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研究生:王鵬翔
研究生(外文):Peng-Shieng Wang
論文名稱:提升γ-胺基丁酸之相關研究
論文名稱(外文):Study on the enhancement of γ-aminobutyric acid production
指導教授:謝佳雯謝佳雯引用關係
指導教授(外文):ChiaWen Hsieh
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:微生物免疫與生物藥學系研究所
學門:生命科學學門
學類:其他生命科學學類
論文種類:學術論文
畢業學年度:102
語文別:中文
論文頁數:106
中文關鍵詞:γ-胺基丁酸芽菜發酵乳酸菌
外文關鍵詞:γ-aminobutyric acidgerminationlactic acid bacteriafermentation
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本研究主要目的為提升 γ-胺基丁酸 (GABA) 之含量,而以兩種不同操作策略著手,首先以芽菜培育方法切入,探討種子浸泡時間與灑水頻率對於芽菜中 GABA 含量之影響。結果顯示,芽菜中之 GABA 含量將隨著種子浸泡時間與發芽程度增加而提升,最終蕎麥芽菜中之 GABA 含量為 100.36 mg/100 g,相較於其本身未發芽種子而言有效提升了二十倍,而紫高麗芽菜中之 GABA 含量為所有芽菜中最高,可高達至 238.76 mg/100 g。
另外在探討微生物發酵生產方式方面,先由台中地區挑選取得兩種醃漬食品 (白菜泡菜與蘿蔔泡菜) 與嘉義地區之動物腸胃道之體液 (牛瘤胃液與雞隻盲腸) 作為微生物篩選來源,而以 gad gene 作為篩選依據,並以豆漿培養評估菌株 GABA 生產能力,最後定序比對 16S rDNA 鑑定菌種後則是探討各類發酵因子添加、培養溫度、厭氣處理、發酵時間與含水量等條件對於生產 GABA 之影響。篩菌結果顯示,來自白菜泡菜汁液中之菌株編號為 I30-1,其含有 gad gene,並進一步於豆漿培養兩天後,能夠有效提升增加 GABA 濃度 518 mg/L,且初步鑑定為 Lactobacillus plantarum。隨後將 I30-1 運用於探討培養基添加物因子,並以脫脂豆粉為原料,結果顯示以添加濃縮糖蜜發酵液(Condensed Molasses Fermentation Solubles, CMS) 為最具影響生產 GABA 之因子,液態發酵兩天後,GABA 濃度可從原脫脂豆粉 31 提升至 165 mg/L。而在探討 CMS 因子濃度與發酵時間上,結果顯示 GABA 濃度將隨著 CMS 添加比例與發酵時間增加而增加,在添加 20% CMS 條件下液態發酵六天,最終 GABA 濃度可達至 805 mg/100 g,相較於原脫脂豆粉而言可有效提升約 35 倍。另外,在培養溫度與厭氣處理探討上,其對於影響 I301 發酵生產 GABA 而言則無明顯差異。最後在探討含水量與最佳化條件固態發酵部分,發酵產物 GABA 濃度則有效提升至介於 600 至 750 mg/100 g ,而倘若能提高含水量將有助於提升 GABA 產量,最終所得之高濃度 GABA 發酵豆粉,未來在動物飼糧添加上將具有極高之發展潛力。

The purpose of this study is to enhance the γ-aminobutyric acid (GABA) production with two different strategies. First the different conditions of soaking and spraying water were investigated on sprouts cultivation system for the accumulation of GABA in sprouts of cereals. Through the treatment of soaking for 12 h and water-spraying at every 3 h at 27℃, we obtained the results of GABA contents of buckwheat sprout was 100.36 mg/100 g, which was twenty times higher than its seeds (4.62 mg/100 g).
