海藻希瓦氏菌发酵产生河豚毒素的提取与检测

【作者】 岳田芳；

【导师】 宫庆礼；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2008， 硕士

【摘要】 近年来,河豚毒素的微生物起源越来越得到人们的认可。海洋微生物发酵法产生河豚毒素,具有原料来源广、成本低,不受季节和原料的限制,不破坏生态平衡的特点,因此人们期望通过细菌发酵实现资源的有效利用和河豚毒素的大量生产。国外早在上世纪80年代就已经着手此项研究,并且取得了骄人的成绩。而我国在这方面的研究还很薄弱。海藻希瓦氏菌最早于1985年由Yuichi Kotati等人从红藻（Jania sp.）表面分离出来,当时被命名为OK-1^T（T=type strain）,研究发现OK-1^T与腐败希瓦氏菌有很多相似的生理、生化特征,而且可以产生河豚毒素。本论文目的在于从结构和生物学功能上证实,海藻希瓦氏菌发酵产生的TTX与河豚组织中的TTX是一致的,为TTX微生物起源假说奠定坚实的实验基础。优化海藻希瓦氏菌生产河豚毒素的发酵条件,细致研究海藻希瓦氏菌产生河豚毒素的分离纯化工作,为可持续生产TTX提供新思路。本文对海藻希瓦氏菌生理生化指标进行了测定。通过测定菌体密度和菌体收获量,研究了部分初始条件及添加不同营养物质对海藻希瓦氏菌生长的影响,采用单因素试验和正交试验对海藻希瓦氏菌生产河豚毒素的发酵条件进行了优化。优化后的培养基最佳配方为:在2216E培养基中添加1.0%葡萄糖、2.5%酵母粉、1.0%磷酸高铁。统计分析,葡萄糖是影响海藻希瓦氏菌菌体收获量的主要因素。采用SCM杯式超滤系统对海藻希瓦氏菌发酵液的过滤除杂进行了研究,考察了操作压力对过滤效果的影响,同时还针对料液的预处理对超滤结果的影响进行了研究,主要措施是调节料液pH值,改变其温度,在其中添加混凝剂。结果表明,操作压力为0.2MP,通量达到最大,截留率也相对较大。料液的pH调至4,加热料液的温度至60℃都能使通量和截留率达到较满意的水平。研究发现在料液中添加50mg/L硫酸铝混凝剂能有效减缓超滤膜污染。本文还考查了分子筛去除分子量较小的杂质的效果,其中蛋白去除率50.8%,有机物去除率82.8%。考查了弱酸性阳离子交换树脂D₁₅₂对TTX的净化作用,结果显示,除色效果很好,糖类、氨基酸类也有很好的去除效果。本文利用薄层层析对发酵产物经分离得到的TTX进行初步定量,结果与小鼠生物检测法结果有良好的相关性。采用直接竞争抑制性酶联免疫吸附试剂盒对发酵产物进行了进一步的定量测定,并最终运用高效液相色谱法对海藻希瓦氏菌发酵产物中的TTX含量进行精确定性、定量。更多还原

【Abstract】 Nowadays,people prefer to accept the theory that bacterial is the origin of tetrodotoxin.The method using marine bacteria producing TTX has lot of advantages, such as wide source,low cost,unlimited season and material,never destroying the ecosystem balance.It is supposed to use marine bacteria fermentation to realize the efficient making use of resource and to produce of adequate TTX.It is reported that foreign scientists have done some work on this research and achieve marked success. But it has few report in our country.Shewanella alga was firstly separated from Jania sp.by Yuichi Kotati,named OK-1^T. Shewanella alga has similar biochemical characteristics with Shewanella putrefaciens. This bacteria can producing TTX.The purpose of this thesis is confirming the TTX origin from bacteria is same as the TTX origin from puffer by mouse assay and HPLC, supporting the postulation of bacteria origin of TTX.The other purpose of this thesis is optimizing the culture medium on Shewanella alga producing tetrodotoxin, optimizing the separation and purification method,offering the new idea for producing TTX.At this experiment,morphological and biochemical characteristics of Shewanella alga was determined.Different culture media ingredient that have effects on the grow of Shewanella alga were studied by determined cells concentration and cells weight.The fermentation parameters was optimized by single factors experiment and orthogonal experiment.The optimum culture medium for Shewanella alga was in medium of glucose 1.0%,yeast powder 2.5%,phosphatic iron 1.0%.After statistic analysis, glucose was considered to be primary factor.Purification study of fermentation solution of Shewanella alga was carried out adopting SCM cup ultrofiltration system. To improve the ultrofiltration process by change operating pressure,adjust pH, change temperature,add coagulation for the solution.Results showed that 0.2MP operating pressure,pH 4,60℃,100mg/L Al₂（SO₄）₃·18H₂O coagulation can increase flux and removal of impurity effectively.Coagulation can ease membrane pollution. Using molecular sieve chromatography to remove small molecular impurity also studied in this thesis.The rate of removal of protein is 50.8%and the rate of removal of UV₂₅₄ is 82.8%.The purification effect of D₁₅₂ macroreticular cation exchange resin showed that D₁₅₂ is good for getting rid of colour,sugar,amino acid.TLC and direct competitive inhibition enzyme-linked immtmosorbent assay was used to determine TTX quality of fermentation substance of Shewanella alga.The accuracy of TLC and ELISA was compared with the traditional mouse bioassay system.The results showed no significant differences between the two methods.Using HPLC to determine TTX quality and character.更多还原