WO2022028107A1 - 一种丝素丝胶蛋白复合溶液的制备方法 - Google Patents

一种丝素丝胶蛋白复合溶液的制备方法 Download PDF

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WO2022028107A1
WO2022028107A1 PCT/CN2021/099614 CN2021099614W WO2022028107A1 WO 2022028107 A1 WO2022028107 A1 WO 2022028107A1 CN 2021099614 W CN2021099614 W CN 2021099614W WO 2022028107 A1 WO2022028107 A1 WO 2022028107A1
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silk
sericin
silk fibroin
solution
water
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王建南
刘苹
裔洪根
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Suzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms

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  • the invention relates to the technical field of biomedical materials, in particular to a preparation method of a silk fibroin sericin composite solution.
  • tissue repair methods include traditional autologous transplantation, allogeneic transplantation and artificial tissue engineering scaffold transplantation.
  • autologous transplantation repair has the best therapeutic effect, it has serious constraints on many factors such as damage to the donor site, insufficient dose and complications; allogeneic transplantation faces problems such as immune rejection and disease transmission; tissue engineering was proposed in the 1990s. , and then the research on tissue engineering scaffold materials has been rapidly developed. Synthetic polymer materials in tissue engineering have the advantages of wide sources, tunable physicochemical properties, and excellent processability.
  • tissue engineering scaffolds Lack of biological activity, poor cytocompatibility, no degradation or degradation products affecting the tissue microenvironment. In situ induced tissue regeneration is considered to be the most ideal mode for repairing defective or diseased tissue, so natural polymers are widely favored in the research of tissue engineering scaffolds due to their excellent bioactivity and degradability, such as collagen, gelatin, and alginate. , hyaluronic acid, chitosan, etc.
  • the mechanical properties of most materials are very poor, and the application research as engineering scaffolds for tissues (such as bones, teeth, etc.) that require high mechanical properties still faces great challenges.
  • Silk fibroin and sericin have excellent cytocompatibility, hemocompatibility and histocompatibility, and controllable molding.
  • tissue engineering scaffold materials they have been widely used in tissue engineering applications such as skin, blood vessels, and bones.
  • the currently studied regenerated silk (silk fibroin, sericin) protein scaffolds also have poor mechanical properties and are difficult to meet the requirements for replacement of hard tissues such as bones and teeth.
  • the macromolecular structure of vellum and sericin is greatly damaged, and the degree of polymerization (molecular weight) of the polymer is an extremely important intrinsic factor that determines the performance of the polymer.
  • the protein in silk has high purity, unique composition, and the preparation method can be relatively simple and mild, that is, it can control the damage to silk macromolecules, unlike collagen. Purification is complicated.
  • the silk fibroin in the inner core of silk accounts for 70-80% of the silk mass.
  • the macromolecules are connected by disulfide bonds with six heavy chains of about 390 kDa and six light chains of about 26 kDa, and then hydrophobic with another glycoprotein/P25 of about 25 kDa.
  • the heavy chain has the basis of the sequence structure to form high-performance materials; the sericin covering the silk accounts for 20-30% of the silk quality, and the molecular weight of sericin is also relatively large, and there are as many as 9 kinds. From the histological point of view, it is pointed out that there are three layers of sericin, namely, sericin, sericin, and sericin. And the internal solubility is getting lower and lower, and the ⁇ conformation increases.
  • the regenerated silk fibroin solutions are all obtained by dissolving strong alkali weak acid salts or enzyme degumming salts. These methods cause the destruction of silk fibroin macromolecules; the regenerated sericin solutions are mainly derived from silkworm cocoons, and the two solutions are generally prepared independently.
  • CN02138129.1 discloses the preparation of a silk fibroin and sericin composite tissue engineering scaffold. The composite material is prepared by mixing pre-prepared regenerated silk fibroin and regenerated sericin in proportion.
  • the degumming method uses a strong base and weak acid salt solvent, and sericin is extracted from the outer part of the silkworm cocoon silk covered with sericin.
  • CN201510353363.8 discloses a bone tissue engineering scaffold material containing silk fibroin and sericin and a preparation method thereof.
  • the regenerated silk fibroin and regenerated sericin are also obtained in steps and then mixed to prepare the scaffold material.
  • the preparation of silk fibroin does not disclose specific process conditions.
  • the sericin is obtained from silk cocoons and treated with organic solvents such as alcohol.
  • the technical problem solved by the present invention is to provide a preparation method of a silk fibroin sericin composite solution, which has the characteristics of high viscosity and high molecular weight.
  • the present application provides a preparation method of a silk fibroin sericin composite solution, comprising the following steps:
  • A) silk is placed in warm water for pretreatment, then placed in 65 ⁇ 95 °C of water for treatment, and dried to obtain the silk of initial treatment;
  • the temperature of the warm water is 35 ⁇ 60° C., and the time of the pretreatment is 1 ⁇ 10 min.
  • the method further includes: taking out the pretreated silk, washing and dehydrating in water at 25-30°C.
  • the treatment time in water at 65-95° C. is 1-8 h.
  • the ratio of the silk to warm water is 1 g: (40-60) ml
  • the ratio of the pretreated silk to water is 1 g: (40-60) ml.
  • the method before the drying after the treatment, further comprises: washing the treated silk in water at 25-30°C and then dehydrating; the drying adopts hot air drying at 40-60°C.
  • the neutral salt solution is an 8-10 M lithium bromide aqueous solution, and the ratio of the initially treated silk to the lithium bromide aqueous solution is 1 g: (20-30) ml.
  • the temperature of the treatment is 50-80°C.
  • the purifying method is specifically:
  • the obtained silk fibroin sericin composite solution is poured into a dialysis bag with a molecular weight cut-off of 10-50 kDa, and placed in deionized water for continuous dialysis for 1-3 days.
  • the present application provides a method for preparing a silk fibroin sericin composite solution, which comprises first placing the silk in warm water for pretreatment, and then placing it in water for treatment to obtain initially treated silk; and then treating it in a neutral salt solution , the silk fibroin sericin composite solution is obtained after purification; the present application adopts the mild degumming and one-step dissolution method to prepare the silk fibroin sericin composite solution.
