碩士 / 國立高雄海洋科技大學 / 水產食品科學研究所 / 96 / The objective of the present study is to obtain collagen peptide from fish scale using enzymatic methods. The soluble enzymes of Protamex, Alcalase, Trypsin, Bromelain, Papain, Flavourzyme, Pepsin and Keratinase were used and compared to their reactivity to fish scale. Papain showed the highest activity for the hydrolysis of fish scale collagen, and followed by Alcalase, Flavourzyme, Bromelain, Protamex, Trypsin and Pepsin. However, hydrolysates of fish scale treated with Flavourzyme were found having higher amount of low MW (1000-100Da) materials. Thus, Alcalase, Papain and Flavourzyme were selected and used in the following studies. The fish scale was then treated with single enzyme system (including Alcalase & Papain) or two enzymes system (including Alcalase plus Flavourzyme and Papain plus Flavourzyme) .
The optimal conditions in single enzyme system for the hydrolysis of fish scale were in the following: both pH 8.0, temperature 55 oC for Alcalase and Papain; enzyme activity, 0.72 U/mL for Alcalase, 210 U/mL for Papain; reaction time, both 1 h for Alcalase and Papain; amount of fish scale used, 5% and 2% for Alcalase and Papain respectively. The optimal conditions in two enzymes system were in the following: Flavourzyme activity, 174 U/mL for Alcalase plus Flavourzyme, 232 U/mL for Papain plus Flavourzyme; amount of fish scale used, 6% for Alcalase plus Flavourzyme, 3% for Papain plus Flavourzyme. concentration of collagen peptide (MW <1.3 kDa) derived from fish scale treated with two enzymes system was 53.9% for Alcalase plus Flavourzyme, 48.9% for Papain plus Flavourzyme.
Three methods for the immobilization of Alcalase were used, ie. Alginate gel, glutaraldehyde-crosslinked chitosan (Chitosan method A) and chitosan microcapsules (Chitosan method B). The optimal conditions for the enzyme immobilized by Alginate gel were in the following: CaCl2 concentration, 0.3 M; setting time, 2 h; Alginate concentration, 2%; enzyme activity, 0.18 U/mL. The optimal conditions for the enzyme immobilized by Chitosan method A were in the following：Chitosan concentration, 3.0%; glutaraldehyde concentration 1.0%; setting time, 12 h; enzyme activity, 2.4 U/mL. The optimal conditions in Chitosan method B were in the following：Chitosan concentration 3 %、Tripolyphosphate concentration 2.5 %、pH 5, enzyme activity, 0.12 U/mL. The optimal pH and temperature of immobilized enzymes from three methods were all pH 8.0 and 50℃. Thermostability of immobilized enzymes was higher than that of soluble enzymes. The immobilized enzymes were then reacted with fish scale at 50 oC for 4 h as one cycle and the reaction was repeated for 8 cycles. The residual activity of them was listed in the following: 17.2 % in Alginate gel method, 49.5 % in Chitosan method A, 53.2% in Chitosan method B. Kinetics studies indicated Km and Vmax of soluble alcalase were 0.075 mM and 250.1 U/mg-min, respectively, while Km and Vmax in immobilized Alcalase with Alginate gel were 0.091 mM and 227.3 U/mg-min, 0.13 mM and 217.3 U/mg-min in that of Chitosan method A, 0.128 mM and 212.8 U/mg-min in that of Chitosan method B. Moreover, recovery of collagen peptide from fish scale treated with free and immobilized enzymes were 56.3 and 33.0% , respectively.
| Identifer | oai:union.ndltd.org:TW/096NKIM8084001 |
| Date | January 2008 |
| Creators | Shuang-Chih Wu, 吳双智 |
| Contributors | Jen-Min Kuo, 郭建民 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 86 |
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