益生菌用於發酵在各種食物已有多年歷史。近年來許多研究指出，藉由益生菌(probiotics)的攝取可平衡腸道菌相並有助於維持身體健康。這項研究的目的是以研究Lactobacillus sp. TKU 012所生產之蛋白酶的純化和定性及其對酸和膽汁耐受力及其應用。由嬰兒吐奶中所篩選出之菌株Lactobacillus sp.TKU012能以烏賊軟骨粉末作為生產蛋白酶之主要營養源。Lactobacillus sp.TKU012生產蛋白酶之較適培養條件為: 將含有1%烏賊軟骨粉末、0.1%K2HPO4、0.05%MgSO4．7H2O之100mL液體培養基(pH9)於250mL之三角錐形中，於25℃振盪培養3天後，可得最大蛋白酶活性。利用Lactobacillus sp.TKU012之蛋白質酶較適生產條件大量培養，所得發酵液經硫酸銨沉澱、DEAE-Sepharose離子交換層析及Sephacryl S-100膠體層析一連串分離步驟，可純化出一種蛋白質酶。TKU012 蛋白酶之生化特性經分析結果，其最適反應溫度50℃、最適反應pH10、熱安定性為＜60℃、pH安定性5-10及利用SDS-PAGE分析出來的分子量約為49kDa;活性會受到PMSF的抑制，屬於絲胺型的蛋白質酶;其活性會受到Cu2+、Ba2+ 和Zn2+ (5mM)的抑制，蛋白質酶的殘餘活性分別只剩53%、44%與32%;且純化後的酵素在0.5(mM)SDS，0.5%(v/v)Tween 40，0.5%(v/v)Triton X-100界面活性劑存在下殘餘活性分別為98%、67%及45%。有機溶劑對酵素活性及安定性影響方面，在甲苯、乙醚、與丙酮(25%, v/v)等有機溶劑下，蛋白質酶仍有50%以上的殘餘活性; 而將酵素與有機溶劑(20%, v/v)於4℃與25℃下，待反應10天後，皆仍然保有75%以上的殘餘活性。
在其它研究，TKU 012在對酸鹼度2.5 磷酸鹽緩衝的這個測試之耐受性上， 它在酸鹼度2.5時 可達到103 cfu/ml。耐膽鹽性試驗方面，在含0.3%牛膽鹽之MRS plus broth 中培養24 小時存活率達90％以上。經過耐酸性(pH2.5)和耐膽鹽性(0.3%)測試，TKU 012模擬消化過程之測試及TKU 012菌株的膽鹽耐受性可達到80%以上。

Probiotics have been used in various fermented foods for many years. Resent reports indicated that digest probiotics are microbial food supplements, which beneficially affect the balance of intestinal microflora and host’s healthy. The purpose of this study was to investigate the purification and characteristics of a protease from Lactobacillus paracasei TKU012and its acid and bile resistant ability discussion . The protease-producing strain, Lactobacillus paracasei TKU012, was isolated from infant Spits milk . The optimized culture medium was composed of 1% squid pen powder(SPP), 0.1%K2HPO4, 0.05%MgSO4．7H2O at pH9. The strain was incubated in 100mL of above liquid medium and kept shaking at 25℃ for 3 days. The TKU012 protease was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose column chromatography and Sephacryl S-100 gel chromatography. The molecular mass of TKU012 protease determined by SDS-PAGE was approximately 49kDa.The optimum temperature, optimum pH , pH stability and thermal stability of TKU012 protease was 50℃, pH 10, pH5-10 and＜60℃ . The protease was characterized as a Serine protease because it was inactivated by PMSF. Additionally,the pure protease retained 98% ,67% ,and 45% of its original activity in the presence of 0.5(mM)SDS, 0.5%Tween 40 and 0.5%Triton X-100, respectively.
 In the presence of organic solvent such as toluene, ethyl ether and acetone, the protease retained more than 50% of its activity. The TKU012 protease retain over 75 % of its activity by pre-incubation in the organic solvent at 4℃ and 25℃ for 10 days.  
    Additionally, TKU012 showed tolerance properties in this test on pH 2.5 phosphate buffer, it could achieve 103 cfu/ml . In the bile resistance test, it viability can achieve higher than 90% survival population in MRS plus broth (include 0.3% bull bile salt) after being incubated for 24hrs.According to the acid and bile resistance test, TKU012 take the imitating digestive tract test and it was higher than 80% survival population.

