WO2020048270A1 - 一种用蜗牛酶制备壳寡糖的方法及其用途 - Google Patents

一种用蜗牛酶制备壳寡糖的方法及其用途 Download PDF

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WO2020048270A1
WO2020048270A1 PCT/CN2019/098907 CN2019098907W WO2020048270A1 WO 2020048270 A1 WO2020048270 A1 WO 2020048270A1 CN 2019098907 W CN2019098907 W CN 2019098907W WO 2020048270 A1 WO2020048270 A1 WO 2020048270A1
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chitosan
chitooligosaccharide
preparing
snail
chitooligosaccharides
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French (fr)
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苏政权
曹华
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广东药科大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the technical field of oligosaccharide preparation, and particularly relates to a method for preparing chitooligosaccharide by using snail enzyme and its use.
  • Chitosan also known as acetochitin, is a compound of chitin de-N-acetyl, which has biodegradability, safety, and good biocompatibility.
  • chitosan is a long-chain macromolecule with poor water solubility under physiological conditions and high solution viscosity, which limits its application as a biological effector.
  • Chitooligosaccharide is a degradation product of glucosamine polymer with a degree of polymerization of less than 10.
  • Chito-oligosaccharides with a polymerization degree of 5 and 6 have anti-oxidant, anti-tumor, immune-enhancing, and intestinal bacteria activation activities.
  • Suitable for clinical treatment can also be used as food additives and health products, has a wide range of applications in the fields of food, medicine and cosmetics.
  • the physical degradation method mainly degrades the chemical bonds in the chitosan molecule by breaking during the irradiation process, such as ultrasonic method, microwave radiation method, etc., the yield is relatively low, the production cost is relatively high, and it is difficult to achieve industrialization.
  • Chemical degradation methods mainly include acid hydrolysis, hydrogen peroxide, and sodium perborate oxidative degradation methods.
  • acid hydrolysis is commonly used to degrade chitosan with hydrochloric acid.
  • the enzymatic hydrolysis method mainly includes specific enzymatic hydrolysis and non-specific enzymatic hydrolysis. Although the former is highly specific for the degradation of chitosan, the current enzyme-producing ability to obtain chitosanase strains is low and the sources are limited.
  • Non-specific enzymes such as a method for preparing chitooligosaccharides using papain freeze-drying, as disclosed in Chinese Patent Document 201711093783.2, can quickly reduce the viscosity of chitosan in a short period of time, resulting in more chitooligosaccharides, but The average molecular weight of the oligosaccharide is too small, which may affect the biological activity of the product.
  • the chitosan oligosaccharides with a certain molecular weight can be obtained by the composite degradation method, the method is complicated and complicated, and it is used in commercial production. Due to restrictions, the stability and quality of the obtained chitooligosaccharides are also limited, which reduces the biological activity and use value of chitooligosaccharides.
  • the purpose of the present invention is to provide a method for preparing chitooligosaccharides by snail enzymes, which mainly aims at the problems existing in the prior art: the physical degradation method has high cost and low yield, the chemical degradation method has multiple steps, and the obtained product has more monosaccharides. 2.
  • the content of chitooligosaccharide is low and a large amount of chemical reagents will pollute the environment.
  • the common enzymolysis method is used to prepare chitooligosaccharide, the degradation efficiency of each single enzyme is not high, and the source of certain specific enzymes is limited, the price is high, and the combined
  • the application of several non-specific enzymes must also consider the problem of interaction degradation, which leads to restrictions on the use of enzymes.
  • the method for preparing chitooligosaccharide using snail enzyme provided by the present invention is simple and rapid, has little environmental pollution, and has good characteristics and ideal molecular weight of the obtained chitooligosaccharide.
  • the technical solution of the present invention is:
  • a method for preparing chitooligosaccharide using snail enzyme comprising the following steps:
  • step S2 Add a snail enzyme solution having a mass concentration of 1.0% to the chitosan solution obtained in step S1, and the mass ratio of the snail enzyme to the chitosan substrate is 1:10, and hydrolyze in a water bath at 35-45 ° C for 2 hours. After 3 hours, inactivate at 100 ° C in a water bath for 5-15 minutes, cool to room temperature, continue stirring, dropwise add a 5% NaOH solution, adjust the pH to 4-6, filter, and collect the filtrate;
  • step S3 Put the filtrate obtained in step S2 into a dialysis device with a regenerated cellulose dialysis bag with a molecular weight cut-off of 3000 Da and an outer layer with a dialysis bag with a molecular weight cut-off of 1000 Da.
  • the dialysis device is dialyzed in distilled water for 24 hours, and the dialysis is changed every 8 hours. Liquid, continuously collecting dialysate, heating and concentrating the collected dialysate on an electric furnace to obtain a concentrated solution;
  • step S4 Spread the concentrated solution obtained in step S3 on a tray with a thickness of 4 ⁇ 1 mm, put it in the refrigerator to pre-freeze, and wait until the condenser temperature drops to -50 ° C to -60 ° C, put it into a dry sublimator, Vacuum dry sublimation. When the temperature rises to 28 ° C, take it out and collect the dry powder.
  • the concentration of the acetic acid-sodium acetate buffer in step S1 is 0.2 mol / L, and the pH value is 4.5.
  • the 1% snail enzyme solution in step S2 is prepared from a 0.2 mol / L acetate-sodium acetate buffer solution with a pH value of 4.5.
  • step S2 the enzymatic hydrolysis temperature in step S2 is 40 ° C.
  • step S2 the enzymolysis time in step S2 is 2.5 h.
  • the molecular weight of the chitooligosaccharide obtained in step S3 is less than 1000 Da and 1000 Da to 3000 Da.
  • step S4 is a program cooling, the temperature is reduced by 1 ° C per minute, and the temperature is reduced to -56 ° C.
  • the present invention also provides the use of the chitooligosaccharide prepared by the method for preparing a chitooligosaccharide with a snail enzyme in the preparation of a medicine or a health product for improving idiopathic pulmonary fibrosis.
