WO2020177455A1 - 一种小分子透明质酸或其盐及其制备方法 - Google Patents

一种小分子透明质酸或其盐及其制备方法 Download PDF

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WO2020177455A1
WO2020177455A1 PCT/CN2019/128316 CN2019128316W WO2020177455A1 WO 2020177455 A1 WO2020177455 A1 WO 2020177455A1 CN 2019128316 W CN2019128316 W CN 2019128316W WO 2020177455 A1 WO2020177455 A1 WO 2020177455A1
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hyaluronic acid
salt
hyaluronidase
small molecule
molecule hyaluronic
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PCT/CN2019/128316
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French (fr)
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韩秀云
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山东安华生物医药股份有限公司
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Priority to JP2020533114A priority Critical patent/JP7105888B2/ja
Priority to EP19842856.7A priority patent/EP3730623A4/en
Publication of WO2020177455A1 publication Critical patent/WO2020177455A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02001—Hyaluronate lyase (4.2.2.1)
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/99—Other carbon-oxygen lyases (4.2.99)

Definitions

  • the present disclosure relates to the technical field of bioengineering, in particular to a small molecule hyaluronic acid or a salt thereof and a preparation method thereof.
  • Hyaluronic acid is an acidic mucopolysaccharide, an unbranched polymer glycosaminoglycan composed of repeating units of N-acetylglucosamine and D-glucuronic acid disaccharide, present in animal tissues In the intercellular substance and the capsule of some bacteria, it is widely used in cosmetics, food and medicine industries.
  • small-molecule hyaluronic acid exhibits very strong biological activity (Hui ZM, a bioactive hyaluronic acid fragment, its manufacturing mehtod, its formulation and its applications, patent number 2014101553593.5.), which can improve skin immune function and eliminate Skin inflammation, promotion of wound healing, treatment of acute and chronic pharyngitis and treatment of gingivitis, etc. It is an activator of immune cells and cytokines.
  • the small molecule hyaluronic acid has good permeability and absorption, and is easily absorbed by the dermis and intestines, and can quickly replenish the hyaluronic acid lost in the body. Therefore, small molecule hyaluronic acid has broad application prospects in the fields of food health, cosmetics and clinical medicine.
  • the degradation methods mainly include physical degradation, chemical degradation and biological degradation.
  • the physical degradation methods mainly include heating, mechanical shearing force, ultraviolet rays, ultrasonic waves, 60C irradiation and gamma-ray radiation, etc., which can degrade HA.
  • the molecular weight prepared by the physical method is higher than 10,000, the resulting molecular weight range distribution is larger, and the product stability is poor.
  • the chemical degradation methods of HA include hydrolysis and oxidative degradation.
  • the hydrolysis methods are divided into acid hydrolysis (HCL) and alkali (NaOH) hydrolysis.
  • the commonly used oxidants for oxidative degradation are sodium hypochlorite and hydrogen peroxide.
  • the relative molecular mass of the degradation product using chemical degradation method can be controlled by changing the addition amount of acid, alkali or oxidant and the reaction time, the degradation cost is low, and it is easy to produce on a large scale, but the chemical degradation method introduces chemical reagents and the reaction conditions are complicated. , It is easy to affect the properties of HA and bring difficulties to the purification of products, and produce a lot of industrial wastewater.
  • Biological enzymatic degradation is the degradation of ⁇ -glycosidic bonds under the action of hyaluronidase.
  • Hyaluronidase also called hyaluronidase
  • hyaluronidase is a glycosidase that can make HA low-molecular-weight. It can also degrade acid mucopolysaccharides in other body tissues. It is widely present in eukaryotes and prokaryotes and has specificity. Physiological activity.
  • the invention patent with application number CN201611127881.9 provides a method for preparing ultra-low molecular weight hyaluronic acid oligosaccharides and their salts by combining solid-liquid biphasic enzymatic hydrolysis and ultrafiltration. Enzymatic hydrolysis of hyaluronic acid is achieved .
  • the current hyaluronidase is usually extracted and prepared from animal tissues, which is expensive and not suitable for industrial applications. Therefore, a low-cost, simple process, suitable for industrialized large-scale production of small-molecule hyaluronic acid method has important application potential.
  • the hyaluronic acid extraction process is mostly the organic reagent precipitation method, which causes environmental pollution, high energy consumption, and low yield of small molecules of hyaluronic acid.
  • spray drying is used to extract hyaluronic acid.
  • the inlet temperature is above 180°C, and the physical or chemical properties of heat-sensitive materials are easily changed, especially for biologically active materials such as bacteria, Active enzymes will be inactivated.
  • the purpose of the present disclosure includes, for example, providing a small molecule hyaluronic acid or a salt thereof and a preparation method thereof to overcome the above-mentioned drawbacks.
  • the method provided in the embodiments of the present disclosure mainly uses hyaluronidase produced by Lactobacillus plantarum and low-temperature spray drying to degrade to obtain small-molecule hyaluronic acid or its salt.
  • Enzymatic hydrolysis reaction solution add hyaluronidase to purified water, start stirring, slowly add solid powder of hyaluronic acid or its salt, and obtain an enzymatic hydrolysis solution through enzymatic hydrolysis.
  • the hyaluronidase is a plant Lactobacillus mutagenic strain CnT012-56 is produced by fermentation; the strain is classified and named Lactobacillus plantarum, and its preservation unit: General Microbiology Center of China Microbial Culture Collection Management Committee, Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing , Preservation date: November 28, 2018, Preservation number: CGMCC NO.16836;
  • Hyaluronidase inactivation heating the enzymatic hydrolysis solution of step (1) to denature and inactivate the hyaluronidase in the enzymatic hydrolysis solution;
  • step (4) Low-temperature spray drying: the small-molecule hyaluronic acid and its salt solution in step (4) are spray-dried at low-temperature to obtain a solid powder of small-molecule hyaluronic acid and its salt with a desired molecular weight.
  • step (1) first add hyaluronidase to the purified water, turn on the stirring, adjust the pH to 5-8, and the temperature to 30-40°C , Slowly add the solid powder of hyaluronic acid or its salt to make the mass volume concentration reach 50-100g/L. After the degradation reaction for 0.5-1.5h, continue to slowly add the solid powder of hyaluronic acid or its salt to make the mass volume concentration Reach 100-200g/L, continue enzymatic hydrolysis for 5-8h.
  • step (1) in the above-described method for the preparation of small molecules of hyaluronic acid or a salt thereof, in step (1) in the addition amount of hyaluronidase per gram of hyaluronic acid or salt thereof is added to 10 2 -10 5 IU The hyaluronidase.
  • the denaturation and inactivation condition of the hyaluronidase in step (2) is that the enzymatic hydrolysis solution is heated to 45-80°C and maintained for 5-60 min .
  • the adsorption condition of the enzymatic hydrolysate in step (3) is 0.01%-1% activated carbon, adsorption for 30-90min, in step (4) It is filtered with a cellulose membrane with a pore size of 0.22 ⁇ m.
