Effect of pH Adjustment on the Color of Tilapia Dark Muscle during Storage and Investigation of Myoglobin Gene in Different Tilapia Species

• Main Authors: Pei-Fen Li, 李佩芬
• Other Authors: Chan-Jen Chow
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/91369814490485339308

碩士 === 國立高雄海洋科技大學 === 水產食品科學研究所 === 95 === Part 1: This study was aimed to maintain the nature color of tilapia dark muscle during storage. Tilapia dark muscle and its myoglobin extract were subjected to estimate color changes (in a model test) during iced and frozen storage after adjusting pH to various values. In addition, alive tilapia sacrificed with or without struggle was filleted and frozen as the ordinary procedure in a processing factory to investigate the effect of pH on color of the tilapia fillets. The myoglobin extracts were prepared with phosphate buffer at pH 5.0-8.0 and individually stored at 0°C or −20°C. The results showed that metMb formation ratio (MetMb%) of all samples increased gradually during iced or frozen storage. We found that formation of metMb in the samples with pH lower than 6.3 was accelerated and the MetMb% of the samples stored at 0°C and −20°C exceeded 50% within 2 days and 14 days, respectively. Moreover, their color CIE a* values decreased gradually during both iced and frozen storage and they changed faster as pH was lower than 6.3. Also significant browning in myoglobin solutions were observed when their pH values were lower than 6.3. However, there was no significant browning occurred when solution pH was higher than 6.5. It indicates that the critical pH for autoxidation of tilapia myoglobin (in solution) is probably at the range of 6.3-6.5. Subsequently, the effect of acids on metMb formation in tilapia dark muscle was investigated. Tilapia dark muscle samples soaked in 10, 5% lactic acid, or 10% sodium carbonate were vacuum packaged and separately stored at 0°C or −20°C. MetMb% of all samples increased gradually during iced or frozen storage. It indicated that the autoxidation rate of the groups treated with 10% lactic acid (pH 4.98) and treated with 5% lactic acid (pH 5.72) were the fastest and second, respectively. Besides, that of control (pH 6.36) and 10% sodium carbonate group (pH 6.72) are the third and slowest, respectively. Meanwhile, the group treated with lactic acids showed the most significant in the decrease of CIE a* value. A significant browning was observed on the appearance of the group treated with 10% lactic acid after 12-h iced storage and the group treated with 5% lactic acid displayed a slightly browned color after 1-d iced storage. Browning on the appearance of the control was not observed until the 9th day. However, no noticeable browning appearance on the group treated with 10% sodium carbonate was seen on the 11th day. During froze storage, the group treated with lactic acid was very subjected to browning while the group treated with sodium carbonate enabled to retard the browning well. Moreover, the fillets treated with 10% sodium carbonate were carried out vacuum packing after freezing, frozen and then packed procedure, and show a better color display than those made by the traditional “packed and then frozen procedure”. In addition to effect of acid solutions, the effects of the slaughter methods and CO treatment on metMb formation in tilapia dark muscle were also inspected. Tilapia fillets were obtained from various treatments. They were (1) struggle group (alive tilapia struggling till death), (2) instantly killed group (iced alive tilapia and sacrificed immediately), and (3) CO treated group (fillet prepared in a factory with CO gas treatment) and the pH of three groups was 6.5, 7.0 and 6.6, respectively. MetMb% in fillets of the struggle group increased faster, 41.3% on the 45th day, while that of the instantly killed group was 22.1% on the 60th day during frozen storage. Comparing to other groups, the dark muscle of fillets from the struggle group exhibited a faster decrease in both CIE a* value and 630 nm/580 nm ratio. The group treated with CO gas showed no significant change in MetMb% during storage at −20°C but it had higher CIE a* value than other two groups（p＜0.05）. However, the CO gas treatment for fish fillets is prohibited by Taiwanese law. To maintain nature color of dark muscle during frozen storage, the decline in pH of fish during post-harvest should be minimized. Moreover the “frozen and then packed procedure” would be beneficial to color preservation of tilapia fillets. Part 2: In order to compare the amino acid sequence of Mb between different tilapia species, total RNA were extracted from dark muscles of mossambicus tilapia (Oreochromis mossambicus), aureus tilapia (O. aureus), niloticus tilapia (O. niloticus) and tilapia hybrid (O. niloticus (♀) × O. aureus (♂)). Reversely transcribed to cDNA, and then gene sequence of myoglobin coding region segments were cloned. Moreover, total length of Mb gene sequence of mossambicus tilapia were determined from total RNA by RLM-RACE (RNA Ligase Mediated Rapid Amplification of cDNA Ends). Deduced amino acid sequences of Mb were completely conserved in different tilapia species, and the identities was 100%. Comparing the alignment of the deduced amino acid sequences of myoglobin in the different tilapia with bigeye tuna, bluefin tuna, chub mackerel, and Atlantic blue marlin, we found that their identities were 78, 78, 75 and 77%, respectively. Furthermore, assessment of the amino acid sequence of myoglobin in the α-helical segments (A, B, C, E, F, G, H) and intersegmental regions between tilapia and bigeye tuna, it indicated that the similarities among α-helical segment C and segment F, as well as intersegment region F-G and region G-H were 100%. Moreover, the sequence identities of α-helical segments B and segment H and intersegment region F-G were quite low only 73.3, 65.0 and 50.0, respectively. Identities of the others segment and intersegment were 82-93%. On the other hand, as nucleotide sequence of mossambicus tilapia myoglobin were determined by RACE, there were 797 base pairs, encoding 147 amino acids, were found in the gene sequence of tilapia Mb. In addition, autoxidation of myoglobin extract solutions from dark muscle of different tilapia species were measured at 25°C. The results showed that autoxidation rate constants of Mb from different tilapia species were very similar. It suggests that autoxidation rate constants of tilapia Mb are similar due to highly conserved structures in different species.