  • Chitosan is a natural cationic polymer made of N-acetyl-D-glucosamine and D-glucosamine through ⁇ -1,4 glycosidic bonds. It is composed of shrimp, crab, insect shell and The product obtained by removing the acetyl group after the chitin in the fungal cell wall is treated with concentrated alkali. There are four types of glycosidic bonds in the chitosan linear molecular structure: GlcNAc-GlcNAc, GlcNAc-GlcN, GlcN-GlcN, and GlcN-GlcNAc.
  • Snail enzyme is a mixed enzyme containing more than 20 kinds of enzymes such as cellulase, pectinase, and protease. Compared with the prior art, the enzymatic effect of using specific or non-specific enzymes is provided.
  • the natural snail provided by the present invention The enzyme degrades chitosan into chitosan oligosaccharides with a constant molecular weight of less than 1000 Da and a molecular weight of 1000-3000 Da.
  • the chitooligosaccharide concentrated solution prepared by snail enzyme is used to reduce the chitooligosaccharide concentrated solution by 1 ° C per minute to -56 ° C by a program cooling method during pre-freezing.
  • the obtained chitosan powder is light yellow. Fine texture, stable molecular structure, can be stored for a long time.
  • snail enzymes are used to prepare chitooligosaccharides, and the chitooligosaccharides having a molecular weight of less than 1000 Da have an average viscosity of 1.46 / mPa.s, and the chitooligosaccharides having a molecular weight of 1000 Da to 3000 Da have an average viscosity of 1.81 / mPa. s, all far less than the viscosity of the raw material chitosan of 40.04 / mPa.s.
  • the chitooligosaccharides obtained by the present invention have activities such as anti-oxidation, anti-tumor, improving immunity and activating intestinal bacteria, and have a wide range of applications in the fields of food, medicine and cosmetics.
  • the chitooligosaccharides prepared by the present invention Sugar has the function of improving idiopathic pulmonary fibrosis and is beneficial to the rehabilitation of patients with idiopathic pulmonary fibrosis.
  • the present invention has the following beneficial effects:
  • the method for preparing chitooligosaccharide using snail enzyme provided by the present invention is simple, fast, and safe, suitable for industrial production, and has good industrial promotion practicability and value.
  • the method for preparing chitooligosaccharides using snail enzymes obtained by the present invention obtains chitooligosaccharides with a molecular weight of less than 1000 Da and a molecular weight of 1000 Da to 3000 Da.
  • the chito oligosaccharides prepared by the present invention can significantly improve specific characteristics.
  • the activation of lung fibroblasts in rat models of idiopathic pulmonary fibrosis has a very good effect on improving idiopathic pulmonary fibrosis and is beneficial to the rehabilitation of patients with idiopathic pulmonary fibrosis.
  • the freeze-drying method used in combination with the present invention can better maintain the physical properties of chitooligosaccharide, and at the same time is conducive to maintaining the physiological activity of chitooligosaccharide, which is convenient for the preservation and further use of the product.
  • the present invention explores and optimizes specific process conditions such as enzymolysis time, enzymolysis temperature, substrate concentration, and enzyme-to-mass ratio of snail enzymatic hydrolysis of chitosan to improve enzymolysis efficiency, and combines freeze-drying method
  • process conditions such as enzymolysis time, enzymolysis temperature, substrate concentration, and enzyme-to-mass ratio of snail enzymatic hydrolysis of chitosan to improve enzymolysis efficiency, and combines freeze-drying method
  • Chitosan batch number used in the present invention 121211A, model: 95% deacetylation, manufacturer: Shandong Aokang Biotechnology Co., Ltd.); snail enzyme (BR), manufacturer: Shanghai Yuanye Biotechnology Co., Ltd .; regenerated cellulose dialysis Bag (cut-off molecular weight 1000Da and 3000Da), manufacturer: Shanghai Anpu Experimental Technology Co., Ltd.
  • Example 1 Method for preparing chitooligosaccharides by snail enzyme
  • step S2 Add a snail enzyme solution having a mass concentration of 1.0% to the chitosan solution obtained in step S1.
  • the mass ratio of the snail enzyme and the chitosan substrate is 1:10.
  • step S3 Put the filtrate obtained in step S2 into a dialysis device with a regenerated cellulose dialysis bag with a molecular weight cut-off of 3000 Da and an outer layer with a dialysis bag with a molecular weight cut-off of 1000 Da. Liquid, continuously collecting dialysate, heating and concentrating the collected dialysate on an electric furnace to obtain a concentrated solution;
  • step S4 Spread the concentrated solution obtained in step S3 on a tray with a thickness of 4 ⁇ 1 mm, put it in the refrigerator and pre-freeze it.
  • the condenser temperature drops to -56 ° C, put it in a drying sublimator and vacuum dry sublimate.
  • the temperature rises to 28 ° C take it out to obtain dry chitooligosaccharide powder with a molecular weight of less than 1000 Da and a molecular weight of 1000 Da to 3000 Da.
  • Example 2 A method for preparing chitooligosaccharides by snail enzyme
  • step S2 Add a snail enzyme solution having a mass concentration of 1.0% to the chitosan solution obtained in step S1.
  • the mass ratio of the snail enzyme and the chitosan substrate is 1:10.
  • step S3 Put the filtrate obtained in step S2 into a dialysis device with a regenerated cellulose dialysis bag with a molecular weight cut-off of 3000 Da and an outer layer with a dialysis bag with a molecular weight cut-off of 1000 Da.
  • the dialyzer is dialyzed in distilled water for 24 hours, and the dialysis is changed every 8 hours. Liquid, continuously collecting dialysate, heating and concentrating the collected dialysate on an electric furnace to obtain a concentrated solution;
  • step S4 Spread the concentrated solution obtained in step S3 on a tray with a thickness of 4 ⁇ 1mm, put it in the refrigerator and pre-freeze it.
  • the condenser temperature drops to -60 ° C, put it in a drying sublimator and vacuum dry sublimation.
  • the temperature rises to 25 ° C. take it out to obtain chitosan dry powder with a molecular weight of less than 1000 Da and a molecular weight of 1000 Da to 3000 Da.
