加热完成后，进行24小时冻干处理，研磨得到粉末，将该粉末进行透析纯化处理，去除多余未反应的柠檬酸，然后再次进行冻干、研磨处理，得到壳聚糖-柠檬酸盐粉末；
称量10g壳聚糖-柠檬酸盐粉末溶解于1L水中，然后加入13g茶黄素，在封闭条件下，90℃下，搅拌反应2小时，然后进行24小时冻干、研磨处理，随后进行透析纯化，除去未反应完全的茶黄素，然后进行冻干、研磨处理，即制备得到壳聚糖衍生物(壳聚糖-茶黄素盐)。
实施例8：壳聚糖衍生物(壳聚糖-表没食子儿茶素没食子酸酯盐)的制备
一种壳聚糖衍生物(壳聚糖-表没食子儿茶素没食子酸酯盐)的制备方法，包括以下步骤：选择分子量为180kDa，脱乙酰度为88％的壳聚糖(经过核磁共振氢谱核验)，称量10g该壳聚糖溶解于1L水中并加入乙酸(乙酸的体积浓度为2.5％)混合，形成混合物，然后在80℃下，加热1小时，加热完成后，进行24小时冻干处理，研磨得到粉末，将该粉末进行透析纯化处理，去除多余未反应的乙酸，然后再次进行冻干、研磨处理，得到壳聚糖-乙酸盐粉末；
称量10g壳聚糖-乙酸盐粉末溶解于1L水中，然后加入6g表没食子儿茶素没食子酸酯，在封闭条件下，70℃下，搅拌反应1.5小时，然后进行24小时冻干、研磨处理，随后进行透析纯化，然后进行冻干、研磨处理，即制备得到壳聚糖衍生物(壳聚糖-表没食子儿茶素没食子酸酯盐)。
实施例9：壳聚糖衍生物(壳聚糖-花青素盐)的制备
一种壳聚糖衍生物(壳聚糖-花青素盐)的制备方法，包括以下步骤：
选择分子量为200kDa，脱乙酰度为88％的壳聚糖(经过核磁共振氢谱核验)，称量10g该壳聚糖溶解于1L水中并加入乳酸(乳酸的体积浓度为2.5％)混合，形成混合物，然后在85℃下，加热2小时，加热完成后，进行24小时冻干处理，研磨得到粉末，将该粉末进行透析纯化处理，去除多余未反应的乳酸，然后再次进行冻干、研磨处理，得到壳聚糖-乳酸盐粉末；
称量10g壳聚糖-乳酸盐粉末溶解于1L水中，然后加入15g花青素，在封闭条件下，70℃下，搅拌反应2小时，然后进行24小时冻干、研磨处理，随后进行透析纯化，然后进行冻干、研磨处理，即制备得到壳聚糖衍生物(壳聚糖-花青素盐)。
实施例10
与实施例1相比，实施例10的区别仅在于，实施例10中壳聚糖-乙酸盐与表没食子儿茶素没食子酸酯进行反应的温度为110℃，反应的时间为1.5小时，其余过程与实施例1相同。
对比例1(现有技术中壳聚糖-表没食子儿茶素没食子酸酯共轭产物的制备方法)
选择分子量为280kDa，脱乙酰度为88％的壳聚糖(经过核磁共振氢谱核验)，称量10g该壳聚糖溶解于1L水中并加入乙酸(乙酸的体积浓度为2％，即乙酸在壳聚糖、水以及乙酸形成的混合物中的体积浓度为2％)混合，搅拌过夜，得到壳聚糖溶液，取100mL上述壳聚糖溶液，加1mol/L 的盐酸调pH至3.5，加入1mL 0.5mol/L过氧化氢和0.025g抗坏血酸，40℃搅拌1小时，加入1g表没食子儿茶素没食子酸酯，40℃下，搅拌反应12小时，然后进行24小时冻干、研磨处理，随后进行透析纯化，除去未反应完全的表没食子儿茶素没食子酸酯，然后进行冻干、研磨处理，即制备得到壳聚糖-表没食子儿茶素没食子酸酯共轭产物。
产品效果测试
1.抗菌效果测试
取实施例1-7、实施例10、对比例1制得的壳聚糖衍生物，测试其对细菌(包括革兰氏阳性菌和革兰氏阴性菌，其中，革兰氏阳性菌包括金黄色葡萄球菌、蜡样芽孢杆菌、植物乳酸杆菌，革兰氏阴性菌包括大肠杆菌、绿脓杆菌)、酵母(糠秕马拉色菌、白色念珠菌)、霉菌(黑曲霉、意大利青霉)的最低抑菌浓度(MIC)(样品接菌浓度单位为ppm，浓度梯度为4000ppm、2000ppm、1000ppm、500ppm)。
培养的体系为市售的营养肉汤(由广东环凯微生物科技有限公司提供，型号为022010)，培养的体系的pH为6，细菌的培养条件为，在温度为36℃下培养7天，酵母和霉菌的培养条件为，在温度为28℃下培养7天，结果如表1所示。
表1：抗菌效果(表1中数据表示MIC，单位ppm)
