WO2010139100A1 - A method for producing maltotriose-beta-cyclodextrin by enzymic method - Google Patents
A method for producing maltotriose-beta-cyclodextrin by enzymic method Download PDFInfo
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- WO2010139100A1 WO2010139100A1 PCT/CN2009/001251 CN2009001251W WO2010139100A1 WO 2010139100 A1 WO2010139100 A1 WO 2010139100A1 CN 2009001251 W CN2009001251 W CN 2009001251W WO 2010139100 A1 WO2010139100 A1 WO 2010139100A1
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- cyclodextrin
- pullulan
- maltotriose
- maltotriosyl
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- C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
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- An enzymatic preparation method of maltotriosyl- ⁇ -cyclodextrin which relates to preparing a branch by using pullulan and ⁇ -cyclodextrin as raw materials through hydrolysis property and reverse synthesis ability of pullulanase
- the method of cyclodextrin belongs to the technical field of cyclodextrin.
- Cyclodextrin is a class of cyclic oligosaccharides which are linked by D-glucopyranose units with ⁇ -(1,4)-glycosidic linkages.
- the hydrophobic outer tubular structure of the cyclodextrin determines its molecular capsule properties, and it can react with many compounds to form inclusion complexes, which exhibit sustained release, solubilization, oxidation resistance, anti-light, and thermal stability. And cover up the odor and other advantages.
- the research on cyclodextrin has become hotter and hotter, and has made considerable achievements in the fields of medicine, food, environmental protection, daily chemical and supramolecular chemistry.
- cyclodextrins can be classified into cyclodextrins such as ⁇ -, ⁇ -, ⁇ -, ⁇ -.
- the more common cyclodextrins are alpha-, beta- and gamma-cyclodextrins consisting of 6, 7 and 8 glucose residues, respectively.
- the ⁇ -cyclodextrin containing 7 glucose residues has a simple production process and low cost, and is the only cyclodextrin product which can be mass-produced and widely used in the industry.
- ⁇ -cyclodextrin has problems of poor water solubility, hemolytic and non-digestive tract safety, which restricts its further application in various aspects.
- Branched cyclodextrin is a strategy to increase the solubility of cyclodextrin water by introducing a glycosyl group.
- the sugar group modification of ⁇ -cyclodextrin can achieve the purpose of increasing water solubility on the one hand, and reduce the matrix on the other hand. Hemolysis and non-digestive safety of cyclodextrins.
- the glycosyl modification of ⁇ -cyclodextrin is generally carried out by chemical method and enzymatic method, and compared with chemical modification, enzymatic modification has the advantages of high specificity and high safety.
- the object of the present invention is to provide a method for preparing branched cyclodextrin, which uses pullulan and ⁇ -cyclodextrin as substrates, and utilizes the hydrolysis property and reverse synthesis of pullulanase to firstly
- the polysaccharide is converted into a higher purity maltotriose, and the maltotriose is grafted onto the parent cyclodextrin to obtain maltotriosyl- ⁇ -cyclodextrin.
- pullulan As a substrate, enzymatic hydrolysis of a-(1,6)-glycosidic bond to break it, preparing higher purity maltotriose, controlling the mass concentration of pullulan to 3% ⁇ 5 %, the concentration of pullulan added is controlled at 100 U/g pullulan, and the pH is about 5.0, the reaction temperature is 45 ° C, hydrolysis is 8 h, too high or too low pH, hydrolysis temperature and substrate concentration will be Decrease the DE value of the hydrolyzate; The enzyme protein was removed by centrifugation and concentrated to a certain volume by rotary evaporation.
- the sugar solution was adjusted to 5.0, and the pullulanase 200 U/g ⁇ -cyclodextrin was added, and then the reaction was incubated at 50 Torr for 48 hours; the enzyme was removed in a boiling water bath, and the enzyme protein was removed by centrifugation, and then filtered through a lOOODa filter to remove the reaction solution.
