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標題: 嗜酸乳桿菌胞外多醣體醱酵製程、純化與結構鑑定
Fermentation Process, Purification and Identification of Extracellular Polysaccharides from Lactobacillus acidophilus
作者: 黃嘉駿
Huang, Jia-Jun
關鍵字: 嗜酸乳桿菌;Lactobacillus acidophilus;胞外多醣體醱酵程序;反應曲面法;多醣體純化;多醣體結構鑑定;Extracellular polysaccharide of fermentation process;Response surface methodology;Purification of polysaccharide;Identification of polysaccharide
出版社: 化學工程學系所
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本研究係採用嗜酸乳桿菌(Lactobacillus acidophilus BCRC 10695)作為評估乳酸菌生產胞外多醣體(Exopolysaccharides, EPS)之菌株。論文分三階段依序探討醱酵程序、多醣體純化和多醣體分子結構鑑定。在碳、氮源種類對乳酸菌生產胞外多醣體之影響上,實驗結果顯示蔗糖可獲得較大的胞外多醣體產量。在氮源的選擇方面,實驗結果表示酵母萃取物可獲得較佳的胞外多醣產量。在EPS分子量分布上,不同碳、氮源胞外多醣體皆有相同的分子量分布,其中高分子量EPS占全部EPS的45%。透過添加界面活性劑有助於提升EPS高分子量比率到60-68%。另外探討不同碳、氮源濃度和界面活性劑對乳酸菌生產EPS之最適化產量條件,結果發現當蔗糖濃度、酵母萃取物濃度和polysorbate 80分別為10.15 g/L、25.0 g/L和2.0 g/L時,培養條件為35℃及96小時,可獲得最佳之EPS理論值(923.7 mg/L),與實驗結果923.6 mg/L非常一致。
在EPS之純化及鑑定上,研究結果顯示調整無菌醱酵液酸鹼值到pH 3,經酒精沉澱可獲得最低的蛋白質對多醣體比值。經酒精沉澱純化多醣體水溶液,再調整pH小於1,可移除蛋白質99.9 %及EPS 53.5 %。活性碳等溫吸附實驗結果顯示,溶液中核酸可減少98.5%。Langmuir等溫吸附曲線預測活性碳最大的吸附能力為250.3 OD260/mL/g。添加QIAGEN protease進行醱酵液中蛋白質水解,實驗結果顯示,在經過活性碳吸附後的樣品組和控制組EPS濃度相同。EPS結構鑑定研究,以HPAEC-PAD分析顯示多醣組成之單醣成份有甘露糖、葡萄糖、半乳糖和核糖。GC-MS結構鑑定分析醣類連接位置,結果顯示部份甲基化醣醇乙酸酯2,3,4,6-tetra-O-methyl-D-Glucitol、1,3,4-tri-O-methyl-, triacetate, D-Mannitol、2,3,4-tri-O-methyl-, triacetate, D-Galactitol、2,3-O- methyl-, triacetate, D-ribitol和4-O-methyl-, pentaacetate, D-Glucitol,醣類連接位置組成分別為t-linked D-Glcp、1,2-linked D-Manp、1,6-linked D-Galp、1,5-linked D-Ribf和1,2,3,6-linked D-Glcp。綜合上述生產製造、分離純化和醣類分子結構鑑定之研究成果有助於提升乳酸菌生產EPS,並可應用於生物醫學研究和生物科技產業生產、製造和開發相關醫學級產物。

In this study, fermentation process, purification and identification of extracellular poly-saccharides (Exopolysaccharides, EPSs) from Lactobacillus acidophilus BCRC 10695 were developed. The effect of carbon sources including glucose, fructose, lactose and sucrose was explored. The submerged culture of L. acidophilus was carried out in a 250 mL shake flask at 35 ℃, 50 rpm and initial pH 6.5 under anaerobic cultivation (flushing nitrogen gas) for 55 h. Biomass, pH, sugar consumption and EPS concentration were sampled and analyzed. Effect of nitrogen sources such as proteose peptone no.3, beef extract and yeast extract were ex-plored. Surfactants including polysorbate 20, polysorbate 80 and span 80 were also investi-gated. It was found that yeast extract and sucrose were the best choices for producing EPS by L. acidophilus. The addition of surfactants effectively promoted 15% to higher molecular weight EPSs. Response surface methodology (RSM) was applied for optimization of cultiva-tion conditions. The optimal concentrations of sucrose, yeast extract and polysorbate 80 were 10.15 g/L, 25 g/L and 2 g/L at 96 h respectively. The best predicted EPS production was 923.7 mg/L, similar to that in the experiment of 923.6 mg/L.
EPS purification was carried out. The harvested broth adjusted pH to 3 displayed the lowest proteins to EPSs ratio (P/E). The collected EPSs further underwent a pH adjustment to less than 1 followed by centrifugation. The protein and EPS amounts could be removed to 99.9 % and 53.5 % respectively. Activated carbon was used to remove 98.5% of nucleic acid. The addition of QIAGEN protease displayed little effect on EPS purification. Chemical structure of the purified EPS was integratedly identified with HPAEC-PAD, GC-MS and HAPEC-PAD. Analysis results showed four sugars including mannose, glucose, galactose and ribose existed. GC-MS analysis results showed that methylation analysis of EPS had 2,3,4,6-tetra-O-methyl-D-Glucitol; 1,3,4-tri-O-methyl-, triacetate D-Mannitol; 2,3,4-tri-O-methyl-, triacetate, D-Galactitol; 2,3-O-methyl-, triacetate, D-ribitol and 4-O-methyl-, pentaacetate, D-Glucitol of partially O-methylated alditol acetates. The sugar linked location was t-linked D-Glcp, 1,2-linked D-Manp, 1,6-linked D-Galp, 1,5-linked D-Ribf, and 1,2,3,6-linked D-Glcp, respectively. The study results about EPS productiron, pu-rification and chemical structrue identification can be appled to the production of the related medical grade products in the biomedical and biotechnologic industry.
其他識別: U0005-0608201323254500
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