高产油脂酵母BS01的分离鉴定及其油脂抗氧化活性评价
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摘要
利用苏丹黑细胞染色和菌泥染色从百色地区土壤中分离得到一株高产油脂酵母菌,编号为BS01。通过形态、生理生化特征及26S r DNA的D1/D2区和ITS序列分析得知,该产油酵母为圆红冬孢酵母(Rhodosporidium toruloides)。菌株BS01油脂含量为48.97%;其油脂具有较强的抗氧化能力,DPPH自由基清除IC50为59.1 mg/m L;脂肪酸成分GC-MS分析表明,其油脂主要成分为油酸、亚油酸、棕榈酸及硬脂酸,其中不饱和脂肪酸占51.29%,与常见植物油脂的组成类似,可以用作生物柴油生产原料。
A high oil-producing yeast strain was isolated from a soil in Baise by methods of sudan black B and Sudan Ⅲ staining,and named as strain BS01.Strain BS01 was identified by a polyphasic taxonomic approaches including morphological,physiological and biochemical characteristics and sequences analysis of 26 S r DNA gene D1/D2 domain and ITS region.It was found that strain BS01 belonged to Rhodosporidium toruloides with 48.97%of lipid in the cell.According to the DPPH radical scavenging assay,the IC50 value was 59.1 mg/m L indicating that the lipid from strain BS01 possessed effective(good) antioxidant activity.In addition,the fatty acid composition of lipid determined by Gas Chromatography-Mass Spectrometer(GC-MS) showed that oleic acid,linoleic acid,palmitic acid and stearic acid were the main components.And the amount of unsaturated fatty acid reached 51.29%,which was equivalent to the amount of plant oils.This result indicated that lipid from oleaginous yeast Rhodosporidium toruloides BS01 was alternative feedstock for producing biodiesel.
A high oil-producing yeast strain was isolated from a soil in Baise by methods of sudan black B and Sudan Ⅲ staining,and named as strain BS01.Strain BS01 was identified by a polyphasic taxonomic approaches including morphological,physiological and biochemical characteristics and sequences analysis of 26 S r DNA gene D1/D2 domain and ITS region.It was found that strain BS01 belonged to Rhodosporidium toruloides with 48.97%of lipid in the cell.According to the DPPH radical scavenging assay,the IC50 value was 59.1 mg/m L indicating that the lipid from strain BS01 possessed effective(good) antioxidant activity.In addition,the fatty acid composition of lipid determined by Gas Chromatography-Mass Spectrometer(GC-MS) showed that oleic acid,linoleic acid,palmitic acid and stearic acid were the main components.And the amount of unsaturated fatty acid reached 51.29%,which was equivalent to the amount of plant oils.This result indicated that lipid from oleaginous yeast Rhodosporidium toruloides BS01 was alternative feedstock for producing biodiesel.
引文
Bai F.Y.,J ia J.H.,and Liang H.Y.,2002,Molecular taxonomi study on the problematic Candida stains based on 26S rD NA D1/D2 domain sequence comparison,Junwu Xitong(My cosystema),21(1):27-32(白逢彦,贾建华,梁慧燕,2002假丝酵母属疑难菌株大亚基rD NA D1/D2区域序列分析及其分类学意义,菌物系统,21(1):27-32)
Bellou S.,Triantaphyllidou I.E.,Aggeli D.,Elazzazy A.M.Baeshen M.N.,and Aggelis G.,2016,Microbial oils as food additives:recent approaches for improving microbial oi production and its polyunsaturated fatty acid content,Curr Opin.Biotechnol.,37:24-35
Edem D.O.,2002,Palm oil:biochemical,physiological,nutri tional,hematological and toxicological aspects:a review Plant Foods for Human Nutrition,57(3-4):319-341
Gen Q.,Wang Q.,and Chi Z.M.,2014,Direct conversion of cas sava starch into single cell oil by co-cultures of the oleagi nous yeast Rhodosporidium toruloides and immobilized amylases-producing yeast Saccharomycopsis fibuligera,Renewable Energy,62:522-526
Hu Y.L.,Zhong T.H.,Chan Z.H.,Liu R.Z.,Luo Z.H.,and Ye DZ.,2014,Breeding high-yield oil-producing strain to use cheap carbon source by UV induced protoplast mutagenesis Weishengwuxue Tongbao(Microbiology China),41(5):881-890(胡艳丽,钟添华,产竹华,刘润忠,骆祝华,叶德赞2014,原生质体紫外诱变选育可利用廉价碳源的高产油新菌株,微生物学通报,41(5):881-890)
