臺灣博碩士論文加值系統

豆漿為亞洲重要的一樣飲品，而其中含有異黃酮的其具有減輕女性更年期症狀的效果，而有文獻指出乳酸菌具有轉化異黃酮提高其生物活性的功能，因其β-葡萄醣苷酶具有催化分解β-醣苷鍵的功能可將豆漿中含醣基的異黃酮去醣基生成高生物活性的異黃酮，文獻指出超音波對於乳酸菌有刺激提升其β-葡萄醣苷酶活性的功能。

本研究用優化後的超音波處理條件處理菌體後經再由添加營養素的方式來提升β-葡萄醣苷酶活性，根據菌株篩選的結果顯示六株中Lactobacillus acidophilus BCRC 10695、Lactobacillus casei BCRC 10696、Lactobacillus bulgaricus BCRC 14080、Streptococcus thermophiles BCRC 13680、Streptococcus thermophiles BCRC 12257、Streptococcus thermophiles BCRC BAA-250以L. acidophilus BCRC 10695具有最高的酵素活性，從其生長曲線得知在12小時到36小時其菌數、酸鹼值、濁度等參數具有較高的穩定度，β-葡萄醣苷酶活性也相對其他點高。

而在超音波條件篩選的接種菌液在37℃下進行不同條件的篩選，振幅 (20% (40W) ~ 60% (80W))，處理時間 (1分鐘 ~ 3分鐘) 以及菌株培養時間 (12小時 ~ 36小時) 等條件篩選，篩選結果以振幅20% (40W)、2分鐘處理時間、菌株培養24小時的條件處理，其菌數、酸鹼值、濁度等參數具有較高的穩定度，β-葡萄醣苷酶活性的提升效果較好。

在培養基營養素添加的實驗中以添加抗壞血酸 (0.5% ~ 0.01%)，生物素 (1000 ~ 1 ppm)以及Tremella flava ATCC 36815黃白木耳胞外多醣 (1.0% ~ 0.01%)。添加結果以結果顯示添加1%及0.5% T. flava ATCC 36815黃白木耳胞外多醣具有提升超音波處理後L. acidophilus BCRC 10695 β-葡萄醣苷酶酵素活性的功能，經過36小時發酵時間所表現出的酵素活性約為控制組的1.78倍和1.81倍，其菌數、酸鹼值、濁度等參數具有較高的穩定度。

最後以37℃、振幅20% (40W)、2分鐘超音波處理培養24小時的接種菌液，後接入添加0.5% T. flava ATCC 36815黃白木耳胞外多醣的豆漿中，其結果顯示添加黃白木耳多醣的超音波處理豆漿中異黃酮在daidzin及daidzein的轉化能力則較控制組為佳。

Soymilk is an important Asian beverage, with high quality protein, isoflavones to reduce the menopausal symptoms, some studies have shown that lactic acid bacteria can convert of glucosides isoflavones into aglycone isoflavones to promote its biological activity, β-glucosidase can catalyze the decomposition of β-glucoside linkers, generating highly bioactive isoflavones, the literature pointed out that ultrasonic treatment on lactic acid bacteria could stimulate its β-glucosidase activity.

In this study, the β-glucosidase activity was increased by the addition of nutrients after ultrasound treatment with the optimized ultrasonic treatment conditions. According to the results of the experiment bacteria screening, six strains of bacteria , L. acidophilus BCRC 10695, L. casei BCRC 10696, L. bulgaricus BCRC 14080, S. thermophiles BCRC 13680, S. thermophiles BCRC 12257, S. thermophiles BCRC BAA-250 where L. acidophilus BCRC 10695 has the highest β-glucosidase activity and is known from its growth curve that incubate 12 hours to 36 hours, it’s bacterial count, pH, turbidity these parameters were most stable and β-glucosidase activity is relatively high relative to other incubate time.

In the ultrasonic conditions screening, the bacteria was treated at 37℃ under different conditions of screening. Amplitude (20% (40W) ~ 60% (80W)), treatment time (1 minute ~ 3 minutes) and bacteria incubate time (12 hours ~ 36 hours) conditions screening. The results showed that the best ultrasonic treatment condition were Amplitude 20% (40W), 2 minutes and the bacteria was incubated 24 hours. The parameters such as the bacteria count, pH and turbidity were most stability, and β-glucosidase activity was promoted.

