Studies on Species Identification of Cyprinidae Fishes and Effects of Processing on Heterocyclic Amines Fomation in Braised Silver Carp with Spring Onion

• Main Authors: Chen, Chien-Hung, 陳建宏
• Other Authors: Hwang, Deng-Fwu
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/24216256912014042617

博士 === 國立臺灣海洋大學 === 食品科學系 === 102 === Food safty is an issue that becomes more important around the global. Recently, for different reasons, including religion, race, allergies, the abuse of endangered species and the heedless use of cheap or toxic fish, previously developed techniques in species identification have been improved in manufactured products. DNA-based methods offer an alternative approach to species identification by using polymerase chain reaction (PCR). In the present studies, mitochondrial DNA (mtDNA) was chosen as the target DNA based on the faster rate of evolution of mtDNA and because its high copy number (approximately 1,600-6,000 molecules per animal cell) compensates for the loss of DNA during food processing. Therefore, we established complete sequences of cytochrome b gene (cytb, 1,141 bp) and cytochrome c oxidase I gene (COI, 1,551 bp) of 10 common Cyprinidae fishes in Taiwan. We employed Cytb gene as a target of species identification, based on the degree of difference between Cytb gene sequences in each carps (11.39-21.30%) is greater than the COI gene sequence (7.35-18.38%). Then, a rapid PCR-RFLP method was used to amplify a 426 bp fragment of cytb using primer set Lnew1/Hnew1, and the fragment was digested with NlaIII, BstXI and EcoRV, then checked by agarose gel electrophoresis (AE). The results show that using this PCR-RFLP technique for the species identification of the 10 Cyprinidae was successful. The method was further applied to investigate 16 commercial samples of processed Cyprinidae products from traditional markets. The results indicate that 9 of the samples were Carassius auratus auratus, Cyprinus carpio carpio, or Ctenopharyngodon idella. The other 6 samples did not belong to the Cyprinidae family according to PCR-RFLP. These samples were further analyzed with the forensically informative nucleotide sequencing (FINS) and BLAST methods. The results show that these 6 processed products were Oreochromis spp., a monophyletic fish group. The last commercial product could not be amplified to obtain a 426 bp fragment, so its species remains unknown. In order to avoid the situation of above, we desinged new primer sets CypbL / CypbH (331 bp) to amplified shorter fragment, and the PCR product was subsequently co-digested with endonuclease (NlaIII and MseI). The PCR-RFLP profiles of the 10 Cyprinidae fish species determined using capillary electrophoresis (CE) were able to successfully differentiate the different fish species. This method was further applied to detect the species used in 24 commercial Cyprinidae-related products. The results identified 4 samples as Cyprinus carpio carpio, 8 samples as Carassius auratus auratus, and 12 samples as Ctenopharyngodon idella. This study shows that performing PCR-RFLP in a CE system is useful for identifying the species of fish from which the processed products originated, and this method can be a potential analytical tool for large and routine food authentication in the future. The braised silver carp with spring onion is a delicious and traditional dish of the Chinese new year’s eve. The dish was fried at high temperature and marinated for a long time, which are may produce a lot of hazardous chemicals. Everyone knows heterocyclic amine compounds (HCAs) are an important class of mutagens and carcinogens, which generated through the Maillard reaction or protein pyrolysis in protein-riched food, such as fish, meat and egg. In order to know HCA levels of this dish, high performance liquid chromatography (HPLC) with photodiode array detector (HPLC-PDAD) was employed to detect the 15 HCAs. We collected the 6 commercial products of braised silver carp with spring onion, and divided into the outer meat, inner meat, ovary and marinade. The results indicated that the average of HCAs in commercial product was 2,960.02 ng/g, and the range was from 1,269.57 ~ 6,879.48 ng/g. Among them, IQ type HCAs (IQ, IQx, MeIQ, MeIQx, PhIP, 4,8-DiMeIQx and 7,8-DiMeIQx) content was less than the non-IQ type HCAs (Harman, Norharman, Glu-P-1, Glu-P-2, AC, MeAC, Trp-P-1 and Trp-P-2). On the other hand, outer meat showed the HCAs level higher than inner meat. The ovary contained higher HCAs content when it was dissected from the fish. We further studied the changes of HCAs content during processes. The fresh silver carp contained a small amount of HCAs (124.34 ng/g). After frying at 140oC for 15 min, the content of HCAs increased to733.36 ng/g that was 5.90 folds to fresh fish. After marinating at 100oC for 4 hr, the amounts of HCAs significantly increased to 4,609.84 ng/g that was 37.07 folds of that of fresh fish and 6.29 folds to that of frying product. We concluded that heating at low temperature for a long time leads to generate greater HCAs level than heating at high temperature for short time. Different types and concentrations of antioxidants in the marinade (0.02% vitamin C, 0.02% BHT, 0.20% vitamin C and 0.20% BHT) was added to know the effects of the inhibition of generation of HCAs. The results showed that the total content of HCAs increased 13.60% when treated with 0.02% vitamin C in the marinade. However, treatment with 0.20% vitamin C or BHT in marinadewas able to inhibit HCAs generation, resulting the HCAs contents to 72.13% and 78.44%, respectively of the control. But addition of 0.02% BHT in marinade showed no significant effect of inhibition. Based on food safety concerns and the inhibitory effect, we suggested that 0.20% vitamin C is the best choice of antioxidants for reducing formation of HCAs in braised silver carp with spring onion.