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研究生:郭志彬
論文名稱:孢子型乳酸菌(Bacilluscoagulans)之研究
論文名稱(外文):A Study for Spore-Forming Lactic Acid Bacteria(Bacillus coagulans)
指導教授:李國陽李國陽引用關係
學位類別:碩士
校院名稱:南台科技大學
系所名稱:化學工程系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:93
中文關鍵詞:乳酸菌孢子型乳酸菌發酵
外文關鍵詞:Lactic acid bacteriaspore-forming lactic acid bacteriaFermentation
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中文摘要
乳酸菌可以產生乳酸,降低腸道中之pH而達到抑制腸內腐敗菌及致病菌之生長,同時它亦可能產生多種抗菌物質可以殺死腸道中之壞菌,在現代忙碌生活裡對我們的胃腸扮演著重要的角色。世界各國有關乳酸菌的相關產品相當多,如A菌、B菌、C菌、微胞保護乳酸菌、孢子型乳酸菌等。上市產品又有優酪乳、乳酸菌粉等健康食品;尤其為耐酸程度頗佳的孢子型乳酸菌更是蔚為風潮。
本實驗以Bacillus coagulans為實驗菌株。研究計畫分成二部分。第一部份目的為探討其較適生長環境,進而提高乾菌重(Cell Concentration);第二部分為產孢條件之研究,目的為提高產孢率。研究中我們探討Bacillus coagulans之較適生長環境(包括生長環境之溫度、pH和較適化的培養基)並利用分批進料法之策略探討不同發酵形態之差異。另外,產孢條件方面,探討溫度與菌液濃度稀釋對產孢之影響,並對此孢子型乳酸菌之耐受性作一系列之研究。
結果顯示,Bacillus coagulans較適生長溫度為37℃和pH6環境下。而葡萄糖濃度的添加對Bacillus coagulans生長影響大,在有添加金屬離子作為培養基時,其乾菌重(Cell Concentration)較高,表示金屬離子確實有助於孢子型乳酸菌之生長;而利用Fedbatch方式發酵確實能提高乾菌重(Cell Concentration)為26g/L。另外,實驗發現其最佳的產孢溫度為45℃,而以1:1之發酵菌液和無菌水之比例產孢的速度最快;再者,對於孢子的耐高溫、耐酸和耐膽鹽程度的研究也都有不錯的效果。孢子在100℃高溫下可存活150分鐘,在90℃下菌體幾乎不會死亡。孢子在pH2環境下可存活120分鐘,菌體在pH4、pH5環境下幾乎不會死亡。另外,孢子對膽鹽的耐受度可達65.4﹪。而孢子型乳酸菌也能有效抑制金黃色葡萄球菌和大腸桿菌(抑菌圈分別為12mm和15mm),而其抵抗抗生素的能力也有其效果存在。
關鍵字:乳酸菌、孢子型乳酸菌、發酵
ABSTRACT
Lactic acid bacteria can inhibit microbial growth by producing lactic acid and decreasing pH value in the intestinal. Meanwhile, lactic acid bacteria can produce various antibiotic substances, and there are also useful for human stomach. On the market, there are many kinds of commodities made by lactic acid bacteria products such as Lactobacillus acidoplilus, Bifidobacterium bifidium, Lactobacillus casei , and spore-forming lactic acid bacteria.
Lactic acid bacteria can be applied to yogurt, powder of lactic acid bacteria, and so on. In particular the spore-forming lactic acid bacteria, it has good tolerance to the acid environment. Bacillus coagulans is the strain to be searched in this study. We’d like to look for high cell density culture, and then research carefully the conditions of fermentation including temperature, pH environment and optimum medium. In addition, the conditions for producing spore were also regulated in this study.
The results showed that the cell concentration are higher in the medium of additive metal ion. It indicates that the medium of additive metal ion can help furthermore growth of Bacillus coagulans, and the fed-batch typed fermentation can increase cell concentration(26g/L) again. Besides, adding steriled water into the bacteria liquid after fermentation(1:1) would shorten the spore producing time, better results were found on the tolerance of higher temperature, and both acid and base environment which were imitated as in gastric juice and bile environment. The spore of Bacillus coagulans can survive for 150min at 100℃ and it can also survive for 120min at pH2, the results also showed that it’s survive ratio at 90℃ was 90﹪, 80﹪at pH4, and 65.4﹪at base environment.
The result presented that the optimum temperature for producing spore was 45℃, the Bacillus coagulans could inhibit both S. aureus and E. coli, and it also could against several kinds of antibiotics.
Keyword:Lactic acid bacteria, spore-forming lactic acid bacteria, Fermentation.
