鳖源胶原蛋白分离纯化及生物学性能的研究

发布时间：2018-01-14 19:33

本文关键词：鳖源胶原蛋白分离纯化及生物学性能的研究　出处：《浙江万里学院》2015年硕士论文　论文类型：学位论文

【摘要】：近年来,胶原蛋白因其良好的生物相容性在生物医药材料的应用中得到广泛的关注,中华鳖作为我国传统营养滋补佳品,其裙边中含有丰富的胶原蛋白,不仅可以作为水产胶原蛋白的良好来源,还能为中华鳖深加工利用提供研究方向。本论文旨在基于对中华鳖裙边基本成分及胶原纤维组织形态的分析,对中华鳖裙边胶原蛋白纯化工艺进行研究,并对获得的纯品表征理化性质和研究生物学性能,为中华鳖深加工高值化利用和鳖源胶原蛋白在生物材料中的应用提供一定的理论基础。本研究结果如下:(1)裙边基本营养成分及胶原纤维组织结构的研究。测定发现中华鳖裙边中氨基酸种类齐全,羟脯氨酸含量高达8.22%;水分为74.90%,总蛋白、脂肪及灰分含量分别为78.20%、1.27%及9.90%;羟脯氨酸法测定中华鳖裙边胶原蛋白为71.20%,约占总蛋白的95%;Van Gieson染色法观察发现裙边胶原纤维含量非常高;苦味酸-天狼星红染色法观察发现裙边中胶原纤维类型主要是I型。(2)裙边胶原蛋白纯化工艺的研究。在实验室建立的裙边胶原蛋白最优提取条件基础上,以胶原蛋白回收率为指标,采用L9(34)正交设计试验优化了影响裙边胶原蛋白粗提液的盐析因素(盐析时间、NaCl浓度、NaCl浓度),得到最佳盐析条件:盐析时间为24h,NaCl浓度2mol/L,胶原蛋白浓度为8g/L,胶原蛋白回收率是80.47%,SDS-PAGE证明盐析能使胶原蛋白得到有效的纯化;并且采用不同截留分子量透析袋(25KD、50KD、100KD)对胶原蛋白粗提液分别进行直接透析24h、48h,SDS-PAGE检验纯化效果发现截留分子量为100KD的透析袋在透析48h后的纯化效果最好,达到了100%纯化。(3)裙边胶原蛋白理化性质的鉴定。以裙边胶原蛋白纯品(STCC)为研究对象,并和马面鱼皮胶原蛋白(HSC)、牛皮胶原蛋白(BSC)的部分理化性质进行比较。SDS-PAGE证明STCC组成形式为[α1(I)]2α2(I),分子量约为350KD,是典型的I型胶原蛋白;紫外图谱显示STCC特征吸收峰在230 nm附近,符合I型胶原蛋白的特征;红外光谱显示STCC特征吸收峰和HSC、BSC基本吻合,说明胶原蛋白在提取和纯化后保留了较为完整的三股螺旋结构,并且不存在物种差异;氨基酸分析发现,STCC中Gly含量最高,约占氨基酸总量的1/3,亚氨基酸(Pro、Hyp)总量高达23%;溶解性实验表明STCC等电点(pI)为6,在NaCl浓度为6%(W/V)时溶解度达到最低点;STCC热变性温度(Td)和熔化温度(Tm)分别为35.1℃和105.14℃,熔化温度(Tm)和HSC的Tm(105.05℃)相近,二者均低于BSC的Tm(110.24℃);扫描电镜图像显示STCC冻干品呈疏松多孔的纤维结构,且孔径均匀。以上结果表明STCC具有良好的水产胶原蛋白特性,可以为其加工开发利用提供一定的理论基础。(4)裙边胶原蛋白生物学性能的研究。分别通过直接水浸法和体外胶原蛋白酶降解法对STCC的吸水性和体外降解性进行了考察,发现STCC吸水力高达12.06g/g,持水力几乎达到100%,且在体外具有可降解性;以SD大鼠为动物模型,对STCC和交联胶原的溶血性、皮肤致敏性进行了研究,发现二者均不会造成SD大鼠溶血和致敏,初步符合生物材料的要求;并建立了SD大鼠肝部创伤模型,以比较STCC、交联胶原和纱布的止血性,发现前两者均具有良好的止血效果,分别在100s、62s时就能达到止血效果,远低于纱布的止血时间260s;以SD大鼠背部创伤皮肤为模型观察STCC、交联胶原和纱布的促愈合作用,STCC在术后7天的愈合率达到90%,而纱布在术后14天才达到愈合率90%,说明STCC能缩短创伤皮肤愈合时间,具有良好的促创伤皮肤愈合性,而交联胶原的促创伤皮肤愈合效果欠佳。STCC表现出了优良的生物学性能,对于其在生物材料中的应用还有待深入研究。
[Abstract]:In recent years, collagen due to its good biocompatibility has received wide attention in the application of biomedical materials in Trionyx sinensis as our traditional nutrition tonic, the skirt is rich in collagen, not only can be used as a good source of aquatic collagen, but also provide research direction for the deep processing and utilization of Chinese soft shelled turtle. This paper aims at based on the analysis of the basic components and collagen fiber morphology of Chinese Trionyx sinensis Calipash, to study the purification process of Chinese Trionyx sinensis Calipash collagen, and to obtain pure product characterization of physicochemical and biological properties research, provide a theoretical basis for application and utilization of collagen soft shelled turtle origin in biological materials for the deep processing of soft shelled turtle high value. The results are as follows: (1) research on skirt basic nutrition and collagen fiber structure. The determination of amino acids in Chinese Trionyx sinensis Calipash Complete, hydroxyproline content of up to 8.22%; 74.90% moisture, total protein, fat and ash content were 78.20%, 1.27% and 9.90%; hydroxyproline determination of Chinese Trionyx sinensis Calipash collagen was 71.20%, accounting for about 95% of the total protein; Van Gieson staining method showed the skirt collagen fiber content is very high; Sirius red staining observation of collagen fiber in skirt type is mainly I. (2) study on the purification process of skirt collagen. Collagen skirt optimal extraction conditions in the laboratory is established based on the collagen recovery index, using L9 (34) orthogonal design test to optimize the influence factors of salting out liquid extract (salting out time skirt collagen the concentration of NaCl, concentration of NaCl), get the best salting out condition: salting out time is 24h, the concentration of NaCl 2mol/L, the concentration of collagen 8g/L, collagen recovery is 80.47%, SDS-PAGE proved that salting out Can make collagen effectively purified; and using different mwcos dialysis bags (25KD, 50KD, 100KD) on collagen extracts were directly 24h 48h SDS-PAGE test, dialysis, purification effect that MWCO 100KD dialysis bag is the best in the purification effect of dialysis after 48h, reached 100% (purification. 