臺灣博碩士論文加值系統

麩胱甘肽 (Glutathione, GSH) 係麩胺酸、半胱胺酸與甘胺酸縮合而成的三胜肽，具有護肝、解毒、抗腫瘤、修護細胞、避免生物體受到氧化損傷、改善皮膚光澤等功能。本研究目的乃以台農 10 號甘藷中分離得到的酵母菌 (Pichia kudriavzevii KR601，簡稱 KR601) 為試驗菌株，探討提升其 GSH 產量的發酵條件。於破菌方法中，KR601 發酵液以檸檬酸調整 pH 為 3.0 沸水加熱 10 分鐘者，獲得的 GSH 含量最高。KR601 以物理性長波 UVA (315~400 nm) 照射誘變後，無法提升 GSH 之產量；短波 UVC (100~280 nm) 照射 150 秒誘變後，GSH 產量提升 13%。試驗菌株以化學誘變劑 N-methyl-N′-nitro-N-nitrosoguanidine (NTG) (2000 µg/mL) 或 H2O2 (1.5 mM) 誘變後，菌株之 GSH 產量分別提升 7% 及 11%。
KR601 的最適碳源為葡萄糖，最適添加濃度為 9%，此時的 GSH 產量為 73.19 ± 1.49 mg/L。最適氮源為酵母萃取液，最適添加濃度為 1%，此時的 GSH 產量為 27.91 ± 0.32 mg/L。最適的前驅物為半胱胺酸 (分析級)，最適的添加濃度為 0.15%，此時的GSH產量為 140.06 ± 1.06 mg/L。以食品級的 L-半胱胺酸鹽酸鹽取代分析級半胱胺酸，最適添加濃度為 0.15%，GSH 產量為 108.44 ± 1.93 mg/L。甲硫胺酸及乳清蛋白無法提升 KR601 之 GSH 產量。
KR601 於 35℃ 培養 6 小時，還原醣及胺基態氮急遽下降，故於饋料發酵中，選擇第 6 小時開始補充營養素。KR601 於 35℃ 批式發酵未補充營養素時，GSH 產量在 24 小時達最高，為 109.52 ± 1.82 mg/L。試驗菌株若於發酵至第 6 小時補充碳源 (9% 葡萄糖)，於培養 32 小時之 GSH 產量達最高，為 147.44 ± 2.74 mg/L，補充碳源可提升 GSH 產量 34.6%。KR601 於發酵至第 6 小時補充氮源 (1% 酵母萃取液)，於培養至 44 小時之 GSH 產量達最高，為 113.36 ± 0.95 mg/L，可提升 GSH 產量 3.5%。試驗菌株若於發酵至第 6 小時補充 0.15% 半胱胺酸，於培養至 12 小時之 GSH 產量最高，為 84.84 ± 1.26 mg/L，GSH 產量減少約 22%。KR601 於發酵過程同時補充碳氮源以及碳氮源加半胱胺酸時，均造成 GSH 產量減少。使用陽離子交換樹脂進行 KR601 發酵液中 GSH 分離純化，以 Amberlite IR120 之回收率較高，可達 61 %。