- Small molecular sugars such as glucose, maltose, maltotriose, etc., in a solution of maltotriosyl- ⁇ -cyclodextrin, precipitated with absolute ethanol, centrifuged to discard the supernatant, and the precipitate is 50 ° C. Drying in vacuo gave a white foamy solid, i.e., maltotriosyl-[beta]-cyclodextrin product.
- the branched cyclodextrin prepared by the method of the present invention has good water solubility and drug solubilization, and the hemolysis effect is lower than that of the parent ⁇ -cyclodextrin.
- the method utilizes pullulan as raw material to prepare maltotriosyl- ⁇ -cyclodextrin, thereby eliminating the refining process and cost in the production of maltotriose, and the conversion rate of cyclodextrin is more than 30%, thereby greatly reducing production. cost.
- the invention realizes the simplification of the preparation process of maltotriosyl- ⁇ -cyclodextrin, the reaction condition is mild and controllable, the production safety is high, and the cost is low; the obtained product has high solubility and high safety.
- the reaction condition is mild and controllable, the production safety is high, and the cost is low; the obtained product has high solubility and high safety.
- the mass concentration ratio of cyclodextrin is 5:1, the pH value of the mixture is adjusted to about 5.0, the pullulanase is added to 200 U/g-cyclodextrin, and then the reaction is kept at 50 ° C for 48 h.
- the boiling water bath is used to kill the enzyme for 15 min. After removing the enzyme protein by centrifugation, it is filtered through a lOOODa filter to remove small molecular sugars such as glucose, maltose, maltotriose, etc. in the reaction solution.
- maltotriosyl- ⁇ -cyclodextrin forms a complex with it.
- the water solubility is 15.3 g/mL, and the water solubility of ⁇ -cyclodextrin and its complex is 0.94 g/mL.
- maltotriosyl- ⁇ -cyclodextrin causes 50% hemolysis of human red blood cells. 10.52 mmol/L, while the mother ⁇ -cyclodextrin caused a 50% hemolysis concentration of human red blood cells of 5.43 mmol/L.
- 5% pullulan solution is used to adjust the pH value to about 5.0, and the pullulan is added.
- the enzyme is lOOU/g pullulan, the super constant temperature water bath controls the reaction temperature at 45 °C, hydrolyzes for 8h, the enzyme is removed by boiling water bath, the enzyme protein is removed by centrifugation, and then concentrated by evaporation to a certain volume to obtain high-purity malt syrup.
- the content of maltotriose was determined by high performance liquid chromatography to be 83%.
- ⁇ -cyclodextrin adjusting the mass concentration ratio of maltotriose to ⁇ -cyclodextrin to 6:1, adjusting the pH value of the mixture to about 5.0, and adding the pullulanase to 200 U/g P-cyclodextrin.
- the reaction was then incubated at 50 ° C for 48 h.
- the enzyme was incubated in a boiling water bath for 15 min, and the enzyme protein was removed by centrifugation, and then filtered through a lOOODa filter to remove small molecular sugars such as glucose, maltose, maltotriose and the like in the reaction solution.
- maltotriosyl- ⁇ -cyclodextrin In the solution of maltotriosyl- ⁇ -cyclodextrin, absolute ethanol was added to precipitate, and the supernatant was discarded by centrifugation. The precipitate was collected and dried under vacuum at 50 ° C to obtain a white foamy solid, maltotriosyl- ⁇ . - Cyclodextrin.
- the solubility at 25 ° C is 0.941 mol / L
- the solubility at 40 ° C is 0.949 mol / L.
- the solubility of ⁇ -cyclodextrin at 25 ° C is 0.016 mol / L
- the solubility at 40 ° C is 0.032 mol / L.
- the water solubility of maltotriosyl- ⁇ -cyclodextrin and its complex is 15.2 g/mL
- the water solubility of ⁇ -cyclodextrin and its complex is 0.94 g/mL.
