碩士 / 國立臺灣海洋大學 / 食品科學系 / 107 / This study is aim to investigate purification and storage of phycobiliproteins from Gracilaria tenuistipitata produced by enzyme-assisted hydrolysis and analyze characteristic and bioactivities of mixed agaro-oligosaccharides from Gracilaria tenuistipitata residue. Using G. tenuistipitata to induce marine bacteria Pseudomonas vesicularis MA103 and Aeromonas salmonicida MAEF108 could produce crude enzymes of agarase, amylase, cellulase, xylanase, ι-carrageenase, λ-carrageenase, and κ-carrageenase. Total enzymatic activities were 13.77 U/mL which produced by MA103 in 3 days, and were 3.49 U/mL which produced by MAEF108 in 2 days. G. tenuistipitata solution was hydrolyzed by crude enzymes solution of 15% MA103 and 15% MAEF108 (M3M8) and 30% MA103 (M3) for 48 hr after homogenizing and ultrasonication. The fluorescence intensity (FI) of phycoerythrin (PE) of M3M8 and M3 increased 19.7 fold and 23.1 fold than control, respectively, and phycocyanin (PC) increased 1.8 fold and 1.9 fold, respectively. Further, G. tenuistipitata hydrolyzed solution by crude enzymes solution were removed < 30 kDa by ultrafiltration (UF) system, and using gel filtration column differentiated by fast protein liquid chromatography (FPLC) to acquire PE, PC, and allophycocyanins (APC). The purity index (PI) of PE, PC, and APC of M3M8 were 3.8, 1.3, and 1.8, respectively and M3 were 3.7, 1.1, and 1.4, respectively. SDS-PAGE chromatogram were shown G. tenuistipitata solution was hydrolyzed by crude enzymes solution and their fractions were α, β (20 kDa) and γ (35 kDa) subunits. In storage test, 4oC was better storage temperature to keep the concentration and fluorescence intensity of PE, 26oC was better storage temperature to keep the concentration of PC and APC, and -80oC was better storage temperature to keep the FI of PC and APC. Moreover, using ion exchange column differentiated by FPLC to acquire PE, and their PI of M3M8 and M3 were both 0.7, and then their fractions were removed < 30 kDa by UF. Using gel filtration to purified the fractions by FPLC, the PI of PE fractions of M3M8 and M3 were 4.4 and 4.3, respectively, and yield were 0.78% and 0.76%, respectively. Using agar to induce marine bacteria P. vesicularis MA103 and A. salmonicida MAEF108 could produce crude enzymes of agarase, activities were 2.97 U/mL and 2.59 U/mL which produced by MA103 and MAEF108 in 2 days, respectively. Mixed agaro-oligosaccharides were gained from G. tenuistipitata residue (GAOS3K) that treated by hot water (121oC/20 min), then collected by UF (< 3 kDa). The yield of GAOS3K-M3M8 and GAOS3K-M3 were 0.62% and 0.66%; The content of sulfate were 10.05% and 9.57%; The content of total phenols were equal to 209.08 and 201.50 μg/mL GAE equivalent. Thin layer chromatography (TLC) chromatogram were shown degree of polymerization (DP) of GAOS3K-M3M8 and GAOS3K-M3 mainly DP 2, DP 4, and DP 6. High performance liquid chromatography (HPLC) chromatogram were shown MW of GAOS3K-M3M8 and GAOS3K-M3 mainly 1,023 Da oligosaccharides, and conjectured DP 6. Fourier transforminfrared spectroscopy (FT-IR) spectra of GAOS3K-M3M8 and GAOS3K-M3 is presented C-O-S, C-O-H, and C-C. In antioxidative activity assays, the DPPH scavenging activity of GAOS3K-M3M8 and GAOS3K-M3 (10 mg/mL) were 27.57% and 40.79%; The Fe2+ chelating ability of GAOS3K-M3M8 and GAOS3K-M3 (5 mg/mL) were 97.36% and 97.38%; The reducing power of GAOS3K-M3M8 and GAOS3K-M3 (10 mg/mL) were equal to 34.0549 and 40.4223 µg/mL Trolox equivalent. In anticoagulant activity assays, which are activated partial thromboplastin time (APTT) and prothrombin time (PT) using rabbit plasma, GAOS3K-M3M8 could prolong the aggregation time and decrease the aggregation level compared to blank. GAOS3K-M3 could prolong the aggregation time and decrease the aggregation level compared to blank in PT and thrombin time (TT) test. The effect of GAOS3K-M3M8 and GAOS3K-M3 on cell viability for B16-F10 cells were both above 85%, and decreased melanin content of B16-F10 cells to 48% and 43%. The tyrosinase inhibition activities of GAOS3K-M3M8 for B16-F10 cells was 14% (10 min), and GAOS3K-M3 was 23% (50 min). Therefore, these results indicated that GAOS3K could perform antioxidative, anticoagulant, and whitening activities.
| Identifer | oai:union.ndltd.org:TW/107NTOU5253043 |
| Date | January 2019 |
| Creators | Chen, Ciao-Yun, 陳巧芸 |
| Contributors | Pan, Chorng-Liang, 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 | 116 |
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