  • Test Example 1 Investigation on the influencing factors of snail enzymatic hydrolysis to produce chitooligosaccharides
  • the enzymolysis time is selected as 0.5h, 1h, 2h, 4h, and 6h.
  • the effect of enzymolysis time on reducing sugar content was examined by measuring the reducing sugar content after the reaction as an evaluation index.
  • the experimental results of the effect of enzymatic hydrolysis time on reducing sugar content are shown in Fig. 3. It can be seen from FIG.
  • the mass concentration of chitosan is 1.0%
  • the mass ratio of enzymatic bottom is 10%
  • the enzymolysis temperature is 50 ° C
  • the pH of 4.5 is the largest when the enzymolysis is 2h.
  • the enzymolysis time 1h, 2h and 4h were selected as the three levels of the orthogonal experiment to investigate the effect of the enzymolysis time on the content of chitooligosaccharide.
  • the enzymolysis temperatures were selected to be 20 ° C, 30 ° C, 40 ° C, and 50 ° C, respectively.
  • the effect of enzymolysis temperature on reducing sugar content was investigated by measuring the reducing sugar content after the reaction as an evaluation index.
  • the experimental results of the effect of the enzymolysis temperature on the reducing sugar content are shown in Fig. 4. It can be seen from FIG.
  • the mass concentration of chitosan is 1.0%
  • the ratio of enzymatic bottom mass is 10%
  • the enzymolysis time is 2h
  • the reducing temperature is 40 ° C under the conditions of enzymolysis temperature of pH 4.5.
  • the content was the largest, and subsequently the reducing sugar content decreased sharply with the increase of the enzymatic hydrolysis temperature.
  • the enzymatic hydrolysis temperatures of 30 ° C, 40 ° C, and 50 ° C were selected as three levels of orthogonal experiments to investigate the effect of the enzymolysis temperature on the content of chitooligosaccharides.
  • the substrate concentrations (mass concentration of chitosan) were selected as 0.5%, 1%, At 1.5%, 2%, 2.5%, and 3%, the effect of substrate concentration on reducing sugar content was examined by measuring the reducing sugar content after the reaction as an evaluation index.
  • the experimental results of the effect of substrate concentration on reducing sugar content are shown in Fig. 5. It can be seen from FIG. 5 that other enzymolysis conditions remain unchanged.
  • the enzymatic bottom mass ratio is 10%
  • the enzymolysis temperature is 40 ° C.
  • the enzymolysis time is 2h
  • the pH is 4.5 when the substrate concentration is 1%. Then, with the increase of substrate concentration, the content of reducing sugar decreased slowly and then stabilized.
  • the enzymatic bottom mass ratios were selected as 5%, 10%, 15%, 20%, At 25% and 30%, by measuring the reducing sugar content after the reaction as an evaluation index, the effect of the enzyme-base mass ratio on the reducing sugar content was examined.
  • the experimental results of the effect of the enzyme base mass ratio on the reducing sugar content are shown in FIG. 6. It can be seen from FIG.
  • the mass concentration of chitosan is 1.0%
  • the temperature of enzymolysis is 40 ° C
  • the time of enzymolysis is 2h
  • the pH is 4.5.
  • the sugar content increased rapidly at 10% enzyme base mass than before, and the reducing sugar content stabilized after 10% without significant change.
  • 5%, 10%, and 15% of the enzyme base mass ratio were selected as the three levels of the orthogonal experiment to investigate the effect of the enzymatic hydrolysis time on the content of chitooligosaccharides.
  • the temperature, the enzyme substrate mass ratio (mass ratio of snail enzyme and chitosan) were selected, the enzymolysis time was used as a variable, and the L9 (3 ⁇ 4) orthogonal test was performed with the reducing sugar content as an index. Optimize the prescription and the results of the orthogonal test are shown in Table 1.
  • a UV-visible spectrophotometer was used to scan the glucosamine hydrochloride spectrum. The results are shown in Figure 1. The curve is 0.20mL, 0.40mL, 0.60mL, 0.80mL, and 1.00mL glucosamine hydrochloride standard solutions from bottom to top. The spectral curve of DNS method. It can be seen from FIG. 1 that the maximum absorption peak wavelength of the glucosamine hydrochloride series standard solution is 485 nm.
  • the regression curve is obtained using the glucosamine concentration (mg / mL) as the abscissa and the absorbance value (mean) as the ordinate.
  • the content of reducing oligosaccharides in the enzymatic hydrolysis solution was used to represent the content of chitooligosaccharides, and the content of chitosan in the enzymatic hydrolysis solution was calculated.
  • the freeze-drying yield is the mass of chitosan (m a ) after weighing constant weight divided by the total mass of chitosan added (m 0 ), multiplied by 100%.
  • m a amount of chitooligosaccharide produced by enzymatic hydrolysis of chitosan (mg)
  • Appropriate amounts of 1000Da and 3000Da chitosan products were weighed, and distilled water was used to prepare a 1% chitosan aqueous solution, and then samples with a molecular weight of less than 1000Da chitosan were made into a 1% aqueous solution.
  • a digital rotary viscometer was used to measure the apparent viscosities of the obtained products with a molecular weight of between 1000 Da and 3000 Da, chitosan oligosaccharides with a molecular weight of less than 1000 Da, and a 1% chitosan solution.
  • the three determination results of the apparent viscosity of the 1% chitosan solution were 39.85, 40.27, and 39.99 mPa.s, respectively, and the average values were 40.04 mPa.s.
  • the measurement results of the chitosan content prepared by the present invention are shown in Table 2.
  • Chito-oligosaccharide raw materials with molecular weights of 1000 Da and 1000-3000 Da obtained from snail enzymes of the present invention were respectively taken, and their average molecular weights were determined by the reducing sugar method.
  • the molecular weight of the chitooligosaccharide prepared by the snail enzyme of the present invention is less than 1000 Da and 1000 Da to 3000 Da.
  • the viscosity is between 1.4 and 1.9 mPa.s, which is far less than 40 mPa.s of chitosan, which meets the chitosan viscosity standard.