  • the hyaluronic acid salt is sodium hyaluronate
  • the pH adjustment adopts hydrochloric acid or sodium hydroxide solution.
  • the final concentration of the small molecule hyaluronic acid or its salt solution in step (4) is 10%-20%.
  • the conditions of the low-temperature spray drying method in step (5) are that the inlet air temperature is 40°C-80°C, and the outlet air temperature is 40°C.
  • the feed rate is 100mL/h-300mL/h.
  • the molecular weight of the hyaluronic acid or its salt used in the enzymatic hydrolysis in step (1) is between 500kDa-700kDa, and the preparation in step (5)
  • the molecular weight of the obtained small molecule hyaluronic acid or its salt is between 1 kDa and 60 kDa.
  • the method for producing hyaluronidase includes the following steps:
  • step (1) Inoculate the seed culture solution in step (1) into a sterilized fermentation medium at a culture temperature of 30°C-37°C, a rotation speed of 0-150 rpm, and culture for 20-48 hours to obtain hyaluronic acid Enzyme fermentation broth;
  • step (3) Perform ultrafiltration with the hyaluronidase supernatant in step (3) of an ultrafiltration membrane with a pore size cutoff molecular weight of 200kDa to obtain a filtrate;
  • the components of the seed culture medium in step (a) are peptone 10g/L, yeast extract 5g/L, and hyaluronic acid 5g/L L, glucose 3g/L, sodium chloride 0.1g/L, ammonium sulfate 2g/L, ferrous sulfate 0.05g/L, magnesium sulfate 0.2g/L, Tween-801mL, pH6.5.
  • the components of the fermentation medium in step (b) are peptone 10g/L, yeast extract powder 5g/L, and hyaluronic acid 5g/L L, glucose 3g/L, sodium chloride 0.1g/L, ammonium sulfate 2g/L, ferrous sulfate 0.05g/L, magnesium sulfate 0.2g/L, Tween-801mL, pH 6.5.
  • the small molecule hyaluronic acid or its salt provided in the embodiments of the present disclosure is prepared by the above-mentioned method.
  • the embodiments of the present disclosure provide the establishment of a hyaluronic acid enzymatic hydrolysis reaction system.
  • the hyaluronidase involved in the embodiments of the present disclosure is derived from Lactobacillus plantarum. As a probiotic, Lactobacillus plantarum can be used in food.
  • the hyaluronidase produced has high safety, and the small molecule hyaluronic acid prepared by it degraded Or its salt is safe and reliable, very suitable for food, cosmetics and pharmaceutical industries.
  • the embodiments of the present disclosure also provide a method for inactivating and purifying the enzymatic hydrolysate.
  • the hyaluronidase inactivation method is to heat up the enzymatic hydrolysate to denature and inactivate the hyaluronidase, and the purification method for the enzymatic hydrolysate is first used Activated carbon is adsorbed, and then the enzymatic hydrolysate is filtered by a cellulose filter membrane. This method is convenient and quick, and achieves efficient inactivation and purification.
  • the embodiments of the present disclosure also provide a method for preparing small-molecule hyaluronic acid solid powder, that is, a low-temperature spray drying method.
  • a method for preparing small-molecule hyaluronic acid solid powder that is, a low-temperature spray drying method.
  • this method has the advantages of high yield, no pollution, low energy consumption, and no residual organic solvent; and compared with the traditional drying method, the spray drying method of this method is lower than 80°C.
  • the minimum temperature of the inlet air is about 40°C. This method is adopted to prepare small molecule hyaluronic acid, which preserves the structural integrity and biological activity of the molecule to the greatest extent.
  • the small molecule hyaluronic acid prepared by the method of the present disclosure has good quality, and the whiteness of the hyaluronic acid solid powder is above 93%, the moisture content can be less than 6.3%, and the molecular weight is between 1kDa-60kDa; The purity of hyaluronic acid is high, and the content can be as high as 97% or more; the yield is high, which can be as high as 90%.
  • the hyaluronidase used in the examples of the present disclosure is derived from Lactobacillus plantarum.
  • the Lactobacillus plantarum is Lactobacillus plantarum CnT012-56 obtained by the following process: a strain is obtained from the fermentation broth of hyaluronic acid.
  • Hyaluronidase-producing Lactobacillus plantarum is then used for mutagenesis breeding with a multifunctional plasma mutagenesis system to obtain a high-hyaluronidase-producing Lactobacillus plantarum CnT012-56.
  • CGMCC China Common Microorganism Collection Management Center
  • the strain was cultured in a fermentation medium for 38 hours, and the hyaluronidase activity in the fermentation broth reached the highest, reaching 6000 IU/mL. After the fermentation broth is centrifuged to remove the bacteria, the microporous membrane is used for impurity removal, and ultrafiltration is purified and concentrated, its activity can reach 70,000IU/mL.
  • the method for producing hyaluronidase using the Lactobacillus plantarum mutagenized strain of the embodiment of the present disclosure includes the following steps:
  • the components of the seed culture medium are peptone 10g/L, yeast extract 5g/L, hyaluronic acid 5g/L, glucose 3g/L, sodium chloride 0.1g/L, ammonium sulfate 2g/L, sulfuric acid Ferrous iron 0.05g/L, magnesium sulfate 0.2g/L, Tween-801mL, pH 6.5; optionally, the fermentation medium components are peptone 10g/L, yeast extract powder 5g/L, hyaluronic acid 5g/ L, glucose 3g/L, sodium chloride 0.1g/L, ammonium sulfate 2g/L, ferrous sulfate 0.05g/L, magnesium sulfate 0.2g/L, Tween-801mL, pH 6.5.
  • the method for detecting the molecular weight of small-molecule hyaluronic acid or its salt is the intrinsic viscosity determination (Wang Yanhou, Wang Fengshan, Guo Xueping, etc. Research on the Intrinsic Viscosity Determination Method of Sodium Hyaluronate[J] . Chinese Pharmaceutical Journal, 2004,39(6):469-471).
  • the basis for the determination of whiteness, moisture content, and purity content is "QB/T 4416-2012 Sodium Hyaluronate, Raw Material for Cosmetics”.
  • the method for preparing small molecule hyaluronic acid or its salt includes the following steps: (1) Enzymatic hydrolysis reaction solution: adding hyaluronidase to purified water, turning on the stirring, and slowly adding The solid powder of hyaluronic acid or its salt is obtained by enzymatic hydrolysis reaction; (2) Hyaluronidase is inactivated: the enzymatic hydrolysis solution of step (1) is heated to make the enzymatic hydrolysis solution (3) Adsorption: Adsorb the enzymatic hydrolysate in step (2) to obtain a mixed solution; (4) Filter and purify: remove the adsorption from the mixed solution in step (3) And denatured hyaluronidase, filtered to obtain pure small molecule hyaluronic acid and its salt solution; (5) low-temperature spray drying: low-temperature spray drying of the small molecule hyaluronic acid and its salt solution in step (4) , To obtain the required molecular weight small molecule h
  • step (1) adopts stepwise addition of hyaluronic acid or its salt solid powder, which can ensure that the hyaluronic acid or its salt solid powder is dissolved and combined with the transparent
  • the action of plasminase ensures the effect of enzymolysis.