  • the pretreatment in warm water and the subsequent treatment in water at a specific temperature make the silk surface
  • the sericin is partially or completely removed, while the silk fibroin macromolecular chain will not be destroyed in the subsequent degumming process during the whole process of the special warm water treatment.
  • the present application uses deionized water, mild degumming, and one-step dissolution to prepare large-molecular-weight and high-viscosity silk fibroin sericin.
  • the method can avoid the damage of the silk fibroin fiber macromolecules, and finally the silk fibroin and sericin in the prepared silk fibroin and sericin composite solution have high viscosity and large molecular weight.
  • the embodiment of the present invention discloses a preparation method of a silk fibroin sericin composite solution, which includes the following steps:
  • A) silk is placed in warm water for pretreatment, then placed in 65 ⁇ 95 °C of water for treatment, and dried to obtain the silk of initial treatment;
  • the silk is placed in warm water for pretreatment, and then placed in water at 65-95° C. for treatment, and then dried to obtain the pre-treated silk; in this process, The sericin of silk is completely or partially dissolved, while the silk fibroin is still in the state of fiber. In water, it is in solution state.
  • the temperature of the warm water is 35-60°C, that is, the silk can be pretreated in warm water at 35°C, 40°C, 45°C, 50°C, 55°C or 60°C, and the ratio of the silk to the warm water is 1 g : (40-60) ml, more specifically, the ratio of the silk to the warm water is 1g: 42ml, 1g: 45ml, 1g: 48ml, 1g: 52ml, 1g: 55ml, 1g: 58ml.
  • the pretreatment is carried out in the above-mentioned warm water at a constant temperature, and the time of the pretreatment is 1-10 minutes, and the above-mentioned pretreatment is always carried out in stirring, so as to ensure that the silk can be initially and completely submerged in the water.
  • the pretreated silk is taken out and washed and dehydrated in deionized water at 25-30°C.
  • the dehydrated silk is treated in water at 65 to 95°C. , 90 °C or 93 °C water treatment.
  • the method that the silk is pretreated in water and then treated in water does not involve any reagent treatment, and can dissolve sericin while protecting the macromolecular structure of silk fibroin from being destroyed.
  • the ratio of the pretreated silk to water is 1g:(40 ⁇ 60)ml, more specifically, the ratio of the pretreated silk to water is 1g:42ml, 1g:45ml, 1g:48ml, 1g : 50ml, 1g: 52ml, 1g: 55ml, 1g: 58ml or 1g: 60ml.
  • the degumming rate of silk sericin can be adjusted according to the different temperature and time of the water, so as to realize the preparation of the composite solution of silk fibroin sericin with different viscosities and different molecular weights; in this application, the water treatment time is preferably 1-8h , so that the silk is partially or completely degummed; more specifically, the water treatment time is 1.5h, 2h, 2.5h, 3h, 3.2h, 3.6h, 4.2h, 4.5h, 5.0h, 5.5h, 6.0h , 6.5h, 6.8h, 7.0h or 7.5h. After the treatment, the treated silk is washed in water at 25-30°C, then dehydrated and dried; the drying adopts hot air drying at 40-60°C.
  • the silk undergoes the above-mentioned treatment to obtain the initially treated silk, which is still in a fibrous state and realizes the protection of silk fibroin, so as to ensure the high molecular weight and high viscosity of silk fibroin sericin.
  • the initially treated silk is then treated in a neutral salt solution and purified to obtain a silk fibroin sericin composite solution.
  • the fibrous silk that is initially treated is dissolved to obtain a composite solution of silk fibroin sericin.
  • the inner core of the silk is silk fibroin, and the periphery is wrapped with different contents of sericin.
  • the neutral salt solution is specifically selected from the lithium bromide aqueous solution in this application, and its concentration is 8-10M, and the ratio of the initially treated silk to the lithium bromide aqueous solution is 1 g: (20-30) ml, more specifically, The ratio of the initially treated silk to the lithium bromide aqueous solution is 1g:22ml, 1g:24ml, 1g:25ml, 1g:27ml or 1g:29ml.
  • the temperature of the treatment is specifically 50-80°C, and more specifically, the temperature of the treatment is specifically 52°C, 57°C, 62°C, 65°C, 67°C, 68°C, 72°C, 75°C or 78°C.
  • the obtained silk fibroin sericin dissolving solution is purified to remove lithium bromide and low molecular weight silk fibroin sericin; the purification method is specifically pouring the obtained silk fibroin sericin complex dissolving solution into Place in a dialysis bag with a molecular weight cut-off of 10-50kDa and place it in deionized water for continuous dialysis for 1-3 days.
  • the invention adopts the mild degumming and one-step dissolving method to prepare the silk fibroin and sericin composite solution, which breaks through the traditional separate preparation of silk fibroin and sericin, which are respectively dissolved and mixed before being used to prepare the composite material. It is important that the molecular weight of silk protein in the composite solution is concentrated above 150kDa, or contains a molecular weight of about 25kDa, while the molecular weight of the silk fibroin solution prepared by the reported conventional method or the solution of silk fibroin sericin dissolved and mixed shows the molecular weight. Continuous distribution, mainly distributed below 85kDa to 15kDa or even smaller.
  • the silk is treated with hot water, so that the silk fibroin macromolecular chain (especially the heavy chain) will not be destroyed during the degumming process; From the outside to the inside of the coating layer, the types, structures and properties of sericin are also different.
  • silk fibroin sericin composite solutions with different compositions and materials with different properties (especially mechanical properties) are obtained. .
  • the viscosity of the silk protein solution of the one-step dissolution method is much higher than that of the conventionally reported mixed solution.
  • the present invention provides a new technology for the preparation of high-performance regenerated silk protein material based on the molecular weight of the polymer, which is the fundamental factor affecting the mechanical properties of the polymer, and will solve the problem of natural biopolymer tissue that can induce regeneration and repair of hard tissue.
  • the degumming rate of the silk treated in steps 1 and 2 was measured by weighing method, and the degumming rate was measured to be 8%.
