The Use of Protein Electrophoretic Techniques in Identifying Raw and Processed Puffer Fish Species

• Main Authors: Tai-Yuan Chen, 陳泰源
• Other Authors: Deng-Fwu Hwang
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/75813992344661973577

博士 === 國立臺灣海洋大學 === 食品科學系 === 92 === 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.