目錄

頁次
封面內頁
簽名頁
授權書
中文摘要………………………………………………………....….i英文摘要………………………………………………….....…..iii
誌謝……………………………………………………….…………..v
目錄………………………………………………………..........vi
圖目錄…………………………………………………………......xi
表目錄………………………………………………………………..xii

第一章 緒論..............................................1
第二章 文獻回顧……………………………………………….2
       2.1益生菌之定義及選擇…………………………………….2
       2.2  益生菌之分類…………………………………………..2
       2.3  乳酸桿菌之基本特性…………………………………..3
       2.4 蛋白酶之水解…………………………………....4
2.4.1蛋白酶分類………………………………………….8
          2.4.2 蛋白酶的一般性質………………….....9
2.5 乳酸菌蛋白酶………….…………………………………9
第三章 材料與方法…………………………………………………11
       3.1 實驗菌株………………………….……………………11
       3.2實驗材料……………………………………………..…11
       3.3實驗儀器……………………………………………….12
        3.4菌株之篩選與分離純化 …………….………….……13
       3.4.1 菌種鑑定……………………………………………..14
       3.5 酵素最適培養條件探討……………..........14
            3.5.1不同碳源的影響…………….………………...14
            3.5.2 碳源濃度的影響……………………….……..14
3.5.3 培養液pH…….………………………………15
3.5.4 培養溫度……………….……......................... 15
3.5.5 培養體積（通氣量）……….………..............15
3.5.6 培養時間…….…………………..………...….15
3.6 蛋白質定量分析…………….………..................15
3.7蛋白酶活性之測定…. .....…...…..…………………….16
3.8蛋白酶酵素之純化分離…….……………...................16
3.8.1 大量培養….……………………....................... 17
3.8.2 硫酸銨沉澱……………………………………17
3.8.3 離子交換樹脂層析法….……………………...17
3.8.4 膠體過濾層析法…………………....................18
3.9 蛋白質電泳分析（Electrophoresis）……...…….....18
3.10 蛋白酶酵素之特性分析………..……………...…..18
3.10.1最適反應pH的測定…………..... ……………..18
3.10.2 pH安定性的測定….. …………………………...19
3.10.3最適反應溫度測定.................. …………………19
3.10.4酵素熱安定的測定........................... ……...19
3.10.5 金屬離子與抑制劑對酵素活性的影響.......... ...19
3.10.6 有機溶劑對酵素之活性及安定性的影響..........20
3.10.7 界面活性劑對酵素活性的影響.......................20
3.11 基質特異性....................................20
3.12利用Lactobacillus paracasei TKU012發酵液進行芥蘭生長測試................................................21
3.12.1芥蘭之預培養.....................................21
3.12.2 促進芥蘭生長試驗............................21
3.13 乳酸菌對酸液、膽鹽的耐受性試驗......................22
3.13.1  耐酸性試驗................................22
3.13.2  耐膽鹽性試驗...............................22
3.13.3  模擬腸道消化過程試驗..........................23
3.13.4  耐鹽試驗…………………………………………..24
3.13.5   pH值與可滴定酸測定…………………………...24
3.13.6 貯藏實驗……………………………………………25
3.13.7溶菌酶實驗…………………………………………25
第四章結果與討論…………………………………………………….26
4.1 蛋白酶生產菌之篩選. ……………………………………..26
4.2 菌株特性……………………………………………………26
4.3碳/氮源的選擇………………………………………………26
4.4 TKU012所生產蛋白酶較適培養條件探討................27
4.5 綜合結果……………………………………………………27
4.6 蛋白酶之純化分離及定性
4.6.1 酵素純化…………………………………………………32
4.6.2 酵素之生化特性分析……………………………………33
4.6.3 分子量測定………………………………………………33
4.6.4 蛋白酶之最適反應溫度…………………………………34
4.6.5 蛋白酶之熱安定性………………………………………34
4.6.6蛋白酶之最適pH 反應…………………………………34
4.6.7 蛋白酶之pH 安定性…………………………………….35
4.6.8 各種金屬離子、鹽類與抑制劑（EDTA、PMSF）對酵素活性之影響. ……………………………………………35
4.6.9 界面活性劑對酵素活性之影響……………………….…36
4.6.10 基質特異性………………………………………….…..36
4.6.11 有機溶劑對蛋白酶活性及安定性影響…………………37
4.7 L.paracasei TKU012 之發酵上清液對芥蘭生長影響…………37
4.7.1乳酸菌對植物生長之影響…………………………………37
4.8乳酸菌之菌體測試.. ……………………..56
4.8.1不同鹽濃度對乳酸菌之影響.. ……………………………….56
4.8.2酸對乳酸菌之影響. …………………………………………..56
4.8.3不同膽鹽濃度對乳酸菌之影響.. …………………………….56
4.8.4模擬腸道試驗. ……………………………………………….57
4.8.5貯藏實驗之乳酸及菌數測定…………………………………58
4.8.6溶菌酶對乳酸菌之影響………………………………………58
第五章  結論………………………………………………………..66
參考文獻…………………………………………………………..…68

