Study On The Producing, Sturcuture And Activity Of Sea Cucumber Fucoidan And Its Enzymatic Degradation Products

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.