Glutathione (GSH) is a tripeptide bearing γ-L-glutamyl-L-cysteinylglycine residues. Functions of glutathione are to reduce oxidative stress, detoxication, liver defends, antitumor, cell repair, and skin color improvement. In this study, Pichia kudriavzevii KR601 isolated from sweet potato will be used a test strain to investigate the fermentation comditions for increasing GSH production. Data obtained showed that KR601 cell disrupted by boiling water at pH 3 for 10 mins showed the highest efficiency to extract GSH. KR601 treated with high wavelength of UVA (320~400 nm) did not change its GSH production. KR601 treated with short wavelength of UVC (100~280 nm) increased its GSH production by 13%, while those treated with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and H2O2 increased their GSH production by 6 and 11%, respectively. Glucose is the best carbon source to KR601 for GSH production, the optimal concentration is 9%, and GSH production is 73.19 ± 1.49 mg/L, while yeast extract is the best nitrogen source, the optimal concentration is 1%, and GSH production is 27.91 ± 0.32 mg/L. Analytical grade cysteine is the best GSH precursor, the optimal concentration is 0.15%, GSH production is 140.06 ± 1.06 mg/L ; the optimal concentration of food grade cysteine is also 0.15%, while GSH production is 108.44 ± 1.93 mg/L. Methione and whey protein did not increase the GSH production of KR601.
Reducing sugar and amino nitrogen decreased rapidly at 6th hr. in the fermentation process of KR601 at 35℃., the 6th hr was thus selected as a point to supplement additional nutrient for KR601. In batch fermentation of KR601 with no supplement of additional nutrient, GSH production reached the highest amount at 24th hr. being 109.52 ± 1.82 mg/L. In feed-batch fermentation of KR601 with supplement of additional carbon source (9% glucose) at 6th hr. GSH production reached the highest amount at 32nd hr. being 147.44 ± 2.74 mg/L with 34.6% increase. KR601 with supplement of additional nitrogen source (1% yeast extract) at 6th hr., GSH production reached the highest amount at 44th hr. being 113.36 ± 0.95 mg/L with 3.5% increase. KR601 with supplement of additional 0.15% cysteine at 6th hr., GSH production reached the highest amount at 12th hr. being 84.84 ± 1.26 mg/L with 22% decrease. KR601 with supplement of additional either carbon source+nitrogen source or carbon source+nitrogen source+cysteine at 6th hr., both decrease their GSH production. In the purification of GSH from fermented liquid of KR601 with Cation exchanger, Amberlite IR120 showed the highest recovery being 61%.

摘要
Abstract
致謝
目錄
圖目錄
表目錄
附圖目錄
附表目錄
壹、前言
貳、文獻整理
一、麩胱甘肽的簡介
二、麩胱甘肽的結構、生理功能特性與應用
(一) 麩胱甘肽的結構
(二) 麩胱甘肽的生理功能特性
(三) 麩胱甘肽的應用
三、麩胱甘肽生產方法
(一) 萃取法
(二) 化學合成法
(三) 生物合成法
四、麩胱甘肽高產菌株選育
(一) 麩胱甘肽生產菌種的選擇
(二) 麩胱甘肽生產菌種的改良
五、優化培養基產生麩胱甘肽
(一) 碳源的影響
(二) 氮源的影響
(三) 微量元素的影響
(四) 前驅物的影響
六、麩胱甘肽的分離純化
參、實驗設計
肆、實驗材料與方法
一、實驗材料
(一) 菌株來源介紹
(二) 培養基
(三) 化學藥品
(四) 陽離子交換樹脂
(五) 儀器設備
二、實驗方法
(一) 菌株活化
(二) 菌株保存
(三) 物理性誘變
(四) 化學性誘變
(五) 麩胱甘肽 (GSH) 及 GSSG 含量測定
(六) 菌體生物量 (Biomass) 分析
(七) 最適培養基配方探討
(八) DNS (3, 5-dinitrosalicylic acid) 還原醣含量測定 (Miller, 1959)
(九) 胺基態氮 (amino nitrogen) 測定 (Church et al., 1983)
(十) 批次發酵與饋料發酵
(十一) 以陽離子交換樹脂進行 GSH 分離純化
(十二) 統計分析
伍、結果與討論
一、微生物破菌方法之探討
(一) 以不同酒精濃度進行破菌
(二) 以不同 pH 值的檸檬酸進行破菌
二、利用菌株誘變提升 GSH 產量
(一) 物理性誘變
(二) 化學性誘變
三、KR601 菌株最適培養基組成
(一) 最適碳源及最適添加濃度
(二) 最適氮源添加濃度
(三) 最適前驅物以及最適添加量探討
四、批次發酵與饋料發酵對 GSH 之提升量
(一) KR601 發酵過程還原醣及胺基態氮的變化
(二) 批式發酵
(三) 饋料發酵
五、以陽離子交換樹脂進行 GSH 分離純化
(一) 以 GSH 標準品進行 Dowex IR100 陽離子交換純化
(二) 以培養液中 GSH 樣品進行 Dowex IR100 陽離子交換純化
(三) 以 GSH 標準品進行 Amberlite IR120 陽離子交換純化
(四) 以培養液中 GSH 樣品進行 Amberlite IR120 陽離子交換純化
陸、結論
柒、參考文獻

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