Kurtzman C.P.,and Robnett C.J.,1998,Identification and phy logeny of ascomycetous yeasts from analysis of nuclea large subunit(26S)ribosomal DNA partial sequences,Anto nie van Leeuwenhoek,73(4):331-371
Ling J.Y.,Nip S.,de Toledo R.A.,Tian Y.,and Shim H.,2016Evaluation of specific lipid production and nutrients remova from wastewater by Rhodosporidium toruloides an biodiesel production from wet biomass via microwave irra diation,Energy,108:185-194
Ling J.Y.,Nip S.,and Shim H.,2013,Enhancement of lipid pro ductivity of Rhodosporidium toruloides in distillery wastewa ter by increasing cell density,Bioresour.Technol.,146301-309
Liu N.,and Liu Y.L.,2010,Application of ribosomal RNA gen for taxonomical classification of yeasts,Zhongguo Nongy Kexue(Scientia Gricultura Sinica),43(22):4701-4708(刘宁,刘延琳,2010,核糖体RNA基因在酵母分类鉴定中的应用,中国农业科学,43(22):4701-4708)
Matsakas L.,Bonturi N.,Miranda E.A.,Rova U.,and Chris takopoulos P.,2015,High concentrations of dried sorghum stalks as a biomass feedstock for single cell oil production by Rhodosporidium toruloides,Biotechnol.Biofuels,8(1):6
Meng X.,Yang J.M.,Xu X.,Zhang L.,Nie Q.J.,and Xian M.2009,Biodiesel production from oleaginous microorgan isms,Renewable Energy,34(1):1-5
Ratledge C.,and Wynn J.P.,2002,The biochemistry and molecu lar biology of lipid accumulation in oleaginous microorgan isms,Advances in Applied Microbiology,51:1-51
Samaram S.,Mirhosseini H.,Tan C.P.,Ghazali H.M.,Bordbar Sand Serjouie A.,2015,Optimisation of ultrasound-assisted extraction of oil from papaya seed by response surface methodology:oil recovery,radical scavenging antioxidant activi ty,and oxidation stability,Food Chemistry,172:7-17
Sitepu I.R.,Garay L.A.,Sestric R.,Levin D.,Block D.E.,Ger man J.B.,and Boundy-Mills K.L.,2014,Oleaginous yeast for biodiesel:current and future trends in biology and pro duction.Biotechnol.Adv.,32(7):1336-1360
Song A.D.,Feng C.,Wang F.Q.,and Xie H.,2009,Research o screening method of the high bio-oil production microorgan isms.Weishengwuxue Tongbao(Microbiology China),36(3):383-388(宋安东,冯冲,王风芹,谢慧,2009,生物油脂高产菌株筛选方法研究,微生物学通报,36(3):383-388)
Uckun Kiran E.,Salakkam A.,Trzcinski A.P.,Bakir U.,and Webb C.,2012,Enhancing the value of nitrogen from rape seed mea l for microbial oil production,Enzyme Microb Technol.,50(6-7):337-342
Wang Q.,Guo F.J.,Rong Y.J.,and Chi Z.M.,2012,Lipid pro duction from hydrolysate of cassava starch by Rhodosporidium toruloides 21167 for biodiesel making,Renewable Ener gy,46:164-168
Wang Z.P.,Fu W.J.,Xu H.M.,and Chi Z.M.,2014,Direct con version of inulin into cell lipid by an inulinase-producing yeast Rhodosporidium toruloides 2F5,Bioresource Technol ogy,161:131-136
Wei X.Y.,Yin L.Q.,Zhong C.,Zhang M.H.,and Niu Y.Z.,2014Advances in the DPPH radical scavenging assay for antioxi dant activity evaluation,Shipin Kexue(Food Science),35(9):317-322(韦献雅,殷丽琴,钟成,章明海,牛应泽,2014DPPH法评价抗氧化活性研究进展,食品科学,35(9)317-322)
Wu S.G.,Hu C.M.,Jin G.J.,Zhao X.,and Zhao Z.B.K.,2010Phosphate-limitation mediated lipid production by Rhodosporidium toruloides,Bioresource Technology,101(15):6124-6129
Wu S.G.,Zhao X.,Shen H.W.,Wang Q.,and Zhao Z.K.,2011Microbial lipid production by Rhodosporidium toruloides un der sulfate-limited conditions,Bioresource Technology,102(2):1803-1807
Wu X.M.,Wu G.Y.,Han Y.,and Zhang P.D.,2003,Fatty acid composition of six edible vegetable oils and their biodiese fuels,Zhongguo Youzhi(China Oils and Fats),28(12):65-67(巫淼鑫,邬国英,韩瑛张培娣,2003,6种食用植物油及其生物柴油中脂肪酸成分的比较研究,中国油脂,28(12):65-67)