  • the dried silk is weighed and dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:25 (g/mL), and treated at 65° C. until the silk is completely dissolved to obtain a Bombyx mori silk fibroin sericin composite solution;
  • the viscosity of sericin (11Pa.s and 40Pa.s) is much higher than that of silk fibroin, and the presence of sericin will increase the viscosity of the solution, so the extreme value of the viscosity of the solution in this example under shearing action is lower than that of Example 1.
  • the degumming rate of the silk treated in steps 1 and 2 was measured by weighing method, and the degumming rate was measured to be 17%.
  • the dried silk is weighed and dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:25 (g/mL), and treated at 65° C. until the silk is completely dissolved to obtain a Bombyx mori silk fibroin sericin composite solution;
  • the degumming rate of the silk treated in steps 1 and 2 was measured by weighing method, and the degumming rate was measured to be 17%.
  • dehydrated silk was placed in deionized water preheated at 90°C and boiled at a constant temperature for 7 hours according to a liquor ratio of 1:50 (g/mL), and the silk was taken out and fully cleaned with deionization at 25-30°C, and dehydrated. Then dry it with hot air at 40-60°C;
  • the dried silk is weighed and dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:25 (g/mL), and treated at 65° C. until the silk is completely dissolved to obtain a Bombyx mori silk fibroin sericin composite solution;
  • the silk fibroin sericin composite solution was poured into a dialysis bag with a molecular weight cut-off of 14kDa, and the wall of the dialysis bag was a semi-permeable membrane. Place the dialysis bag filled with silk fibroin sericin solution in a container filled with deionized water, replace the water in the container with new deionized water every 2 hours, and continue dialysis for 3 days to obtain a purified home.
  • Silk fibroin sericin complex aqueous solution placed into a dialysis bag with a molecular weight cut-off of 14kDa, and the wall of the dialysis bag was a semi-permeable membrane.
  • the degumming rate of the silk treated in steps 1 and 2 was measured by weighing method, and the degumming rate was measured to be 23%.
  • the degumming rate is the same, that is, the sericin is completely removed, and the dialysis molecular weight is the same.
  • the molecular weight of the silk solution degummed by hot water in this example is not only large in molecular weight but also in a concentrated distribution.
  • Conventional sodium carbonate degumming The molecular weight of the silk solution is small and the distribution is very scattered; the larger the molecular weight, the higher the viscosity, and the smaller the molecular weight polydispersity coefficient, the higher the viscosity. times and 12.5 times.
  • the dried silk is weighed and dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:25 (g/mL), and treated at 65° C. until the silk is completely dissolved to obtain a Bombyx mori silk fibroin sericin composite solution;
  • the degumming rate of the silk treated in the first and second steps is measured by the weighing method, and the degumming rate is measured to be 23%.
  • the viscosity value and the viscosity extreme value under the initial shear action measured in this embodiment are Comparative Example 1 40 times and 21 times.
  • the Bombyx mori fibroin dissolving solution is perfused in a dialysis bag with a molecular weight cut-off of 14kDa, the wall of the dialysis bag is a semi-permeable membrane, and the dialysis bag perfused with the Bombyx mori silk fibroin dissolving solution is placed in a container filled with deionized water, The water in the container was replaced with new deionized water every 2 hours, and the dialysis continued for 3 days to obtain the purified silk fibroin aqueous solution;
  • a rotary evaporator to concentrate or dilute silk fibroin with deionized water to obtain an aqueous solution of a certain mass fraction.
  • the molecular weight distribution of the silk fibroin solution is measured by polyacrylamide gel electrophoresis, and it is mainly distributed below 85kDa, although the same A dialysis bag with a molecular weight cut-off of 14 kDa was used, but the content of silk fibroin below 15 kDa was still high.
  • the degumming rate of the silk treated in the first and second steps was measured by the weighing method, and the degumming rate was measured to be 23%.
  • the Bombyx mori fibroin dissolving solution is perfused in a dialysis bag with a molecular weight cut-off of 14kDa, the wall of the dialysis bag is a semi-permeable membrane, and the dialysis bag perfused with the Bombyx mori silk fibroin dissolving solution is placed in a container filled with deionized water, The water in the container was replaced with new deionized water every 2 hours, and the dialysis continued for 3 days to obtain the purified silk fibroin aqueous solution;
  • the dehydrated silk is placed in preheated deionized water at 90°C and cooked at a constant temperature for 7 hours according to the liquor ratio of 1:50 (g/mL), the fibers are discarded, and the sericin aqueous solution is collected and freeze-dried to obtain solid sericin;
  • the molecular weight and viscosity of the silk fibroin-sericin composite aqueous solutions prepared in Examples 1-5 are significantly higher than those of the silk protein solutions obtained by conventional degumming methods in Comparative Examples 1-2.