  • the results of using the reducing sugar method to measure the chitosan oligosaccharides prepared by the snail enzyme of the present invention show that the chitosan oligosaccharides of Examples 1 and 2 having molecular weights less than 1000 Da and 1000 Da to 3000 Da obtained by the dialysis method used in the present invention are measured.
  • the actual average molecular weights obtained were 482 ⁇ 12 Da, 468 ⁇ 20 Da, 1382 ⁇ 46 Da, and 1298 ⁇ 53 Da, indicating that the molecular weight distribution of the chitooligosaccharide prepared by the present invention is uniform.
  • Test example three Effect of chitooligosaccharide on idiopathic pulmonary fibrosis
  • Test subjects Select 40 healthy male SD rats, weighing 250 ⁇ 20g, provided by the Experimental Animal Center of Sun Yat-sen University.
  • Test materials Chitosan oligosaccharide and pirfenidone capsules prepared in Example 1 were purchased from Beijing Contini Pharmaceutical Co., Ltd., and the national standard H20133376.
  • Ten rats were randomly selected from the 50 rats as the control group. The remaining rats were intraperitoneally injected with 3% sodium pentobarbital at a concentration of 30 mg / kg. After anesthesia, the rats were fixed on their backs and the neck was routinely disinfected after skin preparation. Put on a towel, cut open the skin, and bluntly separate the exposed trachea layer by layer. Pass the obliquely into the trachea with a 1mL syringe and quickly inject 0.2 to 0.3ml of bleomycin solution into the lungs. Immediately raise the rat plate and shake it left and right for 2min After that, disinfect the skin and suture the wound again.
  • the 40 rats successfully modeled were randomly divided into the control group, the model group, the pirfenidone group, and the test 1 group.
  • the doses of each group were as follows:
  • Control group intragastric saline of equal volume
  • Pirfenidone group gavage of 10 mg / kg of pirfenidone
  • Test 1 group intragastrically administered 10 ml / kg of chitooligosaccharide prepared in Example 1 (dissolved in normal saline);
  • LN serum laminin
  • the pirfenidone group and the test 1 group can reduce the lung coefficient of the idiopathic pulmonary fibrosis model, and the difference is statistically significant.
  • the nisinone group is equivalent.
  • the test group 1 can also significantly reduce the LN content of the idiopathic pulmonary fibrosis model, indicating that the low-molecular-weight chitooligosaccharide prepared by the present invention has a significant improvement effect on idiopathic pulmonary fibrosis.

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Abstract

