博士 / 國立海洋大學 / 食品科學系 / 87 / ABSTRACT
In the recent decade, food poisoning incidents due to marine toxins such as tetrodotoxin (TTX) and paralytic shellfish poison (PSP) were sporadically occurred in Taiwan. The causative fish and shellfish of these food poisoning incidents were mainly puffer fish, followed by purple clam, gastropod Nassariidae and goby fish. In order to have a clearly understanding of the toxicity, toxin and toxin source of those toxic fish and shellfish, the toxicological studies of cultured puffer Takifugu rubripes, goby fish and starfish Astropecten scoparius, the possible prey of toxic gastropods, have been undertaken.
To monitor toxicity of cultured puffer, T. rubripes specimens were monthly collected from two aquacultural farms in Ilan County and one farm in Taipei County from November 1996 to October 1997. These specimens were dissected into various parts, and their toxicity was determined by the standard tetrodotoxin bioassay. All specimens collected from Ilan County were non-toxic (<10 MU/g), while the specimens collected from Taipei County during the period from January to March 1997 showed certain toxicity in the ovary and liver. The toxicity was as follows: 105 ± 9 (mean ± S.E.) MU/g ovary and 13 ± 3 MU/g liver in January, 88 ± 12 MU/g ovary in February, and 253 ± 55 MU/g ovary and 17 ± 6 MU/g liver in March. The highest toxicity in ovary and liver was 624 MU/g and 119 MU/g, respectively. Further, among ten specimens of the flatworm Stylochus orientalis collected from the Taipei''s farm in February 1997, four worms showed a low toxicity (1-3 MU/specimen). Flatworms contained TTX-related substances as analyzed by HPLC and GC-MS. In the same period of time, the microflora of the digestive gland in the puffer, aquacultural water and sediment were examined. The results showed that the predominant genus was Vibrio and comprised 37-65% and 40-65% of the microflora incubated aerobically and facultative anaerobically, respectively. Furthermore, HPLC and GC-MS analyses demonstrated that some of the bacteria isolated, such as Aeromonas sp., Pseudomonas vesicularis, V. alginolyticus, V. parahaemolyticus and Vibrio sp. produced TTX and/or its related substances.
On the other hand, two paralytic food poisoning incidents due to ingestion of two kinds of fishes included goby Yongeichthys nebulosus and Sillago japonica occurred in Kaohsiung Prefecture in February 1997 and Kaohsiung City in March 1998. After TTX bioassay, the causative fish was identified as Y. nebulosus. The toxicity of retained goby specimens ranged from 2,860-7,650 and 276-950 mouse units (MU), respectively. The toxins were partially purified from the methanolic extracts of toxic fishes by ultrafiltration and Bio-Gel P-2 column chromatography. Cellulose acetate membrane electrophoresis and high performance liquid chromatography analyses demonstrated that the compositions of toxins were tetrodotoxin and anhydrotetrodotoxin. These results indicated that goby fishes inhabited in the estuaries and the downstream portions of rivers and the coast area in Taiwan might contain toxins and cause food poisoning. At the same time, the intensive investigation of toxic goby was also performed from August 1996 to July 1998. More than 300 specimens of twelve species of goby were collected from fifteen locations and examined for toxicity. The specimens of three goby Y. nebulosus, Prachaeturichtys palynema and Radigobius caninus collected from Pingtung, Penghu, Kaohsiung, Miaoli, and Hsinchu were found to contain toxins. The most toxic goby was Y. nebulosus, and its frequency of being found toxic was 93%. Specimens collected from Pingtung Prefecture were most toxic, the toxicity was over 1,000 MU/specimen. The seasonal variation of toxicity in Y. nebulosus was found that the highest toxicity was in winter with the value of 4,998 MU/specimen. The toxin was partially purified from methanolic extract of each toxic goby species by Diaflo YM-1 membrane ultrafiltration and Bio-Gel P-2 column chromatography. HPLC and electrophoresis analyses indicated that each toxin was composed of tetrodotoxin and anhydrotetrodotoxin. In order to elucidate relationship between the toxicity and microflora of the intestine in the goby and environmental materials, the goby Y. nebulosus specimens, seawater and sediment were collected from Penghu Prefecture in September 1998 to assay the aerobic microflora in intestines and environmental materials. The results showed that the predominant genus of goby intestines was Vibrio comprising 46% of the microflora, with V. alginolyticus as the major isolate. The predominant genus in seawater was Pasteurella that comprised 47% of the microflora. Moreover, the predominant genera of sediment were Flavobacterium and other unidentified bacteria that comprised 51% of the microflora. Furthermore, HPLC, UV and GC-MS analyses demonstrated that some isolated bacteria, such as Aeromonas sp., Pseudomonas sp. and V. alginolyticus, could produce TTX and/or its related substances.