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Abstract

本发明提供了一种丝素丝胶蛋白复合溶液的制备方法，包括以下步骤：A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液。本申请采用温和脱胶、一步溶解法制备了丝素丝胶蛋白复合溶液，突破了传统的丝素蛋白和丝胶蛋白分别制备、分别溶解后混合再用于制备复合材料；重要的是，该复合溶液中丝蛋白分子量集中在150kDa以上；而报导的常规方法制备的丝素蛋白溶液或丝素丝胶溶解再混合的溶液分子量大小呈现连续分布、主要分布在85kDa以下直至15kDa甚至更小。

Description

一种丝素丝胶蛋白复合溶液的制备方法
本申请要求于2020年08月06日提交中国专利局、申请号为202010782559.X、发明名称为“一种丝素丝胶蛋白复合溶液的制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医用材料技术领域，尤其涉及一种丝素丝胶蛋白复合溶液的制备方法。
背景技术
随着外科手术的飞速发展，人类疾病的剧增、年轻化及世界人口的老龄化，组织移植物临床需求与治疗面临着越来越大的压力。常见的组织修复方法有传统的自体移植、同种异体移植及人工组织工程支架移植。自体移植修复虽然治疗效果最佳，但存在对供区破坏、取量不足及并发症等诸多因素的严重制约；异体移植面临着免疫排斥反应、疾病传播等问题；20世纪90年代提出了组织工程，随后组织工程支架材料的研究得到了快速的发展。组织工程中的合成高分子材料具有来源广、物化性质可调控、优异加工成型性等优点，如聚酯、聚酸酐、聚乳酸、聚羟基乙酸及其共聚物等多种合成高分子，但材料缺乏生物活性，细胞相容性较差，不降解或降解产物影响组织微环境。原位诱导组织再生被认为是缺损或病变组织修复最理想的模式，因而天然高分子由于具有优异的生物活性和可降解性在组织工程支架研究中广受青睐，如胶原、明胶、海藻酸盐、透明质酸、壳聚糖等。但大多数材料的力学性能很差，作为力学性能要求高的组织(如骨、牙齿等)工程支架的应用研究还存在巨大挑战。
丝素蛋白和丝胶蛋白具有优异的细胞相容性、血液相容性和组织相容性，且可控成型，作为组织工程支架材料已在皮肤、血管、骨骼等组织工程应用中开展了广泛研究，但目前研究的再生蚕丝(丝素、丝胶)蛋白支架材料同样力学性能较差，难以满足骨骼、牙齿等硬组织替换的要求，这是由于现有的脱胶方法和抽提方法对丝素和丝胶大分子结构破坏较大，而高分子的聚合度(分子量)是决定高分子性能的极其重要的内在因素。
与其它天然高分子材料尤其是蛋白质高分子材料不同的是，蚕丝中蛋白的纯度很高，组成独特，制备方法可以比较简单温和，即可以控制对蚕丝大分子 较小的破坏，不像胶原蛋白纯化复杂。
蚕丝内芯的丝素蛋白占蚕丝质量的70～80％，大分子由6条约390kDa的重链和6条约26kDa轻链以二硫键连接，再与另一种约25kDa的糖蛋白/P25疏水作用结合，其中重链具有形成高性能材料的序列结构基础；蚕丝外覆的丝胶蛋白占蚕丝质量的20～30％，丝胶的分子量也比较大，种类有9种之多，涉川明朗从组织学角度指出丝胶分外中内三层，小松计一提出外覆丝胶由外及内存在4种，即丝胶I、丝胶II、丝胶III和丝胶IV，且由外及内溶解性能越来越低，β构象增加。
再生丝素蛋白溶液都以强碱弱酸盐或酶脱胶盐溶解获得，这些方法造成了丝素蛋白大分子的破坏；再生丝胶蛋白溶液主要来源于蚕茧，两种溶液一般独立分离制备。如CN02138129.1公开了一种丝素丝胶蛋白复合组织工程支架的制备，复合材料采用预先制备好的再生丝素蛋白与再生丝胶蛋白按比例进行混合再制备而成，其中丝素纤维的脱胶方法采用了强碱弱酸盐溶剂，丝胶蛋白提取的是蚕茧茧丝外覆丝胶的较外层的部分。又如CN201510353363.8公开了一种含丝素蛋白和丝胶蛋白的骨组织工程支架材料及其制备方法，也是分步分别获得再生丝素蛋白与再生丝胶蛋白后再加以混合制备支架材料，其中丝素蛋白的制备没有公开具体工艺条件，丝胶蛋白的获得来自于蚕茧，并经醇等有机溶剂处理。
发明内容
本发明解决的技术问题在于提供一种丝素丝胶蛋白复合溶液的制备方法，该丝素丝胶蛋白复合溶液具有粘度高、分子量高的特点。
有鉴于此，本申请提供了一种丝素丝胶蛋白复合溶液的制备方法，包括以下步骤：
A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；
B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液。
优选的，所述温水的温度为35～60℃，所述预处理的时间为1～10min。
优选的，所述预处理之后还包括：将预处理后的蚕丝取出后于25～30℃的水中洗净、脱水。
优选的，步骤A)中，所述置于65～95℃的水中处理的时间为1～8h。
优选的，步骤A)中，所述蚕丝与温水的比例为1g：(40～60)ml，预处理后的蚕丝与水的比例为1g：(40～60)ml。
优选的，步骤A)中，所述处理后干燥之前还包括：将处理后的蚕丝于25～30℃的水中清洗后脱水；所述干燥采用40～60℃的热风干燥。
优选的，所述中性盐溶液为8～10M的溴化锂水溶液，所述初处理的蚕丝与所述溴化锂水溶液的比例为1g：(20～30)ml。
优选的，步骤B)中，所述处理的温度为50～80℃。
优选的，所述纯化的方法具体为：
将得到的丝素丝胶蛋白复合溶解液灌注于截留分子量10～50kDa的透析袋内，置于去离子水中持续透析1～3天。
本申请提供了一种丝素丝胶蛋白复合溶液的制备方法，其首先将蚕丝置于温水中预处理，再置于水中处理，得到初处理的蚕丝；再将其于中性盐溶液中处理，纯化后即得到丝素丝胶复合溶液；本申请采用温和脱胶、一步溶解法制备了丝素丝胶蛋白复合溶液，在温水中的预处理以及后续的特定温度水中的处理，使得蚕丝表面的丝胶蛋白部分脱除或全部脱除，而丝素纤维在特定温水处理的全过程中，丝素蛋白大分子链在后续脱胶过程中不会破坏，丝素纤维外层的丝胶蛋白随着包覆层的由外及内，丝胶蛋白的种类、结构和性能也不同，通过控制脱胶程度，得到了不同组成、可制备不同性能(尤其力学性能)材料的丝素丝胶蛋白复合溶液；且保证丝素丝胶蛋白复合溶液高的粘度和分子量。
具体实施方式
为了进一步理解本发明，下面结合实施例对本发明优选实施方案进行描述，但是应当理解，这些描述只是为进一步说明本发明的特征和优点，而不是对本发明权利要求的限制。