3) identification of physical and chemical properties of collagen. The skirt skirt collagen pure (STCC) as the research object, and horse skin collagen (HSC), bovine collagen (BSC) part of the physical and chemical properties of.SDS-PAGE were compared to prove STCC form for [alpha 1 (I)]2 alpha 2 (I), molecular weight about 350KD, is a typical type I collagen; UV spectrum showed the characteristic absorption peak of STCC near 230 nm, in accordance with the features of type I collagen; infrared spectra showed the characteristic absorption peaks of STCC and HSC, BSC are basically consistent, that the collagen in the extraction and purification after Keep a relatively complete three helix structure, and there are no differences between the species; amino acid analysis showed that the content of Gly STCC in the highest, accounted for 1/3 of the total amino acids, amino acid sub (Pro, Hyp) the total amount of up to 23%; the solubility experiments show that the isoelectric point of STCC (pI) was 6, the concentration of NaCl was 6% (W/V) when reaching the lowest solubility; STCC thermal denaturation temperature (Td) and melting temperature (Tm) were 35.1 degrees and 105.14 degrees, the melting temperature (Tm) and HSC Tm (105.05 DEG C) is similar to that of the two was lower than that of BSC Tm (110.24 DEG C); scanning electron microscope images show that STCC freeze-dried fiber product is porous, and uniform pore size. These results indicate that STCC has the characteristics of good aquatic collagen, can provide a theoretical basis for the development and utilization of processing. (4) study on the biological properties of skirt collagen. Through direct water soaking method of STCC and collagen in vitro enzyme degradation Water and in vitro degradation were investigated, found that STCC water absorption capacity of up to 12.06g/g, the water holding capacity of almost 100%, and the in vitro biodegradability; using SD rats as animal model, hemolysis of STCC and collagen cross-linking, skin sensitization was studied, it is found that the two are not caused by SD the rats of hemolysis and sensitization, initially meet biological material requirements; and the establishment of a rat model of liver injury of SD, compared with STCC, and cross-linked collagen hemostatic gauze, found that both has good hemostatic effect and respectively in 100s, 62s can achieve the hemostatic effect, bleeding time is far lower than the 260s gauze in the back skin trauma; SD rat model to observe STCC, promote the healing of cross-linked collagen and gauze, STCC on the 7 day postoperative healing rate reached 90%, and after 14 days to reach the gauze on the healing rate of 90%, indicating that STCC can shorten the wound healing time of skin, with good It is good for promoting skin wound healing. However, the wound healing effect of cross-linked collagen is not good..STCC shows excellent biological properties, and its application in biomaterials remains to be further studied.

【学位授予单位】：浙江万里学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R318.08

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