属于低聚糖制备技术领域，具体涉及一种用蜗牛酶制备壳寡糖的方法及其应用。所提供的用蜗牛酶制备壳寡糖的方法，对蜗牛酶酶解壳聚糖的酶解时间、酶解温度、底物浓度、酶底质量比等具体的工艺条件进行探索和优化，提高酶解效率，结合冷冻干燥法，保持了壳寡糖的性状，便于产品的保存和进一步使用，为蜗牛酶制备壳寡糖的大生产提供依据。制得的壳寡糖还可以显著的改善特发性肺纤维化大鼠模型的纤维细胞活化，对特发性肺纤维化具有很好的改善作用，有利于特发性肺纤维化患者的康复。

Description

一种用蜗牛酶制备壳寡糖的方法及其用途 技术领域
本发明属于低聚糖制备技术领域，具体涉及一种用蜗牛酶制备壳寡糖的方法及其用途。
背景技术
壳聚糖又称乙酰甲壳素，是甲壳素脱N-乙酰基的化合物，具有生物可降解性、安全性、良好的生物兼容性。但壳聚糖是长链大分子，生理条件下水溶性差，溶液黏度大，使其作为生物效应剂的应用受到很大限制。壳寡糖是其降解产物，指聚合度小于10的氨基葡萄糖的聚合物，相对分子质量小，水溶性好，易被分散和吸收，且具备许多与壳聚糖相似的生物学性质，特别是聚合度为5和6的壳寡糖，具有抗氧化、抗肿瘤、免疫增强、活化肠道菌等活性，在体内治疗肿瘤时，不引起急性中毒以及体质量的迅速下降，毒副作用较小，适用于临床治疗，也可作为食品添加剂和保健品，在食品、医药及化妆品领域有着广泛的应用。
目前利用壳聚糖制备壳寡糖的方法主要有3种：物理降解法、化学降解法和酶解法。物理降解法主要通过将壳聚糖分子内的化学键在辐射过程中发生断裂而降解，如超声波法、微波辐射法等，收率较低、生产成本相对较高，实现工业化难度较大。化学降解法主要包括酸解法、过氧化氢及过硼酸钠氧化降解法等，如酸解法常用盐酸降解壳聚糖，虽然适合工业化生产，但是根据EinbuAslak等人对于甲壳素在盐酸中的解聚和乙酰化反应的研究发现酸解法所得产品单糖较多，壳寡糖含量低，生物活性难以体现，且制备过程中需要大量的化学试剂，会严重污染环境。
酶解法主要有专一性酶解和非专一性酶解，前者虽然对壳聚糖的降解专一性强，但目前获得壳聚糖酶菌株的产酶能力较低，且来源有限。非专一性酶，如中国专利文献201711093783.2公开的一种采用木瓜蛋白酶冷冻干燥制备壳寡糖的方法，能在短时间内迅速降低壳聚糖黏度，得到较多的壳寡糖，但是所得壳寡糖平均分子量过小，可能会对产品的生物活性有影响。可见单独使用一种非专一性酶制备壳寡糖时，产品往往黏度较低，寡糖含量少，单糖多，产品质量不好，或者降解反应效率低，难以实际应用，更值得注意的是，专一性酶价格昂贵、难以商业化。
近年来，采用多种非专一性酶联合降解壳聚糖制备壳寡糖成为研究热点。如蛋白酶、纤维素酶等联合应用，一方面能快速降低反应物壳聚糖的黏度，另一方面能获得大量生物活性 高的聚合度为3～7的壳寡糖，但该方法必须使用多种酶，各个酶之间的配比，相互协调作用不一，得到的壳寡糖分子量不一，虽用复合降解法可得到确定分子量的壳寡糖，但该方法繁琐复杂，商业化生产应用受到限制，所得的壳寡糖的稳定性和质量也受限，降低了壳寡糖的生物活性和用价值。
发明内容
本发明的目的在于提供一种用蜗牛酶制备壳寡糖的方法，主要针对现有技术存在的问题：物理降解法成本较高且产率低，化学降解法步骤繁多，所得产品单糖较多、壳寡糖含量低且大量的化学试剂会污染环境，而使用常用的酶解法制备壳寡糖时，各个单酶的降解效率不高且某些专一性酶来源受限、价格昂贵，联合应用几种非专一性酶时又必须考虑相互作用降解问题，导致选用酶时受到限制。本发明提供的用蜗牛酶制备壳寡糖的方法简便、快速，对环境污染小，所得壳寡糖性状良好、分子量理想。
为了解决上述技术问题，本发明的技术方案是：
一种用蜗牛酶制备壳寡糖的方法，包括以下步骤：
S1取壳聚糖原料干燥至恒重，溶解于乙酸-乙酸钠缓冲液中，搅拌溶解，配制成质量浓度为1.0％的壳聚糖溶液；
S2向步骤S1所得壳聚糖溶液中加入质量浓度为1.0％的蜗牛酶溶液，所述蜗牛酶和壳聚糖底物质量比为1:10，于水浴箱35～45℃中酶解2h～3h后，100℃水浴5～15min灭活，冷却至室温，连续搅拌，滴加质量浓度为5％的NaOH溶液，调节pH值至4～6，过滤，收集滤液；
S3将步骤S2所得滤液装入内层装有截留分子量为3000Da再生纤维素透析袋，外层包有截留分子量为1000Da再生纤维素透析袋的透析装置，在蒸馏水中透析24h，每8h换一次透析液，连续收集透析液，将收集的透析液于电炉上加热浓缩，得浓缩液；
S4将步骤S3中所得浓缩液平铺在托盘中，厚度为4±1mm，放入冰箱中预冷冻，待到冷凝器温度降至-50℃～-60℃时，放入干燥升华器中，真空干燥升华，待温度回升至28℃时，取出，收集干粉即得。
进一步地，所述步骤S1中的乙酸-乙酸钠缓冲液浓度为0.2mol/L，pH值为4.5。
进一步地，所述步骤S2中的1％蜗牛酶溶液由pH值为4.5的0.2mol/L的乙酸-乙酸钠缓冲溶液配制而成。
进一步地，所述步骤S2中的酶解温度为40℃。
进一步地，所述步骤S2中的酶解时间为2.5h。
进一步地，所述步骤S3中获得的壳寡糖分子量为小于1000Da和1000Da～3000Da。
进一步地，所述步骤S4中的预冷冻为程序降温，每分钟温度降低1℃，温度降低至-56℃。
此外，本发明还提供了所述的用蜗牛酶制备壳寡糖的方法制备得到的壳寡糖在制备改善特发性肺纤维化药品或保健品中的用途。
壳寡糖是一种由N-乙酰-D-氨基葡萄糖和D-氨基葡萄糖通过β-1，4糖苷键连接而成的天然的阳离子高分子聚合物，是由虾、蟹、昆虫的外壳及真菌细胞壁中的甲壳素经浓碱处理后脱去乙酰基而得到的产物。在壳聚糖直链分子结构中存在4种类型的糖苷键：GlcNAc-GlcNAc、GlcNAc-GlcN、GlcN-GlcN和GlcN-GlcNAc。蜗牛酶是一种含有纤维素酶、果胶酶、蛋白酶等20多种酶的混合酶，相对现有技术使用专一性酶或非专一性酶的酶解效果，本发明提供的天然蜗牛酶将壳聚糖降解为产物质量均恒的分子量小于1000Da和分子量为1000～3000Da的壳寡糖。
本发明采用蜗牛酶制得的壳寡糖浓缩液，在预冷冻时，将壳寡糖浓缩液以程序降温法每分钟降低1℃，直至-56℃，所得的壳寡糖粉末为淡黄色，质地细腻，分子结构稳定，可长期保存。