针对现有技术中，丝素蛋白大分子破坏严重的问题或丝素丝胶复合溶液分别制备的问题，本申请采用去离子水温和脱胶、一步溶解法制备了大分子量高 粘度丝素丝胶蛋白复合溶液，该方法可以避免丝素纤维大分子的破坏，最终使制备的丝素丝胶蛋白复合溶液中的丝素蛋白和丝胶蛋白均具有较高的粘度和大分子量。具体的，本发明实施例公开了一种丝素丝胶蛋白复合溶液的制备方法，包括以下步骤：
A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；
B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液。
在上述丝素丝胶蛋白复合溶液制备过程中，本申请首先蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，即得到初处理的蚕丝；在此过程中，蚕丝的丝胶蛋白全部或部分溶解，而丝素蛋白仍然是纤维状态，若有未溶解的丝胶，则未溶解的丝胶包裹在丝素纤维的外表面，溶解掉的丝胶则存在于水中，呈溶液状态。所述温水的温度为35～60℃，即所述蚕丝可在35℃、40℃、45℃、50℃、55℃或60℃的温水中进行预处理，所述蚕丝与温水的比例为1g：(40～60)ml，更具体地，所述蚕丝与所述温水的比例为1g：42ml、1g：45ml、1g：48ml、1g：52ml、1g：55ml、1g：58ml。所述预处理在上述恒温的温水中进行，所述预处理的时间为1～10min，且上述预处理一直在搅拌中进行，以保证蚕丝可以初步、完全浸没于水中。在预处理完成后，则将预处理后的蚕丝取出后于25～30℃的去离子水中洗净、脱水。脱水后的蚕丝则在65～95℃的水中处理，更具体地，脱水后的蚕丝可在68℃、70℃、72℃、75℃、78℃、80℃、82℃、84℃、87℃、90℃或93℃的水中处理。本申请蚕丝先在水中预处理再在水中处理的方式，不涉及任何试剂处理，可以在保护丝素蛋白大分子结构不被破坏的同时溶解丝胶蛋白。所述预处理后的蚕丝与水的比例为1g：(40～60)ml，更具体地，所述预处理后的蚕丝与水的比例为1g：42ml、1g：45ml、1g：48ml、1g：50ml、1g：52ml、1g：55ml、1g：58ml或1g：60ml。根据水的不同温度以及时间可以调整蚕丝丝胶蛋白的脱胶率，以实现不同粘度和不同分子量丝素丝胶蛋白复合溶液的制备；在本申请中，所述水中处理的时间优选为1～8h，以使蚕丝部分或全部脱胶；更具体地，所述水中处理的时间为1.5h、2h、2.5h、3h、3.2h、3.6h、4.2h、4.5h、5.0h、5.5h、6.0h、6.5h、6.8h、7.0h或7.5h。 在处理之后，则将处理后的蚕丝于25～30℃的水中清洗后脱水、干燥；所述干燥采用40～60℃的热风干燥。蚕丝经过上述处理，得到初处理的蚕丝，该蚕丝仍然呈现纤维状态，实现了丝素蛋白的保护，以利于保证丝素丝胶蛋白的高分子量和高粘度。
按照本发明，然后将初处理的蚕丝在中性盐溶液中处理，纯化，即得到丝素丝胶蛋白复合溶液。在此过程中，将初处理的纤维状的蚕丝溶解，即得到丝素丝胶蛋白复合溶液，此时的蚕丝内芯为丝素纤维，外围包裹有不同含量的丝胶。所述中性盐溶液在本申请中具体选自溴化锂水溶液，其浓度为8～10M，所述初处理的蚕丝与所述溴化锂水溶液的比例为1g：(20～30)ml，更具体地，所述初处理的蚕丝与所述溴化锂水溶液的比例为1g：22ml、1g：24ml、1g：25ml、1g：27ml或1g：29ml。所述处理的温度具体为50～80℃，更具体地，所述处理的温度具体为52℃、57℃、62℃、65℃、67℃、68℃、72℃、75℃或78℃。
在上述处理之后，则将得到的丝素丝胶溶解液进行纯化，以去除溴化锂和小分子量丝素丝胶蛋白；所述纯化的方式具体为将得到的丝素丝胶蛋白复合溶解液灌注于截留分子量10～50kDa的透析袋内，置于去离子水持续透析1～3天。
本发明采用温和脱胶、一步溶解法制备了丝素丝胶蛋白复合溶液，突破了传统的丝素蛋白和丝胶蛋白分别制备、分别溶解后混合再用于制备复合材料。重要的是，该复合溶液中丝蛋白分子量集中在150kDa以上，亦或含有一种25kDa左右的分子量，而报导的常规方法制备的丝素蛋白溶液或丝素丝胶溶解再混合的溶液分子量大小呈现连续分布、主要分布在85kDa以下直至15kDa甚至更小。温和脱胶一步溶解法丝蛋白溶液制备过程中，蚕丝采用热水处理，使得丝素蛋白大分子链(尤其是重链)在脱胶过程中不会被破坏；丝素纤维外层的丝胶蛋白随着包覆层的由外及内，丝胶蛋白的种类、结构和性能也不同，通过控制脱胶程度，得到了不同组成、可制备不同性能(尤其力学性能)材料的丝素丝胶蛋白复合溶液。该一步溶解法的丝蛋白溶液粘度远远高于常规报导的混合溶液。更重要的是，本发明从高分子的分子量这个影响聚合物力学性能的根本因素出发，提供了高性能再生蚕丝蛋白材料的制备新技术，将解决可诱导硬组织再生修复的天然生物聚合物组织工程支架力学性能不足的卡脖子技 术难题。
为了进一步理解本发明，下面结合实施例对本发明提供的丝素丝胶蛋白复合溶液的制备方法进行详细说明，本发明的保护范围不受以下实施例的限制。
实施例1
1.以经过煮茧缫制的家蚕生丝为原料，取家蚕生丝1公斤，按照浴比1:50(g/mL)量取50L去离子水预热至60℃，放入生丝恒温60℃搅拌处理1～10分钟，然后取出用25～30℃的去离子将丝洗净、脱水；
2.将上述脱水后的蚕丝按照浴比1:50(g/mL)置于90℃预热的去离子水中控制恒温煮1小时，取出含有丝胶的蚕丝用25～30℃的去离子充分清洗干净，脱水后用40～60℃的热风进行干燥；
3.称取干燥后的蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得到家蚕丝素丝胶蛋白复合溶解液；
4.将丝素丝胶蛋白复合溶解液灌注于截留分子量为14kDa的透析袋内，透析袋壁是半透膜；将灌注了丝素丝胶蛋白溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素丝胶蛋白复合水溶液。
5.采用旋转蒸发器浓缩丝素丝胶蛋白获得同一质量分数的复合水溶液，即得到大分子量的高粘度的家蚕丝素丝胶蛋白复合水溶液。使用AR2000流变仪测得丝素丝胶蛋白复合水溶液的初始剪切作用下的粘度值和粘度极值为3.5Pa.s和10Pa.s。
