碩士 / 國立臺灣海洋大學 / 食品科學系 / 100 / The aim of this study is to investigate M. nitidum via hot-water-extract, and cellulase, and then crude polysaccharides (PS) degrading enzymes from Pseudomonas vesicularis MA103 and Aeromonas salmonicida MAEF108 (induced with M. nitidum PS) for enzyme hydrolysis to separate oligosaccharides (OS) with different degree of polymerization (DP), and using Chlorsulfonic acid- N, N-dimethylformamide (DMF) method to increase sulfate contents of these OS, and then examine the physiological activity effects of their antioxidant activity, anticoagulant and anti-platelet activity of human plasma, and anti-viral activity. After M. nitidum was extracted by hot-water to obtain PS, added the cellulase hydrolysis as well as the crude polysaccharides (PS) degrading enzymes from P. vesicularis MA103 and A. salmonicida MAEF108 (induced with M. nitidum PS) for enzyme hydrolysis. Total sugar content of M. nitidum PS increased from 2.05 mg/mL to 19.35 mg/mL, and 20.34 mg/mL. Reducing sugar content of M. nitidum PS increased from 1.98 mg/mL to 18.67 mg/mL, and 18.76 mg/mL, respectively. Oligosaccharides hydrolysates with molecular mass < 3 kDa were collected by Ultra-filtration system, then though Sephadex G-10 gel filtration chromatography (GPC) and high performance liquid chromatography (HPLC) to obtain nine peaks. The 4-7 peaks are named as the Mon-C7, Mon-C4, and Mon-C2/3, respectively according to their degree of polymerization. After using chlorsulfonic acid- N,N-dimethylformamide (DMF) method to increase sulfate contents of these OS, the sulfate content of Mon-UF, Mon-C7, Mon-C4, and Mon-C2/3 increased from 0.15%-1.95% to 20.04%-22.73%, and designated as Mon-UF-DMF, Mon-C7-DMF, Mon-C4-DMF, and Mon-C2/3-DMF. In the evaluation of the antioxidation effect of M. nitidum OS and oversulfated OS, DPPH free radical scavenging and the ability of chelating on Fe (II) ion of OS and oversulfated OS did not exist significant activity. However, the reducing power could not found except that Mon-C4 existed 3.49 μg/mL of vitamin C in reducing power. In the experiment on anticoagulant activities of human plasma, activated partial thromboplastin time (APTT) of Mon-C7-DMF (20 mg/mL) was 77 sec, which is longer than that of clinical examination normal value of APTT 30-50 sec. The average absorbance value of OD660nm was 0.450, about 16% lower than the blank. In the experiment on prothrombin time (PT) analysis, M. nitidum OS and oversulfated OS were no obvious delayed in PT, but there were a tendency for reduction on Coagulation degree while oversulfated OS concentration was 20 mg/mL. In platelet aggregation analysis, 5 mg/mL and 20 mg/mL of Mon-UF-DMF, will help to reduce the platelet aggregation response of volunteer A and volunteer B, respectively, compared with Mon-UF (5 mg/mL and 20 mg/mL) showing the percentage of agglutination was decreased by 13.48%-15% (volunteer A) and 20%-24.97% (volunteer B). The Mon-C7-DMF helped volunteer A and volunteer B to inhibit platelet aggregation level. The reduction of platelet aggregation level of volunteer A and volunteer B were about 4.94%-7.10% 13.59%-15.23%, respectively. In anti-virus test, inhibition of Japanese encephalitis virus (JEV) adsorption BHK-21 cells response. The results showed that in the prevention experiment, M. nitidum oversulfated OS performed higher survival rate of host cell. In contrast, inhibition of JEV propagation reaction had not significant cell survival rate. The aforementioned outcome means that M. nitidum oversulfated OS cannot inhibit the propagation of the virus, but it may interfere with the JEV to attach to the BHK-21 cells surface. After comparing M. nitidum OS and oversulfated OS showed better effect on the anticoagulant and anti-platelet agglutination experiments than OS. The results can serve as the basis for the development of a novel health food to prevent thromboembolic disease in the future.
| Identifer | oai:union.ndltd.org:TW/100NTOU5253087 |
| Date | January 2012 |
| Creators | Jung-Fang Cheng, 鄭蓉芳 |
| Contributors | Chorng-Liang Pan, 潘崇良 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 94 |
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