Xu J.Y.,Zhao X.B.,Wang W.C.,Du W.,and Liu D.H.,2012Microbial conversion of biodiesel byproduct glycerol to tria cylglycerols by oleaginous yeast Rhodosporidium toruloide and the individual effect of some impurities on lipid produc tion,Biochemical Engineering Journal,65:30-36
Yang X.B.,Jin G.J.,Gong Z.W.,Shen H.B.,Bai F.W.,and Zhao Z.B.K.,2014,Recycling biodiesel-derived glycerol by th oleaginous yeast Rhodosporidium toruloides Y4 through th two-stage lipid production process,Biochemical Engineer ing Journal,91:86-91
Yang X.B.,Jin G.J.,Wang Y.Y.,Shen H.W.,and Zhao Z.B.K.2015,Lipid production on free fatty acids by oleaginous yeasts under non-growth conditions,Bioresour.Technol.,193557-562
Yu X.,Tian J.,Xie H.B.,Shen H.W.,and Wang Q.,2014,Th integrated production of microbial lipids and bio-SiO 2from rice husks by an organic electrolytes pretreatment technolo gy,Bioresour.Technol.,153:403-407
Zhao X.,Wu S.G.,Hu C.M.,Wang Q.,Hua Y.Y.,and Zhao Z.BK.,2010,Lipid production from Jerusalem artichoke b Rhodosporidium toruloides Y4,J.Ind.Microbiol.Biotech nol.,37(6):581-585
Zhou W.W.,Wang W.R.,Li Y.H.,and Zhang Y.K.,2013,Lipid production by Rhodosporidium toruloides Y2 in bioethano wastewater and evaluation of biomass energetic yield,Bior esource Technology,127:435-440
Bellou S.,Triantaphyllidou I.E.,Aggeli D.,Elazzazy A.M.Baeshen M.N.,and Aggelis G.,2016,Microbial oils as food additives:recent approaches for improving microbial oi production and its polyunsaturated fatty acid content,Curr Opin.Biotechnol.,37:24-35
Edem D.O.,2002,Palm oil:biochemical,physiological,nutri tional,hematological and toxicological aspects:a review Plant Foods for Human Nutrition,57(3-4):319-341
Gen Q.,Wang Q.,and Chi Z.M.,2014,Direct conversion of cas sava starch into single cell oil by co-cultures of the oleagi nous yeast Rhodosporidium toruloides and immobilized amylases-producing yeast Saccharomycopsis fibuligera,Renewable Energy,62:522-526
Hu Y.L.,Zhong T.H.,Chan Z.H.,Liu R.Z.,Luo Z.H.,and Ye DZ.,2014,Breeding high-yield oil-producing strain to use cheap carbon source by UV induced protoplast mutagenesis Weishengwuxue Tongbao(Microbiology China),41(5):881-890(胡艳丽,钟添华,产竹华,刘润忠,骆祝华,叶德赞2014,原生质体紫外诱变选育可利用廉价碳源的高产油新菌株,微生物学通报,41(5):881-890)
Kurtzman C.P.,and Robnett C.J.,1998,Identification and phy logeny of ascomycetous yeasts from analysis of nuclea large subunit(26S)ribosomal DNA partial sequences,Anto nie van Leeuwenhoek,73(4):331-371
Ling J.Y.,Nip S.,de Toledo R.A.,Tian Y.,and Shim H.,2016Evaluation of specific lipid production and nutrients remova from wastewater by Rhodosporidium toruloides an biodiesel production from wet biomass via microwave irra diation,Energy,108:185-194
Ling J.Y.,Nip S.,and Shim H.,2013,Enhancement of lipid pro ductivity of Rhodosporidium toruloides in distillery wastewa ter by increasing cell density,Bioresour.Technol.,146301-309
Liu N.,and Liu Y.L.,2010,Application of ribosomal RNA gen for taxonomical classification of yeasts,Zhongguo Nongy Kexue(Scientia Gricultura Sinica),43(22):4701-4708(刘宁,刘延琳,2010,核糖体RNA基因在酵母分类鉴定中的应用,中国农业科学,43(22):4701-4708)
Matsakas L.,Bonturi N.,Miranda E.A.,Rova U.,and Chris takopoulos P.,2015,High concentrations of dried sorghum stalks as a biomass feedstock for single cell oil production by Rhodosporidium toruloides,Biotechnol.Biofuels,8(1):6
Meng X.,Yang J.M.,Xu X.,Zhang L.,Nie Q.J.,and Xian M.2009,Biodiesel production from oleaginous microorgan isms,Renewable Energy,34(1):1-5