6.采用旋转蒸发器浓缩或去离子水稀释丝素丝胶蛋白获得一定质量分数的复合水溶液，采用聚丙烯酰胺凝胶电泳法测得复合溶液的分子量分布主要是＞150kDa，少量的50～100kDa，另有25kDa左右的蛋白链。
7.采用称重法对第1、2步处理后的蚕丝进行脱胶率测定，测得脱胶率为8％。
实施例2
1.以经过煮茧缫制的家蚕生丝为原料，取家蚕生丝1公斤，按照浴比1:50(g/mL)量取50L去离子水预热至60℃，放入生丝恒温60℃搅拌处理1～10分钟，然后取出用25～30℃的去离子将丝洗净、脱水；
2.将上述脱水后的蚕丝按照浴比1:50(g/mL)置于90℃预热的去离子水中控制恒温煮4小时，取出含有丝胶的蚕丝用25～30℃的去离子充分清洗干净，脱水后用40～60℃的热风进行干燥；
3.称取干燥后的蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得家蚕丝素丝胶蛋白复合溶解液；
4.将丝素丝胶蛋白复合溶解液灌注于截留分子量为14kDa的透析袋内，透析袋壁是半透膜，将灌注了丝素丝胶蛋白溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素丝胶蛋白复合水溶液。
5.采用旋转蒸发器浓缩丝素丝胶蛋白获得同一质量分数的复合水溶液，即得到大分子量的高粘度的家蚕丝素丝胶蛋白复合水溶液。使用AR2000流变仪测得丝素丝胶蛋白复合水溶液的初始剪切作用下的粘度值和粘度极值为4.5Pa.s和9Pa.s。丝胶蛋白的粘度(11Pa.s和40Pa.s)远大于丝素蛋白的，丝胶的存在会增加溶液的粘度，所以本实施例溶液在剪切作用下的粘度极值小于实施例1。
6.采用旋转蒸发器浓缩或去离子水稀释丝素丝胶蛋白获得一定质量分数的复合水溶液，采用聚丙烯酰胺凝胶电泳法测得复合溶液的分子量分布主要是＞180kDa，另有25kDa左右的蛋白链。
7.采用称重法对第1、2步处理后的蚕丝进行脱胶率测定，测得脱胶率为17％。
实施例3
1.以经过煮茧缫制的家蚕生丝为原料，取家蚕生丝1公斤，按照浴比1:50(g/mL)量取50L去离子水预热至60℃，放入生丝恒温60℃搅拌处理1～10分钟，然后取出用25～30℃的去离子将丝洗净、脱水；
2.将上述脱水后的蚕丝按照浴比1:50(g/mL)置于90℃预热的去离子水中控制恒温煮4小时，取出含有丝胶的蚕丝用25～30℃的去离子充分清洗干净，脱水后用40～60℃的热风进行干燥；
3.称取干燥后的蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得家蚕丝素丝胶蛋白复合溶解液；
4.将丝素丝胶蛋白复合溶解液灌注于截留分子量为50kDa的透析袋内，透析袋壁是半透膜，将灌注了丝素丝胶蛋白溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素丝胶蛋白复合水溶液。
5.采用旋转蒸发器浓缩丝素丝胶蛋白获得同一质量分数的复合水溶液，即得到大分子量的高粘度的家蚕丝素丝胶蛋白复合水溶液。使用AR2000流变仪测得丝素丝胶蛋白复合水溶液的初始剪切作用下的粘度值和粘度极值为8.0Pa.s和12Pa.s。与实施例2相比，丝素丝胶复合溶液的粘度值显著增加是因为溶液分子量增大、小分子量减少(去除了25kDa左右的蛋白链)。与实施例5相比，丝胶的存在增加了溶液的粘度。
6.采用旋转蒸发器浓缩或去离子水稀释丝素丝胶蛋白获得一定质量分数的复合水溶液，采用聚丙烯酰胺凝胶电泳法测得复合溶液的分子量分布主要是＞180kDa。
7.采用称重法对第1、2步处理后的蚕丝进行脱胶率测定，测得脱胶率为17％。
实施例4
1.以经过煮茧缫制的家蚕生丝为原料，取家蚕生丝1公斤，按照浴比1:50(g/mL)量取50L去离子水预热至60℃，放入生丝恒温60℃搅拌处理1～10分钟，然后取出用25～30℃的去离子将丝洗净、脱水；
2.将上述脱水后的蚕丝按照浴比1:50(g/mL)置于90℃预热的去离子水中控制恒温煮7小时，取出蚕丝用25～30℃的去离子充分清洗干净，脱水后用40～60℃的热风进行干燥；
3.称取干燥后的蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得家蚕丝素丝胶蛋白复合溶解液；
4.将丝素丝胶蛋白复合溶解液灌注于截留分子量为14kDa的透析袋内，透析袋壁是半透膜。将灌注了丝素丝胶蛋白溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素丝胶蛋白复合水溶液。
5.采用旋转蒸发器浓缩丝素丝胶蛋白获得同一质量分数的复合水溶液， 即得到大分子量的高粘度的家蚕丝素丝胶蛋白复合水溶液。使用AR2000流变仪测得丝素丝胶蛋白复合水溶液的初始剪切作用下的粘度值和粘度极值为2.0Pa.s和5Pa.s。
6.采用旋转蒸发器浓缩或去离子水稀释丝素丝胶蛋白获得一定质量分数的复合水溶液，采用聚丙烯酰胺凝胶电泳法测得复合溶液的分子量分布主要是＞200kDa，另有25kDa左右的蛋白链。
7.采用称重法对第1、2步处理后的蚕丝进行脱胶率测定，测得脱胶率为23％。
与比较例1常规脱胶法相比，脱胶率相同，即脱尽了丝胶，且透析截留分子量相同，但是本实施例热水脱胶的丝溶液的分子量不仅分子量大且分布很集中，常规碳酸钠脱胶的丝溶液分子量小且分布很散；分子量越大粘度越大，分子量多分散系数越小粘度越大，本实施例测量的初始剪切作用下的粘度值和粘度极值为比较例1的20倍和12.5倍。
实施例5
1.以经过煮茧缫制的家蚕生丝为原料，取家蚕生丝1公斤，按照浴比1:50(g/mL)量取50L去离子水预热至60℃，放入生丝恒温60℃搅拌处理1～10分钟，然后取出用25～30℃的去离子将丝洗净、脱水；
2.将脱水后的蚕丝按照浴比1:50(g/mL)置于90℃预热的去离子水中控制恒温煮7小时，取出蚕丝用25～30℃的去离子充分清洗干净，脱水后用40～60℃的热风进行干燥；
3.称取干燥后的蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得家蚕丝素丝胶蛋白复合溶解液；
4.将丝素丝胶蛋白复合溶解液灌注于截留分子量为50kDa的透析袋内，透析袋壁是半透膜，将灌注了丝素丝胶蛋白溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素丝胶蛋白复合水溶液。
