南极细菌Pseudoalteromonas sp.3-3-1-2胞外多糖发酵条件优化及免疫活性研究

发布时间：2018-09-14 10:39

【摘要】：南极细菌长期生活在酷寒、强辐射的极端环境中,因而形成了独特的生理生化特征,进化出与众不同的极端环境适应机制。胞外多糖(exopolysaccharide, EPS)是微生物在生长代谢过程中产生的、且分泌到胞外的可溶性多糖及多糖复合物,在细胞间信息交换、细胞保护、细胞粘附和环境适应性等方面具有重要作用。由于极端环境中的EPS所具有的独特的生物活性使其在食品加工、新药研发和新型饲料开发等方面具有非常重大的应用潜力。研究了在基础饲料中添加不同含量(0、0.5、1.0、1.5和2.0 g/kg)南极细菌Pseudoalteromonas sp.3-3-1-2产 EPS对黄盖鲽(Pseudopleuronectes yokohamae)生长和免疫活性的影响。结果表明,当基础饲料中EPS添加量为1.5 g/kg时,试验组黄盖鲽的体重和体长均高于对照组,呈显著性差异(P0.05)。添加量为1.0和1.5 g/kg试验组的脾脏和后肠淋巴细胞呼吸爆发能力和对照组相比有明显提高,呈极显著性差异(P0.01)；脾脏淋巴细胞增殖能力也有明显提高,呈显著性差异(P0.05)。和对照组相比,添加量为1.5 g/kg试验组的脾脏蛋白含量明显提高,呈显著性差异(P0.05)；脾脏和肝脏酸性磷酸酶(ACP)活力明显提高,呈显著性差异(P0.05)。添加量为1.5和2.0 g/kg试验组的肝脏蛋白含量和总超氧化物歧化酶(T-SOD)活力明显提高,呈显著差异(P0.05)。攻毒实验表明,试验组黄盖鲽均获得了一定的免疫保护,且添加量为1.5 g/kg试验组的免疫保护率最高(50.0%)。由此得出,在基础饲料中添加南极细菌Pseudoalteromonas sp.3-3-1-2产EPS可促进黄盖鲽生长,提高特异性免疫活力和非特异性免疫活力,提高黄盖鲽抗病能力,且添加量以1.5 g/kg效果最佳。利用单因子试验和响应面法对南极细菌Pseudoalteromonas sp.3-3-1-2产胞外多糖条件进行了优化。单因子试验表明,菌株3-3-1-2产胞外多糖的最佳碳源和氮源分别为蔗糖和硝酸钾,其最佳添加量分别为4%和1%；最适培养温度为15℃；最佳培养基盐度和初始pH分别为45和9；对产糖有显著影响的单因素为碳源(蔗糖)、氮源(KN03)和温度。通过Box-Behnken试验设计和Design-Expert响应面对上述因素进行分析,确定优化的菌株3-3-1-2产胞外多糖的发酵条件为：蔗糖43.1 g/L、硝酸钾9.6 g/L和温度17℃；经条件优化后菌株3-3-1-2发酵液的粗胞外多糖产量可达3.15 g/L。对菌株Pseudoalteromonas sp.3-3-1-2产EPS进行了DEAE Sepharose fastflow和G-50的初步分离纯化,对获得的较纯成分EPS-1进行进一步的纯度鉴定和分子量的测定,并对其进行了单糖组分分析,以期获得其多糖分子结构。
[Abstract]:Extracellular polysaccharides (EPS) are produced by microorganisms in the process of growth and metabolism, and secrete extracellular soluble polysaccharides and polysaccharide complexes in fine grains. Intercellular information exchange, cell protection, cell adhesion and environmental adaptability play an important role. EPS in extreme environments has great potential applications in food processing, new drug research and development and new feed development due to its unique biological activities. The effects of EPS production by Antarctic bacteria Pseudoalteromonas sp.3-3-1-2 on the growth and immune activity of Pseudopleuronectes yokohamae were studied. The results showed that the body weight and length of flatfish in the experimental group were significantly higher than those in the control group (P 0.05) when the EPS content in the basal diet was 1.5 g/kg. Compared with the control group, the spleen and hind intestine lymphocyte respiratory burst ability and the spleen lymphocyte proliferative ability of the 5 g/kg test group were significantly improved (P 0.01), and the spleen lymphocyte proliferative ability was significantly improved (P 0.05). Compared with the control group, the spleen protein content of the 1.5 g/kg test group was significantly increased. The content of liver protein and the activity of total superoxide dismutase (T-SOD) in the 1.5 and 2.0 g/kg groups were significantly higher than those in the control group (P 0.05). It was concluded that the addition of Antarctic bacteria Pseudoalteromonas sp.3-3-1-2 to the basic diet could promote the growth of flatfish, enhance the specific and non-specific immune activities, and enhance the disease resistance of flatfish, and the dosage of 1.5 g/kg was the best. The optimum conditions for producing extracellular polysaccharides by Antarctic bacteria Pseudoalteromonas sp.3-3-1-2 were studied by single factor test and response surface methodology. The optimum fermentation conditions for exopolysaccharide production of strain 3-3-1-2 were determined as follows: sucrose 43.1 g/L, potassium nitrate 9.6 g/L and temperature 17, respectively. The crude exopolysaccharide yield of strain 3-3-1-2 fermentation broth could reach 3.15 g/L after optimized conditions. The EPS produced by strain Pseudoalteromonas sp.3-3-1-2 was purified by DEAE Sepharose fastflow and G-50. The purity and molecular weight of the purified EPS-1 were further identified and determined. In order to obtain the molecular structure of polysaccharides.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816

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