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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/14573

Title: 應用蛋白質電泳技術鑑定生鮮河魨和河魨香魚片之魚種
The Use of Protein Electrophoretic Techniques in Identifying Raw and Processed Puffer Fish Species
Authors: Tai-Yuan Chen
陳泰源
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 蛋白質;電泳;魚種鑑定;河魨;香魚片
Protein;SDS-PAGE;IEF;2DE;Puffer fish;Species identification
Date: 2003
Issue Date: 2011-06-30T07:39:58Z
Abstract: ABSTRACT (in Chinese) 台灣河魨產量最多之種類為無毒種克氏兔頭魨 (Lagocephalus gloveri),又稱為黑鯖河魨,主要做為香魚片之原料。但有些有毒河魨種如外型酷似黑鯖河魨的月尾兔頭魨 (L. lunaris) 和橫紋多紀魨 (Takifugu oblongus),亦常為加工業者誤用做為香魚片之原料,因而引發數起誤食有毒河魨或有毒香魚片之案例,有必要採用有效鑑定方法來區分有毒與無毒之河魨。本實驗採取三種蛋白質電泳技術 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE; native-isoelectric focusing, N-IEF;和immobilized pH gradients-two dimensional electrophoresis, IPG-2DE) 分析各種河魨肌肉蛋白質組成,以探討這些技術應用於生鮮河魨鑑定上之可行性。本實驗亦探討比較不同染色法 (Coomassie blue/silver 複染色,銀染色或Coomassie blue染色) 對SDS-PAGE和N-IEF蛋白質電泳帶解析度之影響。另嘗試以此三種蛋白質電泳技術鑑定香魚片之來源魚種。從SDS-PAGE蛋白質圖譜中發現水溶性、鹽溶性、SDS可溶性和尿素可溶性蛋白質皆有種別特異性蛋白質存在,且都是小分子量蛋白質(< 30 kD)。Coomassie blue/silver複染色可提高SDS-PAGE蛋白質電泳帶解析度,尤其是小分子量蛋白質,故可提高對河魨之辨種能力。當種別特異性蛋白質不明顯存在時,蛋白質密度組成有利於精確的魚種鑑定。從N-IEF蛋白質圖譜中可發現河魨肌肉水溶性蛋白質主要為中酸性蛋白質,介於等電點isoelectric point (pI) 範圍5.85-8.65。種別特異性蛋白質廣泛存在N-IEF蛋白質圖譜中,並可區分成三個區間,分別為pI 3.50-5.20、pI 5.85-6.55 和 pI 7.35-8.15。因為酸性蛋白質區間之河魨種別特異性蛋白質大部分皆為單一電泳帶,所以最適合應用於魚種鑑定。IPG-2DE蛋白質圖譜搭配Coomassie blue染色可偵測到171-260個蛋白質。在等電點3.5-7.0 和分子量7.4-45.0 kD這個區域可有效地區分所有河魨魚種,這主要是因為小分子量酸性蛋白質具有種別特異性。在香魚片魚種鑑定方面,毒成分經HPLC和GC-MS鑑定為河魨毒和脫水河魨毒,毒量為9-18 MU/g (mouse units/g);以SDS-PAGE分析香魚片之尿素可溶性蛋白質,發現其種別特異性蛋白質與毒鯖河魨L. lunaris相符合,來源魚種鑑定為L. lunaris。因此,這三種蛋白質電泳技術 (SDS-PAGE, N-IEF和IPG-2DE) 皆能有效且正確地應用於區分生鮮河魨魚種。而且以SDS-PAGE分析尿素可溶性蛋白質可用以鑑定河魨香魚片之來源魚種。
ABSTRACT Due to morphological similarities, the manufacturers and consumers may have difficulty in distinguishing nontoxic Lagocephalus gloveri from L. lunaris, a species which accumulates lethal level of TTX in its muscle. Therefore, serious food poisoning incidents due to ingestion of toxic puffer fish or the toxic dried dressed fish fillets have occasionally occurred in Taiwan. From the viewpoint of food protection and public safety, it is critical to accurately identify the non-toxic puffer fish from the toxic species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), native-isoelectric focusing (N-IEF), and immobilized pH gradients-two dimensional electrophoresis (IPG-2DE) were employed in this study to validate the feasibility of using these techniques to develop species-specific muscle protein profiles for raw puffer fish species identification. On the other hand, species identification of dried dressed fish fillet was studied using these three protein electroprotic techniques. Species-specific bands of sarcoplasmic, myofibrillar, SDS-soluble, and urea-soluble proteins were found in the molecular weight region below 30 kD of the SDS-PAGE patterns. Coomassie blue/silver double staining provided better protein banding patterns for discrimination of different puffer fish species than Coomassie blue staining alone. In some cases, especially for Coomassie blue stained urea-soluble proteins, the density of the protein profiles facilitate precise identification. Evaluation of the N-IEF patterns showed that the majority of water-soluble puffer fish muscle proteins fell in the region with isoelectric point (pI) values of 5.85-8.65. The characteristic species-specific protein bands were found in all the three regions of pI 3.50-5.20, pI 5.85-6.55, and pI 7.35-8.15. Among them, the pI 3.50-5.20 was the most suitable region for identifying species-specific proteins. Coomassie blue staining was shown to be adequate for revealing the protein profiles for the identification of puffer fish species. In viewing the IPG-2DE patterns, it was noted that puffer fish muscle proteins that fell in the region with pI values of 3.5-7.0 and molecular weights of 7.4-45.0 kD were good for species comparison. The more acidic proteins of lower molecular weights showed species-specific characteristics. From toxin examination on dried dressed fish fillets, three fish fillets were found to be toxic and the toxicity ranged from 9 to 18 mouse units per g. The toxin preparation was further purified and identified as tetrodotoxin and anhydrotetrodotoxin by HPLC and GC-MS. This study also shows that SDS-PAGE analysis on the 8 M urea extracts of muscle protein is suitable for identifying species of processed puffer fish and could be used to detect adulteration or substitution. Therefore, species identification of raw puffer fish can be achieved by comparison of their characteristic SDS-PAGE, N-IEF, and IPG-2DE protein pattern profiles. Moreover, SDS-PAGE analysis on the urea-soluble proteins could be used to identify the origin puffer fish species of the dried dressed fish fillets.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0D88320005
http://ntour.ntou.edu.tw/ir/handle/987654321/14573
Appears in Collections:[食品科學系] 博碩士論文

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