5.采用旋转蒸发器浓缩丝素丝胶蛋白获得同一质量分数的复合水溶液，即得到大分子量的高粘度的家蚕丝素丝胶蛋白复合水溶液。使用AR2000流变仪测得丝素丝胶蛋白复合水溶液的初始剪切作用下的粘度值和粘度极值为 4.0Pa.s和8.5Pa.s。与实施例4相比，丝素丝胶复合溶液的粘度值显著增加是因为溶液分子量增大、小分子量减少(去除了25kDa左右的蛋白链)。
6.采用旋转蒸发器浓缩或去离子水稀释丝素丝胶蛋白获得一定质量分数的复合水溶液，采用聚丙烯酰胺凝胶电泳法测得复合溶液的分子量分布主要是＞200kDa。
7.采用称重法对第1、2步处理后的蚕丝进行脱胶率测定，测得脱胶率为23％，本实施例测量的初始剪切作用下的粘度值和粘度极值为比较例1的40倍和21倍。
比较例1常规纯丝素溶液制备方法
1.以经过煮茧缫制的家蚕生丝为原料，将家蚕生丝按照浴比1:50(g/mL)放入浓度为0.1～0.06％的碳酸钠水溶液中，于98～100℃处理三次，每次处理30分钟，然后取出用25～30℃的去离子水将丝充分清洗干净，脱水后用40-60℃的热风进行干燥；
2.称取上述干燥后的家蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得家蚕丝素蛋白溶解液；
3.将家蚕丝素蛋白溶解液灌注于截留分子量为14kDa的透析袋内，透析袋壁是半透膜，将灌注了家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液；
4.然后采用旋转蒸发器浓缩丝素蛋白获得同一质量分数的水溶液，使用AR2000流变仪测得家蚕丝素蛋白溶液的初始剪切作用下的粘度值和粘度极值为0.1Pa.s和0.4Pa.s。
5.采用旋转蒸发器浓缩或去离子水稀释丝素蛋白获得一定质量分数的水溶液，采用聚丙烯酰胺凝胶电泳法测得丝素蛋白溶液的分子量分布分散，并且主要分布于85kDa以下，虽然同样使用了截留分子量为14kDa的透析袋，但15kDa以下的丝素蛋白含量仍很多。
6.采用称重法对第1、2步处理后的蚕丝进行脱胶率测定，测得脱胶率为23％。
比较例2常规脱胶制备丝素溶液、再与丝胶蛋白溶液混合的复合溶液
1.以经过煮茧缫制的家蚕生丝为原料，将家蚕生丝按照浴比1:50(g/mL)放入浓度为0.1～0.06％的碳酸钠水溶液中，于98～100℃处理三次，每次处理30分钟，然后取出用25～30℃的去离子水将丝充分清洗干净，脱水后用40～60℃的热风进行干燥；
2.称取干燥后的家蚕丝按1:25(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至蚕丝完全溶解，得家蚕丝素蛋白溶解液；
3.将家蚕丝素蛋白溶解液灌注于截留分子量为14kDa的透析袋内，透析袋壁是半透膜，将灌注了家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液；
4.另取经过煮茧缫制的家蚕生丝1公斤，按照浴比1:50(g/mL)量取50L去离子水预热至60℃，放入生丝恒温60℃搅拌处理1～10分钟，然后取出用25～30℃的去离子将丝洗净、脱水；
5.脱水后的蚕丝按照浴比1:50(g/mL)置于90℃预热的去离子水中控制恒温煮7小时，弃去纤维，收集丝胶水溶液冷冻干燥得固体丝胶；
6.然后采用旋转蒸发器浓缩第3步的丝素蛋白水溶液并加入与实施例2等同含量的丝胶、溶解混合均匀，调制与所有实施例相同质量分数的丝素丝胶复合水溶液，使用AR2000流变仪测得溶液的初始剪切作用下的粘度值和粘度极值为0.45Pa.s和1.07Pa.s。
实施例1～5制备的丝素丝胶蛋白复合水溶液的分子量和粘度显著高于对比例1～2常规脱胶法获得的丝蛋白溶液。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而 是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (9)

  1. 一种丝素丝胶蛋白复合溶液的制备方法，包括以下步骤：
    A)将蚕丝置于温水中进行预处理，再置于65～95℃的水中处理，干燥，得到初处理的蚕丝；
    B)将所述初处理的蚕丝在中性盐溶液中处理，将得到的丝素丝胶蛋白复合溶解液再纯化，得到丝素丝胶蛋白复合溶液。
  2. 根据权利要求1所述的制备方法，其特征在于，所述温水的温度为35～60℃，所述预处理的时间为1～10min。
  3. 根据权利要求1所述的制备方法，其特征在于，所述预处理之后还包括：将预处理后的蚕丝取出后于25～30℃的水中洗净、脱水。
  4. 根据权利要求1所述的制备方法，其特征在于，步骤A)中，所述置于65～95℃的水中处理的时间为1～8h。
  5. 根据权利要求1所述的制备方法，其特征在于，步骤A)中，所述蚕丝与温水的比例为1g：(40～60)ml，预处理后的蚕丝与水的比例为1g：(40～60)ml。
  6. 根据权利要求1所述的制备方法，其特征在于，步骤A)中，所述处理后干燥之前还包括：将处理后的蚕丝于25～30℃的水中清洗后脱水；所述干燥采用40～60℃的热风干燥。
  7. 根据权利要求1所述的制备方法，其特征在于，所述中性盐溶液为8～10M的溴化锂水溶液，所述初处理的蚕丝与所述溴化锂水溶液的比例为1g：(20～30)ml。
  8. 根据权利要求1所述的制备方法，其特征在于，步骤B)中，所述处理的温度为50～80℃。
  9. 根据权利要求1所述的制备方法，其特征在于，所述纯化的方法具体为：
    将得到的丝素丝胶蛋白复合溶解液灌注于截留分子量10～50kDa的透析袋内，置于去离子水中持续透析1～3天。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114790300A (zh) * 2022-04-29 2022-07-26 中国科学院青岛生物能源与过程研究所 一种丝素蛋白纳米粒的制备方法
CN115247198A (zh) * 2022-06-14 2022-10-28 上海中匡生物科技有限公司 一种制备丝素多肽的方法及其应用
CN119736791A (zh) * 2025-03-04 2025-04-01 杭州万事利丝绸数码印花有限公司 一种蚕丝保胶工艺及高丝胶蚕丝

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111875691B (zh) * 2020-08-06 2022-05-17 苏州大学 一种丝素丝胶蛋白复合溶液的制备方法