Besides, fermentation by microorganism is a good way for approaching. The 57 isolates were selected from pickle vegetables and digestive tract of animal. Then, the glutamate decarboxylase encoded gene gad were analyzed by polymerase chain reaction and the identification analysis was made by the sequencing of 16S rDNA. The ability of producing GABA from the selected lactic acid bacteria (LAB) was confirmed with homemade soybean milk. After all, single-factor experimental designs were adopted to select the key factors that influence the GABA production and carried out expected fermentation results. The screened results showed that I301 was identified as Lactobacillus plantarum which was carried the gad gene and possessed highest GABA-producing ability among the 57 tested isolates. It was found that condensed molasses fermentation solubles (CMS), fermentation days and water contents were the key factors of affecting GABA production. According to the optimal condition for solid state fermentation, the GABA content could be higher than 750 mg/100 g.
目錄
誌謝…………………………………………………………………………… I
中文摘要……………………………………………………………………… II
英文摘要……………………………………………………………………… IV
目錄…………………………………………………………………………… V
圖目錄………………………………………………………………………… IX
表目錄………………………………………………………………………… XI
壹、前言........................................... 1
一、研究動機與目的…………………………………………………… 1
二、研究策略…………………………………………………………… 2
貳、文獻回顧………………………………………………………………… 3
一、γ-胺基丁酸……………………………………………………… 3
(一) 分子特性…………………………………………………… 3
(二) 生物體合成與代謝路徑…………………………………… 5
(三) 生理活性與功能…………………………………………… 7
(四) GABA 生產方式…………………………………………… 8
二、種子發芽…………………………………………………………… 9
(一) 定義與條件特性…………………………………………… 9
(二) 種子中 GABA 富化 (enrichment) 之方法……………… 10
三、黃豆………………………………………………………………… 11
(一) 簡介………………………………………………………… 11
(二) 加工利用…………………………………………………… 12
四、乳酸菌……………………………………………………………… 15
(一) 益生菌之定義與條件……………………………………… 15
(二) 乳酸菌之基本代謝特性…………………………………… 17
(三) 乳酸菌之分類與分佈……………………………………… 21
(四) 影響乳酸菌發酵生產 GABA 之條件因子………………… 22
(五) 乳酸菌發酵生產 GABA 之現況與應用…………………… 24
參、材料與方法……………………………………………………………… 26
一、實驗大綱…………………………………………………………… 26
二、實驗方法…………………………………………………………… 32
(一) GABA 之定量分析………………………………………… 32
(二) GABA 之確效試驗………………………………………… 35
(三) 芽菜培育系統……………………………………………… 37
(四) 豆漿培養基之製備………………………………………… 38
(五) 乳酸菌酛之篩選製備與菌落數測定……………………… 39
(六) 菌種鑑定…………………………………………………… 40
(七) 菌體基因體的純化………………………………………… 41
(八) 瓊脂膠體電泳……………………………………………… 43
(九) 聚合酶連鎖反應…………………………………………… 44
(十) 還原醣測定………………………………………………… 51
(十一) 蛋白質濃度測定………………………………………… 55
肆、結果與討論……………………………………………………………… 57
一、GABA 定量分析方法之建立……………………………………… 57
(一) 偵測極限與定量極限……………………………………… 57
(二) 檢量線……………………………………………………… 57
(三) 精密度……………………………………………………… 60
(四) 準確度……………………………………………………… 60
(五) 選擇性……………………………………………………… 60
二、芽菜培育系統之建立……………………………………………… 64
(一) 黃豆與蕎麥芽菜之 GABA 含量比較……………………… 65
(二) 不同長度蕎麥芽菜之GABA含量比較…………………… 67
(三) 芽菜培育系統之建立與不同品系芽菜之GABA含量比較 69
三、乳酸菌發酵生產 GABA…………………………………………… 72
(一) 薄層層析法………………………………………………… 72
(二) gad gene 之分析篩選……………………………………… 75
(三) 豆漿培養基生產 GABA 能力試驗………………………… 81
(四) 培養基添加因子篩選試驗………………………………… 84
(五) 有效因子添加比例與溫度對於生產GABA之影響試驗… 87
(六) 厭氣處理與發酵時間對於生產GABA之影響試驗…… 90
(七) 含水量對於生產 GABA 之影響試驗………………… 95
(八) 最適化固態發酵之 GABA 產量比較…………………… 97
伍、結論……………………………………………………………………… 99
陸、參考文獻………………………………………………………………… 101
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