Study On The Transformation And Separation And Purification Of Steviol Glycosides By Penicillium Pinophilum

Steviol glycoside and stevia phenolic acid are important secondary metabolites and functional ingredients of Stevia rebaudiana.Steviol glycoside is a high-potency,low-caloric natural sweetener.What's more,the structure transformation of stevioside(SS)and rebaudioside C(RC),and the production of rare steviol glycosides are conspicuous.Rebaudioside A(RA)has better taste than SS and RC.Therefore,SS and RC are used as a raw material for structural modification.The structure of rare glycosides rubusoside(RS)and dulcoside A(DA)are respectively lacking a gluosyl in site C13 compared with SS and RC.However,the flavor and pharmacological activities of RS and DA are better than SS and RC.Thus,it is significant to transform SS and RC into RS and DA.In this work,the microorganisms Penicillium pinophilum and Staphylococcus saprophyticus XJ-3 were screend to transform SS and RC into RS and DA.Further researches were focused on the factors which influenced the transformation effect of Penicillium pinophilum:enzyme production type,substrate concentration,temperature and pH.The results showed that conversion rate of SS and RC reached to 99.11%and 55.63%respectively after converted for 48h by enzyme from Penicillium pinophilum(cultivated by solid medium).The transformation conditions:steviol glycoside 2%,55?,pH5.0.To separate the transformation compounds,the results showed that polyamide could separate RS from other steviol glycoside,macroporous resin AB-8 could separate DA and RC.The RS adsorbed by polyamide was eluted by ethanol aqueous solution,after being concentrated and dried,finally,the purity of RS reached to 91.30%.The DA adsorbed by macroporous resin AB-8 was eluted by ethanol aqueous solution,after being concentrated and dried,finally,the purity of DA reached to 83.15%.The separation results of enzyme,which produced by Penicillium pinophilum,showed that SS invertase pruduced by Penicillium pinophilum was salted out by 80%saturation ammonium sulfate.Then RC invertase could be salted out by 50%or 80%saturation ammonium sulfate.However,the SDS-PAGE figure showed that the molecular weight of the proteins(salted out by 50%and 80%ammonium sulfate)were different.In addition,gel filtration chromatography experiment showed that the transformation of RC and SS were carried out by different enzymes.The conclusion:molecular weight of RC converting enzyme was smaller than that of SS converting enzyme,which could be obtained according to the principle of molecular sieve and the order of the two peak.The results of the purification of SS and RA and RC by recrystallization showed that when methanol solution contained 4%water recrystallized 30%content of steviol glycoside for 36h under 4?,90.70%purity of SS could be obtained and the crystallization rate was 26.71%.Then,the mother liquid which filtering out the SS crystal was recrystallized by 45%ethyl acetate-55%methanol solution,and the crystal of RA with purity of 84.93%could be obtained.Finally,the mother liquid which filtering out the RA crystal was dried by rotary evaporation,and RC with purity of 61.30%could be obtained.The work also analyzed the content of steviol glycoside and phenolic acid extracted from different cultivars of stevia rebaudiana.The results showed that cultivars 10 and cultivar 5 were the best cultivar of SS and RC suppling.Cultivar 10 contained 13.72%SS and 8.76%phenolic acid.Cultivar 5 contained 7.10%SS and 6.73%phenolic acid.In addition,the effect of storage time on the concentration of steviol glycoside and phenolic acid were investigated.The results showed that stevia phenolic acids decomposed rapidly with time while the content of steviol glycoside remained at the same level.