|Abstract: ||本研究自海水吳郭魚養殖池中篩選出5株具有合成果聚糖能力之細菌 (M1、M3、M6、M11、及 T1)，比較其果聚糖產生量及所產果聚糖之分子量分佈情形。鑑定較高產量果聚糖生產菌株 T1，探討不同碳源、氮源、蔗糖濃度、起始 pH 值、及培養溫度對於 T1 菌果聚糖產量之影響，該菌於不同蔗糖濃度及培養溫度時，所產果聚糖分子量之變化，並探究 T1 菌株所產果聚糖作為益生素之特性。此外，添加不同含量 T1 菌株所產果聚糖於點帶石斑(Epinephelus coioides H.) 飼料中，評估其對於魚隻成長、腸道細菌數、魚肉組成份、免疫指數、及抵抗病原菌之影響。 結果顯示，在含有 12% 蔗糖培養液中菌株 M1、M3、及M6 於 48 h 所產果聚糖的濃度為 20.35-23.01 g/L。M11菌株則於培養 48 h 才產出 5.43 g/L 果聚糖。T1 菌株於培養 48 h 果聚糖產量達29.15 g/L。有關所產果聚糖之分子量方面，M1、M3、M6、M11、及 T1 等 5 菌株所產之主要果聚糖分子量分別為 10.79、10.78、10.31、1.78、及 14.71 KDa。各菌株所產出主要果聚糖以酸液熱水解處理後，發現其單糖組成份中均以果糖為主；分析其 1H NMR 及 13C NMR 圖譜，顯示與文獻上其他論文報告中所述果聚糖的鍵結資料相當接近，因此推斷該 5 株菌所產果糖聚合物為 β(2-6) 鍵結型態的果聚糖。高產量果聚糖生產菌株 T1 經生化特性及 16S rRNA 分析鑑定為 Bacillus licheniformis，並命名為 B. licheniformis FRI MY-55。 在測試的 8 種不同碳源、氮源、及起始 pH 值之培養液中，B. licheniformis FRI MY-55 分別以含蔗糖 (20%)、大豆腖 (0.5%)、及 pH 7.0 之培養液條件下果聚糖產量較高。以 18-55°C 不同培養溫度 (含 20% 蔗糖) 試驗結果顯示，B. licheniformis FRI MY-55以 23°C及 30°C 培養者在 36 h 有較高濃度果聚糖，分別為 66.84 g/L 及 62.81 g/L。培養在含 1-50% 蔗糖培養液 (40°C) 試驗結果顯示，B. licheniformis FRI MY-55 之果聚糖產量依培養液中蔗糖含量提高而上升，其中以含有 40% 及50% 蔗糖培養液培養 36 h 及96 h，可得到多量果聚糖 (分別為131.75 g/L 及 145.94 g/L)。其生產速率為 3.92 g/L/h (含有 30% 蔗糖培養液，24 h)，高於目前一般高產量果聚糖的菌株。所產果聚糖的分子量方面，提高培養溫度 (自18-55°C) 或培養液中的蔗糖濃度 (自10-50%)，B. licheniformis FRI MY-55所產果聚糖的分子量約自13 KDa降為3 KDa。有關 B. licheniformis FRI MY-55 所產果聚糖之益生素試驗顯示，對 16 株益生菌益生活性分數 (prebiotic activity score) 皆出現正值，顯示該果聚糖具有益生素之特性。 點帶石斑經餵食不同含量B. licheniformis FRI MY-55所產果聚糖 (0%、0.5%、1.0%、2.5%、及 5.0%) 飼料 12 週後，最終體重及增重率以餵食含 2.5% 果聚糖組最高，且與對照組 (0%)有明顯差異 (p < 0.05)。魚隻腸道中總好氣性生菌數及弧菌數方面，餵食添加果聚糖各組石斑魚腸道中的總好氣性生菌數及弧菌數均明顯較對照組 (0%) 為低 (p < 0.05)。免疫指數及活存率方面，點帶石斑飼餵含不同量果聚糖飼料12週並以病原菌 Vibrio harveyi攻擊後，血清中總蛋白量、球蛋白、溶菌酶 (lysozyme) 活性、及活存率均以餵食含 2.5% 果聚糖飼料的魚隻中最高，且統計上與對照組 (0%) 有明顯差異 (p < 0.05)。有關各組魚肉組成分析結果顯示，添加 1.0%、2.5%、及 5.0% 果聚糖各組魚體組成中粗蛋白含量明顯高於對照組 (0%) (p < 0.05)。本研究顯示，飼料中添加2.5% 果聚糖可以有效促進點帶石斑的成長及疾病抵抗能力。|
Five bacterial strains, M1, M3, M6, M11, and T1 with levan-producing capability were isolated from brackish water tilapia culture pound. The effects of carbon and nitrogen sources, initial pH of the medium, sucrose concentration, and temperature on levan production by the higher levan-producing strain, T1, effects of sucrose concentration and temperature on the molecular weight of levan; and prebiotic effect of levan were investigated. In addition, the effects of levan produced by T1 on growth performance, intestinal count of viable bacteria, immune status, pathogen resistance, and body composition of orange-spotted grouper (Epinephelus coioides H.) were evaluated. In the medium containing 12% sucrose at 28°C, strains M1, M3, and M6 produced 20.35-23.01 g/L levan after 48 h incubation. The amount of levan produced after 48 h cultivation