  • the amount of hyaluronidase added in step (1) is 10 2 -10 5 IU hyaluronidase per gram of hyaluronic acid or its salt. This ratio is mainly because too high hyaluronidase addition will cause hyaluronidase or its salt to be hydrolyzed too quickly, resulting in uneven molecular weight of the product, and too low hyaluronidase addition cannot guarantee the enzymatic effect .
  • a cellulose filter membrane with a pore size of 0.22 ⁇ m can be used to filter out the activated carbon and hyaluronidase to the utmost extent to ensure Purity of subsequent products.
  • hydrochloric acid or sodium hydroxide solution is used to adjust the pH, which on the one hand ensures the safety of the final product, and on the other hand can reduce the production cost.
  • the final concentration of the small molecule hyaluronic acid or its salt solution in step (4) is 10%-20%, which is compatible with the low-temperature spray drying method, Ensure the normal operation of the subsequent drying process.
  • the above-mentioned hyaluronic acid solution is spray-dried at low temperature to prepare small-molecule hyaluronic acid solid powder.
  • the inlet air temperature of the cryogenic spray is 40°C
  • the outlet air temperature is 40°C
  • the flow rate is 100 mL/h.
  • the whiteness of the prepared small molecule hyaluronic acid solid powder is 95%, the moisture content is 6.3%, the molecular weight is 60kDa, the hyaluronic acid content is 97%, and the yield is 89%.
  • the above-mentioned hyaluronic acid solution is spray-dried at low temperature to prepare small-molecule hyaluronic acid solid powder.
  • the inlet air temperature of the cryogenic spray is 50°C
  • the outlet air temperature is 40°C
  • the flow rate is 100 mL/h.
  • the whiteness of the prepared small molecular hyaluronic acid solid powder is 96%, the moisture content is 7.3%, the molecular weight is 50kDa, the hyaluronic acid content is 96%, and the yield is 90%.
  • the above-mentioned hyaluronic acid solution is spray-dried at low temperature to prepare small-molecule hyaluronic acid solid powder.
  • the inlet air temperature of the cryogenic spray is 60°C
  • the outlet air temperature is 40°C
  • the flow rate is 150 mL/h.
  • the whiteness of the prepared small-molecule hyaluronic acid solid powder is 95%, the moisture content is 8.2%, the molecular weight is 40kDa, the hyaluronic acid content is 97%, and the yield is 90%.
  • the above-mentioned hyaluronic acid solution is spray-dried at low temperature to prepare small-molecule hyaluronic acid solid powder.
  • the inlet air temperature of the cryogenic spray is 65°C
  • the outlet air temperature is 40°C
  • the flow rate is 100 mL/h.
  • the whiteness of the prepared small molecule hyaluronic acid solid powder is 93%, the moisture content is 7.2%, the molecular weight is 30kDa, the hyaluronic acid content is 98%, and the yield is 91%.
  • the above-mentioned hyaluronic acid solution is spray-dried at low temperature to prepare small-molecule hyaluronic acid solid powder.
  • the inlet air temperature of the cryogenic spray is 70°C
  • the outlet air temperature is 40°C
  • the flow rate is 200 mL/h.
  • the whiteness of the finished product small molecule hyaluronic acid solid powder is 93%, the moisture content is 8.3%, the molecular weight is 20kDa, the hyaluronic acid content is 98%, and the yield is 88%.
  • the above-mentioned hyaluronic acid solution is spray-dried at low temperature to prepare small-molecule hyaluronic acid solid powder.
  • the inlet air temperature of the cryogenic spray is 75°C
  • the outlet air temperature is 40°C
  • the flow rate is 250 mL/h.
  • the whiteness of the prepared small molecule hyaluronic acid solid powder is 93%, the moisture content is 7.8%, the molecular weight is 10kDa, the hyaluronic acid content is 99%, and the yield is 89%.
  • the above-mentioned hyaluronic acid solution is spray-dried at low temperature to prepare small-molecule hyaluronic acid solid powder.
  • the inlet air temperature of the cryogenic spray is 80°C
  • the outlet air temperature is 40°C
  • the flow rate is 300 mL/h.
  • the whiteness of the prepared small molecule hyaluronic acid solid powder is 94%, the moisture content is 6.9%, the molecular weight is 1kDa, the hyaluronic acid content is 99%, and the yield is 90%.
  • Comparative Example 1 and Comparative Example 2 It can be seen from Comparative Example 1 and Comparative Example 2 that the product quality and product yield of hyaluronic acid obtained by the traditional alcohol precipitation method are worse than those of the low-temperature spray drying method.
  • the method for preparing small molecule hyaluronic acid or its salt uses hyaluronidase produced by Lactobacillus plantarum to prepare small molecules through enzymatic hydrolysis, enzyme inactivation, adsorption, filtration purification and low-temperature spray drying.
  • Hyaluronic acid or its salt the process is simple, the hyaluronidase inactivation method used is convenient and quick, which helps to achieve high-efficiency inactivation and purification; the low-temperature spray drying used has high yield, no pollution, and energy The advantages of low consumption, no organic solvent residues, and maximum preservation of molecular structural integrity and biological activity; the small molecule hyaluronic acid has high whiteness, low moisture content, high content, high yield, and can meet the needs of Different molecular weight requirements; Lactobacillus plantarum endows the product with higher safety and lower production cost, effectively improving the industrial production and application potential of small molecule hyaluronic acid or its salt.

