Study On Microbial Fermentation Of Deodorizer Distillate For Production Microbial Sterols

Sterol is a kind of monohydric secondary alcohol of full cyclopentane hydrogen skeleton,which given four-ring steroid nucleus.Depending on its origins,sterols can be divided into three types: animal sterols,plant sterols and microbial sterols.Sterol has a good biological activities,such as decreasing cholesterol,diminishing inflammation,resisting oxidation.It is widely used in medical,food and cosmetics.Deodorizer distillate is a by-product produced in the deodorization step of vegetable oil refining.In this study,deodorizer distillate was fermented with C.tropicalis 1253,the types of sterols in the extracts were indentified.The fermentation conditions of sterol-rich cells and the extraction conditions of the organic solvents were researched.The main results are as follows:?1?Separation,purification and identification of microbial sterols from C.tropicalis 1253.The appropriate conditions of saponification were: mixing the yeast cells with saponifier?20% NaOH: 95% ethanol= 5:3,V:V?in 1:50,saponifing in a water bath at 90°C for 3 h.The purity of sterols was 6.6% by using this method.Selected six organic solvents to extract sterols,such as cyclohexane,n-hexane,ethyl acetate,n-heptane,aceton and petroleum.The extraction effect of n-hexane was the best,the purity of sterols were 6.45%.The optimal extracting time and temperature were 45 min and 20°C.Dissolving the crude sterol from extractings in ethanol,placing it in the refrigerator at 4°C,and letting it stand for 24 h to make it appear flocculation.After the suction filtration,removing the solvent by distillation.The sterol purity of the extract was as much as 6.95%,which increased by 5.3% than before.Finally by using the Gas Chromatography-Mass Spectrometer?GC-MS?and High Performance Liquid Chromatography?HPLC?identification,it was found that the main sterols synthesized by C.tropicalis 1253 were sitosterol and campesterol,however the ergosterol was less.Squalene could be produced by C.tropicalis 1253 simultaneously.?2?The optimization of culture condition of sterol-rich yeast cells.When the carbon source was the compound of deodorization distillate and linoleic acid and the nitrogen source was the compound of urea and beef extract,the sterol content was the highest.Glycine and vitamin H could promote the bacteria sterol synthesis obviously.The seven kinds of emulsifiers which were tested could promote the growth of yeast.Sodium dodecyl benzene sulfonate?SDBS?could promote the synthesis of sterols dramaticlly,the sterol content was as high as 5.63%.MgSO₄?ZnSO₄?KNO₃ were beneficial to the growth of yeast.Among of them MgSO₄ was the best one.And the ZnSO₄ could promote the synthesis of sterols obviously.Through the orthogonal test,the optimal constitute of mineral salt were: ZnSO₄ 0.2 g/L,MgSO₄ 0.1 g/L,KNO₃ 0.2 g/L.After the verification experiment,the sterol content of yeast cells was 5.81%.?3?The research of the yeast cell wall-breaking and sterol extraction.The best conditions for breaking cell wall were: cell density 10%,homogeneous pressure 800 bar,broken numbers 30 times.In this condition,the wall broken rate was 74%.The yeast cells were broken by the High Pressure Homogenizer firstly,then using n-hexane to extract sterols in 5 L extraction pot.Through the experiment,the optimal condition of extract sterol were: solid-liquid ratio?yeast: n-hexane,m/v?1:15,extraction time 40 min,extraction temperature 30°C,bacterial suspension pH 6.0,extraction 3 times.On this occasion,the yield of sterol extract was 3.74 mg/g,and the purity of the sterols was 3.3%.The extraction ratio of mechanical wall-breaking+ organic solvent was very low.It mainly due to the cell wall broken rate was low.