目  次
摘要……………………………………………………………………………………....i
英文摘要………………………………………………………………………………...ii
誌謝……………………………………………………………………………………..iii
目次……………………………………………………………………………………..iv
表目錄…………………………………………………………………………………...v
圖目錄…………………………………………………………………………………..vi
第一章 前言…………………………………………………………………………...1
乳酸菌的簡介…………………………………………………………………….1
第二章 文獻回顧……………………………………………………………………...5
2.1 乳酸菌的定義與分類………………………………………………………..5
2.2 乳酸菌在腸道中的作用機制………………………………………………..9
2.3 乳酸菌的特殊性……………………………………………………………10
2.4 乳酸菌的應用………………………………………………………………12
2.5 孢子型乳酸菌(Bacillus coagulans)的認識……………………………..15
2.6 何謂孢子……………………………………………………………………15
2.7 孢子型乳酸菌(Bacillus coagulans)的鑑定特徵………………………..16
2.8孢子型乳酸菌在腸道的特殊性…………………………………………….16
2.9孢子型乳酸菌的特點……………………………………………………….16
2.10 孢子型乳酸菌的應用……………………………………………………..17
2.11 乳酸菌的抑菌作用………………………………………………………..23
2.12 膽鹽對乳酸菌之影響……………………………………………………..24
第三章 研究目的…………………………………………………………………….28
第四章 研究架構…………………………………………………………………….30
第五章 研究方法…………………………………………………………………….31
5.1 實驗藥品……………………………………………………………………31
5.2 培養基之配方………………………………………………………………32
5.3 實驗菌株……………………………………………………………………33
5.4 實驗儀器……………………………………………………………………33
5.4.1 發酵儀器…………………………………………………………….33
5.4.2 分析儀器…………………………………………………………….33
5.5 實驗方法與步驟…………………………………………………………...34
5.5.1 菌種的篩選……………………………………………………….34
5.5.2 Bacillus coagulans的生長影響…………………………………..34
A. 溫度對Bacillus coagulans生長之影響………………………35
B. pH值環境對Bacillus coagulans生長之影響……………….35
C. 震盪和攪拌對Bacillus coagulans生長之影響………………35
5.5.3 Bacillus coagulans 的最適培養與成分需求…………………….36
A.培養基成分對Bacillus coagulans的生長影響……………….36
B.兩蛋白腖對發酵之影響………………………………………..37
C.金屬離子對Bacillus coagulans成長分析…………………….38
5.5.4 利用Fed batch提高菌量…………………………………………39
5.5.5 Bacillus coagulanst產孢條件尋求……………………………….42
A.利用濃度改變提生產孢率…………………………………….42
B.利用改變溫度提升產孢率……………………………………..42
5.5.6 Bacillus coagulans的耐受性試驗………………………………..43
A. Bacillus coagulans的耐高溫試驗…………………………….43
B. Bacillus coagulans的耐酸性試驗…………………………….43
C. Bacillus coagulans對膽鹽的耐受性………………………….43
D.抗生素對Bacillus coagulans生長之影響…………………….44
E. Bacillus coagulans的抑菌作用……………………………….44
第六章 結果與討論………………………………………………………………….46
6.1 Bacillus coagulans菌株的初步確認………………………………….46
6.2 Bacillus coagulans的生長分析……………………………………….50
A. 溫度對Bacillus coagulans生長之影響……………………………50
B. pH值環境對Bacillus coagulans生長之影響……………………50
C. 震盪和攪拌對Bacillus coagulans生長之影響……………………53
6.3 Bacillus coagulans的最適培養與成分需求………………………….56
A. 培養基成分對Bacillus coagulans的生長分析……………………56
B. 兩蛋白腖對發酵之影響……………………………………………64
C. 金屬離子對Bacillus coagulans成長分析…………………………65
6.4 利用Fed batch提高發酵菌量…………………………………………68
6.5 Bacillus coagulans產孢條件尋求…………………………………….70
A.利用濃度改變提生產孢率………………………………………….70
B.利用改變溫度提升產孢率…………………………………………..72
6.6 Bacillus coagulans的耐受性試驗…………………………………….73
A.溫度對Bacillus coagulans之影響………………………………….73
B. Bacillus coagulans的耐酸性試驗………………………………….74
C. Bacillus coagulans對膽鹽的耐受性…………………………….…75