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Abstract

提供一种小分子透明质酸或其盐及其制备方法。使用菌种保藏号为CGMCC NO.16836的植物乳杆菌发酵以生产制得透明质酸酶溶液，该透明质酸酶溶液酶解分子量介于500kDa-700kDa透明质酸或其盐，制得的小分子透明质酸或其盐的分子量介于1kDa-60kDa；小分子透明质酸酶解液经酶灭活、活性炭吸附除杂、过滤微孔膜滤、低温喷雾干燥得到小分子透明质酸或其盐。所得产品安全性高、透皮吸收性好、极大保证了分子结构的完整性及生物活性，具有更广泛的应用。

Description

一种小分子透明质酸或其盐及其制备方法
相关申请的交叉引用
本公开要求于2019年03月05日提交中国专利局的申请号为CN201910164881.3、名称为“一种制备小分子透明质酸或其盐的方法”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及生物工程技术领域，具体地涉及一种小分子透明质酸或其盐及其制备方法。
背景技术
透明质酸(hyaluronic acid，HA)，是一种酸性黏多糖，由N-乙酰氨基葡糖和D-葡糖醛酸双糖重复单位构成的无分支高分子糖胺聚糖，存在于动物组织细胞间质和某些细菌的荚膜中，被广泛应用于化妆品、食品和医药等行业领域。
近年来研究发现，小分子透明质酸表现出非常强的生物活性(Hui ZM，a bioactive hyaluronic acid fragment，its manufacture mehtod，its formulation and its applications，Patent number 2014101553593.5.)，具有提高皮肤免疫功能、消除皮肤炎症、促进创口愈合、治疗急慢性咽喉炎和治疗牙龈炎等作用，是免疫细胞和细胞因子的激活剂。小分子透明质酸渗透吸收性好，极易被真皮和肠道吸收，可以迅速补充体内流失的透明质酸。因此，小分子透明质酸在食品保健、化妆品以及临床医疗领域具有广阔的应用前景。
随着小分子透明质酸的研究与应用的推广，近年来，国内外开始重视HA的降解及其降解产物的制备研究。目前，降解的方法主要有物理降解、化学降解和生物降解三大类。物理降解方法主要有加热、机械剪切力、紫外线、超声波、60C照射和γ-射线辐射等因素，均可使HA发生降解。但是，物理法制备的分子量高于1万，产生的分子量范围分布较大，产品稳定性较差。HA的化学降解方法有水解法和氧化降解法，水解法分为酸水解(HCL)和碱(NaOH)水解，氧化降解常用的氧化剂为次氯酸钠和过氧化氢。尽管使用化学降解方法降解产物的相对分子质量可以通过改变酸、碱或氧化剂的加入量和反应时间来控制，降解成本较低，易于大规模生产，但化学降解法引入了化学试剂，反应条件复杂，易给HA性质产生影响和产品的纯化带来困难，且产生大量的工业废水。生物酶法降解是在透明质酸酶的作用下β-糖苷键断裂而发生的降解。酶法降解温和，是制备小分子透明质酸的最佳技术手段。透明质酸酶又叫玻璃酸酶，是可使HA发生低分子化作用的糖苷酶，也能降解其他机体组织中的酸性粘多糖，其广泛存在于真核生物和原核生物中，具有特异性的生理学活性。
如申请号为CN201611127881.9的发明专利提供了一种固液双相酶解与超滤联用制备 超低分子量透明质酸寡糖及其盐的方法，通过酶法实现对透明质酸酶解。但是，目前的透明质酸酶通常是从动物组织中提取制备，价格昂贵，不适合工业化应用。因此，一种低成本、工艺简单、适合工业化大规模生产的小分子透明质酸的方法具有重要的应用潜力。
同时透明质酸提取工艺多为有机试剂沉淀法，此方法造成环境污染，能耗大，并且提取小分子的透明质酸收率低。此外还有运用喷雾干燥，进行透明质酸提取，但是一般的喷雾干燥机，进风温度在180℃以上，热敏性的物料，物理性质或化学性质易发生变化，特别是生物活性材料，如细菌、活性酶等会失活。
发明内容
本公开的目的包括，例如，提供一种小分子透明质酸或其盐及其制备方法，以克服上述缺陷。
本公开实施例提供的方法主要通过植物乳杆菌产生的透明质酸酶配合低温喷雾干燥，降解得到小分子透明质酸或其盐。
本公开的实施例，例如可以以如下方式来实现：
根据本公开的实施例提供的制备小分子透明质酸或其盐的方法，包括以下步骤：
(1)酶解反应液：向纯化水中加入透明质酸酶，开启搅拌，缓缓加入透明质酸或其盐的固体粉末，通过酶解反应得到酶解液，所述透明质酸酶为植物乳杆菌诱变菌株CnT012-56发酵生产制得；该菌株分类命名为Lactobacillus plantarum，其保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，保藏日期：2018年11月28日，保藏编号：CGMCC NO.16836；
(2)透明质酸酶灭活：对步骤(1)的所述酶解液进行升温处理，使所述酶解液中的透明质酸酶变性失活；
(3)吸附：对步骤(2)中的所述酶解液进行吸附，得到混合液；
(4)过滤纯化：从步骤(3)混合液中去除吸附剂以及变性透明质酸酶，过滤得到纯净的小分子透明质酸及其盐溶液；
(5)低温喷雾干燥：对步骤(4)的所述小分子透明质酸及其盐溶液进行低温喷雾干燥，得到所需分子量的小分子透明质酸及其盐的固体粉末。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(1)中首先向纯化水中加入透明质酸酶，开启搅拌，调节pH为5-8，温度为30-40℃，缓缓加入透明质酸或其盐的固体粉末，使质量体积浓度达到50-100g/L，降解反应0.5-1.5h后，继续缓缓加入透明质酸或其盐固体粉末，使质量体积浓度达到100-200g/L，继续酶解5-8h。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(1)中所述透明质酸酶的添加量为每克透明质酸或其盐添加10 2-10 5IU的所述透明质酸酶。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(2)中所述透明质酸酶的变性失活条件为酶解液升温至45-80℃下维持5-60min。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(3)中所述酶解液的吸附条件为0.01％-1％活性炭，吸附30-90min，步骤(4)中采用孔径为0.22μm的纤维素滤膜过滤。
可选地，在上述制备小分子透明质酸或其盐的方法中，透明质酸盐为透明质酸钠，pH调节采用盐酸或氢氧化钠溶液。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(4)中所述小分子透明质酸或其盐溶液的最终浓度为10％-20％。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(5)中所述低温喷雾干燥法的条件为进风温度为40℃-80℃，出风温度为40℃，进料速度为100mL/h–300mL/h。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(1)中所述酶解使用的透明质酸或其盐的分子量介于500kDa-700kDa，步骤(5)中制得的所述小分子透明质酸或其盐的分子量介于1kDa-60kDa。
可选地，在上述制备小分子透明质酸或其盐的方法中，所述透明质酸酶的生产方法包括以下步骤：