本发明用蜗牛酶制备壳寡糖的方法制备得到的壳寡糖分子量为小于1000Da壳寡糖的平均粘度为1.46/mPa.s，分子量为1000Da～3000Da的壳寡糖平均粘度为1.81/mPa.s，均远小于原料壳聚糖为40.04/mPa.s的粘度。本发明得到的壳寡糖有抗氧化、抗肿瘤、提高免疫力和活化肠道菌等活性，在食品、医药及化妆品领域有着广泛的应用，另外通过实验我们还发现，本发明制备的壳寡糖具有改善特发性肺纤维化的功能，有利于待发性肺纤维化患者的康复。
与现有技术相比，本发明具有以下有益效果：
(1)本发明提供的用蜗牛酶制备壳寡糖的方法简便、快速、安全，适合产业化生产，具有良好的工业推广实用性和价值。
(2)本发明提供的用蜗牛酶制备壳寡糖的方法得到了分子量小于1000Da和分子量为1000Da～3000Da的壳寡糖，通过实验我们发现，本发明制得的壳寡糖可以显著的改善特发性肺纤维化大鼠模型的肺纤维细胞活化，对特发性肺纤维化具有很好的改善作用，有利于特发性肺纤维化患者的康复。
(3)本发明结合采用的冷冻干燥法能够较好地保持壳寡糖的物理性状，同时有利于壳寡糖生理活性的保持，便于产品的保存和进一步使用。
(4)本发明对蜗牛酶酶解壳聚糖的酶解时间、酶解温度、底物浓度、酶底质量比等具体的工艺条件进行探索和优化，提高酶解效率，并结合冷冻干燥法，进行了一系列的试验研究和筛选，为蜗牛酶制备壳寡糖的大生产提供依据。
附图说明
图1氨基葡萄糖盐酸盐标准系列溶液的光谱扫描图谱；
图2氨基葡萄糖盐酸盐标准曲线；
图3酶解时间-还原糖含量变化曲线；
图4酶解温度-还原糖含量变化曲线；
图5底物浓度-还原糖含量变化曲线；
图6酶底质量比-还原糖含量变化曲线。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的保护范围之内。
本发明中使用的壳聚糖批号：121211A，型号：脱乙酰度95％，厂家：山东奥康生物科技有限公司)；蜗牛酶(BR)，厂家：上海源叶生物科技有限公司；再生纤维素透析袋(截留分子量1000Da和3000Da)，厂家：上海安谱实验科技股份有限公司。
实施例1、一种用蜗牛酶制备壳寡糖的方法
S1取壳聚糖原料干燥至恒重，溶解于pH值4.5的0.2mol/L乙酸-乙酸钠缓冲液中，搅拌溶解，配制成质量浓度为1.0％的壳聚糖溶液；
S2向步骤S1所得壳聚糖溶液中加入质量浓度为1.0％的蜗牛酶溶液，所述蜗牛酶和壳聚糖底物质量比为1:10，于40℃水浴箱中酶解2.5h后，100℃水浴10min灭活，冷却至室温，连续搅拌，滴加质量浓度为5％的NaOH溶液，调节pH值至4.5，过滤，收集滤液；
S3将步骤S2所得滤液装入内层装有截留分子量为3000Da再生纤维素透析袋，外层包有截留分子量为1000Da再生纤维素透析袋的透析装置，在蒸馏水中透析24h，每8h换一次透 析液，连续收集透析液，将收集的透析液于电炉上加热浓缩，得浓缩液；
S4将步骤S3中所得浓缩液平铺在托盘中，厚度为4±1mm，放入冰箱中预冷冻，待到冷凝器温度降至-56℃时，放入干燥升华器中，真空干燥升华，待温度回升至28℃时，取出，即得分子量小于1000Da和分子量为1000Da～3000Da的壳寡糖干粉。
实施例2、一种用蜗牛酶制备壳寡糖的方法
S1取壳聚糖原料干燥至恒重，溶解于pH值4.5的0.2mol/L乙酸-乙酸钠缓冲液中，搅拌溶解，配制成质量浓度为1.0％的壳聚糖溶液；
S2向步骤S1所得壳聚糖溶液中加入质量浓度为1.0％的蜗牛酶溶液，所述蜗牛酶和壳聚糖底物质量比为1:10，于40℃水浴箱中酶解2h后，100℃水浴15min灭活，冷却至室温，连续搅拌，滴加质量浓度为5％的NaOH溶液，调节pH值至6，过滤，收集滤液；
S3将步骤S2所得滤液装入内层装有截留分子量为3000Da再生纤维素透析袋，外层包有截留分子量为1000Da再生纤维素透析袋的透析装置，在蒸馏水中透析24h，每8h换一次透析液，连续收集透析液，将收集的透析液于电炉上加热浓缩，得浓缩液；
S4将步骤S3中所得浓缩液平铺在托盘中，厚度为4±1mm，放入冰箱中预冷冻，待到冷凝器温度降至-60℃时，放入干燥升华器中，真空干燥升华，待温度回升至25℃时，取出，即得分子量小于1000Da和分子量为1000Da～3000Da的壳寡糖干粉。
试验例一、蜗牛酶酶解制备壳寡糖的影响因素考察
1.酶解时间单因素实验
在固定壳聚糖质量浓度为1.0％，酶底质量比为10％，酶解温度为50℃，pH值为4.5的条件下，选取酶解时间分别为0.5h、1h、2h、4h、6h、8h时，通过测定反应后还原糖含量为评价指标，考察酶解时间对还原糖含量的影响。酶解时间对还原糖含量的影响的实验结果如图3所示。由图3可知，其他酶解条件不变，壳聚糖质量浓度为1.0％，酶底质量比为10％，酶解温度50℃，pH4.5的条件下酶解2h时还原糖含量最大，随后随着酶解时间的增长，还原糖含量下降后又趋于平稳。根据单因素实验结果分析后，选择酶解时间1h、2h和4h作为正交实验的三个水平来考察酶解时间对壳寡糖含量的影响。
2.酶解温度单因素实验
在固定壳聚糖质量浓度为1.0％，酶底质量比为10％，酶解时间为2h，pH值为4.5的条 件下，选取酶解温度分别为20℃、30℃、40℃、50℃、60℃和70℃时，通过测定反应后还原糖含量为评价指标，考察酶解温度对还原糖含量的影响。酶解温度对还原糖含量的影响的实验结果如图4所示。由图4可知，其他酶解条件不变，壳聚糖质量浓度为1.0％，酶底质量比为10％，酶解时间为2h，pH4.5的条件下酶解温度为40℃时还原糖含量最大，随后随着酶解温度的增加，还原糖含量急剧下降。根据单因素实验结果分析后，选择酶解温度30℃、40℃和50℃为正交实验的三个水平来考察酶解温度对壳寡糖含量的影响。
3.底物浓度单因素实验
在固定酶底质量比为10％，酶解温度为40℃，酶解时间为2h，pH值为4.5的条件下，选取底物浓度(壳聚糖质量浓度)分别为0.5％、1％、1.5％、2％、2.5％和3％时，通过测定反应后还原糖含量为评价指标，考察底物浓度对还原糖含量的影响。底物浓度对还原糖含量的影响的实验结果如图5所示。由图5可知，其他酶解条件不变，酶底质量比为10％，酶解温度40℃，酶解时间为2h，pH值为4.5的条件下底物浓度为1％时还原糖含量最大，随后随着底物浓度的增加，还原糖含量缓慢下降后又趋于平稳。