Ratledge C.,and Wynn J.P.,2002,The biochemistry and molecu lar biology of lipid accumulation in oleaginous microorgan isms,Advances in Applied Microbiology,51:1-51
Samaram S.,Mirhosseini H.,Tan C.P.,Ghazali H.M.,Bordbar Sand Serjouie A.,2015,Optimisation of ultrasound-assisted extraction of oil from papaya seed by response surface methodology:oil recovery,radical scavenging antioxidant activi ty,and oxidation stability,Food Chemistry,172:7-17
Sitepu I.R.,Garay L.A.,Sestric R.,Levin D.,Block D.E.,Ger man J.B.,and Boundy-Mills K.L.,2014,Oleaginous yeast for biodiesel:current and future trends in biology and pro duction.Biotechnol.Adv.,32(7):1336-1360
Song A.D.,Feng C.,Wang F.Q.,and Xie H.,2009,Research o screening method of the high bio-oil production microorgan isms.Weishengwuxue Tongbao(Microbiology China),36(3):383-388(宋安东,冯冲,王风芹,谢慧,2009,生物油脂高产菌株筛选方法研究,微生物学通报,36(3):383-388)
Uckun Kiran E.,Salakkam A.,Trzcinski A.P.,Bakir U.,and Webb C.,2012,Enhancing the value of nitrogen from rape seed mea l for microbial oil production,Enzyme Microb Technol.,50(6-7):337-342
Wang Q.,Guo F.J.,Rong Y.J.,and Chi Z.M.,2012,Lipid pro duction from hydrolysate of cassava starch by Rhodosporidium toruloides 21167 for biodiesel making,Renewable Ener gy,46:164-168
Wang Z.P.,Fu W.J.,Xu H.M.,and Chi Z.M.,2014,Direct con version of inulin into cell lipid by an inulinase-producing yeast Rhodosporidium toruloides 2F5,Bioresource Technol ogy,161:131-136
Wei X.Y.,Yin L.Q.,Zhong C.,Zhang M.H.,and Niu Y.Z.,2014Advances in the DPPH radical scavenging assay for antioxi dant activity evaluation,Shipin Kexue(Food Science),35(9):317-322(韦献雅,殷丽琴,钟成,章明海,牛应泽,2014DPPH法评价抗氧化活性研究进展,食品科学,35(9)317-322)
Wu S.G.,Hu C.M.,Jin G.J.,Zhao X.,and Zhao Z.B.K.,2010Phosphate-limitation mediated lipid production by Rhodosporidium toruloides,Bioresource Technology,101(15):6124-6129
Wu S.G.,Zhao X.,Shen H.W.,Wang Q.,and Zhao Z.K.,2011Microbial lipid production by Rhodosporidium toruloides un der sulfate-limited conditions,Bioresource Technology,102(2):1803-1807
Wu X.M.,Wu G.Y.,Han Y.,and Zhang P.D.,2003,Fatty acid composition of six edible vegetable oils and their biodiese fuels,Zhongguo Youzhi(China Oils and Fats),28(12):65-67(巫淼鑫,邬国英,韩瑛张培娣,2003,6种食用植物油及其生物柴油中脂肪酸成分的比较研究,中国油脂,28(12):65-67)
Xu J.Y.,Zhao X.B.,Wang W.C.,Du W.,and Liu D.H.,2012Microbial conversion of biodiesel byproduct glycerol to tria cylglycerols by oleaginous yeast Rhodosporidium toruloide and the individual effect of some impurities on lipid produc tion,Biochemical Engineering Journal,65:30-36
Yang X.B.,Jin G.J.,Gong Z.W.,Shen H.B.,Bai F.W.,and Zhao Z.B.K.,2014,Recycling biodiesel-derived glycerol by th oleaginous yeast Rhodosporidium toruloides Y4 through th two-stage lipid production process,Biochemical Engineer ing Journal,91:86-91
Yang X.B.,Jin G.J.,Wang Y.Y.,Shen H.W.,and Zhao Z.B.K.2015,Lipid production on free fatty acids by oleaginous yeasts under non-growth conditions,Bioresour.Technol.,193557-562
Yu X.,Tian J.,Xie H.B.,Shen H.W.,and Wang Q.,2014,Th integrated production of microbial lipids and bio-SiO 2from rice husks by an organic electrolytes pretreatment technolo gy,Bioresour.Technol.,153:403-407
Zhao X.,Wu S.G.,Hu C.M.,Wang Q.,Hua Y.Y.,and Zhao Z.BK.,2010,Lipid production from Jerusalem artichoke b Rhodosporidium toruloides Y4,J.Ind.Microbiol.Biotech nol.,37(6):581-585
Zhou W.W.,Wang W.R.,Li Y.H.,and Zhang Y.K.,2013,Lipid production by Rhodosporidium toruloides Y2 in bioethano wastewater and evaluation of biomass energetic yield,Bior esource Technology,127:435-440