- maltotriosyl- ⁇ -cyclodextrin caused 50% hemolysis of human red blood cells to be 10.61 mmol/L
- maternal ⁇ -cyclodextrin caused 50% hemolysis of human red blood cells to be 5.43 mmol/L.
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Abstract
Provided is a method for producing maltotriosyl beta-cyclodextrin, which comprises the following steps: using pulullan polysaccharide as zymolyte, converting pulullan polysaccharide into maltotriose by using pullulanase to hydrolyze the alpha -(1,6) glycosidic bond of pulullan polysaccharide, adjusting the concentration of maltotriosyl and beta-cyclodextrin to a proper ratio, and then linking maltotriosyl and beta-cyclodextrin to produce maltotriosyl beta-cyclodextrin through the reverse synthetic ability of pulullanase.
Description
一种麦芽三糖基 -β-环糊精的酶法制备方法 技术领域 Enzymatic preparation method of maltotriosyl-β-cyclodextrin
一种麦芽三糖基 -β-环糊精的酶法制备方法， 涉及一种以普鲁兰多糖和 β - 环糊精为原料， 通过普鲁兰酶的水解特性和逆向合成能力， 制备分支环糊精的 方法， 属于环糊精技术领域。 An enzymatic preparation method of maltotriosyl-β-cyclodextrin, which relates to preparing a branch by using pullulan and β-cyclodextrin as raw materials through hydrolysis property and reverse synthesis ability of pullulanase The method of cyclodextrin belongs to the technical field of cyclodextrin.
背景技术 Background technique
环糊精 （Cyclodextrin) 是由 D-吡喃型葡萄糖单元以 α - ( 1、 4) -糖苷键连 接而成的一类环状低聚糖。 环糊精内疏水外亲水的筒状结构决定了它具有分子 胶囊的特性， 可以和许多化合物作用生成包合物， 从而表现出缓释、 增溶、 抗 氧化、 抗光照、 热稳定性增加和掩盖异味等优点。 近年来对环糊精的研究愈来 愈热， 己在医药、 食品、 环保、 日化和超分子化学领域取得了相当的成就。 Cyclodextrin is a class of cyclic oligosaccharides which are linked by D-glucopyranose units with α-(1,4)-glycosidic linkages. The hydrophobic outer tubular structure of the cyclodextrin determines its molecular capsule properties, and it can react with many compounds to form inclusion complexes, which exhibit sustained release, solubilization, oxidation resistance, anti-light, and thermal stability. And cover up the odor and other advantages. In recent years, the research on cyclodextrin has become hotter and hotter, and has made considerable achievements in the fields of medicine, food, environmental protection, daily chemical and supramolecular chemistry.
根据葡萄糖单元数目的不同， 环糊精可以分为 α-、 β -、 γ -、 δ -、 ε-等环糊精。 比较常见的环糊精是分别由 6、 7和 8个葡萄糖残基组成的 α-， β-和 γ-环糊精。 其中含有 7个葡萄糖残基的 β-环糊精生产工艺简单， 成本较低， 是目前工业上 唯一能大量生产且应用较广的环糊精产品。但是， β-环糊精具有水溶性较差、溶 血性以及非消化道安全性的问题， 制约了其在各方面的进一步应用。 分支环糊 精便是通过引入糖基来提高 环糊精水溶解度的一种策略， 通过 β-环糊精的糖 基改性一方面可以达到增加水溶性的目的， 另一方面则可以降低母体环糊精的 溶血效应和非消化道安全性。 β-环糊精的糖基改性一般通过化学法和酶法两种方 法， 相对于化学法改性， 酶法改性则具有专一性强， 安全性高等优点。 Depending on the number of glucose units, cyclodextrins can be classified into cyclodextrins such as α-, β-, γ-, δ-, ε-. The more common cyclodextrins are alpha-, beta- and gamma-cyclodextrins consisting of 6, 7 and 8 glucose residues, respectively. The β-cyclodextrin containing 7 glucose residues has a simple production process and low cost, and is the only cyclodextrin product which can be mass-produced and widely used in the industry. However, β-cyclodextrin has problems of poor water solubility, hemolytic and non-digestive tract safety, which restricts its further application in various aspects. Branched cyclodextrin is a strategy to increase the solubility of cyclodextrin water by introducing a glycosyl group. The sugar group modification of β-cyclodextrin can achieve the purpose of increasing water solubility on the one hand, and reduce the matrix on the other hand. Hemolysis and non-digestive safety of cyclodextrins. The glycosyl modification of β-cyclodextrin is generally carried out by chemical method and enzymatic method, and compared with chemical modification, enzymatic modification has the advantages of high specificity and high safety.
