Study On The Production Of Trehalose By Yeast

In this dissertation, the methods for quantitative determination of trehalose in yeastextract, the breeding of trehalose producer mutant and its fermentation conditions of trehaloseand so on were studied. The main contents and results obtained are as follows:(1) The strains of Saccharomyces cerevisiae preserved in our laboratory were tested todetermine the capacity of trehalose production, which found that the strain SC2was veryfavorable for trehalose synthesis, and can accumulate intracellular trehalose of48mg/g dryweight. Compared to other cells broking methods, it was found the dry heat method was morefavorable for the yeast strain. Then the method of quantitative determination of trehalose inyeast extract was studied, which found that the method of DNS and anthrone sulfuric acidmethod with higher reliability than the other methods.(2) The strain SC2was treated gradually by UV and DES，directed screened usingsodium chloride, glucose and ethanol gradient plate, then the strain SA12was obtained, whichcan accumulate intracellular trehalose of64mg/g dry weight, improved by33%.(3) The seed medium component and culture condition of the strain SA12wereoptimized, the optimum medium component as: glucose40g/L, yeast extract1g/L, peptone20g/L, K2HPO43g/L. The optimum culture conditions as: initial pH5.5, liquid volume80mL/500mL conical flask. The fermentation medium and culture conditions were alsooptimized, the optimum medium as: glucose81g/L, corn steep liquor52g/L, yeast extract1g/L, K2HPO40.9g/L, NaH2PO41.5g/L, MgSO4·7H2O0.26g/L; the optimum cultureconditions as: initial pH5.5, liquid volume100mL/500mL conical flask, seed age12h. Underthe optimized conditions of fermentation, the trehalose intracellular content reached149mg/gdry weight and the yield3.1g/L.(4) The conditions of inducing were studied and it was found that the final intracellulartrehalose content reach52%when added40g/L NaCl in46h than that without the saltaddition, and the trehalose intracellular content and yield reached227mg/g dry weight and4.82g/L respectively. After the period of logarithmic growth, adding2%～8%(v/v) ethanol inthe fermentation medium, there was no positive effect on yeast intracellular trehalose content.However as the added ethanol concentration increasing, the trehalose content decreasedseverely. In process of fermentation, after the heat shock of37℃,39℃and41℃at67h,68hand69h respectively, the intracellular trehalose content increased12%and the higher thetemperature of heat shock, the shorter time needed to the maximum trehalose content.(5) The fed-batch fermentation of the strain SA12was studied and it was found that theoptimal initial glucose concentration was40g/L, the optimal feeding time was12h, and theoptimal DO control strategy as controled at20%～30%between0h and16h and at0～10% between16h and72h. Under the optimized fermentation conditions with NaCl stress, the stainSA12was cultured in the7L fermentator, the cell biomass could reach23.3g/L, theintracellular trehalose content could reach229mg/g dry weight, and the total output was5.4g/L in7L fermentator.(6) Based on the nonlinear fitting curve of Logistic model, DoseResp model, Boltzmannmodel and SGompertz model, the dynamics models of strain growth, substrate consumptionand product yield were obtained and the fitting parameters and curves could reflect the batchculture process of trehalose.