牛皮胶原的提取及分子量分布研究

发布时间：2018-06-23 08:59

本文选题：牛皮边角料 + 胶原　；参考：《郑州大学》2017年硕士论文

【摘要】：作为动物体内含量最多、分布最广的一种蛋白质,胶原具有生物相容性好、可降解、免疫原性低等优点,可应用于生物医药、食品、化妆品等诸多领域。胶原的应用与其分子量等息息相关。本文以牛皮边角料为原料,首先对原料进行预处理达到除杂和脱毛的目的。随后,利用单因素实验探索了胃蛋白酶酶解牛皮提取胶原的工艺,以胶原提取率为参数分析了料液比(牛皮质量与0.5 mol/L醋酸溶液体积比)、酶用量、温度、初始pH和酶解时间五个变量对提取胶原的影响。借助傅立叶红外光谱(FTIR)和扫描电子显微镜(SEM)表征了所提取胶原的结构与形貌。借助聚丙烯酰胺凝胶电泳(SDS-PAGE)研究了不同条件下提取胶原的分子量分布,借助紫外可见分光光度计(UV-vis)在波长为313 nm下研究了所提取胶原的体外成纤能力,并利用差示扫描量热法(DSC)表征了其热变性行为,初步建立了从牛皮中提取胶原的条件与胶原主要性能的关系,为胶原的提取和应用提供了一定的理论依据。预处理后的牛皮检测结果表明:预处理对牛皮固含量的影响较小。牛皮pH处于中性范围,热变性温度与文献报道中相符合,为50.3°C。单因素实验表明:其它条件一定时,仅料液比改变,随醋酸溶液体积的增加,胶原提取率逐渐提高;酶用量增大时,胶原提取率也随之增大,酶用量从1.5%到2.5%时提取率提高最为显著;温度对胶原的提取影响突出,温度过高会导致提取率显著下降;初始pH低时胶原提取率偏低,但当pH增大到2.0以上,提取率在设定pH梯度范围内所受影响微弱;酶解时间得到的结果是,随酶解时间的延长,胶原提取率呈递增趋势,但酶解达到一定时间后,很难通过改变酶解时间显著提高其提取率。对所提取试样的红外光谱、数码照片和扫描电镜图研究表明:得到的产物为鲜亮纯白的胶原,呈片层状结构。提取条件对所提取胶原结构影响不大。由不同条件下提取出胶原的电泳结果可知:胶原的分子量大小及分布受料液比、酶用量、溶液初始pH和酶解时间影响较小,所有电泳图都存在α条带且绝大多数条带在66.2 kDa以上;提取温度改变会使胶原分子量发生显著变化,50°C时胶原的α条带消失,分子量分布更宽,因此温度是控制胶原分子量的一个关键因素。与煮沸的胶原试样对比发现,料液比、酶用量、溶液初始pH和酶解时间四种因素条件下得到的胶原均具有体外成纤能力,其体外成纤能力强弱略有差异;在不同的温度下提取胶原的体外成纤能力方面,仅50°C条件下提取的胶原丧失了体外成纤能力。DSC结果可知:胶原的热变性温度整体在45°C以上。酶用量、初始pH和酶解时间对胶原变性温度影响不大;但牛皮质量一定时,随醋酸溶液体积增大,胶原热变性温度降低;当温度低于37°C时,胶原的热变性温度受提取温度影响较小,当温度高于37°C后胶原的热变性温度降到50°C以下。
[Abstract]:As one of the most abundant and widely distributed proteins in animals, collagen has the advantages of good biocompatibility, biodegradability and low immunogenicity. It can be used in many fields such as biomedicine, food, cosmetics and so on. The application of collagen is closely related to its molecular weight. In this paper, the raw material is used as raw material, the raw material is pretreated to achieve the purpose of removing impurity and depilation. Then, the process of extracting collagen from bovine skin by pepsin enzymatic hydrolysis was studied by single factor experiment. The ratio of material to liquid (the volume ratio of bovine skin to 0.5 mol / L acetic acid solution), the amount of enzyme and the temperature were analyzed based on the extraction rate of collagen. Effects of initial pH and enzymatic hydrolysis time on collagen extraction. The structure and morphology of the extracted collagen were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The molecular weight distribution of collagen extracted under different conditions was studied by using polyacrylamide gel electrophoresis (SDS-PAGE), and the in vitro fibrinogenic ability of the extracted collagen was studied by UV-vis spectrophotometer (UV-vis) at the wavelength of 313 nm. The thermal denaturation behavior was characterized by differential scanning calorimetry (DSC). The relationship between the extraction conditions and the main properties of collagen was preliminarily established, which provided a theoretical basis for the extraction and application of collagen. The results showed that pretreatment had little effect on the solid content of leather. The pH of the skin is in the neutral range, and the thermal denaturation temperature is consistent with that reported in the literature, which is 50.3 掳C. The results of single factor experiments showed that the extraction rate of collagen increased with the increase of the volume of acetic acid solution, and the extraction rate of collagen increased with the increase of enzyme dosage. The extraction rate of collagen increased most significantly when the enzyme dosage was from 1.5% to 2.5. The effect of temperature on the extraction of collagen was obvious, and the extraction rate decreased significantly when the temperature was too high. The extraction rate of collagen was low when the initial pH was low, but when pH increased to more than 2.0, The extraction rate is weak in the range of pH gradient, the result of enzymatic hydrolysis time is that the extraction rate of collagen increases with the extension of enzymatic hydrolysis time, but when the enzymatic hydrolysis reaches a certain time, the extraction rate of collagen increases with the increase of hydrolysis time. It is difficult to improve the extraction rate by changing the enzymatic hydrolysis time. The infrared spectra, digital photographs and scanning electron microscopy of the extracted samples showed that the obtained products were pure white collagen with lamellar structure. The extraction conditions had little effect on the structure of collagen extracted. The electrophoretic results of collagen extracted under different conditions showed that the molecular weight and distribution of collagen were less affected by the ratio of material to liquid, the amount of enzyme, the initial pH of solution and the time of enzymatic hydrolysis. There were 伪 bands in all electrophoretic patterns and most of them were above 66.2 kDa. The molecular weight of collagen disappeared and the molecular weight distribution was wider when the molecular weight of collagen disappeared at 50 掳C when the extraction temperature changed significantly. Therefore, temperature is a key factor to control the molecular weight of collagen. Compared with boiled collagen samples, the collagen obtained under four conditions: the ratio of material to liquid, the amount of enzyme, the initial pH of solution and the time of enzymatic hydrolysis all had the ability to form fiber in vitro, and the ability of fibrin formation in vitro was slightly different. The results of DSC showed that the thermal denaturation temperature of collagen was above 45 掳C. The amount of enzyme, initial pH and enzymatic hydrolysis time had little effect on the denaturation temperature of collagen, but the thermal denaturation temperature of collagen decreased with the increase of acetic acid solution volume when the weight of the skin was constant, and when the temperature was lower than 37 掳C, the denaturation temperature of collagen decreased. The thermal denaturation temperature of collagen was less affected by the extraction temperature. When the temperature was higher than 37 掳C, the thermal denaturation temperature of collagen decreased below 50 掳C.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ936

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