(a)将Lactobacillus plantarum CnT012-56菌种接种到已灭菌的种子培养基中，培养温度为30℃-37℃、转速为0-100rpm，培养8-24h，获得种子培养液；
(b)将步骤(1)中的所述种子培养液接种到已灭菌的发酵培养基中，培养温度为30℃-37℃、转速为0-150rpm，培养20-48h，获得透明质酸酶发酵液；
(c)对步骤(2)中的所述透明质酸酶发酵液进行离心，获得上清液；
(d)利用孔径截留分子量为200kDa的超滤膜队步骤(3)中的所述透明质酸酶上清液进行超滤，获得滤过液；
(e)利用孔径截留分子量为20kDa的超滤膜对步骤(4)中的所述滤过液进行超滤，除去小分子杂质，得到纯化的透明质酸酶。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(a)中所述种子培养基的组分是蛋白胨10g/L，酵母浸粉5g/L，透明质酸5g/L，葡萄糖3g/L，氯化钠0.1g/L，硫酸铵2g/L，硫酸亚铁0.05g/L，硫酸镁0.2g/L，吐温-801mL，pH6.5。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(b)中所述发酵培养基的组分是蛋白胨10g/L，酵母浸粉5g/L，透明质酸5g/L，葡萄糖3g/L，氯化钠0.1g/L，硫酸铵2g/L，硫酸亚铁0.05g/L，硫酸镁0.2g/L，吐温-801mL，pH 6.5。
本公开的实施例提供的小分子透明质酸或其盐是通过上述方法制备的。
本公开实施例的有益效果包括，例如：
(1)本公开实施例提供了一种透明质酸酶解反应体系的建立。本公开实施例所涉及透明质酸酶来源于植物乳杆菌，植物乳杆菌作为一种益生菌可应用于食品中，所产透明质酸酶具有高安全性，其降解制备的小分子透明质酸或其盐安全可靠，非常适用于食品、化妆品及医药行业。
(2)本公开实施例还提供了酶解液灭活纯化的方法，透明质酸酶灭活方法为将酶解液升温使透明质酸酶变性失活，酶解液纯化方法为先用用活性炭吸附，然后采用纤维素滤膜过滤酶解液，该方法方便快捷，实现了高效灭活纯化。
(3)本公开实施例还提供了制备小分子透明质酸固体粉末的方法，即，低温喷雾干燥法。相较于传统醇沉淀方式，该方法具有收率高、无污染、能耗低、无有机溶剂残留的优点；并且相比于传统的干燥法，该方法的喷雾干燥在物料温度低于80℃下完成，其进风温度最低为40℃左右，采取该方法制备小分子透明质酸，最大限度保存了分子的结构完整和生物活性。
(4)同时本公开实施例的方法制备的小分子透明质酸质量好，且透明质酸固体粉末的白度在93％以上，水分含量可小于6.3％，分子量介于1kDa-60kDa；小分子透明质酸的纯度高，含量可以高达97％以上；收率高，可以高达90％。
具体实施方式
为了使本技术领域的人员更好地理解本公开中的技术方案，下面将结合本公开实施例对技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本公开的一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本公开保护的范围。
本公开的实施例所用透明质酸酶来自于植物乳杆菌，可选地，该植物乳杆菌是通过以下过程获得的植物乳杆菌CnT012-56：从染菌透明质酸发酵液中分离得到一株产透明质酸酶的植物乳杆菌，然后利用多功能等离子体诱变系统进行诱变育种得到一株高产透明质酸酶的植物乳杆菌CnT012-56。
该菌种由中国普通微生物菌种保藏管理中心(CGMCC)保藏，保藏日期：2018年11月28日，保藏编号：CGMCC NO.16836；确定为植物乳杆菌，命名为Lactobacillus plantarum CnT012-56。该菌株在发酵培养基中培养38小时，其发酵液中透明质酸酶的活力达到最高，可达6000IU/mL。发酵液经离心去除菌体、微孔滤膜除杂、超滤纯化浓缩后，其活力可达70000IU/mL。
可选地，利用本公开实施例的植物乳杆菌诱变菌株来生产透明质酸酶的方法，包括以下步骤：
(1)将Lactobacillus plantarum CnT012-56菌种接种到已灭菌的种子培养基中，培养温度为30℃-37℃、转速为0-100rpm，培养8-24h，获得种子培养液；
(2)将种子培养液接种到已灭菌的发酵培养基中，培养温度为30℃-37℃、转速为0-150rpm，培养20-48h，获得透明质酸酶发酵液；
(3)离心上述透明质酸酶发酵液，获得上清液；
(4)利用孔径截留分子量为200kDa的超滤膜来超滤上述透明质酸酶上清液，获得滤过液；
(5)利用孔径截留分子量为20kDa的超滤膜来超滤上述滤过液，除去小分子杂质，得到纯化的透明质酸酶；
可选地，种子培养基的组分是蛋白胨10g/L，酵母浸粉5g/L，透明质酸5g/L，葡萄糖3g/L，氯化钠0.1g/L，硫酸铵2g/L，硫酸亚铁0.05g/L，硫酸镁0.2g/L，吐温-801mL，pH6.5；可选地，发酵培养基组分是蛋白胨10g/L，酵母浸粉5g/L，透明质酸5g/L，葡萄糖3g/L，氯化钠0.1g/L，硫酸铵2g/L，硫酸亚铁0.05g/L，硫酸镁0.2g/L，吐温-801mL，pH 6.5。
可选地，在本公开实施例中，小分子透明质酸或其盐的分子量的检测方法为特性黏度法测定(王彦厚，王凤山，郭学平，等.玻璃酸钠特性黏度测定方法的研究[J].中国药学杂志，2004,39(6)：469-471)。
可选地，在本公开实施例中，白度、水分含量、纯度含量的测定依据为《QB/T 4416-2012化妆品用原料透明质酸钠》。
可选地，本公开实施例提供的述制备小分子透明质酸或其盐的方法，包括以下步骤：(1)酶解反应液：向纯化水中加入透明质酸酶，开启搅拌，缓缓加入透明质酸或其盐的固体粉末，通过酶解反应得到酶解液；(2)透明质酸酶灭活：对步骤(1)的所述酶解液进行升温处理，使所述酶解液中的透明质酸酶变性失活；(3)吸附：对步骤(2)中的所述酶解液进行吸附，得到混合液；(4)过滤纯化：从步骤(3)混合液中去除吸附剂以及变性透明质酸酶，过滤得到纯净的小分子透明质酸及其盐溶液；(5)低温喷雾干燥：对步骤(4)的所述小分子透明质酸及其盐溶液进行低温喷雾干燥，得到所需分子量的小分子透明质酸及其盐的固体粉末。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(1)采用分步加入透明质酸或其盐固体粉末，可以确保透明质酸或其盐固体粉末溶解以及与透明质酸酶作用，保证酶解效果。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(1)中透明质酸酶添加量为每克透明质酸或其盐加10 2-10 5IU透明质酸酶，采用该比例主要是由于过高的透明质酸酶添加量会导致透明质酸或其盐酶解过快，造成产物分子量不均，而过低的透明质酸酶添加 量无法保证酶解效果。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(4)中采用孔径为0.22μm的纤维素滤膜过滤可以最大限度的将活性炭和透明质酸酶过滤掉，保证后续产品的纯度。
可选地，在上述制备小分子透明质酸或其盐的方法中，采用盐酸或氢氧化钠溶液来调节pH，一方面保证最终产物的安全性，另一方面可以降低生产成本。
可选地，在上述制备小分子透明质酸或其盐的方法中，步骤(4)中小分子透明质酸或其盐溶液最终浓度为10％-20％，该浓度与低温喷雾干燥法相匹配，保证后续干燥过程的正常运行。