发明内容 Summary of the invention
本发明的目的是提供了一种分支环糊精的制备方法， 即以普鲁兰多糖和 β - 环糊精为底物， 利用普鲁兰酶的水解特性和逆向合成作用， 先将普鲁兰多糖转 化为较高纯度的麦芽三糖， 再将麦芽三糖接枝到母体 环糊精上， 从而得到麦 芽三糖基 - β -环糊精。 The object of the present invention is to provide a method for preparing branched cyclodextrin, which uses pullulan and β-cyclodextrin as substrates, and utilizes the hydrolysis property and reverse synthesis of pullulanase to firstly The polysaccharide is converted into a higher purity maltotriose, and the maltotriose is grafted onto the parent cyclodextrin to obtain maltotriosyl-β-cyclodextrin.
本发明的技术方案： 一种麦芽三糖基 -β-环糊精的酶法制备方法， 以普鲁兰 多糖和 β-环糊精为底物， 利用普鲁兰酶的水解特性和逆向合成能力， 首先将普 鲁兰多糖水解为麦芽三糖， 然后将其接枝到 β-环糊精上； 步骤为： Technical Solution of the Invention: An enzymatic preparation method of maltotriosyl-β-cyclodextrin, using pullulan and β-cyclodextrin as substrates, utilizing hydrolysis characteristics and reverse synthesis of pullulanase The ability to first hydrolyze pullulan into maltotriose and then graft it onto the β-cyclodextrin; the steps are:
( 1 ) 以普鲁兰多糖为底物， 酶法水解 a- ( 1、 6) -糖苷键使其断裂， 制备 较高纯度的麦芽三糖，控制普鲁兰多糖质量浓度为 3%〜5%，加入的普鲁兰酶浓 度控制在 100U/g普鲁兰多糖， 在 pH值 5.0左右、 反应温度 45°C时， 水解 8h， 过高或过低的 pH、水解温度和底物浓度都会降低水解液的 DE值； 沸水浴灭酶，
离心除去酶蛋白后旋转蒸发浓縮至一定体积。 (1) Using pullulan as a substrate, enzymatic hydrolysis of a-(1,6)-glycosidic bond to break it, preparing higher purity maltotriose, controlling the mass concentration of pullulan to 3%~5 %, the concentration of pullulan added is controlled at 100 U/g pullulan, and the pH is about 5.0, the reaction temperature is 45 ° C, hydrolysis is 8 h, too high or too low pH, hydrolysis temperature and substrate concentration will be Decrease the DE value of the hydrolyzate; The enzyme protein was removed by centrifugation and concentrated to a certain volume by rotary evaporation.