4.酶底质量比对反应的影响
在固定壳聚糖质量浓度为1.0％，酶解温度为40℃，酶解时间为2h，pH为4.5的条件下，选取酶底质量比分别为5％、10％、15％、20％、25％和30％时，通过测定反应后还原糖含量为评价指标，考察酶底质量比对还原糖含量的影响。酶底质量比对还原糖含量的影响的实验结果如图6所示。由图6可知，其他酶解条件不变，壳聚糖质量浓度为1.0％，酶解温度40℃，酶解时间为2h，pH4.5的条件下，随着酶底质量比的增加，还原糖含量在10％酶底质量比前迅速升高，在10％后还原糖含量趋于平稳，变化不明显。根据单因素实验结果分析后，选择酶底质量比5％、10％和15％作为正交实验的三个水平来考察酶解时间对壳寡糖含量的影响。
5.正交试验优选制备方法
根据上述1-4中结果，选取温度、酶底物质量比(蜗牛酶和壳聚糖质量比)，酶解时间作为变量，进行L9(3^4)正交试验，以还原糖含量作为指标优化处方，正交试验结果如表1所示。
表1：L9(3^4)正交试验极差分析
Figure PCTCN2019098907-appb-000001
Figure PCTCN2019098907-appb-000002
注：K 1、K 2、K 3分别为每次实验各因素的水平的实验结果之和；R 1、R 2、R 3分别为各因素各水平结果的平均值；R 极为各因素各水平结果的平均值的差值。
由表1可知，正交试验极差分析结果显示，极差最大值对应的因素即影响还原糖含量(壳寡糖含量)的最大因素为酶底质量比，其次为酶解时间，影响最小的为酶解温度。最优的试验方案为A 2B 2C 2，即酶解温度为40℃、酶底质量比为10％和酶解时间为2h。
试验例二、壳寡糖含量测定
1、氨基葡萄糖盐酸盐吸收波长扫描
分别于10mL比色管中加入0.00mL、0.20mL、0.40mL、0.60mL、0.80mL和1.00mL质量浓度为0.1％氨基葡萄糖标准溶液，再分别加入1.00mL、0.80mL、0.60mL、0.40mL、0.20mL和0.00mL的蒸馏水，之后分别加入1mLDNS试剂，沸水浴10min显色，取出自然冷却至室温后分别用蒸馏水定容至10mL；
用紫外-可见分光光度计对氨基葡萄糖盐酸盐进行光谱扫描，结果如图1，曲线由下到上依次为0.20mL、0.40mL、0.60mL、0.80mL和1.00mL氨基葡萄糖盐酸盐标准溶液DNS法的光谱曲线。由图1可知，氨基葡萄糖盐酸盐系列标准溶液的最大的吸收峰波长为485nm。
2、氨基葡萄糖盐酸盐标准曲线绘制
以0号管为参比，波长485nm，测定不同浓度梯度氨基葡萄糖盐酸盐紫外吸收强度(n＝3)，绘制标砖曲线，如图2。
由图2可知，以氨基葡萄糖浓度(mg/mL)为横坐标，吸光度值(均值)为纵坐标，作回归曲线，得回归方程y＝10.041x-0.0162，R 2＝0.9987，线性良好。
根据此标准曲线，采用酶解液中还原糖的含量来表示壳寡糖含量，从而计算酶解溶液中的壳寡糖含量。
3、壳寡糖含量测定
取实施例1和2制备得到的壳寡糖500μL，置于10mL比色管中，加蒸馏水至1mL，再加入1mLDNS试剂，沸水浴10min显色，取出自然冷却至室温，蒸馏水定容至10mL，以空白管为参比，测定吸光度值，计算壳寡糖的含量。壳寡糖的产率计算：
壳寡糖产率及相关计算结果如表2所示。
冷冻干燥产率，即为称量恒重后的壳寡糖质量(m a)除以加入的壳聚糖总质量(m 0)，乘以100％。
Figure PCTCN2019098907-appb-000003
式中：
η％：冷冻干燥产率
m a：壳聚糖酶解产生的壳寡糖的量(mg)
m 0：壳聚糖的量(mg)
4、壳寡糖产品的定性鉴别
分别称量适量的1000Da和3000Da的壳寡糖产品，用蒸馏水配制成1％壳寡糖水溶液，再称量小于1000Da分子量壳寡糖样品制成1％水溶液。采用数字式旋转粘度计分别测定所得产品1000Da～3000Da分子量壳寡糖、小于1000Da分子量壳寡糖样品、1％壳聚糖溶液的表观粘度。1％壳聚糖溶液表观粘度的三次测定结果分别为39.85、40.27、39.99mPa.s，平均值为40.04mPa.s，本发明制得的壳寡糖含量测定结果如表2所示。
5、还原糖法测壳寡糖原料药的平均分子量
分别取本发明用蜗牛酶制得的壳寡糖的分子量1000Da和1000～3000Da的壳寡糖原料药，用还原糖法测定其平均分子量。
6、试验结果
壳寡糖含量、水分和产品鉴定结果如表2。
表2.壳寡糖鉴定结果
Figure PCTCN2019098907-appb-000004
由表2结果可看出，本发明用蜗牛酶制得的壳寡糖，经透析袋分离后，得到的分子量小于1000Da和1000Da～3000Da的壳寡糖产率均在21～26％之间，该方法制得的壳寡糖重复性好(RSD<5％)，所制得的分子量小于1000Da和1000Da～3000Da的壳寡糖质量分布较均横。
本发明用蜗牛酶制得的分子量小于1000Da和1000Da～3000Da壳寡糖的粘度为1.4～1.9mPa.s之间，远小于壳聚糖的40mPa.s，符合壳寡糖粘度标准。
使用还原糖法测定本发明用蜗牛酶制得的壳寡糖的结果显示，本发明采用的透析法制得的分子量小于1000Da和1000Da～3000Da的实施例1和实施例2的壳寡糖，所测得的实际平均分子量为482±12Da、468±20Da和1382±46Da、1298±53Da，说明本发明制得的壳寡糖分子量分布均匀。
试验例三、壳寡糖对特发性肺纤维化的影响试验
1、试验对象：选取健康雄性SD大鼠40只，体重为250±20g，由中山大学实验动物中心提供。
2、试验材料：实施例1制备的壳寡糖，吡非尼酮胶囊，购于北京康蒂尼药业有限公司，国药准字H20133376。
3、特发性肺纤维化动物模型建立：
从50只大鼠中随机挑选10只作为对照组大鼠，剩余的大鼠腹腔注射3％浓度为30mg/kg的戊巴比妥钠，麻醉后仰卧固定鼠台，颈部备皮后常规消毒铺巾，切开皮肤后逐层钝性分离暴露气管，以1mL的注射器斜行刺入气管内，向肺内快速注射博来霉素溶液0.2～0.3ml，立 即竖起鼠板，左右摇晃2min后，再次消毒皮肤缝合伤口。
4、试验方法：