Figure PCTCN2021082319-appb-000005
Figure PCTCN2021082319-appb-000006
Figure PCTCN2021082319-appb-000007
备注：表1中“/”表示没有抑菌效果。
从表1可以看出，本发明实施例1-7、实施例10制得的壳聚糖衍生物相对对比例1具有更好的抗菌效果。而对比例1制得的壳聚糖-表没食子儿茶素没食子酸酯共轭产物对霉菌没有抗菌效果。从实施例1与实施例10的数据可以看出，壳聚糖-乙酸盐与表没食子儿茶素没食子酸酯进行反应的温度对制得的壳聚糖衍生物的抗菌效果具有一定的影响。其余实施例制得的壳聚糖衍生物的抗菌效果与实施例1的效果类似。
2.稳定性测试
取实施例1制得的壳聚糖衍生物，测试其经不同温度条件处理后的抗菌效果，进一步用来测试实施例1制得的壳聚糖衍生物的稳定性。培养的体系以及培养条件与上面相同(细菌的培养条件为，在温度为36℃下培养7天，酵母和霉菌的培养条件为，在温度为28℃下培养7天)。不同温度条件具体分为：常温25℃处理1小时、高压湿热处理(101KPa，121℃，15分钟)、常压水浴121℃处理1小时、常压油浴180℃处理1小时。抗菌结果如表2所示。
表2：抗菌效果(表2中数据表示MIC，单位ppm)
Figure PCTCN2021082319-appb-000008
Figure PCTCN2021082319-appb-000009
从表2可以看出，经过高压湿热处理1小时、常压水浴121℃处理1小时、常压油浴180℃处理1小时等条件处理后，实施例1制得的壳聚糖衍生物对革兰氏阳性菌、革兰氏阴性菌和酵母的抗菌效果与常温25℃保持一致，表明本发明实施例制得的壳聚糖衍生物具有良好的耐高温稳定性。另外，发明人还意外的发现，实施例1制得的壳聚糖衍生物经过高压湿热处理1小时、常压水浴121℃处理1小时、常压油浴180℃处理1小时等条件处理后，实施例1制得的壳聚糖衍生物对霉菌的抗菌效果意外的增强了，取得了一种意料不到的技术效果。其余实施例制得的壳聚糖衍生物具有类似上述实施例1制得的壳聚糖衍生物类似的耐高温稳定性。
3.抗氧化性测试
取本发明实施例1-6制得的壳聚糖衍生物，测试其抗氧化性(抗氧化性的强弱用DPPH自由基清除率来衡量，DPPH指1,1-二苯基-2-三硝基苯肼)，结果如表3所示。
表3：抗氧化性效果
样品 DPPH自由基清除率(％)
壳聚糖 0
儿茶素 54
实施例2 50
表儿茶素 68
实施例3 59
表儿茶素没食子酸酯 78
实施例5 65
表没食子儿茶素 73
实施例4 61
表没食子儿茶素没食子酸酯 84
实施例1 72
没食子儿茶素没食子酸酯 74
实施例6 60
对比例1 73
从表3可以看出，实施例1-6制得的壳聚糖衍生物仍然具有较好的抗氧化性。
4.在食品防腐保鲜中的应用效果
取实施例1制得的壳聚糖衍生物，测试其在食品防腐保鲜中的应用效果。实验过程如下：
(1)冻牛肉解冻后，切块，每块300-400g；
(2)实验分组，具体5个组，分别为空白组、壳聚糖组、表没食子儿茶素没食子酸酯组、实施例1制得的壳聚糖衍生物组以及山梨酸钾组，
(3)每组配制2％质量浓度的食盐水，分别加入壳聚糖(食盐水中壳聚糖的浓度为3000ppm)、表没食子儿茶素没食子酸酯(食盐水中表没食子儿茶素没食子酸酯的浓度为3000ppm)、实施例1制得的壳聚糖衍生物(食盐水中实施例1制得的壳聚糖衍生物的浓度为3000ppm)、山梨酸钾(食盐水中山梨酸钾的浓度为75ppm)，(山梨酸钾组记录食盐水原始pH值，加柠檬酸调pH至5.0后，再加入山梨酸钾，并搅拌均匀，有利于发挥山梨酸钾的抗菌作用)，于锅中加热，放入2大块牛肉块，大火烧开后，转小火炖制1小时左右，具体火候以牛肉软烂但不散开为宜；
(4)将煮熟的牛肉分切成20g左右小块，每2块装入一袋，直接热封封口，每组封14袋；
(5)冷藏放置，第0天测试菌落总数，每次检测取2个平行样，之后测试第1天、第2天、第3天、第4天以及第7天的菌落总数；