(2) 调节麦芽三糖与 环糊精的质量浓度之比为 5:1〜6:1， 控制麦芽三糖 浓度为 l-1.2g/mL， 如实验可以选用 20mL 糖液， 调节其中麦芽三糖的量为 20-24g, 再加入 4g P _环糊精， 由于 环糊精水溶性较差， 混合体系中的 β-环 糊精不能完全溶解，呈现过饱和状态。调节糖液 ρΗ为 5.0，添加普鲁兰酶 200U/g β-环糊精，然后在 50Ό保温反应 48h;沸水浴灭酶，离心除去酶蛋白后，用 lOOODa 滤膜过滤， 以除去反应液中的小分子糖类如葡萄糖、 麦芽糖、 麦芽三糖等， 在 麦芽三糖基 -β-环糊精的溶液中， 加入无水乙醇沉析， 离心弃掉上清液， 沉析物 50°C真空干燥得到白色泡沬状固体， 即麦芽三糖基 -β-环糊精产品。 (2) Adjust the ratio of the mass concentration of maltotriose to cyclodextrin to 5:1~6:1, and control the concentration of maltotriose to be 1-1.2g/mL. If the experiment can choose 20mL sugar solution, adjust the malt three. The amount of sugar is 20-24 g, and 4 g of P-cyclodextrin is added. Since the cyclodextrin is poorly water-soluble, the β-cyclodextrin in the mixed system is not completely dissolved and exhibits a supersaturated state. The sugar solution was adjusted to 5.0, and the pullulanase 200 U/g β-cyclodextrin was added, and then the reaction was incubated at 50 Torr for 48 hours; the enzyme was removed in a boiling water bath, and the enzyme protein was removed by centrifugation, and then filtered through a lOOODa filter to remove the reaction solution. Small molecular sugars such as glucose, maltose, maltotriose, etc., in a solution of maltotriosyl-β-cyclodextrin, precipitated with absolute ethanol, centrifuged to discard the supernatant, and the precipitate is 50 ° C. Drying in vacuo gave a white foamy solid, i.e., maltotriosyl-[beta]-cyclodextrin product.
本发明的有益效果： 本发明方法制备得到的分支环糊精具有较好的水溶性 和药物增溶性， 而且溶血效应低于母体 β-环糊精。 该方法以普鲁兰多糖为原料 制备麦芽三糖基 -β-环糊精， 省却了麦芽三糖生产中的精制过程和费用， 环糊精 的转化率达到 30%以上，从而大大降低了生产成本。本发明实现了麦芽三糖基 -β- 环糊精制备过程的简单化， 反应条件温和可控， 生产安全性高， 成本较低的优 点； 其所获得的产品具有溶解度高、 安全性高的特点， 适用于食品、 医药、 化 妆品、 添加剂、 香精香料、 环保以及分析检测等领域。 Advantageous Effects of the Invention: The branched cyclodextrin prepared by the method of the present invention has good water solubility and drug solubilization, and the hemolysis effect is lower than that of the parent β-cyclodextrin. The method utilizes pullulan as raw material to prepare maltotriosyl-β-cyclodextrin, thereby eliminating the refining process and cost in the production of maltotriose, and the conversion rate of cyclodextrin is more than 30%, thereby greatly reducing production. cost. The invention realizes the simplification of the preparation process of maltotriosyl-β-cyclodextrin, the reaction condition is mild and controllable, the production safety is high, and the cost is low; the obtained product has high solubility and high safety. Features, suitable for food, medicine, cosmetics, additives, flavors and fragrances, environmental protection and analytical testing.