下面结合具体实施例对本公开的技术方案做进一步说明：
实施例1
向5L纯化水中加入5mL透明质酸酶，开启搅拌，调节pH为5.0，温度为30℃，缓缓加入250g分子量为500kDa的透明质酸钠，加料速度一定要慢，以使物料溶解均匀。可选地，降解反应30min后，继续缓缓加入250g分子量为500kDa的透明质酸钠。酶解反应5h后至分子量60kDa，可选地，将酶解液升温至50℃并维持1h使得透明质酸酶变性失活。可选地，向上述酶解液中加入5g活性炭，吸附60min，经过支撑过滤纸板过滤，再经孔径为0.22μm的纤维素滤膜过滤得到纯净的透明质酸溶液。可选地，将上述透明质酸溶液进行低温喷雾干燥以制备小分子透明质酸固体粉末。可选地，低温喷雾的进风温度为40℃，出风温度为40℃，流速为100mL/h。制得的成品小分子透明质酸固体粉末的白度为95％，水分含量为6.3％，分子量为60kDa，透明质酸的含量为97％，收率为89％。
实施例2
向5L纯化水中加入6mL透明质酸酶，开启搅拌，调节pH为5.5，温度为32℃，缓缓加入300g分子量为650kDa的透明质酸钠，加料速度一定要慢，以使物料溶解均匀。可选地，降解反应40min后，继续缓缓加入400g分子量650kDa的透明质酸钠。酶解反应6h后至分子量50kDa，可选地，将酶解液升温至50℃并维持50min以使透明质酸酶变性失活。可选地，向上述酶解液中加入5g活性炭，吸附60min，经过支撑过滤纸板过滤，再经孔径为0.22μm的纤维素滤膜过滤得到纯净的透明质酸溶液。可选地，将上述透明质酸溶液进行低温喷雾干燥以制备小分子透明质酸固体粉末。可选地，低温喷雾的进风温度为50℃，出风温度为40℃，流速为100mL/h。制得的成品小分子透明质酸固体粉末的白度为96％，水分含量为7.3％，分子量为50kDa，透明质酸的含量为96％，收率为90％。
实施例3
向5L纯化水中加入7mL透明质酸酶，开启搅拌，调节pH为6.0，温度为34℃，缓缓加入400g分子量为700kDa的透明质酸钠，加料速度一定要慢，以使物料溶解均匀。可选 地，降解反应50min后，继续缓缓加入300g分子量为700kDa的透明质酸钠。酶解反应6.5h后至分子量40kDa，可选地，将酶解液升温至50℃并维持1h以使透明质酸酶变性失活。可选地，向上述酶解液中加入5g活性炭，吸附60min，经过支撑过滤纸板过滤，再经孔径为0.22μm的纤维素滤膜过滤得到纯净的透明质酸溶液。可选地，将上述透明质酸溶液进行低温喷雾干燥以制备小分子透明质酸固体粉末。可选地，低温喷雾的进风温度为60℃，出风温度为40℃，流速为150mL/h。制得的成品小分子透明质酸固体粉末的白度为95％，水分含量为8.2％，分子量为40kDa，透明质酸的含量为97％，收率为90％。
实施例4
向5L纯化水中加入8mL透明质酸酶，开启搅拌，调节pH为6.5，温度为36℃，缓缓加入400g分子量为500kDa的透明质酸钠，加料速度一定要慢，以使物料溶解均匀。可选地，降解反应60min后，继续缓缓加入400g分子量为500kDa的透明质酸钠。酶解反应7h后至分子量30kDa，可选地，将酶解液升温至50℃并维持1h以使透明质酸酶变性失活。可选地，向上述酶解液中加入5g活性炭，吸附60min，经过支撑过滤纸板过滤，再经孔径为0.22μm的纤维素滤膜过滤得到纯净的透明质酸溶液。可选地，将上述透明质酸溶液进行低温喷雾干燥以制备小分子透明质酸固体粉末。可选地，低温喷雾的进风温度为65℃，出风温度为40℃，流速为100mL/h。制得的成品小分子透明质酸固体粉末的白度为93％，水分含量为7.2％，分子量为30kDa，透明质酸的含量为98％，收率为91％。
实施例5
向5L纯化水中加入8mL透明质酸酶，开启搅拌，调节pH为7.0，温度为38℃，缓缓加入400g分子量为650kDa的透明质酸钠，加料速度一定要慢，以使物料溶解均匀。可选地，降解反应70min后，继续缓缓加入400g分子量650kDa的透明质酸钠。酶解反应8h后至分子量20kDa，可选地，将酶解液升温至50℃并维持1h以使透明质酸酶变性失活。可选地，向上述酶解液中加入5g活性炭，吸附60min，经过支撑过滤纸板过滤，再经孔径为0.22μm的纤维素滤膜过滤得到纯净的透明质酸溶液。可选地，将上述透明质酸溶液进行低温喷雾干燥以制备小分子透明质酸固体粉末。可选地，低温喷雾的进风温度为70℃，出风温度为40℃，流速为200mL/h。制得的成品小分子透明质酸固体粉末的白度为93％，水分含量为8.3％，分子量为20kDa，透明质酸的含量为98％，收率为88％。
实施例6
向5L纯化水中加入9mL透明质酸酶，开启搅拌，调节pH为8.0，温度为40℃，缓缓加入500g分子量为650kDa的透明质酸钠，加料速度一定要慢，以使物料溶解均匀。可选地，降解反应80min后，继续缓缓加入500g分子量为650kDa的透明质酸钠。酶解反应7.5h后至分子量10kDa，可选地，将酶解液升温至50℃并维持1h以使透明质酸酶变性失活。可 选地，向上述酶解液中加入5g活性炭，吸附60min，经过支撑过滤纸板过滤，再经孔径为0.22μm的纤维素滤膜过滤得到纯净的透明质酸溶液。可选地，将上述透明质酸溶液进行低温喷雾干燥以制备小分子透明质酸固体粉末。可选地，低温喷雾的进风温度为75℃，出风温度为40℃，流速为250mL/h。制得的成品小分子透明质酸固体粉末的白度为93％，水分含量为7.8％，分子量为10kDa，透明质酸的含量为99％，收率为89％。
实施例7
向5L纯化水中加入10mL透明质酸酶，开启搅拌，调节pH为6.5，温度为38℃，缓缓加入400g分子量为700kDa的透明质酸钠，加料速度一定要慢，以使物料溶解均匀。可选地，降解反应90min后，继续缓缓加入500g分子量为700kDa的透明质酸钠。酶解反应6h后至分子量7kDa-9kDa，可选地，将酶解液升温至50℃并维持1h以使透明质酸酶变性失活。可选地，向上述酶解液中加入5g活性炭，吸附60min，经过支撑过滤纸板过滤，再经孔径为0.22μm的纤维素滤膜过滤得到纯净的透明质酸溶液。可选地，将上述透明质酸溶液进行低温喷雾干燥以制备小分子透明质酸固体粉末。可选地，低温喷雾的进风温度为80℃，出风温度为40℃，流速为300mL/h。制得的成品小分子透明质酸固体粉末的白度为94％，水分含量为6.9％，分子量为1kDa，透明质酸的含量为99％，收率为90％。
此外，采用传统的醇沉淀方式而非本公开实施例的低温喷雾干燥法来制备小分子透明质酸固体粉末。
对比例1
向5L纯化水中加入10mL透明质酸酶，开启搅拌，调节pH为6.5，温度为38℃，缓缓加入400g分子量为700kDa的透明质酸钠，加料速度一定要慢，以使物料溶解均匀。降解反应90min后，继续缓缓加入500g分子量为700kDa的透明质酸钠。酶解反应6h后至分子量7kDa-9kDa，将酶解液升温至50℃并维持1h以使透明质酸酶变性失活。向上述酶解液中加入5g活性炭，吸附60min，经过支撑过滤纸板过滤，再经孔径为0.22μm的纤维素滤膜过滤得到纯净透明质酸溶液。将所得纯净透明质酸溶液加入4倍酒精中进行反沉，沉淀过程开启搅拌。沉淀结束，静置一小时，去除上清液，加入一倍体积的酒精脱水三次，离心，去除上清液，将固形物真空干燥制得小分子透明质酸固体粉末。小分子透明质酸粉末的白度为89％，水分含量为8.9％，分子量为9kDa，透明质酸的含量为95％，收率为78％。
对比例2