D. 抗生素對Bacillus coagulans生長之影響…………………………76
E. Bacillus coagulans的抑菌作用…………………………………….78
第七章 結論………………………………………………………………………….80
第八章 參考文獻…………………………………………………………………….82
第九章 附錄………………………………………………………………………….88
A 鑑定方法……………………………………………………………………..88
B 革蘭氏染色方法 …………………………………………………………….91
C 過氧化氫酵素檢測(catalase)……………………………………………..92
D 氧化酵素檢測(oxidase)……………………………………………………93
表目錄
表1.1 目前研究發表及市售產品中之菌株………………….……………………….4
表2.1 乳酸菌的型態發酵形式及好氣條件生長情形………………………………..8
表2.2 各種食品加工時利用之乳酸菌種類…………………………………………13
表2.3 目前美國食品藥物管理局認定是安全的菌種………………………………20
表2.4 在抗生素添加飼料中生菌劑 (孢子製劑)之安定性……………………..21
表2.5 目前已被用於生菌劑置備的菌種…………………………………………...22
表5.1 液態培養基的配製……………………………………………………………34
表5.2 液態培養基的配製……………………………………………………………34
表5.3 固態培養基的配製……………………………………………………………35
表6.1 孢子型乳酸菌初步鑑定結果…………………………………………………48
表6.2 乳酸菌耐膽鹽之能力…………………………………………………………75
表6.3 抗生素對乳酸菌之影響………………………………………………………77
表6.4 大腸桿菌和金黃色葡萄球菌對Bacillus coagulans之敏感度………………79
表6.5 大腸桿菌和金黃色葡萄球菌對乳酸菌之敏感度……………………………79
圖目錄
圖1.1 乳酸菌之生態 3
圖2.1 正常乳酸發酵之代謝途徑 6
圖2.2 混合乳酸發酵之代謝途徑 7
圖2.3 甘胺膽鹽之結構式,為結合型膽鹽 26
圖2.4 牛磺膽鹽之結構式,為結合型膽鹽 26
圖2.5 膽酸結構式,為去結合型膽鹽 27
圖5.1 批次培養發酵裝置圖 40
圖5.2 連續饋料培養發酵裝置圖……………………………………………………41
圖6.1 孢子型乳酸菌在顯微鏡下所觀察到的型態…………………………………47
圖6.2 孢子型乳酸菌在液態培養基下所呈現的顏色………………………………47
圖6.3 孢子型乳酸菌經革蘭氏染色之結果(革蘭氏陽性)………………………48
圖6.4 Bacillus coagulans在Biolog中之96孔洞標準反應……………………….49
圖6.5 受測Bacillus coagulans96孔洞反應與標準反應之比較鑑定結果…………49
圖6.6 各發酵溫度對乾菌重(Cell Concentration)之影響……………………….51
圖6.7 不同pH值對孢子型乳酸菌生長之影響…………………………………….52
圖6.8 振盪與未振盪對Bacillus coagulans生長之影響(搖瓶)…………………54
圖6.9 通氣與未通氣對Bacillus coagulans生長之影響(5L發酵)……………..55
圖6.10 37℃搖瓶成長曲線……………………………………………………………60
圖6.11 以Glucose、Peptone、Malt extract、Yeast extract為培養基之批式發
酵37℃生長曲線(5L發酵)………………………………………………….60
圖6.12 Glucose之添加濃度對Bacillus coagulans生長之影響(搖瓶)…………61
圖6.13 Peptone之添加濃度對Bacillus coagulans生長之影響(搖瓶)…………61
圖6.14 Yeast extract之添加濃度對Bacillus coagulans生長之影響(搖瓶)……62
圖6.15 Malt extract之添加濃度對Bacillus coagulans生長之影響(搖瓶)…….62
圖6.16 培養基組成的添加與否對Bacillus coagulans生長之影響(5L發酵)…63
圖6.17 Peptone與Tryptone的添加對Bacillus coagulans生長之影響(5L發酵)
……………………………………………………………………………….64
圖6.18 37℃生長曲線圖。培養基為Yeast extract(20g/L)、Tryptone(15g/L)
、Glucose(20g/L)、MgSO4(0.2g/L)、KH2PO4(1g/L)、MnCl2(
0.2g/L)…………………………………………………………………….66
圖6.19 加入金屬離子對Bacillus coagulans生長之影響(搖瓶)….……………66
圖6.20 不同比例金屬離子之對Bacillus coagulans生長之影響(搖瓶)……….67
圖6.21 以Glucose、Tryptone、Yeast extract、MgSO4、KH2PO4、MnCl2為培
養基之批式發酵對乾菌重(Cell Concentration)之影響(5L發酵)………67
圖6.22 同樣濃度下Batch與Fed-batch對Bacillus coagulans之影響(5L發酵)69
圖6.23 溫度對未產孢菌體之影響…………………………………………………..71
圖6.24 產孢溫度對產孢率之影響…………………………………………………..71
圖6.25 不同稀釋比例對產孢速度的影響…………………………………………..72
圖6.26 溫度對孢子型乳酸菌存活率之影響……………………….……………….73
圖6.27 pH值對孢子型乳酸菌存活率之影響………………………………………74
圖9.1 Biolog鑑定之前處理……………..…………………………………………89
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