具体实施方式 detailed description
实施例 1 Example 1
反应体系中， 选用 3%普鲁兰多糖溶液， 调节 ρΗ值 5.0左右， 加入普鲁兰 酶为 100U/g普鲁兰多糖， 超级恒温水浴控制反应温度在 45° (：， 水解 8h， 沸水 浴灭酶， 离心除去酶蛋白后旋转蒸发浓缩至一定体积， 制得高纯度麦芽三糖浆， 经高效液相色谱测定麦芽三糖含量为 85%。 加入 β-环糊精， 调节麦芽三糖与 β- 环糊精的质量浓度之比为 5:1， 调节混合液 ρΗ值 5.0左右， 添加普鲁兰酶为 200U/g -环糊精， 然后在 50°C保温反应 48h。沸水浴灭酶 15min， 离心除去酶蛋 白后， 用 lOOODa滤膜过滤， 以除去反应液中的小分子糖类如葡萄糖、 麦芽糖、 麦芽三糖等。 在麦芽三糖基 -β-环糊精的溶液中， 加入无水乙醇沉析， 离心弃掉 上清液， 收集沉淀后， 50°C真空干燥得到白色泡沬状固体即麦芽三糖基 -β-环糊 精。 25°C其溶解度为 0.945mol/L， 40°C其溶解度为 0.951mol/L; 而 25Όβ-环糊 精溶解度为 0.016mol/L， 40°C溶解度为 0.032mol/L。 以洋地黄毒甙为例， 麦芽三 糖基 -β-环糊精与其形成复合物的水溶解度为 15.3 g/mL， β-环糊精与其形成复合 物的水溶解度为 0.94 g/mL。 37 °C时， 麦芽三糖基 -β-环糊精引起人红血球 50% 溶血的浓度为 10.52mmol/L， 而母体 β-环糊精引起人红血球 50%溶血的浓度为 5.43 mmol/L。 In the reaction system, 3% pullulan solution was selected, the pH value was adjusted to about 5.0, the pullulanase was added to 100 U/g pullulan, and the super constant temperature water bath was controlled at 45 ° (:, hydrolysis for 8 h, boiling water bath The enzyme was removed, the enzyme protein was removed by centrifugation, and concentrated to a certain volume by rotary evaporation to obtain a high-purity maltose syrup. The content of maltotriose was determined by high performance liquid chromatography to be 85%. β-cyclodextrin was added to adjust maltotriose and β. - The mass concentration ratio of cyclodextrin is 5:1, the pH value of the mixture is adjusted to about 5.0, the pullulanase is added to 200 U/g-cyclodextrin, and then the reaction is kept at 50 ° C for 48 h. The boiling water bath is used to kill the enzyme for 15 min. After removing the enzyme protein by centrifugation, it is filtered through a lOOODa filter to remove small molecular sugars such as glucose, maltose, maltotriose, etc. in the reaction solution. In the solution of maltotriosyl-β-cyclodextrin, The ethanol was precipitated, the supernatant was discarded by centrifugation, and the precipitate was collected, and dried under vacuum at 50 ° C to obtain a white foamy solid, maltotriosyl-β-cyclodextrin. The solubility at 25 ° C was 0.945 mol/L. The solubility at 40 ° C is 0.951 Mol/L; and the solubility of 25Όβ-cyclodextrin is 0.016mol/L, and the solubility at 40°C is 0.032mol/L. Taking digitalis scorpion as an example, maltotriosyl-β-cyclodextrin forms a complex with it. The water solubility is 15.3 g/mL, and the water solubility of β-cyclodextrin and its complex is 0.94 g/mL. At 37 °C, maltotriosyl-β-cyclodextrin causes 50% hemolysis of human red blood cells. 10.52 mmol/L, while the mother β-cyclodextrin caused a 50% hemolysis concentration of human red blood cells of 5.43 mmol/L.
