Seaweed Shewanella Fermentation Tetrodotoxin Extraction And Detection

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.