天然多糖微载体的制备工艺及微囊化细胞生长代谢研究
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摘要
生物微胶囊技术是一种很有潜力的新兴细胞固定化方法,微囊化细胞培养在发酵、制药、细胞移植、人工器官等领域具有良好的应用前景。但目前的生物微胶囊存在膜材料成本高,制备工艺复杂,稳定性差,传递限制等问题,针对上述问题,本文以天然多糖海藻酸钠和壳聚糖作为微胶囊制备材料,采用脉冲电场制备工艺制备壳聚糖/海藻酸盐微胶囊,对其工艺条件和微胶囊的性能进行研究,考察了壳聚糖/海藻酸盐微囊化细胞培养对细胞生长代谢的影响,为壳聚糖/海藻酸盐微囊化技术在细胞培养领域的应用奠定理论与实践基础。
主要结论如下:
1.系统考察了壳聚糖/海藻酸盐微胶囊的稳定性和通透性能。海藻酸钠溶液浓度对微胶囊的形态影响较大,20g/L为最佳浓度;采用壳聚糖与海藻酸盐凝胶珠进行成膜反应,增加壳聚糖分子量及溶液浓度,微胶囊稳定性提高,但通透性能降低;适当延长成膜反应时间,微胶囊膜稳定性提高。以锌离子和钡离子为凝胶浴制备的微胶囊稳定性优于以钙离子为凝胶浴制备的微胶囊,但通透性能不如海藻酸钙微胶囊。微胶囊在中性和酸性环境中的稳定性要优于碱性环境。
2.壳聚糖和海藻酸钠具有良好的生物相容性。微囊化对原核细胞和真核细胞生长代谢的影响是一致的,由于微囊膜对底物和代谢产物传递有一定的扩散阻力,微囊化细胞的生长代谢稍滞后于游离细胞,但微胶囊能够消除底物抑制,给细胞的生长提供一个相对稳定的微环境,使细胞生长的稳定期延长。
3.微囊化细胞在批次培养中底物消耗速率加快,批次培养时最大菌浓和代谢产物的最高产量均高于初次培养和游离培养;壳聚糖/海藻酸盐微胶囊具有足够的膜强度,能够承受长时间的发酵培养,可重复使用,适合于固定化细胞高密度培养。
4.微胶囊的制备条件对微囊化细胞的代谢有一定影响。正交分析结果表明,微囊化酿酒酵母乙醇产量的影响因素显著性依次为:壳聚糖分子量、壳聚糖浓度、成膜时间和pH值。正交试验确定的最优工艺条件为:壳聚糖分子量5万、壳聚糖浓度2.0 g/L、成膜时间15min和培养基pH=6.0,在该条件下微囊化酿酒酵母的乙醇产量可达到13.5g/L,高于其他条件下的产量。
The microencapsulation is an emerging cell immobilization method, the microencapsulated cell culture has good application prospect in fermentation, drugs manufacture, cell transplant, artificial internal organ and other fields. However, there are some problems in the application of microencapsulation, such as the high cost of membrane materials, the complicated preparation procedure, the lower stability and mass diffusion restriction. In order to solve above problems, this paper used natural polysaccharide (Chitosan and sodium alginate) as the micro-carrier preparation materials, the chitosan/alginate microcapsules were prepared by the impulsive electrostatic preparation process. The stability and permeability of chitosan/alginate microcapsules were studied first. Then the growth and metabolism properties of microencaspsulated cell were studied, establishing the theory and the practice foundation for the chitosan/alginate microencapsulation technology in the cell culture domain's application. Experimental results were showed as follow:
1. The stability and permeability of chitosan/alginate microcapsules has been systematically studied. The concentration of sodium alginate solution has an important effct on the shape of microcapsules. 20g/L is the best concentration. By increasing the concentration and molecular weight of Chitosan, the stability of microcapsules enhanced, but the permeability reduced. Lengthening properly the membrane formation time, the stability of microcapsules also enhanced. The stability of microcapsules using BaCl_2 and ZnCl_2 as gel solution was better than ones using CaCl_2, but the permeability was worse. Different pH value had a great effect on the stability of microcapsules. The stability of microcapsule in neutral and the acidic environment was better than one in alkalinity environment.
2. The microcapsule materials (Chitosan and sodium alginate) have good biocompatibility. The influence of microencapsulation on the prokaryotic cell and eukaryotic cell's growth and metabolism was consistent. Because the microcapsule membrane has certain diffusional resistance to the transmission of substrate and metabolite, compareing with free cell, the growth and metabolism of microencapsulated cell lagged slightly. But the microcapsule can eliminate the substrate inhibition, providing a relative stabile and even micro environment to cell's growth, which making stabilization period of cell growth prolong.
3. The substrate consumption rate of the microencapsulated cell speeds up in the batch culture. The maximum cell biomass and the maximum ethanol production in the batch culture were higher than the initial culture and the free culture. The Chitosan/alginate microcapsule has the enough membrane strength, which can withstand the long time fermentation and has good reutilization, suiting in the immobilized cell's high density fermentation.
