【作者】 常耀光；

【导师】 薛长湖；

【作者基本信息】 中国海洋大学 ， 水产品加工及贮藏工程， 2010， 博士

【摘要】 海参是一种传统海产珍品,营养价值高,生理活性丰富。目前我国海参加工产业虽发展迅速,但水平仍低。对海参活性物质的深入研究,是实现海参精深加工及低值海参高值化的关键科学问题,也是提高海参加工产业整体水平的必由之路。以此为背景,本文以低值参为原料,以海参活性物质中缺乏研究的海参岩藻聚糖硫酸酯(SC-FUC)为研究对象展开探究。选取四种低值参：海地瓜、白乳参、黑海参及蝶参,提取其体壁中的海参多糖,并以离子交换层析分离纯化出SC-FUC。通过对四种SC-FUC得率、蛋白含量、分子量、单糖组成的分析,确定得率最高(4.07%)的海地瓜SC-FUC作为深入研究的对象。利用凝胶柱层析继续纯化,得到高纯度海地瓜SC-FUC,该多糖仅由岩藻糖组成,分子量为1656.7kDa,硫酸根含量为26.79±3.8%。酶解法是降解多糖最为理想的方法,限于相应糖苷酶的缺乏,海地瓜SC-FUC之前无法实现酶解。本文以海地瓜SC-FUC为诱导底物,从胶州湾近海海域海水中筛选得到一株海洋细菌CZ1127,经生理生化鉴定及16s rDNA分析确定为黄杆菌科细菌,是首株可利用多种海参来源岩藻聚糖硫酸酯的微生物。CZ1127能够稳定产生海参岩藻聚糖硫酸酯酶(FUCase),该酶主要分泌于CZ1127胞内,经HPSEC、FACE及TLC确认系内切酶。酶学性质研究表示CZ1127 FUCase最适反应温度为28℃,最适反应pH为7.0,热稳定性较差,但4℃下稳定性良好,pH5.5-9.0环境下酶活稳定,Cd2+、Mn2+、Ni2+、Zn2+、Hg2+、Cu2+对酶活有抑制,Ca2+、Mg2+对酶活的影响不大。CZ1127FUCase的获取,首次实现了海地瓜SC-FUC的酶解。同时,该酶对于其他多种海参如白乳参、黑海参、仿刺参的SC-FUC亦有降解作用,为国内外首次发现。以CZ1127 FUCase降解海地瓜SC-FUC,建立海地瓜SC-FUC的可控酶解模型,通过数据拟合,得到一以分子量对数为应变量以加酶量及反应时间为自变量的二元二次方程,回归系数良好。酶解制备寡糖混合物,进而以凝胶柱层析进行纯化,得到六个寡糖纯品,包括三糖、四糖、七糖及八糖。以ESI-MS及ESI-MS/MS解析寡糖片段结构,以1H NMR、COSY、TOCSY、NOESY解析低分子量多糖(SC-LMF)结构,并结合多糖1HNMR信息,综合确定海地瓜SC-FUC主要结构为[→3-α-L-fuc-2,4(OSO3)-1→3-α-L-fuc-1→3-α-L-fuc-1→3-α-L-fuc-1→]n,这是海地瓜SC-FUC的首个结构模型。采用大鼠模型考察海地瓜SC-FUC的乙醇型胃溃疡预防活性。海地瓜SC-FUC预防乙醇型胃溃疡活性的量效关系显著,剂量达100mg/kg bw(连续灌胃5天)可明显抑制胃溃疡发生,溃疡抑制率为66.53%。500mg/kg bw海地瓜体壁干粉溃疡抑制率达69.44%,海地瓜SC-FUC是海地瓜抗胃溃疡作用的功效成分。通过组织切片观察、化学指标测定及western blotting分析,阐明海参SC-FUC及海地瓜粉的作用机理包括抗氧化、抑制胃粘膜基质水解、抗炎等多个方面。SC-FUC的乙醇型胃溃疡预防活性与其分子量密切相关。利用CZ1127FUCase将海地瓜SC-FUC降低至适当分子量,可显著提高多糖原有活性。508kDa及54kDa SC-LMF 100mg/kg.bw给药1次起到的预防作用优于原始多糖同剂量给药5次的效果。乙醇型胃溃疡预防活性的阐明,为SC-FUC及SC-LMF的应用提供了崭新的方向,也突显了CZ1127 FUCase良好的应用前景。综上,本文以海地瓜SC-FUC为研究对象,获得了海洋细菌来源的海参岩藻聚糖硫酸酯酶,实现了海地瓜SC-FUC的首次酶解,建立了海地瓜SC-FUC可控酶解模型,纯化得到了酶解系列寡糖片段,解析了海地瓜SC-FUC的结构,证实了海地瓜SC-FUC的乙醇型胃溃疡预防活性,并进一步通过酶解改性提高了其功效。本文以SC-FUC酶解为关键点及主要创新点,是首个关于海参岩藻聚糖硫酸酯酶解产物的研究。本文结果为海参岩藻聚糖硫酸酯的开发利用提供了理论基础,并有望促进海参精深加工及低值海参高值化利用的发展。更多还原

