本文關(guān)鍵詞：鱉源膠原蛋白分離純化及生物學(xué)性能的研究　出處：《浙江萬里學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 中華鱉 裙邊膠原蛋白 純化 理化性質(zhì) 生物學(xué)性能 生物材料

【摘要】：近年來,膠原蛋白因其良好的生物相容性在生物醫(yī)藥材料的應(yīng)用中得到廣泛的關(guān)注,中華鱉作為我國傳統(tǒng)營養(yǎng)滋補(bǔ)佳品,其裙邊中含有豐富的膠原蛋白,不僅可以作為水產(chǎn)膠原蛋白的良好來源,還能為中華鱉深加工利用提供研究方向。本論文旨在基于對中華鱉裙邊基本成分及膠原纖維組織形態(tài)的分析,對中華鱉裙邊膠原蛋白純化工藝進(jìn)行研究,并對獲得的純品表征理化性質(zhì)和研究生物學(xué)性能,為中華鱉深加工高值化利用和鱉源膠原蛋白在生物材料中的應(yīng)用提供一定的理論基礎(chǔ)。本研究結(jié)果如下:(1)裙邊基本營養(yǎng)成分及膠原纖維組織結(jié)構(gòu)的研究。測定發(fā)現(xiàn)中華鱉裙邊中氨基酸種類齊全,羥脯氨酸含量高達(dá)8.22%;水分為74.90%,總蛋白、脂肪及灰分含量分別為78.20%、1.27%及9.90%;羥脯氨酸法測定中華鱉裙邊膠原蛋白為71.20%,約占總蛋白的95%;Van Gieson染色法觀察發(fā)現(xiàn)裙邊膠原纖維含量非常高;苦味酸-天狼星紅染色法觀察發(fā)現(xiàn)裙邊中膠原纖維類型主要是I型。(2)裙邊膠原蛋白純化工藝的研究。在實驗室建立的裙邊膠原蛋白最優(yōu)提取條件基礎(chǔ)上,以膠原蛋白回收率為指標(biāo),采用L9(34)正交設(shè)計試驗優(yōu)化了影響裙邊膠原蛋白粗提液的鹽析因素(鹽析時間、NaCl濃度、NaCl濃度),得到最佳鹽析條件:鹽析時間為24h,NaCl濃度2mol/L,膠原蛋白濃度為8g/L,膠原蛋白回收率是80.47%,SDS-PAGE證明鹽析能使膠原蛋白得到有效的純化;并且采用不同截留分子量透析袋(25KD、50KD、100KD)對膠原蛋白粗提液分別進(jìn)行直接透析24h、48h,SDS-PAGE檢驗純化效果發(fā)現(xiàn)截留分子量為100KD的透析袋在透析48h后的純化效果最好,達(dá)到了100%純化。(3)裙邊膠原蛋白理化性質(zhì)的鑒定。以裙邊膠原蛋白純品(STCC)為研究對象,并和馬面魚皮膠原蛋白(HSC)、牛皮膠原蛋白(BSC)的部分理化性質(zhì)進(jìn)行比較。SDS-PAGE證明STCC組成形式為[α1(I)]2α2(I),分子量約為350KD,是典型的I型膠原蛋白;紫外圖譜顯示STCC特征吸收峰在230 nm附近,符合I型膠原蛋白的特征;紅外光譜顯示STCC特征吸收峰和HSC、BSC基本吻合,說明膠原蛋白在提取和純化后保留了較為完整的三股螺旋結(jié)構(gòu),并且不存在物種差異;氨基酸分析發(fā)現(xiàn),STCC中Gly含量最高,約占氨基酸總量的1/3,亞氨基酸(Pro、Hyp)總量高達(dá)23%;溶解性實驗表明STCC等電點(pI)為6,在NaCl濃度為6%(W/V)時溶解度達(dá)到最低點;STCC熱變性溫度(Td)和熔化溫度(Tm)分別為35.1℃和105.14℃,熔化溫度(Tm)和HSC的Tm(105.05℃)相近,二者均低于BSC的Tm(110.24℃);掃描電鏡圖像顯示STCC凍干品呈疏松多孔的纖維結(jié)構(gòu),且孔徑均勻。以上結(jié)果表明STCC具有良好的水產(chǎn)膠原蛋白特性,可以為其加工開發(fā)利用提供一定的理論基礎(chǔ)。(4)裙邊膠原蛋白生物學(xué)性能的研究。分別通過直接水浸法和體外膠原蛋白酶降解法對STCC的吸水性和體外降解性進(jìn)行了考察,發(fā)現(xiàn)STCC吸水力高達(dá)12.06g/g,持水力幾乎達(dá)到100%,且在體外具有可降解性;以SD大鼠為動物模型,對STCC和交聯(lián)膠原的溶血性、皮膚致敏性進(jìn)行了研究,發(fā)現(xiàn)二者均不會造成SD大鼠溶血和致敏,初步符合生物材料的要求;并建立了SD大鼠肝部創(chuàng)傷模型,以比較STCC、交聯(lián)膠原和紗布的止血性,發(fā)現(xiàn)前兩者均具有良好的止血效果,分別在100s、62s時就能達(dá)到止血效果,遠(yuǎn)低于紗布的止血時間260s;以SD大鼠背部創(chuàng)傷皮膚為模型觀察STCC、交聯(lián)膠原和紗布的促愈合作用,STCC在術(shù)后7天的愈合率達(dá)到90%,而紗布在術(shù)后14天才達(dá)到愈合率90%,說明STCC能縮短創(chuàng)傷皮膚愈合時間,具有良好的促創(chuàng)傷皮膚愈合性,而交聯(lián)膠原的促創(chuàng)傷皮膚愈合效果欠佳。STCC表現(xiàn)出了優(yōu)良的生物學(xué)性能,對于其在生物材料中的應(yīng)用還有待深入研究。
[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.

【學(xué)位授予單位】：浙江萬里學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R318.08

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