Studies On Formation Of Trehalose By Permeabilized Yeast Cells

Trehalose is a natural functional disaccharide.It is non-reducing and stabilized to heatand acid.It also can protect biologic molecule,such as DNA and protein.So trehalose could bewidely used in foodstuff、pharmaceuticals、cosmetic、and agriculture.Trehalose is uses arerestricted due to the difficulty in producing it.Yeast is a major strain of trehaloseproduction.Trehalose is accumulated at the end of the reproductive stages and under extremeenvironmental conditions. Because the trehalose is intracellular product,its productivity islimited.In this paper,the permeabilization method of alcohol yeast cell to chang cellmembrane permeability, reduction of trehalose accumulation as a result of feedbackinhibition,in order to achieve the purpose of improving production of trehalose.The mainresearch contents are as follows:the measuring method for trehalose,the optimization ofmedium and culture of conditions,and the purification of the intracellar trelose.The method of trehalose measuring was studied.It includes the confirmation of the legthof boiling time,the confirmation of the length of spectrum of measurement and the standardcurve of intracellar trehalose.The results showed the optimal boiling time is8minutes.Theoptimal length of spectrum is620nm,the formula of calculating trehalose in yeast were gotfrom the stantard curve of trehalose,sulfuric acid concentration5mL.In addition,the use ofindustrial production in the DNS method used to derermine sample content of reducing sugarand total sugar,algae indirectly calculate the extracellular trehalose content,the menthod issimple and low cost.The permeabilization of alcohol yeast cell was studied using different organic solventsand detergents. The optimum conditions were as followed:1g wet yeast cells treated by15mL0.5%（v/v） Tween-60for45min at30℃.The cells in suspension were converted intopermeabilized cells.Accumulation of trehalose in medium raised to0.917g·L^-1.In order to enhance the content of the trehalose accumulation of Permeabilized alcoholyeast,single-factor and response surface methodology were used to optimize the fermentationmedium. The optimal fermentation medium was obtained as followings:sucrose concentration3.25%, level of yeast extract1.47%, sodium chloride concentration2.74%.Under these conditions,the yield of trehalose reached0.9319g·L^-1.The best operation conditions fortrehalose fermentation system were80mL of liquid volime,0.8g of wet yeast inoculums,pH6.0,30℃of temperature.The yield of trehalose in this operation conditions was up to0.978g·L^-1.Their extraction process was optimized by single factor and orthogonal experiments.Thebest reaction conditions are：concentration of TCA0.8M,reaction temperature90℃，ratio ofdry yeast weight to distilled water volum1:25and extraction time60min.