Study On Microbial Fermentation Of Deodorizer Distillate For Production Sterols

Sterol is one class of steroids which are widely existed in the nature.Sterol is a kind of monohydric secondary alcohol of full cyclopentane hydrogen skeleton,which given four-ring steroid nucleus.Depending on its origin,sterols can be divided into three types:animal sterols,plant sterols and microbial sterols.Vegetable oil deodorizer distillate(VODD)is byproduct produced in the deodorization step of vegetable oil refining.VODD is very complicated,and mainly contains free fatty acids(FFAs),phytosterol,vitamin E,glyceride and other components.Phytosterol is produced mainly from VODD.The aim of this project is to ferment peanut oil deodorizer distillate(PODD)with microorganisms,to concentrate PODD by consuming FFAs as the cells grew,and to obtain sterol-rich yeast cells simultaneously.When PODD was fermented with Candida tropicalis 1253,the cells grew poorly and consumption of FFAs became lower as PODD concentration increased in the culture medium,which was related to lack of oxygen supply.The optimal PODD in the culture medium was 4.0%.In this work,it was found that inorganic nitrogen sources had a better fermentation results than organic nitrogen sources,and the urea was the best nitrogen source.Ca2+,Fe2+,Co2+,Mn2+ and Zn2+ significantly inhibited the cell growth.Especially Ca2+ had the strongest inhibition effect among mineral salts tested.Only Mg2+ stimulated obviously the cell growth of C.tropicalis1253.The optimal culture medium for the cell growth was:PODD 40.0g/L,ureal.0 g/L,MgSO4 0.2 g/L,K2HPO4 2.0 g/L and KH2PO42.0 g/L.The optimum fementation condition was:pH6.0,inoculum size 10%,temperatue 30℃,shaking speed 200r/min and fermentation period 120h.After fermentation under the optimum conditions,the cell biomass reached 6.38 g/100mL(wet weight).FFAs concentration in PODD decreased from 66.3%to 13.37%.The highest consumption rate was 79.84%,and phytosterol concentration in PODD increased from 14.9%to 28.43%.PODD was successfully concentrated with microbial fermentation.After fermentation of PODD with C.tropicalis 1253,it was found that the cells obtained were rich in sterols.In order to obtain sterol-rich cells as high as possible,the fermentation conditions was investigate in this work.The results suggested that when FFAs were used as a carbon source,C.tropicalis 1253 synthesize a large amount of sterols.Inorganic nitrogen sources made sterols in the cells higher than organic nitrogen sources,and urea was the best nitrogen source.Ca2+,Li+,Fe2+,Co2+and Zn2+ significantly inhibited synthesis of sterols.However,Mg2+ stimulated obviously synthesis of sterols.The optimal culture medium for sterols synthesis was:PODD 40.0 g/L,urea 1.4 g/L,MgSO4 0.1 g/L,K2HPO4 2.0 g/L and KH2PO4 2.0 g/L.The optimum fermentation condition for sterols synthesis was:pH7.0,inoculum size 11%,temperature 30℃ shaking speed 200 r/min,fermentation period 98 h.After fermentation,the cell biomass of C.tropicalis 1253 was 6.01 g/100mL(wet weight)and the content of total sterols was as high as 4.22%(wet weight).Extraction of sterols in the cells of C.tropicalis 1253 was carried out by saponification-organic solvent extraction.The sterol extraction rate with wet cells was 11.19%higher than that of dry cells,and its reason was that the wet cells were easier to be broken and saponified.The cells were broken by acid,alkali,enzyme,ultrasonic wave,repeated freezing and melting respectively.It was found that alkali treatment was the best effective to break cell walls.Alkali(sodium hydroxide)not only effectively breaks cell walls,but also saponify PODD.N-Hexane,normal heptane,cyclohexane,ethyl acetate,petroleum ether and ethyl ether were used as solvents to extract sterols.It was found that ethyl acetate had the highest rate of sterol extraction,62.98%.The optimal saponifier was the mixture of 20%KOH:95%ethanol = 5:3,v/v.The optimal conditions for extraction of sterols were:mixing the wet yeast cells with saponifier in 1:50,saponifing in a water bath at 80℃ for 3.5 h,and then removing ethanol by distillation,adding ethyl acetate after the flask was cooled to room temperature,extracting for 15min and setting for 2 h,separating the supernatant,and then removing the solvent by distillation.The content of ergosterol in the crude sterol extract was determined by HPLC.It was found that crude sterol extract contained 1.29%ergosterol.This result indicated that C.tropicalis 1253 could synthesize a large amount of sterols,but ergosterol was not the major sterol.The kind and structure of other sterols synthesized in the cells need to be further identified.