【Abstract】 Sea cucumber is a traditional valuable aquatic product in China, and possesses a series of biological functions and high nutritional value. Sea cucumber processing industry in China still maintains a low level, although it was developing fast presently. Research on the field of sea cucumber biological materials is the key of sea cucumber fine-processing and value-increment, and also is the way in promoting the whole level of sea cucumber processing industry.On this background, low-price sea cucumber was chosen as material, and sea cucumber fucoidan (SC-FUC) which was lack of study among biological materials of sea cucumber was employed as the subject of this research.Four kinds of low-price sea cucumber (Bohadschia marmorata, Acaudina Molpadioides, Holothuria atra, Cucumaria frondosa) were chosen. Sea cucumber polysaccharide was extracted, and SC-FUC was furthermore purified. Through comparing yield, protein content, molecular mass and sugar composition of those SC-FUCs, A. Molpadioides SC-FUC which possesses the highest yield was finally chosen for the further study. The high purity SC-FUC was achieved utilizing gel filtration chromatography. A. Molpadioides SC-FUC is only composed of fucose, its molecular mass is 1656.7kDa, and its sulfate content is 26.79±3.8%。Enzymatic degrading is the ideal method for polysaccharide degradation, however the enzymatic degradation of A. Molpadioides SC-FUC cannot be realized limited by the lack of respective enzymes. In this contest, we isolated a marine bacterium strain named CZ1127 from coastal seawater of the Jiaozhou Bay using A. Molpadioides SC-FUC as inducing substrate. After 16S rDNA and physiological analyses, the strain was confirmed to belong to family Flavobacteriaceae. CZ1127 also is the first microorganism which can utilizing various SC-FUCs. Fucoidanase can be secreted by CZ1127, and furthermore is confirmed to be a endo-acting enzyme utilizing HPSEC, FACE and TLC. Results of enzymatic property study show that the optional reactive temperature of CZ1127 fucoidanase is 28℃,the optional pH is 7.0, it possess a poor thermal stability but is stable at 4℃, and it is stable under a environment of 5.5-9.0 pH. Activity of CZ1127 fucoidanase is inhibited by Cd2+, Mn2+, Ni2+, Zn2+, Hg2+and Cu2+, and not affected by Ca2+and Mg2+. The acquisition of CZ1127 fucoidanase firstly realized the enzymatic degradation of A. Molpadioides SC-FUC. Meanwhile, various SC-FUCs such as Bohadschia marmorata, Holothuria atra and Apostichopus japonicus SC-FUC can be degraded by this enzyme, and furthermore CZ1127 fucoidanase is the first enzyme possess above ability.The controllable enzymatic degrading model of A. Molpadioides SC-FUC was established by the numerical fitting. The model is a binary quadratic equation which employ logarithm of molecular mass as dependent variable, enzyme dosage and reaction time as independent variables. Oligosaccharide mixture was produced, and furthermore was purified to achieve series oligosaccharide fractions. As the result,6 oligosaccharides containing tri-, tetra-, hepta-and octasaccharide were obtained. Utilizing ESI-MS and ESI-MS/MS to elucidate the structure of oligosaccharide, utilizing 1HNMR, COSY, TOCSY and NOESY to elucidate the structure of low molecular fucoidan(SC-LMF), and combing the information of SC-FUC polysaccharide1H NMR spectra, the main structure of A. Molpadioides SC-FUC was confirmed to be [→3-a-L-fuc-2,4(OSO3)-1→3-a-L-fuc-1→3-a-L-fuc-1→3-a-L-fuc-1→]n.This is the first structure model of A. Molpadioides SC-FUC.Rat model was employed to evaluate the anti ethanol-inducing gastric ulcer activity of SC-FUC. There is an obvious dose dependency of A. Molpadioides SC-FUC anti-ulcer activity, and administration of SC-FUC at 100mg/kg b.w. could effectively relieve the lesion (inhibitor rate 66.53%). Administration of A. Molpadioides powder at 500mg/kg b.w. also inhibits the gastric ulcer, and it confirms that SC-FUC is the effective factor of sea cucumber on anti-ulcer activity. Result of histological study, chemical analysis and western blotting analysis show that the mechanism of anti-ulcer effect of SC-FUC and sea cucumber contain antioxidant, gastric matrix hydrolysis inhibiting and anti-inflammation.Anti-ulcer activity of SC-FUC is related to its molecular mass. Degrading to appreciate molecular mass will be significantly promoted original activity of SC-FUC. The effect of once administration of 508kDa and 54kDa SC-LMF at 100mg/kg.bw are better than 5 times administration of SC-FUC at the same dose. The elucidation of anti-ulcer activity provide a new area for SC-FUC and SC-LMF, and emphasize the application promising of CZ1127 FUCase.Above all, we employed A. Molpadioides SC-FUC as research object, obtained the marine bacterium original sea cucumber fucoidanase, firstly realized the enzymatic degradation of A. Molpadioides SC-FUC, established the enzymatic degrading model of A. Molpadioides SC-FUC, purified oligosaccharide fractions, elucidated the structure of A. Molpadioides SC-FUC, confirm its anti ethanol-inducing gastric ulcer activity, and promoted its function utilizing enzymatic degradation. SC-FUC enzymatic degradation is the key point and main novelty of this paper, and this is the first study on the SC-FUC enzymatic degraded products. Those results provide a theoretical base for SC-FUC application, and will contribute to development of sea cucumber fine processing and price increment.更多还原