| Summary: | 碩士 === 國立高雄海洋科技大學 === 水產食品科學研究所 === 102 === In this study, one of the defatted protein (RP) was prepared from fish roe. After we hydrolyzed the fish roe protein with enzyme and defatted with hexane, the peptides were named roe protein hydrolysates (RH). In the functional and properties analysis of RP, the protein solubility, buffer capacity, isothermal moisture, antioxidant activity and amino acid composition were determined. RH was investigated its antioxidant activity, anti-tyrosinase activity and anti-proliferation of cancer cell Ca9-22. Moreover, the purified and highest anti-radical and anti-tyrosinase activity of peptide fractions were screened, and their amino acid composition was analyzed. The results showed that RP had the least protein solubility at pH 6 but had the highest buffer capacity at pH 2 and pH 12. When it was stored in the relative humidity of 75%, the moisture content was less than 5%. The major molecular weight of SDS-PAGE was distributed among 16-30 and 40-97 kDa. After ultrafiltration (MWCO : 5 kDa), the molecular weight of ultrafiltrated RH (URH) was between 204.2 and 2531 Da. To screen the peptides in URH with the highest anti-radical or anti-tyrosinase activity, ion exchange chromatography and gel filtration chromatography were employed for purification. The fractions with higher anti-radical activity were URH-A-II and URH-A-III while the fractions with higher anti-tyrosinase activity were URH-C-II and URH-C-III. In Lineweaver-Burk plot analysis of URH-C-III, it belongs to non-competitive inhibition of tyrosinase activity. Furthermore, in the anti-proliferation experiment, the URH treatment had an effect on growth and morphology of oral cancer Ca9-22 cells. Based on the amount of adenosine triphosphate (ATP) in cell, URH reduced cell viability in a dose-dependent manner. Moreover, it showed that URH treatment significantly increased the Sub-G1 population of cell cycle, inducing intracellular ROS levels and causing membrane depolarization of mitochondria in Ca9-22 cells. To investigate apoptotic phenomena induced by treatment with URH, Ca9-22 cells were double stained with annexin V-FITC and propidium iodide (PI). However, the result indicated that anti-proliferative effect on the cells may not be casused by apoptosis.
|
|---|