向5L纯化水中加入8mL透明质酸酶，开启搅拌，调节pH为6.5，温度为36℃，缓缓加入400g分子量为500kDa的透明质酸钠，加料速度一定要慢，以使物料溶解均匀。降解反应60min后，继续缓缓加入400g分子量为500kDa的透明质酸钠。酶解反应7h后至分子量30kDa，将酶解液升温至50℃并维持1h以使透明质酸酶变性失活。向上述酶解液中加 入5g活性炭，吸附60min，经过支撑过滤纸板过滤，再经孔径为0.22μm的纤维素滤膜过滤得到纯净的透明质酸溶液。将所得纯净透明质酸溶液加入4倍酒精中进行反沉，沉淀过程开启搅拌。沉淀结束，静置一小时，去除上清液，加入一倍体积的酒精脱水三次，离心，去除上清液，将固形物真空干燥制得小分子透明质酸固体粉末。小分子透明质酸粉末的白度为87％，水分含量为9.2％，分子量为30kDa，透明质酸的含量为94％，收率为79％。
从对比例1和对比例2可以看出，采用传统的醇沉淀方式得到的透明质酸的产品质量以及产品收率都要比低温喷雾干燥法要差。
尽管通过优选实施例的方式对本公开进行了详细描述，但本公开并不限于此。在不脱离本公开的精神和实质的前提下，本领域普通技术人员可以对本公开的实施例进行各种等效的修改或替换，而这些修改或替换都应在本公开的涵盖范围内；此外，任何熟悉本技术领域的技术人员在本公开揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本公开的保护范围之内。因此，本公开的保护范围应所述以权利要求的保护范围为准。
工业实用性
本公开实施例提供的制备小分子透明质酸或其盐的方法，利用植物乳杆菌产生的透明质酸酶通过酶解、酶灭活、吸附、过滤纯化和低温喷雾干燥等步骤来制备小分子透明质酸或其盐，该方法工艺流程简单，所采用的透明质酸酶灭活方法方便快捷，有助于实现高效灭活纯化；所采用的低温喷雾干燥具有收率高、无污染、能耗低、无有机溶剂残留的优点，并且最大限度地保存了分子的结构完整和生物活性；所的小分子透明质酸白度高，水分含量低，含量高，收率高，且能够满足对不同分子量的需求；植物乳杆菌赋予所得产品更高的安全性和更低的生产成本，有效提高了小分子透明质酸或其盐的工业化生产以及应用潜力。

Claims (13)

  1. 一种制备小分子透明质酸或其盐的方法，其特征在于，包括以下步骤：
    (1)酶解反应液：向纯化水中加入透明质酸酶，开启搅拌，缓缓加入透明质酸或其盐的固体粉末，通过酶解反应得到酶解液，所述透明质酸酶为植物乳杆菌诱变菌株CnT012-56发酵生产制得，该菌株分类命名为Lactobacillus plantarum，其保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，保藏日期：2018年11月28日，保藏编号：CGMCC NO.16836；
    (2)透明质酸酶灭活：对步骤(1)的所述酶解液进行升温处理，使所述酶解液中的透明质酸酶变性失活；
    (3)吸附：对步骤(2)中的所述酶解液进行吸附，得到混合液；
    (4)过滤纯化：从步骤(3)混合液中去除吸附剂以及变性透明质酸酶，过滤得到纯净的小分子透明质酸及其盐溶液；
    (5)低温喷雾干燥：对步骤(4)的所述小分子透明质酸及其盐溶液进行低温喷雾干燥，得到所需分子量的小分子透明质酸及其盐固体粉末。
  2. 根据权利要求1所述制备小分子透明质酸或其盐的方法，其特征在于：步骤(1)中首先向纯化水中加入透明质酸酶，开启搅拌，调节pH为5-8，温度为30-40℃，缓缓加入透明质酸或其盐的固体粉末，使质量体积浓度达到50-100g/L，降解反应0.5-1.5h后，继续缓缓加入透明质酸或其盐的固体粉末，使质量体积浓度达到100-200g/L，继续酶解5-8h。
  3. 根据权利要求1所述制备小分子透明质酸或其盐的方法，其特征在于：步骤(1)中所述透明质酸酶的添加量为每克透明质酸或其盐添加10 2-10 5IU的所述透明质酸酶。
  4. 根据权利要求1所述制备小分子透明质酸或其盐的方法，其特征在于：步骤(2)中所述透明质酸酶的变性失活条件为酶解液升温至45-80℃下并维持5-60min。
  5. 根据权利要求1所述制备小分子透明质酸或其盐的方法，其特征在于：步骤(3)中所述酶解液的吸附条件为0.01％-1％活性炭，吸附30-90min，步骤(4)中采用孔径为0.22μm的纤维素滤膜进行所述过滤。
  6. 根据权利要求2所述制备小分子透明质酸或其盐的方法，其特征在于：所述透明质酸盐为透明质酸钠，pH调节采用盐酸或氢氧化钠溶液。
  7. 根据权利要求1所述制备小分子透明质酸或其盐的方法，步骤(4)中所述小分子透明质酸或其盐溶液的最终浓度为10％-20％。
  8. 根据权利要求1所述制备小分子透明质酸或其盐的方法，其特征在于：步骤(5) 中所述低温喷雾干燥的条件为进风温度为40℃-80℃，出风温度为40℃，进料速度为100mL/h–300mL/h。
  9. 根据权利要求1所述制备小分子透明质酸或其盐的方法，其特征在于：步骤(1)中所述酶解使用的透明质酸或其盐的分子量介于500kDa-700kDa，步骤(5)中制得的所述小分子透明质酸或其盐的分子量介于1kDa-60kDa。
  10. 根据权利要求1-9中任一项所述的制备小分子透明质酸或其盐的方法，其特征在于，所述透明质酸酶的生产方法包括以下步骤：
    (a)将Lactobacillus plantarum CnT012-56菌种接种到已灭菌的种子培养基中，培养温度为30℃-37℃、转速为0-100rpm，培养8-24h，获得种子培养液；
    (b)将步骤(1)中的所述种子培养液接种到已灭菌的发酵培养基中，培养温度为30℃-37℃、转速为0-150rpm，培养20-48h，获得透明质酸酶发酵液；
    (c)对步骤(2)中的所述透明质酸酶发酵液进行离心，获得上清液；
    (d)利用孔径截留分子量为200kDa的超滤膜队步骤(3)中的所述透明质酸酶上清液进行超滤，获得滤过液；
    (e)利用孔径截留分子量为20kDa的超滤膜对步骤(4)中的所述滤过液进行超滤，除去小分子杂质，得到纯化的透明质酸酶。
  11. 根据权利要求10中所述的制备小分子透明质酸或其盐的方法，其特征在于，步骤(a)中所述种子培养基的组分是蛋白胨10g/L，酵母浸粉5g/L，透明质酸5g/L，葡萄糖3g/L，氯化钠0.1g/L，硫酸铵2g/L，硫酸亚铁0.05g/L，硫酸镁0.2g/L，吐温-80 1mL，pH6.5。
  12. 根据权利要求10-11中任一项所述的制备小分子透明质酸或其盐的方法，其特征在于，步骤(b)中所述发酵培养基的组分是蛋白胨10g/L，酵母浸粉5g/L，透明质酸5g/L，葡萄糖3g/L，氯化钠0.1g/L，硫酸铵2g/L，硫酸亚铁0.05g/L，硫酸镁0.2g/L，吐温-80 1mL，pH 6.5。
  13. 一种小分子透明质酸或其盐，其特征在于，所述小分子透明质酸或其盐是通过权利要求1-12中任一项所述的制备小分子透明质酸或其盐的方法制备的。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112189765A (zh) * 2020-09-30 2021-01-08 华熙生物科技股份有限公司 含透明质酸的宠物磨牙棒组合物、制法及其应用
CN114250212A (zh) * 2021-12-14 2022-03-29 华熙生物科技股份有限公司 一种透明质酸酶冻干制剂及其制备方法
