正交试验法提取鼠尾腱胶原工艺的优化

doi:10.13481/j.1671-587x.20150437

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (04): 858-863.doi: 10.13481/j.1671-587x.20150437

正交试验法提取鼠尾腱胶原工艺的优化

宋唯一¹, 刘国民², 袁海波³, 彭传刚², 徐坤¹, 殷德辉¹, 曹洪倩¹, 李娟¹

1. 吉林大学公共卫生学院卫生检验教研室, 吉林长春 130021;
2. 吉林大学第二医院骨科, 吉林长春 130041;
3. 吉林大学第一医院呼吸内科, 吉林长春 130021

收稿日期:2014-12-05 发布日期:2015-08-01
通讯作者: 李娟,教授,博士研究生导师(Tel:0431-85619419,E-mail:li_juan@jlu.edu.cn);刘国民,副教授(Tel:0431-88796674,E-mail:liuyedao123@163.com) E-mail:li_juan@jlu.edu.cnliuyedao123@163.com
作者简介:宋唯一(1989-),男,黑龙江省佳木斯市人,在读医学硕士,主要从事骨组织工程方面的研究。
基金资助:
国家自然科学基金资助课题(81473018);吉林大学横向联合资助课题(3R2149483429)

Optimization of extraction of rat tail tendon collagen process by orthogonal test

SONG Weiyi¹, LIU Guomin², YUAN Haibo³, PENG Chuangang², XU Kun¹, YIN Dehui¹, CAO Hongqian¹, LI Juan¹

1.
1. Department of Health Inspection, School of Public Health, Jilin University, Changchun 130021, China;
2. Department of Orthopedics, Secend Hospital, Jilin University, Changchun 130041, China;
3. Department of Respiratory Disease, First Hospital, Jilin University, Changchun 130021, China

Received:2014-12-05 Published:2015-08-01

摘要/Abstract

摘要：

目的: 探讨浸提时间、乙酸浓度及料液比对鼠尾腱胶原提取的影响,建立并优化鼠尾腱胶原的提取工艺,提高胶原提取效率。方法: 选取健康SD大鼠鼠尾,采取酸溶法提取鼠尾腱胶原,观察不同浸提时间(12、24、48、72和96 h)、乙酸浓度(0.10、0.25、0.50、0.75和1.00 mol·L^-1)及料液比(g:mL)(1:5、1:10、1:20、1:40和1:80)对胶原提取率的影响,利用正交试验判断浸提时间、乙酸浓度及料液比等影响因素的最佳工艺参数。通过SDS-PAGE电泳实验、单宁酸沉淀实验和傅里叶红外光谱实验等对提取的胶原进行鉴定。结果: 单因素分析,胶原提取率在一定参数范围内随浸提时间、乙酸浓度及料液比的增加而增加。正交试验,鼠尾腱胶原提取的最佳工艺参数为浸提时间72 h、乙酸浓度0.5 mol·L^-1、料液比为1:40。SDS-PAGE电泳显示,样品为Ⅰ型胶原,单宁酸沉淀实验显示胶原降解程度低,傅里叶红外光谱显示胶原结构完整。样品氨基酸含量测定符合胶原蛋白的成分特征。中性胶原溶液在37℃时能够形成固态胶原凝胶,胶原凝胶经过碳化二亚胺(EDC)交联后,扫描电镜下显示内部相互连接,构成孔径适中的蜂窝状多孔结构。结论: 成功建立并优化鼠尾腱胶原蛋白的提取工艺,得出浸提时间、乙酸浓度及料液比等因素的最佳工艺参数,有效提高了鼠尾腱胶原的提取效率。

关键词: 鼠尾腱, 胶原蛋白, 胶原海绵, 提取工艺, 正交试验法

Abstract:

Objective To investigate the effects of extraction time,concentration of acetic acid and solid-liquid ratio on the extraction of rat tail tendon collagen,and to establish and optimize the extraction process of rat tail tendon collagen,and to increase the extraction efficiency. Methods The healthy SD rats tail were selected,the rat tail tendon collagen was extracted by acid-soluble method,the influence of different extraction time (12,24,48,72,96 h),acetic acid concentrations (0.10,0.25,0.50,0.75,1 mol·L^-1) and solid-liquid ratios (1:5,1:10,1:20,1:40,1:80) in the extraction ratio of collagen was observed. The optimal extraction parameters were explored by orthogonal test to judge the extraction time,the concentration of acetic acid and solid-liquid ratio,etc. The extracted collagen was identified by SDS-PAGE electrophoresis,tannic acid precipitation test and Fourier transform infrared spectroscopy test etc. Results The univariate analysis results showed that the collagen extraction rates were increased with the increasing of the extraction time,the concentration of acetic acid and the liquid ratio within a certain range of parameters. The optimal parameters were the extraction time of 72 h,the concentration of acetic acid of 0.5 mol·L^-1,the solid-liquid ratio of 1:40.SDS-PAGE electrophoresis showed that the samples were mainly collagen typeⅠ.Tannic acid precipitation test showed a low degree of degradation of collagen.The extract was confirmed by infrared spectral analysis to be typeⅠcollagen with intact structure.After cross-linking of collagen,the SEM figure showed that the internal interconnection formed a porous honeycomb structure,and the pore size was suitable. Conclusion The extraction process of rat tail tendon collagen is successfully established and optimized.The optimum process parameters of the extraction time,the acetic acid concentration and the material liquid ratio are gained,which can improve the extraction efficiency of rat tail tendon collagen.

Key words: rat tail tendon, collagen, collagen sponge, extraction process, orthogonal test

中图分类号:

Q503

宋唯一, 刘国民, 袁海波, 彭传刚, 徐坤, 殷德辉, 曹洪倩, 李娟. 正交试验法提取鼠尾腱胶原工艺的优化[J]. 吉林大学学报(医学版), 2015, 41(04): 858-863.

SONG Weiyi, LIU Guomin, YUAN Haibo, PENG Chuangang, XU Kun, YIN Dehui, CAO Hongqian, LI Juan. Optimization of extraction of rat tail tendon collagen process by orthogonal test[J]. Journal of Jilin University Medicine Edition, 2015, 41(04): 858-863.

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正交试验法提取鼠尾腱胶原工艺的优化

Optimization of extraction of rat tail tendon collagen process by orthogonal test

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