实施例 2 Example 2
反应体系中， 选用 5%普鲁兰多糖溶液， 调节 pH值 5.0左右， 加入普鲁兰
酶为 lOOU/g普鲁兰多糖， 超级恒温水浴控制反应温度在 45 °C， 水解 8h， 沸水 浴灭酶， 离心除去酶蛋白后旋转蒸发浓缩至一定体积， 制得高纯度麦芽三糖浆， 经高效液相色谱测定麦芽三糖含量为 83%。 加入 β-环糊精， 调节麦芽三糖与 β- 环糊精的质量浓度之比为 6:1， 调节混合液 ρΗ值 5.0左右， 添加普鲁兰酶为 200U/g P-环糊精， 然后在 50°C保温反应 48h。沸水浴灭酶 15min， 离心除去酶蛋 白后， 用 lOOODa滤膜过滤， 以除去反应液中的小分子糖类如葡萄糖、 麦芽糖、 麦芽三糖等。 在麦芽三糖基 -β-环糊精的溶液中， 加入无水乙醇沉析， 离心弃掉 上清液， 收集沉淀后， 50°C真空干燥得到白色泡沫状固体即麦芽三糖基 -β-环糊 精。 25°C其溶解度为 0.941mol/L， 40°C其溶解度为 0.949mol/L。 而 25°C β-环糊 精溶解度为 0.016mol/L， 40°C溶解度为 0.032mol/L。 以洋地黄毒甙为例， 麦芽三 糖基 -β-环糊精与其形成复合物的水溶解度为 15.2 g/mL， β-环糊精与其形成复合 物的水溶解度为 0.94 g/mL。 37 °C时， 麦芽三糖基 -β-环糊精引起人红血球 50% 溶血的浓度为 10.61mmol/L, 而母体 β-环糊精引起人红血球 50%溶血的浓度为 5.43mmol/L。
In the reaction system, 5% pullulan solution is used to adjust the pH value to about 5.0, and the pullulan is added. The enzyme is lOOU/g pullulan, the super constant temperature water bath controls the reaction temperature at 45 °C, hydrolyzes for 8h, the enzyme is removed by boiling water bath, the enzyme protein is removed by centrifugation, and then concentrated by evaporation to a certain volume to obtain high-purity malt syrup. The content of maltotriose was determined by high performance liquid chromatography to be 83%. Adding β-cyclodextrin, adjusting the mass concentration ratio of maltotriose to β-cyclodextrin to 6:1, adjusting the pH value of the mixture to about 5.0, and adding the pullulanase to 200 U/g P-cyclodextrin. The reaction was then incubated at 50 ° C for 48 h. The enzyme was incubated in a boiling water bath for 15 min, and the enzyme protein was removed by centrifugation, and then filtered through a lOOODa filter to remove small molecular sugars such as glucose, maltose, maltotriose and the like in the reaction solution. In the solution of maltotriosyl-β-cyclodextrin, absolute ethanol was added to precipitate, and the supernatant was discarded by centrifugation. The precipitate was collected and dried under vacuum at 50 ° C to obtain a white foamy solid, maltotriosyl-β. - Cyclodextrin. The solubility at 25 ° C is 0.941 mol / L, and the solubility at 40 ° C is 0.949 mol / L. The solubility of β-cyclodextrin at 25 ° C is 0.016 mol / L, and the solubility at 40 ° C is 0.032 mol / L. Taking digoxigenin as an example, the water solubility of maltotriosyl-β-cyclodextrin and its complex is 15.2 g/mL, and the water solubility of β-cyclodextrin and its complex is 0.94 g/mL. At 37 °C, maltotriosyl-β-cyclodextrin caused 50% hemolysis of human red blood cells to be 10.61 mmol/L, while maternal β-cyclodextrin caused 50% hemolysis of human red blood cells to be 5.43 mmol/L.
