Studies on the Conditions of Levan Production byBacillus licheniformis FRI MY-55 in High-Sucrose Medium and Growth Performance and Enhancing Immune Responseon Grouper of Levan

Autor: Mei-Ying Huang, 黃美瑩
Rok vydání: 2015
Druh dokumentu: 學位論文 ; thesis
Popis: 103
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.
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