浮萍粉做為紅尼羅吳郭魚飼料蛋白源之評估及利用重組表達芽孢枯草桿菌E20木聚醣酶以改善浮萍粉之利用率

Evaluation of duckweed (Lemna minor) meal as protein source for red tilapia (Oreochromis sp.) and applying the recombinant xylanases from Bacillus subtilis E20 to improve the utilization of duckweed meal

蔡錦燕(Tsai Chin-Yen)

指導教授 ： 葉信平

國立屏東科技大學/農學院/水產養殖系所/博士(2019年)

https://doi.org/10.6346/NPUST201900241

摘要

本研究旨在評估使用浮萍（Lemna minor）粉做為紅尼羅的飼料原料的可能性。浮萍粉的粗蛋白質和粗脂肪含量分別為24.2±0.39％和2.93±0.26％。水解胺基酸組成分中，浮萍粉含必需胺基酸高達53.7%。依據商業吳郭魚飼料僅含有5％魚粉之基準及粗蛋白和粗脂質設計為23％和7％的原則進行等氮及等油脂之實驗飼料之配製。實驗飼料製作時，使用浮萍粉進行對照組中之植物混合原料取代，取代之比率分別為20％、40％和60％，相當於飼料中之浮萍粉量為16.5％、33％和49.5％ (命名為D20、D40和D60)，而沒有添加浮萍粉的飼料則作為對照組。在飼餵56天實驗飼料後，各實驗組之存活率、體組成分或對病原性產氣單胞菌(Aeromonas hydrophila)的扺抗力均無顯著性差異。魚隻攝食D40(含有33％浮萍粉)，末重與增重明顯低於對照組及D20，但其飼料效率與對照組及D20組則無顯著性差異。藉由broken-line analysis顯示，紅尼羅飼料中浮萍的最大取代為30％。因此浮萍具有成為紅尼羅飼料蛋白質來源的潛力，為了增加浮萍粉的利用率，實驗選殖並重組外泌表達來自枯草芽孢桿菌E20之木聚醣酶基因。
枯草芽孢桿菌E20 分離自發酵大豆產品納豆，已被應用於各種海洋和淡水水生動物，具有促進生長和提升免疫力之功效。本實驗自枯草芽孢桿菌E20中選殖出木聚醣酶基因，再於枯草芽孢桿菌RIK1285中重組外泌表達。經鑑定為BsXynE20基因後，確定其具有213個胺基酸，並包含28個訊號胜肽，經切割後成為185個胺基酸的作用蛋白，預估分子量為20.25kDa，屬於醣水解酶家族11的木聚醣酶。將BsXynE20基因植入pBE-S表現載體中，並利用枯草芽孢桿菌RIK1285外泌生產重組BsXynE20。透過西方點墨法以及酶譜分析證實培養液中可偵測到具有木聚醣酶活性之重組BsXynE20，其最佳反應pH及溫度分別為5.0-6.0和60℃。隨後將重組BsXynE20預處理之浮萍粉進行吳郭魚飼料製備，結果證實可顯著提升紅尼羅的成長表現。因此，重組BsXynE20的使用能有效提高浮萍做為吳郭魚飼料之營養利用率，能有助於吳郭魚之養殖。

關鍵字

浮萍粉 ； 枯草芽孢桿菌E20 ； 成長表現 ； 外泌表達 ； 紅尼羅 ； 木聚醣酶

並列摘要

This study aimed to evaluate the applicability of using duckweed (Lemna minor) meal as a feed ingredient for red tilapia. The crude protein and crude lipid contents of duckweed meal were 24.2±0.39% and 2.93±0.26%, respectively. Hydrolyzed amino acid composition analysis showed that duckweed meal contained high levels (53.7%) of essential amino acids. A plant-based diet containing 5% fish meal as an attractant was used in this study. Crude protein and crude lipids were established at 23% and 7%, respectively, in diet used in our experiments. Duckweed meal was used to replace 20, 40, and 60% of the mixed-plant portion based on crude protein levels, resulting in 16.5%, 33%, and 49.5% duckweed meal in diets, designated D20, D40, and D60 diets, respectively. The diet without duckweed meal was used as the control diet. No significant differences in survival, body composition, or resistance to pathogen Aeromonas hydrophila were recorded among treatments after 56 days of feeding. Fish fed the D40 diet (containing 33% of duckweed meal) were not significantly different in feed efficiency but decreased in final weight and weight gain compared to the control and D20 diets. The maximal level of duckweed in the experimental red tilapia diet was 30% based on feed efficiency estimated by broken-line analysis. Duckweed meal can be a potential protein source when used as a feed ingredient in red tilapia diets. In order to improve the utilization of duckweed meal, the xylanase gene of Bacillus subtilis E20 were cloned and secretive expression.
Bacillus subtilis E20, an isolate from natto – a Japanese fermented soy product – is used in a wide variety of marine and freshwater aquatic species capable of enhancing growth and immunity of target life. This study describes the cloning, expression and characterization of xylanase gene isolated from B. subtilis E20 in B. subtilis RIK1285. The BsXynE20 gene was identified to encode a 213‐amino‐acid protein for a glycoside hydrolase family 11 xylanase containing a 28‐residue signal peptide whose cleavage yields a 185‐residue mature protein with a predicted molecular weight of 20.25 kDa. The full length of the BsXynE20 gene was subcloned into the pBE‐S expression vector for secretory production of recombinant BsXynE20 in B. subtilis RIK1285. Western blot and zymogram analysis, respectively, revealed that recombinant BsXynE20 can be secreted into culture medium and is a functional xylanase. The xylanase exhibited optimal activity at pH 5.0–6.0 and 60°C. Using recombinant BsXynE20‐pretreated duckweed meal for feed preparation improved the growth performance of tilapia. Thus, the use of BsXynE20 may promote the nutrition utilization of duckweed meal as a feed ingredient in tilapia diet which can be beneficial for tilapia aquaculture.

並列關鍵字

Duckweed meal ； Bacillus subtilis E20 ； growth performance ； secretory expression ； red tilapia ； xylanase

參考文獻

姜樹興 (2014)。動物營養學原理。華香園出版社，台北市。150-151。

許元勳 (2003)。現代生物科技的新寵兒－神奇納豆菌。生物產業。14: 53- 59。

須見洋行 (2002)。納豆激酶的驚奇效果。青春出版社，台北市。50-51。

鄭斌、詹希美 (2005)。信號肽序列及其蛋白質表達中的影用。生物技術通訊。16: 296- 298。

Abrahmsén, L., Moks, T., Nilsson, B., Hellman, U., Uhlén, M. (1985) Analysis of signals for secretion in the staphylococcal protein A gene. EMBO J., 4: 3901- 3906.