Claims
1、一种麦芽三糖基 -β-环糊精的酶法制备方法，其特征是以普鲁兰多糖和 β- 环糊精为底物， 利用普鲁兰酶的水解特性和逆向合成能力,， 首先将普鲁兰多糖 水解为麦芽三糖， 然后将其接枝到 β-环糊精上； 步骤为： 1. An enzymatic preparation method of maltotriosyl-β-cyclodextrin characterized by using pullulan and β-cyclodextrin as substrates, utilizing hydrolysis characteristics and reverse synthesis ability of pullulanase First, the pullulan is hydrolyzed to maltotriose, which is then grafted onto the β-cyclodextrin; the steps are:
( 1 ) 以普鲁兰多糖为底物， 酶法水解 α- ( 1、 6) -糖苷键使其断裂， 制备 较高纯度的麦芽三糖，控制普鲁兰多糖质量浓度为 3%〜5%，加入的普鲁兰酶浓 度控制在 100U/g普鲁兰多糖， 在 pH值 5.0、 反应温度 45°C时， 水解 8h， 沸水 浴灭酶， 离心除去酶蛋白后旋转蒸发浓缩至一定体积； (1) Using pullulan as a substrate, enzymatic hydrolysis of α-(1,6)-glycosidic bond to break it, preparing higher purity maltotriose, controlling the mass concentration of pullulan to 3%~5 %, the concentration of pullulanase added is controlled at 100 U/g pullulan, and the pH is 5.0, the reaction temperature is 45 ° C, hydrolysis is carried out for 8 h, the enzyme is removed by boiling water bath, the enzyme protein is removed by centrifugation, and concentrated to a certain volume by rotary evaporation. ;
(2)调节麦芽三糖与 β -环糊精的质量浓度之比为 5:1〜6:1，控制麦芽三糖 浓度为 l-1.2g/mL， 调节糖液 pH为 5.0， 添加普鲁兰酶 200U/g β-环糊精， 然后 在 50°C保温反应 48h; 沸水浴灭酶， 离心除去酶蛋白后， 用 lOOODa滤膜过滤， 以除去反应液中的小分子糖类， 在麦芽三糖基 -β-环糊精的溶液中， 加入无水乙 醇沉析， 离心弃掉上清液， 沉析物 50°C真空干燥得到白色泡沫状固体， 即麦芽 三糖基 -β-环糊精产品。
(2) adjusting the mass concentration ratio of maltotriose to β-cyclodextrin to 5:1~6:1, controlling the concentration of maltotriose to 1-1.2g/mL, adjusting the pH of the sugar solution to 5.0, adding Pulu Blue enzyme 200U / g β-cyclodextrin, and then incubated at 50 ° C for 48h; boiling water bath enzyme, centrifuged to remove enzyme protein, filtered with lOOODa filter to remove small molecules of sugar in the reaction solution, in malt The solution of trisaccharide-β-cyclodextrin is precipitated by adding absolute ethanol, and the supernatant is discarded by centrifugation. The precipitate is dried under vacuum at 50 ° C to obtain a white foamy solid, ie, maltotriosyl-β-ring. Dextrin products.
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| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
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| CN103014097B (en) * | 2012-11-20 | 2014-02-19 | 江南大学 | Maltotriose preparation method with starch as raw material and special fungal alpha-amylase thereof |
| CN107177647B (en) * | 2017-05-16 | 2019-11-26 | 江南大学 | A kind of enzymatic treatment maltodextrin prepares the method and its application of branched cyclodextrin |
| CN110747245B (en) * | 2019-11-29 | 2021-07-27 | 江南大学 | Method for preparing malt oligosaccharide syrup by using complex enzyme |
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| JPH0515368A (en) * | 1991-07-05 | 1993-01-26 | Nagase Seikagaku Kogyo Kk | New pullulanase and production thereof |
| JPH0759585A (en) * | 1993-08-20 | 1995-03-07 | Akebono Brake Res & Dev Center Ltd | Production of pullulan oligosaccharide |
| KR100270911B1 (en) * | 1998-02-03 | 2000-11-01 | 박호군 | Klebsiella sp. producing pullulanase and process for preparation of maltosyl beta0cyclodextrin |
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| CN101555507A (en) * | 2009-05-18 | 2009-10-14 | 苏理 | Method for using pulullan to prepare high-purity maltotriose |
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| JPH0515368A (en) * | 1991-07-05 | 1993-01-26 | Nagase Seikagaku Kogyo Kk | New pullulanase and production thereof |
| JPH0759585A (en) * | 1993-08-20 | 1995-03-07 | Akebono Brake Res & Dev Center Ltd | Production of pullulan oligosaccharide |
| KR100270911B1 (en) * | 1998-02-03 | 2000-11-01 | 박호군 | Klebsiella sp. producing pullulanase and process for preparation of maltosyl beta0cyclodextrin |
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| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
| US11878079B2 (en) | 2017-04-14 | 2024-01-23 | Capsugel Belgium Nv | Pullulan capsules |
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