by strain M11 was 5.43 g/L. Strain T1 produced 29.15 g/L levan after 48 h incubation. The main molecular weight of levans produced by the isolates M1, M3, M6, M11, and T1 were 10.79, 10.78, 10.31, 1.78, and 14.71 KDa, respectively. The hydrolysates of derived from acid and heating treatments of the levan were mainly fructose. The 1H NMR and 13C NMR spectra of the products of the 5 isolates were very similar to peak positions for levan produced. Based on obtain data, the fructans produced by the 5 isolates were found to be the levan containing β (2-6)-linked backbone. The strain T1 with high levan-producing abilities was identified as Bacillus licheniformis and named B. licheniformis FRI MY-55. The maximum levan yield obtained by B. licheniformis FRI MY-55 in the presence of 20% sucrose, 0.5% soytone, and an initial pH of 7.0, respectively. Testing temperature range of 18-55°C, the maximum levan yield after 36 h was 66.84 g/L at 23°C and 62.81 g/L at 30°C. Levan production by this strain increased proportionally with an increase in sucrose concentration of the medium. The maximum levan yield was 131.75 g/L and 145.94 g/L in 40% and 50% sucrose-containing media after 36- and 96-h cultivation at 40°C. The maximum levan production rate in this strain was 3.92 g/L/h in a 30% sucrose-containing medium, which is the highest rate reported for any strain till date. The molecular weight of produced levan decreased from around 13 to 3 KDa when temperature was increased from 18°C to 55°C or sucrose concentration from 10% to 50%. The produced levan had prebiotic effect. Orange-spotted grouper were fed diets supplemented with levan produced by B. licheniformis FRI MY-55 at concentrations of 0 (control), 0.5%, 1.0%, 2.5%, and 5.0% for 12 weeks. The final weight and percent weight gain were significantly higher in the 2.5% levan-supplemented group than in the control group (0%) (p < 0.05). All levan-supplemented diets significantly decreased the count of total viable aerobic bacteria and Vibrio spp. in the intestines of groupers (p < 0.05). Serum total protein levels, globulin levels, lysozyme activity, and survival rate of orange-spotted grouper after challenge with V. harveyi were significantly higher in the 2.5% levan-supplemented group than the control group (0%) (p < 0.05). This study also found that the 1.0%, 2.5%, and 5.0% levan-supplemented diets significantly increased the crude protein level in the body composition of orange-spotted grouper. Overall, the results of this study indicate that dietary levan (2.5%) could be an effective method for enhancing the growth performance and disease resistance in orange-spotted grouper.