将建模成功的40只大鼠随机分为对照组、模型组、吡非尼酮组、试验1组，各组给药量如下：
对照组：灌胃等体积的生理盐水；
模型组：灌胃等体积的生理盐水；
吡非尼酮组：灌胃10mg/kg的吡非尼酮；
试验1组：灌胃10ml/kg的实施例1制备的壳寡糖(生理盐水溶解)；
试验期间大鼠自由饮水，标准饮食。每周测量大鼠体重一次。于试验开始给药后的第4周处死大鼠。采用股动脉放血法处死大鼠，戊巴比妥钠麻醉后，将大鼠仰卧固定于鼠台上，开胸显露心肺，取下肺脏后用生理盐水反复清洗，用电子天平称量体重和肺重，计算肺系数(肺脏重量/体重×100％)，使用试剂盒检测大鼠的血清层粘连蛋白(LN)含量。
5、试验结果：
5.1、实施例1制备的壳寡糖对特发性肺纤维化模型大鼠肺系数和LN含量的影响如表3所示。
表3 实施例1制备的壳寡糖对特发性肺纤维化模型大鼠肺系数和LN含量的影响
组别 肺系数 LN含量(pg/mL)
对照组 0.44±0.023 80.86±10.50
模型组 0.57±0.04 138.64±16.33
吡非尼酮组 0.47±0.04 * 85.24±11.56 **
试验1组 0.49±0.06 * 101.78±12.14 *
与模型组相比， *P<0.05， **P<0.01。
由表3可知，与模型组相比，吡非尼酮组和试验1组均可以降低特发性肺纤维化模型的肺系数，差异具有统计学意义，且试验1组的降低效果与吡非尼酮组相当，同时，试验1组也可以显著降低特发性肺纤维化模型的LN含量，说明本发明制得的低分子壳寡糖对特发性肺纤维化具有显著的改善作用。

Claims (8)

  1. 一种用蜗牛酶制备壳寡糖的方法，其特征在于，包括以下步骤：
    S1取壳聚糖原料干燥至恒重，溶解于乙酸-乙酸钠缓冲液中，搅拌溶解，配制成质量浓度为1.0％的壳聚糖溶液；
    S2向步骤S1所得壳聚糖溶液中加入质量浓度为1.0％的蜗牛酶溶液，所述蜗牛酶和壳聚糖底物质量比为1:10，于水浴箱35～45℃中酶解2h～3h后，100℃水浴5～15min灭活，冷却至室温，连续搅拌，滴加质量浓度为5％的NaOH溶液，调节pH值至4～6，过滤，收集滤液；
    S3将步骤S2所得滤液装入内层装有截留分子量为3000Da再生纤维素透析袋，外层包有截留分子量为1000Da再生纤维素透析袋的透析装置，在蒸馏水中透析24h，每8h换一次透析液，连续收集透析液，浓缩，得浓缩液；
    S4将步骤S3中所得浓缩液平铺在托盘中，厚度为4±1mm，放入冰箱中预冷冻，待到冷凝器温度降至-50℃～-60℃时，真空干燥升华，待温度回升至28℃时，取出，收集干粉即得。
  2. 如权利要求1所述的用蜗牛酶制备壳寡糖的方法，其特征在于，所述步骤S1中的乙酸-乙酸钠缓冲液浓度为0.2mol/L，pH值为4.5。
  3. 如权利要求1所述的用蜗牛酶制备壳寡糖的方法，其特征在于，所述步骤S2中的1％蜗牛酶溶液由pH值为4.5的0.2mol/L的乙酸-乙酸钠缓冲溶液配制而成。
  4. 如权利要求1所述的用蜗牛酶制备壳寡糖的方法，其特征在于，所述步骤S2中的酶解温度为40℃。
  5. 如权利要求1所述的用蜗牛酶制备壳寡糖的方法，其特征在于，所述步骤S2中的酶解时间为2.5h。
  6. 如权利要求1所述的用蜗牛酶制备壳寡糖的方法，其特征在于，所述步骤S3中获得的壳寡糖分子量小于1000Da和1000Da～3000Da。
  7. 如权利要求1所述的用蜗牛酶制备壳寡糖的方法，其特征在于，所述步骤S4中的预冷冻为程序降温，每分钟温度降低1℃。
  8. 如权利要求1～7任一所述的用蜗牛酶制备壳寡糖的方法制备得到的壳寡糖在制备改善特发性肺纤维化药品或保健品中的用途。
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CN1417342A (zh) * 2001-11-06 2003-05-14 中国科学院大连化学物理研究所 利用蜗牛酶降解天然产物及多糖的方法
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CN107739418A (zh) * 2017-11-08 2018-02-27 广东药科大学 一种采用木瓜蛋白酶冷冻干燥制备壳寡糖的方法
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Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1417342A (zh) * 2001-11-06 2003-05-14 中国科学院大连化学物理研究所 利用蜗牛酶降解天然产物及多糖的方法
CN106011127A (zh) * 2016-06-25 2016-10-12 陈毅忠 一种利用固定化蜗牛酶制备壳寡糖的方法
CN109136307A (zh) * 2018-09-06 2019-01-04 广东药科大学 一种用蜗牛酶制备壳寡糖的方法及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ZHANG, TAO ET AL.: "Study on Chitooligosaccharide Production by Chitosan Hydrolysis with Snailase", WEST CHINA JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 25, no. 3, 15 June 2010 (2010-06-15), pages 313 - 315 *
ZHOU, LULU ET AL.: "Chitosan Aerosol Inhalation Alleviates Lipopolysaccharideinduced Pulmonary Fibrosis in Rats", EXPERIMENTAL LUNG RESEARCH, vol. 40, 16 October 2014 (2014-10-16), pages 467 - 473 *

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