河魨毒生產菌產毒能力與基因圖譜資料之探討

Studies on Toxin-Producing Ability and Gene Map of Tetrodotoxin- Producing Bacteria

林春蘭(Chun lan, Lin)

指導教授 ： 謝承紘 ； 謝宥諒

亞洲大學/健康學院/保健營養生技學系碩士班/碩士(2010年)

摘要

河魨毒 (tetrodotoxin, TTX) 為海洋生物毒之ㄧ，係一種低分子量 (320 Da) 的非蛋白質之神經性劇毒，具有10餘種衍生物。目前關於含有河魨毒的生物體中河魨毒的來源，大致上有兩種說法，一種是由生物本身體內生合成的內因說，另一種是經由攝食寄生或共生細菌而蓄積在體內的外因說。自1986年由學者Noguchi等人從Xanthid crab的腸道分離出具有河魨毒生產菌後，陸續有多位學者由當地河魨腸道中分離出可生產河魨毒之弧菌Vibrio alginolyticus；由數種海洋標準菌株中，亦檢測出多種微生物V. parahaemolyticus, V. anguillarum, V. cholerae及V. costicols均具有河魨毒生產能力。綜合上述結果，河魨TTX及其衍生物之來源，推論係生息在其腸內之微生物生產所致。
為了解在台灣海域之河魨所含有細菌種類，分別於宜蘭大溪漁港和台中港採集黑鯖、滑背、毒鯖河魨之腸壁，使用ORI培養基檢測好氣性生菌數，在ORI培養基的生菌數分別為1.07 × 105 CFU/g、8.01 × 104 CFU/g、9.54 × 107 CFU/g。總共分別分離出286、316、202株，初步依菌落外觀型態分類，使用ORI broth大量培養，經震盪培養3天後，以離心收集菌體。將菌體以超音波破碎機破碎經純化後，以HPLC鑑定河魨毒，初步篩得6株TTX生產菌，進而探討培養條件對河魨毒生產菌產產毒能力之影響，以ORI broth為基礎培養基，分別改變pH值、NaCl濃度和溫度，以試驗菌株在3項條件下之河魨毒生產能力，結果顯示，菌株LiWC263、LLWF001、LLWF013、LLWF049、LLYF008之河魨毒生產性分別在pH 8, 3% NaCl, 25℃有較佳之河魨毒生產量。菌株LiWF271則是在pH7, 3% NaCl, 25℃有較佳之河魨毒生產量。
此外，利用16S rDNA基因定序法，進行河魨毒生產菌基因探討與分析，鑑別不同菌株在系統分類所在的位置，分析菌株與菌株之間基因的差異性。結果顯示分離出6株河魨毒生產菌經16S rDNA定序，利用GCG資料庫系統分析共找到相似性之微生物2屬6株 (Shewanell chilikensis, Shewanella waksmanii, Vibrio ichthyoenteri, Vibrio alginolyticus, Vibrio crassostreae, Vibrio splendidus, Vibrio gigantis和Vibrio chagasii.) 。以UPGMA所建構的遺傳相關樹狀圖，顯示菌株與菌株間的差異很明顯，有效的區分菌種中菌株之差異性。

關鍵字

河魨毒 ； 河魨毒生產菌 ； 16S rDNA

並列摘要

Tetrodotoxin (TTX) is one of the best known marine toxins, because of it’s an extremely potent, low-molecular weight, and non-protein neurotoxin. TTX exists with its several analogs. It was controversial whether TTX in the TTX-bearing organisms was exogenous or endogenous origin. Endogenous is produced by the TTX-bearing organisms itself or exogenous is accumulate TTX via the food chain and could produced by some bacteria or other microorganisms. Noguchi et al. succeeded in isolating the TTX-producing bacteria from the intestines of a Xanthid crab since its first discovery published in 1986. Vibrio alginolyticus was isolated as the TTX-producing bacteria from the intestines of several puffer fish species. It was demonstrated that many species of marine bacteria can produce the TTX including the V. parahaemolyticus, V. anguillarum, V. cholera and V. costicols. These findings indicated that animals containing TTX may have absorbed and accumulated TTX and/or related substances produced by those bacteria.
In order to understand the TTX-producing bacteria species, we test the bacteria isolated from the intenstinal wall of L. lunaris, L. inermis and L. glovei which collected from Ilan and Taichung seacoast. Then using the ORI (Ocean Research Institute) agar broth for the aerobic plate count. Aerobic counts were 1.07×105 CFU/g, 8.01×104 CFU/g and 9.54×105 CFU/g. there are 286, 316 and 202 strains of each species isolated and characterized for classificating their species. After the ORI broth culture for 3 days, these bacteria were then to freeze-dried, TTX extraction and HPLC analysis. To elucidate the optimum conditions of bacteria for producing the TTX, the effect of various culture conditions on TTX formation was studied by using six strains isolated from the puffer fish species. Optimum pH value and NaCl concentration for toxin formation of the strain LiWC63, LLWF001, LLWF013, LLWF049 and LLYF008 were pH 8 and 3% NaCl which were incultured at 25℃. But the optimun pH value and NaCl concentration for toxin formation of the LiWF271 were pH 7 and 3% NaCl which were incultured at 25℃.
The six TTX-producing bacteria were isolated and identified by sequencing their partial 16S rDNA sequence. Based on the partial 16S rDNA alignment, the 6 strain were homogeneous as Shewanell chilikensis, S. waksmanii, V. ichthyoenteri, V. alginolyticus, V. crassostreae, V. splendidus, V. gigantis and V. chagasii. Phylogeny tree based on the 16S rDNA of the six strains was constructed by UPGMA, and were clearly differentiated on the basis of their 16S rDNA sequence.

並列關鍵字

Tetrodotoxin ； Tetrodotoxin-producing bacteria ； 16S rDNA

參考文獻

黃登福。1999a。海洋生物毒和生理活性物質。Journal of Chinese Medicine. 10: 23-50。

黃登福。1994。台灣魚貝類食品中之海洋生物毒。營養學會誌，19:85-99。

Chen, H. C., Chen, S. S., Wu, C.Y., Chai, T. 1981. Bacterial flora of fish holds of home water and distans-water fishing vessels. In recent advances in food science and technology. Hua Shiang Yuan Publ. Co. Taipei, R.O.C. P245-261.

Hwang, D. F., Kao, C. Y., Yang, H. C., Jeng, S. S., Noguchi, T., Hashimoto, K. 1992. Toxicity of puffer in Taiwan. Bulletin of the Japanese Society of Scientific Fisheries, 58: 1541-1547.

Austin, B., Calomiris, J. J., Walker, J. D., Colwell, R. R. 1977. Numerical taxonomy and ecology of petroleum-degrading bacteria. Applied and Environmental Microbiology, 34:60-68.