臺灣博碩士論文加值系統

在本研究中，使用之乳酸菌為本實驗室從黑豆萃取水 (BSWEs) 中篩選分離之菌株，分離後送菌種鑑定，確定此菌株為發酵乳桿菌 (Lactobacillus fermentum)。本研究以不同接菌量 （1％、5％、10％ (v/v））起始濃度之發酵乳桿菌菌液發酵，添加不同濃度酵母萃取物 (YE) （0、2.5、5.0 g/L），麥芽萃取物 (ME)（0、2.5、5.0 g/L） 和蔗糖 （0、 2、5、8、10 g/L）之 BSWEs，並探討發酵乳桿菌發酵 BSWEs 之最適條件，研究發酵乳桿菌的 OD600 值和總生菌數（定義為活細胞計數； CFU/mL 的VCCs）。經測試後選擇以 10％（v/v）起始濃度之發酵乳桿菌菌液用於後續研究。對於添加不同比例濃度之 YE 和 ME 之 BSWEs 的生長曲線測試中，在 BSWEs 中添加的 2.5 g/L YE 和 5.0 g/L ME 為所有濃度中，最佳最適合於發酵乳桿菌生長之濃度比例。對於添加不同比例濃度蔗糖之 BSWEs 的生長曲線測試中，發現添加2 g/L蔗糖的發酵乳桿菌的 OD600 值與添加 5、8和10 g/L蔗糖的 OD600 值非常相似。經過測試後選用 BSWEs 中添加 2.5 g/L YE、5 g/L ME 和 2 g/L 蔗糖為發酵發酵乳桿菌生長之最佳濃度，研究發酵時間對發酵乳桿菌 viable cell counts (VCCs) 之影響。發現發酵乳桿菌的 VCCs （第 7 小時約 7×107 CFU/mL），第 6 ~ 9 小時 VCCs 值隨著發酵時間的增加而增加至最高值 （約 5.4 ~ 5.5×108 CFU/mL），在第 9 小時培養後，VCCs 值隨著發酵時間的增加而開始減少。本研究將以 10 % 起始菌液濃度之發酵乳桿菌發酵添加 5.0 ME + 2.5 YE g/L和 2 g/L 蔗糖之 BSWEs，並探討發酵時間對發酵後 BSWEs 上清液的抗氧化活性 (DPPH 清除活性、TPC和RP) 的影響。清除DPPH自由基能力的結果顯示以第12小時的發酵液其 EC50為所有小時數之發酵液中最少的濃度，其濃度為 1.08 mg/mL；BSWEs 發酵後上清液之 Total phenolic contest (TPC) (定義為沒食子酸當量 (gallic acid equivalent;GAE)) 與 Reducing power (RP) (定義為Vitamin C當量(vitamin C equivalent; Vit.C)) 會隨著發酵時間增加而逐漸下降。在 TPC 的部分，結果顯示發酵第 6 小時 ，稀釋濃度 5 mg/mL 時的發酵液，其總多酚含量達到 0.52 mg GAE /mL 為低濃度中最佳之結果；在 RP 結果得知，發酵第 0 小時，稀釋濃度 10 mg/mL 之發酵液其 RP 可達到最高值，其值為 0.23 mg Vit.C/mL，但發酵第 6 小時之發酵液其 RP 可平穩地維持高值。總而發酵乳桿菌在添加 5.0 ME + 2.5 YE g/L和 2 g/L 蔗糖之 BSWEs 中具有良好的生長，並且可以增加 BSWEs 本身具有的營養價值及功能特性。