CN117535363A (zh) * 2023-12-27 2024-02-09 常州加源医药科技研发有限公司 一种功能性透明质酸的生产方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109897876B (zh) * 2019-03-05 2020-11-06 山东安华生物医药股份有限公司 一种制备小分子透明质酸或其盐的方法
CN110408607B (zh) * 2019-09-02 2021-05-18 山东安华生物医药股份有限公司 一种植物乳杆菌生产透明质酸酶发酵优化工艺
CN110982862B (zh) * 2019-12-27 2022-05-06 华熙生物科技股份有限公司 一种大规模制备高纯度不饱和透明质酸二糖的方法
CN111632015A (zh) * 2020-06-24 2020-09-08 山东贯天下生物科技有限公司 一种鸡冠提取物及其提取方法和应用
CN112553273B (zh) * 2020-12-26 2022-12-09 河北华曙新合药业有限公司 一种超小分子量透明质酸钠的制备工艺
CN113425662A (zh) * 2021-08-16 2021-09-24 湖州伊婷生物科技有限公司 透明质酸与铁皮石斛酵母发酵产物组合物及化妆品

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009060888A (ja) * 2007-08-09 2009-03-26 Kikkoman Corp ヒアルロン酸加水分解酵素及び該酵素を用いた低分子化ヒアルロン酸の製造方法
CN102690847A (zh) * 2012-04-13 2012-09-26 华熙福瑞达生物医药有限公司 酶切法制备寡聚透明质酸盐的方法及所得寡聚透明质酸盐和其应用
CN103255187A (zh) * 2012-02-21 2013-08-21 华熙福瑞达生物医药有限公司 一种低分子透明质酸盐、其制备方法及用途
CN108220364A (zh) * 2016-12-09 2018-06-29 华熙福瑞达生物医药有限公司 一种固液双相酶解与超滤联用制备超低分子量透明质酸寡糖及其盐的方法
CN109897876A (zh) * 2019-03-05 2019-06-18 山东安华生物医药股份有限公司 一种制备小分子透明质酸或其盐的方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101507733B (zh) * 2009-04-02 2011-03-30 北京中生奥普寡肽技术研究所 纳米小分子透明质酸及其制备方法
JP5587049B2 (ja) * 2010-06-21 2014-09-10 株式会社 糖質科学研究所 低分子ヒアルロン酸の製造方法
CA2835016A1 (en) * 2011-05-30 2012-12-06 Novozymes Biopharma Dk A/S Spray drying of high molecular weight hyaluronic acid
CN106309471A (zh) * 2015-07-02 2017-01-11 惠觅宙 一种低分子生物活性透明质酸的应用及其制剂
JP6209580B2 (ja) * 2015-11-11 2017-10-04 株式会社らいむ 紫外線障害低減剤、紫外線障害低減剤の製造方法、培地用添加剤、微生物希釈液用添加剤、培地、微生物希釈液、外用剤および化粧品
CN108410928B (zh) * 2018-01-31 2021-04-06 彭文平 一种高浓度小分子透明质酸的制备方法及其应用
CN110408607B (zh) * 2019-09-02 2021-05-18 山东安华生物医药股份有限公司 一种植物乳杆菌生产透明质酸酶发酵优化工艺

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009060888A (ja) * 2007-08-09 2009-03-26 Kikkoman Corp ヒアルロン酸加水分解酵素及び該酵素を用いた低分子化ヒアルロン酸の製造方法
CN103255187A (zh) * 2012-02-21 2013-08-21 华熙福瑞达生物医药有限公司 一种低分子透明质酸盐、其制备方法及用途
CN102690847A (zh) * 2012-04-13 2012-09-26 华熙福瑞达生物医药有限公司 酶切法制备寡聚透明质酸盐的方法及所得寡聚透明质酸盐和其应用
CN102876748A (zh) * 2012-04-13 2013-01-16 华熙福瑞达生物医药有限公司 酶切法制备寡聚透明质酸盐的方法及所得寡聚透明质酸盐和其应用
CN108220364A (zh) * 2016-12-09 2018-06-29 华熙福瑞达生物医药有限公司 一种固液双相酶解与超滤联用制备超低分子量透明质酸寡糖及其盐的方法
CN109897876A (zh) * 2019-03-05 2019-06-18 山东安华生物医药股份有限公司 一种制备小分子透明质酸或其盐的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BUHREN, B.A. ET AL.: "Hyaluronidase: from Clinical Applications to Molecular and Cellular Mechanisms", EUROPEAN JOURNAL OF MEDICAL RESEARCH., vol. 21, 13 February 2016 (2016-02-13), pages 1 - 7, XP021233587, DOI: 10.1186/s40001-016-0201-5 *
See also references of EP3730623A4
WANG YANHOUWANG FENGSHANGUO XUEPING ET AL.: "Study on Determination Method of Intrinsic Viscosity of Sodium Hyaluronate [J", CHINESE PHARMACEUTICAL JOURNAL, vol. 39, no. 6, 2004, pages 469 - 471

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112189765A (zh) * 2020-09-30 2021-01-08 华熙生物科技股份有限公司 含透明质酸的宠物磨牙棒组合物、制法及其应用
CN112189765B (zh) * 2020-09-30 2022-08-26 华熙生物科技股份有限公司 含透明质酸的宠物磨牙棒组合物、制法及其应用
CN114250212A (zh) * 2021-12-14 2022-03-29 华熙生物科技股份有限公司 一种透明质酸酶冻干制剂及其制备方法
CN117535363A (zh) * 2023-12-27 2024-02-09 常州加源医药科技研发有限公司 一种功能性透明质酸的生产方法

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