As known, same gastropods inhabiting in the coastal waters in Pingtung Prefecture were found to contain TTX and paralytic shellfish poison (PSP). To elucidate the food chain of TTX and PSP in these toxic gastropods, the toxicity of marine animals inhabiting in the coastal waters of southern Taiwan was examined. It was first found that a starfish Astropecten scoparius showed lethal potency. Hence, paralytic toxicity was seasonally detected in every 10 specimens of the starfish A. scoparius from Pingtung in Taiwan from January 1996 to December 1997. The highest toxicity of specimen collected in 1996, expressed as TTX, was 5,938 MU. The average toxicity of these specimens was 1,679 MU. But, the pattern of starfish toxicity in 1997 was somewhat different from that collected in 1996. The average highest specimen toxicity was 16,821MU found in November 1997. The toxin obtained from specimens collected in February, April, July and November 1997 was partially purified by YM-2 membrane ultrafiltration and Bio-Gel P-2 column chromatography. Electrophoresis, HPLC and GC-MS analyses showed that the toxins of starfish obtained from April 1997 were composed of 88% TTX and 12% PSP. The toxin of starfish collected in the other three months was found to be TTX only.
Furthermore, in order to elucidate the intoxification mechanism of TTX in starfish A. scoparius, the compositions in the digestive gland of starfish were examined. It was found that the compositions in the digestive gland of less toxic starfish were mainly bivalves with Veremolpa scabra as the major species. As to the compositions in the digestive gland of more toxic specimens, collected in October and November 1997, were mainly species of gastropods, Umborium suturale and Natica pseustes. The toxicities of U. suturale, V. scabra and N. pseustes found in digestive gland of starfish were 65 MU/g, 33 MU/g and 28 MU/g, respectively. The toxins in these three mollusks were TTX and anh-TTX. The other specimens of U. suturale and N. pseustes, re-collected from the coastal waters of Pingtung Prefecture, were also found to be toxic with average toxicity value of 20±3 MU/g and 77±7 MU/g, respectively. However, V. scabra specimens were found to be nontoxic. It indicated that the starfish A. scoparius might accumulate the toxin from V. scabra and N. pseustes. Furthermore, to elucidate relationship between the toxicity and microflora in starfish viscera, the starfish A scoparius inhabiting in the coastal waters of Pingtung Prefecture was collected in November 1997 for determination of aerobic and facultative anaerobic microflora in the viscera. The results showed that the predominant bacterial genus in the viscera and other parts was Vibrio that comprised 38% and 47% of the aerobic microflora, respectively. The major isolates were V. alginolyticus and V. parahaemolyticus. On the other hand, the predominant genus of viscera was also Vibrio that comprised 51% of facultative anaerobic microflora. Moreover, HPLC, UV and GC-MS analyses demonstrated that bacteria isolated from aerobic microflora, such as Aeromonas sp., V. alginolyticus A011, A018 and A035, and V. parahaemolyticus A026 and A047 could produce TTX and/ or its related substances. TTX-producing bacteria were also found in the facultative anaerobic microflora, such as Aeromonas sp., Alcaligenes sp., Pasteurella multocida, V. alginolyticus and V. parahaemolyticus. In order to compare the TTX-producing ability of bacteria incubated aerobically and facultative anaerobically, two strains of TTX-producing bacteria V. alginolyticus AF089 and V. parahaemolyticus AF067 were enriched in BHI broth aerobically and facultative anaerobically, and then the amount of toxin was analyzed. The results showed that both strains grew better and produced more toxin in facultative anaerobic incubation than aerobic condition. Moreover, the TTX-producing ability of V. alginolyticus AF089 was much better than that of V. parahaemolyticus AF067 incubated either in aerobic or in facultative anaerobic conditions.
In conclusion, Taiwanese cultured puffer T. rubripes, goby Y. nebulosus, and starfish A. scoparius all showed an ability to accumulate high toxicity of TTX or PSP. Moreover, the toxin sources of these three aquatic animals might be closely related to food chain and the intrinsic TTX-producing bacteria.
| Identifer | oai:union.ndltd.org:TW/087NTOU0253045 |
| Date | January 1999 |
| Creators | Shin-Jung Lin, 林欣榮 |
| Contributors | Deng-Fwu Hwang, 黃登福 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 259 |
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