In this study, the water extract from 1:10 in w/w of black soybean (Glycine max (L) Merr. Tainan No. 5) water was determined to be the culture medium by Lactobacillus fermentum isolated from the black soybean water extracts (BSWEs) in our laboratory. The effects of fermentation time, starter concentration (1%, 5%, 10% (v/v)) and different concentration addition of yeast extract (YE) (0, 2.5, 5.0 g/L ), malt extract (ME) (0, 2.5, 5.0 g/L) and sucrose (0, 2, 5, 8, 10g/L) on the OD600 value and cell viability (defined as viable cell counts; VCCs in CFU/mL) of L. fermentum were investigated. For BSWEs only without any addition, the OD600 of L. fermentum increased with increasing the starter concentration. The 10% (v/v) of the starter was selected for subsequent studies. For the different concentration addition of yeast extract and malt extract, the optimal concentrations of 2.5 g/L YE and the 5 g/L ME added in BSWEs were most suitable for L. fermentum growth. For the different sucrose concentration addition, it was found that the OD600 values for L. fermentum for addition of 2 g/L sucrose were very similar to those for addition of 5, 8, and 10 g/L sucrose. The optimal concentration of 2.5 g/L YE , 5 g/L ME and 2 g/L sucrose added in BSWEs was used to study the effect of fermentation time on VCCs of L. fermentum. The VCCs of L. fermentum (about 7 × 107 CFU/mL at the 0th hr) increased up to the saturation value (about 5.42 ~ 5.49 × 108 CFU/mL) with increasing fermentation time during the first 6th ~ 9th hr. After the 9th hr cultural time, the VCCs of L. fermentum began to decrease with increasing fermentation time. In this study, added 5.0 ME + 2.5 YE g/L and 2 g/L sucrose BSWEs to Lactobacillus fermentum with 10% initial bacterial concentration, and explored the antioxidant activity of fermentation time on BSWEs supernatant after fermentation (DPPH radical scavenging, TPC and RP). The result of scavenging DPPH free radicals showed that the EC50 of the fermentation broth at the 12th hr was the lowest concentration in the fermentation at all hours, the concentration was 1.08 mg/mL; the TPC of the supernatant after BSWEs fermentation (defined as pharose The gallic acid equivalent (GAE) and RP (defined as Vitamin C equivalent; Vit. C) gradually decrease as the fermentation time increases. In the TPC section, the results showed that the fermentation broth at the 6th hr of fermentation and the dilution concentration of 5 mg/mL had a total polyphenol content of 0.52 mg GAE /mL, which was the best result in the low concentration; At the 0th hr, the fermentation broth with a dilution of 10 mg/mL had the highest RP value of 0.23 mg Vit.C/mL, but the RP of the fermentation broth at the 6th hr of fermentation could maintain a high value smoothly.

摘要....................................................................i
Abstract................................................................iii
誌謝....................................................................v
目錄....................................................................vi
表目錄...................................................................xii
圖目錄..................................................................xiii
簡寫說明.................................................................xiv
第一章 緒論..............................................................1
1.1 前言................................................................1
1.2 研究目的.............................................................2
第二章 文獻回顧...........................................................4
2.1 益生菌的介紹..........................................................4
2.1.1 益生菌的定義........................................................4
2.1.2 益生菌功效..........................................................5
2.2乳酸菌介紹.............................................................7
2.2.1 乳酸菌定義..........................................................7
2.2.2 乳酸菌的分類........................................................9
2.2.3 乳酸菌的應用........................................................10
2.3 發酵乳桿菌介紹........................................................13
2.3.1 發酵乳桿菌簡介......................................................13
2.3.2發酵乳桿菌功效.......................................................14
2.3.3 發酵乳桿菌應用......................................................15
2.4黑豆介紹..............................................................16
2.4.1 黑豆簡介...........................................................16
第三章 材料與方法.........................................................20
3.1 實驗材料與設備........................................................20
3.1.1 實驗材料來源........................................................20
3.1.2 實驗儀器............................................................21
3.2實驗條件........................................................22
3.2.1 菌種篩選........................................................22
3.2.2 菌種保存........................................................22
3.2.3 菌種活化........................................................22
3.2.4 培養流程........................................................23
3.3分析方法........................................................25
3.3.1微生物分析 (總生菌數測定)...........................................25
3.3.2發酵過程 pH 值變化.................................................25
3.3.3發酵過程糖度值變化..................................................25
3.4抗氧化的測定........................................................25
3.4.1 黑豆水萃取物的製備.................................................25
3.4.2 清除 DPPH 自由基能力之測定.........................................26
3.4.3 還原力測定........................................................27
3.4.4 總多酚類化合物含量測定..............................................28
3.5 統計分析........................................................28
第四章 結果與討論........................................................29
4.1 L. fermentum發酵黑豆萃取水的生長變化.............................29
4.1.1 10% (v/v)起始菌量L.f發酵添加不同濃度的 YE、ME 和蔗糖之 BSWEs OD600 及 pH的變化影響........................................................29
4.1.2 10% (v/v) 起始菌量 L. fermentum 發酵添加 5.0 g/L ME + 2.5 g/L YE
2 g/L 蔗糖之 BSWEs 之 OD600、總生菌數、pH和糖度的變化..................33
4.2 黑豆萃取水發酵液之抗氧化活性的變化.................................35
4.2.1 DPPH 自由基清除試驗...........................................35
4.2.2 總酚類化合物含量試驗............................................38
4.2.3還原能力試驗......................................................40
4.3抗氧化試驗關係......................................................42
4.3.1 DPPH 清除率與總酚類化合物含量關係..................................42
4.3.2 DPPH 清除率與還原能力關係..........................................42
4.3.3還原能力與總酚類化合物含量關係.......................................42
第五章 結論........................................................46
參考文獻........................................................47
附錄一........................................................54
附錄二........................................................55
附錄三........................................................56
附錄四........................................................57
附錄五........................................................58
Extended Abstract.............................................59

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