(6)试验重复两轮，最终结果，以两次独立试验数据的平均值为准。上述监测菌落总数结果如表4所示。
表4：菌落总数(单位：log cfu/mL)
Figure PCTCN2021082319-appb-000010
从表4可以看出，在食品存放7天后，空白组菌落总数已经达到了7.96log cfu/mL，山梨酸 钾组为4.12log cfu/mL，而实施例1制得的壳聚糖衍生物组，还可以保持在1.02log cfu/mL。可见，实施例1制得的壳聚糖衍生物具有良好的防腐保鲜效果。其余实施例制得的壳聚糖衍生物也具有类似的防腐保鲜效果。
单独将壳聚糖和表没食子儿茶素没食子酸酯，添加到上述食品中并不具有良好的抗菌性能。
另外，由于本发明制得的壳聚糖衍生物具有良好的耐高温稳定性，以及良好的抗菌性和抗氧化性，因此，可广泛应用在药品、化妆品或涂料中。
5.溶解效果
在室温25℃下，本发明实施例1制得的壳聚糖衍生物在水中的溶解度为10g/100g水，壳聚糖的溶解度小于0.01g/100g水，对比例1制得的壳聚糖-表没食子儿茶素没食子酸酯共轭产物的溶解度小于0.01g/100g水。

Claims (15)

  1. 一种壳聚糖衍生物，其特征在于，包括黄烷-3-醇结构和壳聚糖结构；所述黄烷-3-醇结构与壳聚糖结构之间通过离子键连接。
  2. 根据权利要求1所述的壳聚糖衍生物，其特征在于，所述黄烷-3-醇结构选自儿茶素、表儿茶素、没食子儿茶素、表没食子儿茶素、表儿茶素没食子酸酯、没食子儿茶素没食子酸酯、表没食子儿茶素没食子酸酯、花青素或茶黄素中的至少一种。
  3. 根据权利要求1所述的壳聚糖衍生物，其特征在于，所述壳聚糖结构的脱乙酰度为50-100％。
  4. 根据权利要求1所述的壳聚糖衍生物，其特征在于，所述壳聚糖衍生物的分子量为1kDa到5000kDa。
  5. 根据权利要求1所述的壳聚糖衍生物，其特征在于，所述壳聚糖衍生物选自壳聚糖-儿茶素盐、壳聚糖-表儿茶素盐、壳聚糖-表没食子儿茶素盐、壳聚糖-表儿茶素没食子酸酯盐、壳聚糖-表没食子儿茶素没食子酸酯盐、壳聚糖-没食子儿茶素没食子酸酯盐或壳聚糖-茶黄素盐中的至少一种。
  6. 根据权利要求5所述的壳聚糖衍生物，其特征在于，所述壳聚糖-表没食子儿茶素没食子酸酯盐的结构式为，
    Figure PCTCN2021082319-appb-100001
    其中，n的取值为1-10000。
  7. 权利要求1-6中任一项所述的壳聚糖衍生物的制备方法，其特征在于，包括以下步骤：将壳聚糖与酸液混合，加热，获得壳聚糖-酸盐，然后加入黄烷-3-醇，搅拌，反应，制得所述壳聚糖衍生物。
  8. 根据权利要求7所述的制备方法，其特征在于，所述壳聚糖的分子量为1kDa到4000kDa。
  9. 根据权利要求7所述的制备方法，其特征在于，所述壳聚糖的脱乙酰度为50-100％。
  10. 根据权利要求7所述的制备方法，其特征在于，所述酸液选自盐酸、甲酸、乳酸、柠檬酸或乙酸的溶液。
  11. 根据权利要求7所述的制备方法，其特征在于，所述酸液的体积浓度为0.5-10％；所述壳聚糖与酸液的质量体积比为(5-20)g:(5-30)mL。
  12. 根据权利要求7所述的制备方法，其特征在于，所述加热的温度为30-300℃；所述加热的时间为10-500分钟。
  13. 根据权利要求7所述的制备方法，其特征在于，所述黄烷-3-醇的加入量与所述壳聚糖-酸盐的质量比为(0.1-5):1；所述反应的温度为30-300℃；所述反应的时间为10-500分钟。
  14. 根据权利要求7所述的制备方法，其特征在于，包括以下步骤：
    将壳聚糖与酸液混合，加热，然后第一次除杂，获得壳聚糖-酸盐，然后加入黄烷-3-醇，搅拌，反应，第二次除杂，制得所述壳聚糖衍生物。
  15. 权利要求1-6中任一项所述的壳聚糖衍生物在制备药品、食品、化妆品或涂料中的应用。
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