Study On The Enrichment Of Ceramide By Saccharomyces Kluyveri Yeast And Its Effect

Ceramide is a kind of lipid substance which is widely found in eukaryotic cells.At present,most of the researches on ceramide are concentrated in plants and mammals,and the production feasibility of ceramide is rarely examined from the viewpoint of microbial fermentation.In this study,four strains of Saccharomyces kluyveri were used as test strains and theoptimal conditions for the growth of Saccharomyces kluyveri yeast were determined by plate count method.The total lipids,polarity,neutral lipids,and ceramide contents of fourstrains of Saccharomyces kluyveri were compared and analyzed,and the highest ceramide content of Saccharomyces kluyveri was screened.The effects of environmental conditions and medium components on the enrichment of ceramide by Saccharomyces kluyveri were further studied.On this basis,combined with thin layer chromatography(TLC),high-phase liquid chromatography(HPLC),silica gel column layers the molecularstructure of the ceramide monomer compound was identified by gas chromatography-mass spectrometry(GC-MS).The results were showed as follows:(1)By studying the culture conditions of four strains of Saccharomyces kluyveri,the optimum culture conditions for the growth of Saccharomyces kluyveri yeast were determined.Under the same culture conditions,four strains of Saccharomyces kluyveri cerevisiae were used for fermentation and the total lipids,polarity,neutral lipids and ceramide contents were compared and analyzed.The results showed that the temperature of 28°C,time of 72 h,inoculum volume of 10.0%,and liquid volume of 100 m L/250 m L were the optimum conditions for the culture of four strains of Saccharomyces kluyveri.The total lipid content of strain No.10955 was the highest after fermentation.The No.1892 strain had the lowest total lipid content;in the total lipidcomposition of the four strains of Saccharomyces kluyveri,the proportion of neutral lipids was slightly higher than that of polar lipids,No.1892 strain had a higher neutral lipid content than the other three strains had,and No.10955 strain had a higher polar lipid content than theother three strains did;Thin-layer chromatography showed polar lipids had functional components such as ceramide(CM),phosphatidylethanolamine(PE),phosphatidylcholine(PC),triglyceride(TG)and ceramide the content of was:strain 10955>strain 10847>strain 1685>strain 1892,in which 10955 strain had the highest ceramide content,which reached 0.39mg/g(2)In order to improve the production of ceramide by Saccharomyces kluyveri,the effects of culture time,culture temperature,beet molasses addition,different concentrations of peptone and diff erent concentrations of ethanol on the enrichment of ceramide were studied.The results showed th at among the five influencing factors,the increase of beet molasses addition(8.0%)and ethanol c oncentration(12.0%)could effectively increase the intracellular ceramide content.Under these condi tions,the ceramide content increased by 1.8 and 1.6 times,respectively.(3)A relatively pure ceramide fraction was obtained by gradient elution on a silica gel column using chloroform:methanol as eluent.Thin-layer chromatography,high-phase liquid chromatographyand gas chromatography-mass spectrometry were used.Combined use of qualitative and quantitative ceramide components.The results showed that in the proportion of chloroform,chloroform/methanol(90:10,v/v),(80:20,v/v),(70:30,v/v),(50:50,v/v)and methanol elution,,higher purity ceramides can be isolated in chloroform/methanol(50:50,v/v);And with the formula of C₄₄H₈₉NO_5,named 2 hydroxy-N-(1,3,4-trihydroxynonadecan-2-yl)pentadecaneamide,ceramide compounds were identified to be gotten.This result laid the foundation for the future Saccharomyces kluyveri yeast as a new source of extraction and development of ceramides.