4. The effect of microencapsulation technology on the metabolism of Saccharomyces cerevisiae was studied. The orthogonal experimental results showed that the significant factor was molecular weight of Chitosan. Under the condition that the molecular weight of Chitosan 50,000, concentration of Chitosan 2.0 mg/mL, membrane formation time 15min, the culture medium pH value 6.0, the ethanol output may achieve 13.5g/L, which was higher than one under other conditions.
主要结论如下:
1.系统考察了壳聚糖/海藻酸盐微胶囊的稳定性和通透性能。海藻酸钠溶液浓度对微胶囊的形态影响较大,20g/L为最佳浓度;采用壳聚糖与海藻酸盐凝胶珠进行成膜反应,增加壳聚糖分子量及溶液浓度,微胶囊稳定性提高,但通透性能降低;适当延长成膜反应时间,微胶囊膜稳定性提高。以锌离子和钡离子为凝胶浴制备的微胶囊稳定性优于以钙离子为凝胶浴制备的微胶囊,但通透性能不如海藻酸钙微胶囊。微胶囊在中性和酸性环境中的稳定性要优于碱性环境。
2.壳聚糖和海藻酸钠具有良好的生物相容性。微囊化对原核细胞和真核细胞生长代谢的影响是一致的,由于微囊膜对底物和代谢产物传递有一定的扩散阻力,微囊化细胞的生长代谢稍滞后于游离细胞,但微胶囊能够消除底物抑制,给细胞的生长提供一个相对稳定的微环境,使细胞生长的稳定期延长。
3.微囊化细胞在批次培养中底物消耗速率加快,批次培养时最大菌浓和代谢产物的最高产量均高于初次培养和游离培养;壳聚糖/海藻酸盐微胶囊具有足够的膜强度,能够承受长时间的发酵培养,可重复使用,适合于固定化细胞高密度培养。
4.微胶囊的制备条件对微囊化细胞的代谢有一定影响。正交分析结果表明,微囊化酿酒酵母乙醇产量的影响因素显著性依次为:壳聚糖分子量、壳聚糖浓度、成膜时间和pH值。正交试验确定的最优工艺条件为:壳聚糖分子量5万、壳聚糖浓度2.0 g/L、成膜时间15min和培养基pH=6.0,在该条件下微囊化酿酒酵母的乙醇产量可达到13.5g/L,高于其他条件下的产量。
The microencapsulation is an emerging cell immobilization method, the microencapsulated cell culture has good application prospect in fermentation, drugs manufacture, cell transplant, artificial internal organ and other fields. However, there are some problems in the application of microencapsulation, such as the high cost of membrane materials, the complicated preparation procedure, the lower stability and mass diffusion restriction. In order to solve above problems, this paper used natural polysaccharide (Chitosan and sodium alginate) as the micro-carrier preparation materials, the chitosan/alginate microcapsules were prepared by the impulsive electrostatic preparation process. The stability and permeability of chitosan/alginate microcapsules were studied first. Then the growth and metabolism properties of microencaspsulated cell were studied, establishing the theory and the practice foundation for the chitosan/alginate microencapsulation technology in the cell culture domain's application. Experimental results were showed as follow:
1. The stability and permeability of chitosan/alginate microcapsules has been systematically studied. The concentration of sodium alginate solution has an important effct on the shape of microcapsules. 20g/L is the best concentration. By increasing the concentration and molecular weight of Chitosan, the stability of microcapsules enhanced, but the permeability reduced. Lengthening properly the membrane formation time, the stability of microcapsules also enhanced. The stability of microcapsules using BaCl_2 and ZnCl_2 as gel solution was better than ones using CaCl_2, but the permeability was worse. Different pH value had a great effect on the stability of microcapsules. The stability of microcapsule in neutral and the acidic environment was better than one in alkalinity environment.
2. The microcapsule materials (Chitosan and sodium alginate) have good biocompatibility. The influence of microencapsulation on the prokaryotic cell and eukaryotic cell's growth and metabolism was consistent. Because the microcapsule membrane has certain diffusional resistance to the transmission of substrate and metabolite, compareing with free cell, the growth and metabolism of microencapsulated cell lagged slightly. But the microcapsule can eliminate the substrate inhibition, providing a relative stabile and even micro environment to cell's growth, which making stabilization period of cell growth prolong.
3. The substrate consumption rate of the microencapsulated cell speeds up in the batch culture. The maximum cell biomass and the maximum ethanol production in the batch culture were higher than the initial culture and the free culture. The Chitosan/alginate microcapsule has the enough membrane strength, which can withstand the long time fermentation and has good reutilization, suiting in the immobilized cell's high density fermentation.
4. The effect of microencapsulation technology on the metabolism of Saccharomyces cerevisiae was studied. The orthogonal experimental results showed that the significant factor was molecular weight of Chitosan. Under the condition that the molecular weight of Chitosan 50,000, concentration of Chitosan 2.0 mg/mL, membrane formation time 15min, the culture medium pH value 6.0, the ethanol output may achieve 13.5g/L, which was higher than one under other conditions.
引文
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