CN115531284A (zh) * 2022-09-15 2022-12-30 江苏华佳丝绸股份有限公司 一种含丝胶蛋白的蚕丝面膜的制备方法
CN119264477B (zh) * 2024-09-24 2026-04-24 苏州大学 一种可见光固化丝素/丝胶蛋白水凝胶的制备方法

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02233128A (ja) * 1989-03-03 1990-09-14 Agency Of Ind Science & Technol 酸素透過膜
CN1229362A (zh) * 1997-06-18 1999-09-22 农林水产省蚕丝昆虫农业技术研究所所长代表日本国 以丝心蛋白和丝胶蛋白为主的创伤用被敷材料及其制法
CN1407992A (zh) * 1999-12-09 2003-04-02 独立行政法人农业生物资源研究所 制备功能丝心蛋白的方法及该蛋白的用途
KR101121040B1 (ko) * 2011-08-25 2012-03-26 경북대학교 산학협력단 고분자량 실크 피브로인을 함유하는 혈당 및 콜레스테롤 강하용 조성물 및 그 제조방법
CN103290085A (zh) * 2013-05-13 2013-09-11 湖州新天丝生物技术有限公司 一种蚕丝蛋白粉及其制备方法
CN110003518A (zh) * 2019-04-16 2019-07-12 苏州大学 一种活性丝素蛋白多孔材料或活性丝素蛋白膜及其制备方法
CN111671980A (zh) * 2020-08-06 2020-09-18 苏州大学 一种仿生复合骨支架及其制备方法
CN111821514A (zh) * 2020-08-06 2020-10-27 苏州大学 一种丝素丝胶蛋白复合膜及其制备方法
CN111870740A (zh) * 2020-08-06 2020-11-03 苏州大学 一种复合蛋白膜及其制备方法
CN111875691A (zh) * 2020-08-06 2020-11-03 苏州大学 一种丝素丝胶蛋白复合溶液的制备方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1181892C (zh) * 2002-08-14 2004-12-29 苏州大学 组织工程支架用多孔材料及其制备方法
CN103613652B (zh) * 2013-11-15 2015-12-30 苏州大学 一种丝素蛋白的提纯方法
CN104984393B (zh) * 2015-06-24 2017-10-20 重庆理工大学 一种骨组织工程支架材料及其制备方法
CN105482137A (zh) * 2015-12-31 2016-04-13 常州市长宇实用气体有限公司 一种漏斗网蛛蛛丝复合丝素蛋白膜的制备方法
CN108392435A (zh) * 2018-05-15 2018-08-14 西南大学 一种蚕丝面膜

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02233128A (ja) * 1989-03-03 1990-09-14 Agency Of Ind Science & Technol 酸素透過膜
CN1229362A (zh) * 1997-06-18 1999-09-22 农林水产省蚕丝昆虫农业技术研究所所长代表日本国 以丝心蛋白和丝胶蛋白为主的创伤用被敷材料及其制法
CN1407992A (zh) * 1999-12-09 2003-04-02 独立行政法人农业生物资源研究所 制备功能丝心蛋白的方法及该蛋白的用途
KR101121040B1 (ko) * 2011-08-25 2012-03-26 경북대학교 산학협력단 고분자량 실크 피브로인을 함유하는 혈당 및 콜레스테롤 강하용 조성물 및 그 제조방법
CN103290085A (zh) * 2013-05-13 2013-09-11 湖州新天丝生物技术有限公司 一种蚕丝蛋白粉及其制备方法
CN110003518A (zh) * 2019-04-16 2019-07-12 苏州大学 一种活性丝素蛋白多孔材料或活性丝素蛋白膜及其制备方法
CN111671980A (zh) * 2020-08-06 2020-09-18 苏州大学 一种仿生复合骨支架及其制备方法
CN111821514A (zh) * 2020-08-06 2020-10-27 苏州大学 一种丝素丝胶蛋白复合膜及其制备方法
CN111870740A (zh) * 2020-08-06 2020-11-03 苏州大学 一种复合蛋白膜及其制备方法
CN111875691A (zh) * 2020-08-06 2020-11-03 苏州大学 一种丝素丝胶蛋白复合溶液的制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TIAN WEI: "Preparation and Mineralization of Silk Fibroin/Sercin Blended Materials", MASTER THESIS, TIANJIN POLYTECHNIC UNIVERSITY, CN, 15 February 2021 (2021-02-15), CN , XP055895521, ISSN: 1674-0246 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114790300A (zh) * 2022-04-29 2022-07-26 中国科学院青岛生物能源与过程研究所 一种丝素蛋白纳米粒的制备方法
CN115247198A (zh) * 2022-06-14 2022-10-28 上海中匡生物科技有限公司 一种制备丝素多肽的方法及其应用
CN119736791A (zh) * 2025-03-04 2025-04-01 杭州万事利丝绸数码印花有限公司 一种蚕丝保胶工艺及高丝胶蚕丝

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