圖 目 錄

頁次
圖4.1 L. paracasei TKU 012 之顯微照片……………………..28
圖4.2 L. paracasei TKU 012之16S rDNA部份序列……………...29
圖4.3 L. paracasei TKU 012 之API鑑定系統分析……………..30
圖4.4不同碳/氮源對於 TKU012生產蛋白酶活性之影響…........31
圖4.5 L. paracasei TKU 012培養於烏賊軟骨培養基生產蛋白酶之生長曲線圖…………………………….……………………….....39
圖4.6 L. paracasei TKU 012所生產蛋白酶之純化分離流程圖…....40
圖4.7 DEAE-Sepharose CL-6B 之蛋白酶層析圖譜…............41
圖4.8 Sephacryl S-100 之蛋白酶層析圖譜………....………….42
圖 4.9蛋白酶於SDS-PAGE 之分子量……………………………....44
圖4.10蛋白酶之最適反應溫度及熱安定性……………………....45
圖4.11蛋白酶之最適反應pH及pH安定性……………….…........46
圖4.12有機溶劑對蛋白酶安定性之影響………………………....50 
圖4.13有機溶劑對蛋白酶活性之影響……………………........51
圖4.14  L. paracasei TKU 012 發酵上清液對芥蘭生長之影響.......55
圖 4.15  L. paracasei TKU 012培養在不同鹽濃度下之生長變化…60
圖4.16 L. paracasei TKU 012培養在不同膽鹽濃度下之生長變化...62

表 目 錄

頁次
表2.1  Lactobacillus的來源 ……………………………..…….5
表2.2以不同乳酸菌發酵於各項產品上之整理………………………6
表2.3乳酸桿菌於食品、醫療保健及環保的應用……………………7
表2.4 乳酸桿菌及乳酸球菌之蛋白酶分類…………….……..…...….10
表4.1  L. paracasei TKU012生產蛋白酶之較適條件……………31
表4.2  L. paracasei TKU012蛋白酶純化總表 ………………….43
表4.3 各種化學藥品對蛋白酶之影響…………………………….47
表4.4 各種界面活性劑對蛋白酶之影響………………..….......48
表4.5 蛋白酶之基質特異性…………………………….........49
表4.6 各種乳酸菌所生產蛋白酶之特性比較……..............52
表4.7 L.paracasei TKU012 之發酵液對芥蘭生長影響………..54
表4.8 L. paracasei TKU012於不同pH下之生長變化………….61
表4.9. L. paracasei TKU012於不同膽鹽濃度下之菌數的生長變化.63
表4.10 L. paracasei TKU012之模擬腸道試驗.................63
表4.11 L. paracasei TKU012之貯藏試驗....................64
表4.12 L. paracasei TKU012之耐溶菌酶試驗...... ..... ....65

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