臺灣博碩士論文加值系統

本研究的主要目的為分類與鑑定荔枝分離乳酸菌的菌種，同時研究分離菌株Weissella confusa E 所生產細菌素的特性。
研究初期主要依據16S rDNA基因，透過RFLP (restriction fragment length polymorphism)及定序來進行乳酸菌的初步分類與鑑定，其次嘗試利用rpoA RFLP來鑑定Leu. mesenteroides與Leu. pseudomesenteroides二個親緣性較高的乳酸菌菌種。此外，利用包括Fast protein liquid chromatography (FPLC)，sep-pak C18 cartridges, RP-HPLC (Reversed-phase High performance liquid chromatographic)等步驟來純化Weissella confusa E 所生產的細菌素後，分析該細菌素的相關特性。
結果顯示荔枝分離到的104株乳酸菌共可分為6個群組，其中又以Leuconostoc的數目最多。結果顯示利用內切酶AccII或AluI裁切rpoA基因時可以有效區分Leu. mesenteroides與Leu. Pseudomesenteroides二菌種。在細菌素分析方面，結果顯示W. confusa E 所生產的細菌素大小約為3426.7 Da。結果亦顯示此細菌素對Listeria monocytogenes有抑菌效果。因分子大小相異於已知的W. confusa細菌素，因此推測有可能為新型的細菌素。

In this study, lactic acid bacteria (LAB) isolates were characterized and identified from litchi fruit. Strain Weissella confusa E isolated from litchi fruit was found to have bacteriocin-producing ability. The examination of characteristic of bacteriocin produced by W. confusa E was performed.
During the initial phase of research, based upon 16S rDNA gene sequence, classification and identification of LAB was done through 16S rDNA RFLP (restriction fragment length polymorphism) and 16S rDNA sequencing. On the other hand, rpoA RFLP was used in order to differentiate the two high-affinity LAB: Leuconostoc mesenteroides and Leu. Pseudomesenteroides. Furthermore, purification of the bacteriocin that produced by W. confusa E was performed with the use of FPLC (Fast protein liquid chromatography), sep-pak C18 cartridges, and RP-HPLC (Reversed-phase High performance liquid chromatographic). Characteristic of the bacteriocin was further examined
The 104 LAB isolated from litchi fruit were initially divided into five groups and Leuconostoc sp. was the most common species found. The results revealed that the restriction endonuclease AccII or AluI could cleave rpoA gene fragment, which could distinguish Leu. Mesenteroides and Leu. Pseudomesenteroide efficiently. Based on the bacteriologic test analysis, the molecular size of the bacteriocin, as determined by MALDI-TOF MS, revealed a distinctive peak corresponding to a molecular mass of 3426.7 Da. The data indicated the inhibitory activities of bacteriocin against Listeria monocytogenes. Because of the lack of identical results were found in the previous studies and its different molecular size compared to known bacteriocins from Weissella confusa, a novel bacteriocins were identified in this study.

中文摘要 1
Abstract 2
第一章 序論 3
第二章 實驗材料與方法 6
2-1荔枝乳酸菌之分類與鑑定 6
2-2菌株來源，保存與培養 7
2-3 指示菌培養 7
2-4細菌素生長之最佳溫度 8
2-5 細菌素的生長曲線 8
2-6細菌素的純化 9
2-7抗菌性圖譜 12
2-8 熱穩定性以及酵素分解實驗 12
2-9 Mass Spectrometry 13
第三章 結果 13
第四章 討論 17
參考文獻 20

 
圖目錄
Figure 1.16S rDNA RFLP patterns of HaeIII, AluI, and Mspl digests from Groups A-F. Lane M, size marker; H, HeaIII digested pattern; A, AluI digested pattern; M, Mspl digested pattern. 25
Figure 2. rpoA RFLP patterns of AccII and AluI digests from Group B1 and Group B2. Lane M, size marker; AccII, AccII digested pattern; AluI, AluI digested pattern. 26
Figure 3. Production of bacteriocin during the growth of Weissella confusa E. 27
Figure 4. Bacteriocin purification flow chart. 28
Figure 5. Elution pattern of the sample after two steps of purification, including FPLC (Strong anion-exchange chromatography) and sep-pak C18 cartridges by reversed-phase high-performance liquid chromatography on a C18 reverse-phase column. 29
Figure 6. Elution pattern of the sample after three steps of purification, including FPLC (Strong anion-exchange chromatography), sep-pak C18 cartridges, and first RP-HPLC by reversed-phase high-performance liquid chromatography on a C18 reverse-phase column. 30
Figure 7. MALDI mass spectrum of sample after four steps of purification, including FPLC (Strong anion-exchange chromatography), sep-pak C18 cartridges, first RP-HPLC, and second RP-HPLC. 31
Figure 8. Elution pattern of the sample after two steps of purification, including FPLC (Hydrophobic interaction chromatography) and sep-pak C18 cartridges by reversed-phase high-performance liquid chromatography on a C18 reverse-phase column. 32
Figure 9. Elution pattern of the sample after three steps of purification, including FPLC (Hydrophobic interaction chromatography), sep-pak C18 cartridges, and first RP-HPLC by reversed-phase high-performance liquid chromatography on a C18 reverse-phase column. 33
Figure 10. MALDI mass spectrum of sample after four steps of purification, including FPLC (Hydrophobic interaction chromatography), sep-pak C18 cartridges, first RP-HPLC, and second RP-HPLC. 34
Figure 11. Elution pattern of the sample after two steps of purification, including FPLC (Strong cation-exchange chromatography) and sep-pak C18 cartridges by reversed-phase high-performance liquid chromatography on a C18 reverse-phase column. 35

Figure 12. Elution pattern of the sample after three steps of purification, including FPLC (Strong cation-exchange chromatography), sep-pak C18 cartridges, and first RP-HPLC by reversed-phase high-performance liquid chromatography on a C18 reverse-phase column. 36
Figure 13. MALDI mass spectrum of sample after four steps of purification, including FPLC (Strong cation-exchange chromatography), sep-pak C18 cartridges, first RP-HPLC, and second RP-HPLC. 37
Figure 14. Elution pattern of the sample after three steps of purification, including FPLC (Strong cation-exchange chromatography), FPLC (Hydrophobic interaction chromatography), and sep-pak C18 cartridges by reversed-phase high-performance liquid chromatography on a C18 reverse-phase column. 38
Figure 15. Elution pattern of the sample after four steps of purification, including FPLC (Strong cation-exchange chromatography), FPLC (Hydrophobic interaction chromatography), sep-pak C18 cartridges, and first RP-HPLC by reversed-phase high-performance liquid chromatography on a C18 reverse-phase column. 39
Figure 16. MALDI mass spectrum of sample after five steps of purification, including FPLC (Strong cation-exchange chromatography), FPLC (Hydrophobic interaction chromatography), sep-pak C18 cartridges, first RP-HPLC, and second RP-HPLC. 40
表目錄
Table 1. Analysis results and characteristics of isolates. 41
Table 2. Analysis results and characteristics of isolates. 42
Table 3. Inhibition spectra of the bacteriocin produced by W. confusa E. 43
Table 4. Inhibitory spectra of bacteriocin from W. confusa E. 44
Table 5. Heat and Enzyme stability test of the bacteriocin 45

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