In the media nutrient addition experiments to add ascorbic acid (0.5% ~ 0.01%)、biotin (1000 ppm ~ 1 ppm) and T. flava ATCC 36815 extracellular polysaccharide (1.0% ~ 0.01%)。The results showed that the addition of 1% and 0.5% T. flava ATCC 36815 extracellular polysaccharide had the effect of promote the β-glucosidase activity of L. acidophilus BCRC 10695 after ultrasonic treatment, after 36 hours of fermentation time The activity of the enzyme was about 1.78 times and 1.81 times of the control. The parameters such as the bacterial count, pH and turbidity were more stable .

The cells were incubated for 24 hours at 37 ° C and ultrasound treatment 20% (40W) for 2 minutes and then incubated to soymilk contains 0.5% T. flava ATCC 36815 extracellular polysaccharide. The results showed that the conversion ability of isoflavones in daidzin and daidezin was better than the control group.

中文摘要……………………………………………………………………………..i
Abstract…………………………………………………………………………….ii
壹、前言……………………………………………………………………………..1
貳、文獻回顧…………………………………………………………………………2
一、益生菌(probiotics) ………………………………………………………………2
(一) 益生菌定義…………………………………………………………...2
(二) 益生菌的益生機制及條件…………………………………………...2
(三) 乳酸菌介紹…………………………………………………………...2
(四) 乳酸菌的生理機能…………………………………………………...3
二、黃豆(soybeam) …………………………………………………………………..8
三、豆漿 (Soymilk) ………………………………………………………………….8
四、異黃酮 (isoflavone) ………………………………………………………………8
(一) 異黃酮結構…………………………………………………………...8
(二) 異黃酮生理活性…………………………………………………….11
(三) 乳酸菌β-葡萄醣苷酶 (β-glucosidase) 的分泌…………………….12
五、超音波…………………………………………………………..........................14
(一) 超音波定義………………………………………………………….14
(二) 超音波作用原理…………………………………………………….15
(三) 超音波的應用……………………………………………………….17
(四) 乳酸菌的超音波處理效應…………………………………………18
參、材料與方法…………………………………………………………………….25
一、實驗架構……………………………………………………………………….25
二、實驗材料及試劑配制………………………………………………………….26
(一) 實驗原料…………………………………………………………….26
(二) 實驗菌株…………………………………………………………….26
(三) 實驗藥品及培養基………………………………………………….26
(四) 實驗設備…………………………………………………………….27
(五) 培養基及試劑配制………………………………………………….28
三、實驗方法………………………………………………………………………..29
(一) 菌種活化…………………………………………………………….29
(二) 接種菌液製備………………………………………………………30
(三) 生長曲線…………………………………………………………….30
(四) 菌液濁度測量………………………………………………………30
(五) 酸鹼值測量…………………………………………………………30
(六) 胞外β-葡萄糖苷酶活性測量………………………………………30
(七) 黃白木耳胞外多醣萃取……………………………………………31
(八) 探針式超音波處理條件篩選………………………………………31
(九) L. acidophilus BCRC 10695培養時間篩選…………………………31
(十) 培養基添加不同營養素……………………………………………32
(十一) 菌體顯微結構分析……………………………………………….32
(十二) 豆漿製備………………………………………………………….32
(十三) 異黃酮成分分析………………………………………………….32
(十四) 統計分析………………………………………………………….33
肆、結果與討論……………………………………………………………………..39
一、實驗菌株β-glucosidase活性篩選………………………………………..39
二、L. acidophilus BCRC 10695生長曲線…………………………………………45
三、超音波處理L. acidophilus BCRC 10695-振幅篩選……………………………50
四、超音波處理 L. acidophilus BCRC 10695-超音波作用時間篩選……………56
五、超音波處理L. acidophilus BCRC 10695-菌株培養時間篩選…………………62
六、菌體電子顯微鏡照攝顯微結構分析…………………………………………..68
七、培養基添加不同營養素之探討………………………………………………..70
(一) 抗壞血酸添加……………………………………………………….70
(二) 黃白木耳 (T. flava ATCC 36815) 胞外多醣………………………76
(三) 生物素……………………………………………………………….82
八、L. acidophilus BCRC10695豆漿發酵…………………………………………89
伍、結論……………………………………………………………………………103
陸、未來展望………………………………………………………………………104
柒、參考文獻………………………………………………………………………105

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