少根根霉多样化的生长动力学模型
|
摘要
【背景】少根根霉物种内菌株间生理生化指标存在差异,相应的遗传背景不明,不利于少根根霉生产发酵的进一步应用。【目的】探究不同少根根霉菌株温度-生长动力学模型间的差异,为其群体遗传研究奠定基础,为生产菌株的筛选提供依据。【方法】选取来自欧亚各地的纯种少根根霉为实验材料,通过形态学鉴定,以及ITS和IGSrDNA分子系统发育重建进行分子鉴定,采用培养基平板培养直接测量法进行温度-生长动力学分析。【结果】少根根霉温度-生长动力学模型呈现丰富的多样性,与各形态和分子系统发育变种基本不具有相关性。菌株间温度-生长速度曲线具有显著性差异。少根根霉生长抑制低温范围、最适生长温度范围、生长抑制高温范围和致死高温范围分别为4-9、30-37、40-49和40-52°C。菌株XY00454和XY00469具有良好的高温适应性和较快的生长速度,有开发成为工业发酵菌的潜力。【结论】少根根霉物种仍然处于剧烈的演化之中,种内形态、分子和生理分化较为活跃,但尚未形成任何独立的种群。根据温度适应性的数据可以筛选出发酵生产潜力菌株。
[Background] Strains of Rhizopus arrhizus vary in physiological and biochemical indicators, but relative genetic background is unclear, hindering their further applications in fermentation. [Objective] This study explored temperature-growth kinetic models among strains of R. arrhizus in order to lay a foundation for investigating population genetics and for screening materials potential in production. [Methods] R. arrhizus isolated from Asia and Europe were first identified by morphology and then by molecular phylogeny reconstructed with ITS and IGS rDNA. Finally, their temperature-growth kinetics was analyzed by directly measuring colonial diameters on medium plates. [Results] The temperature-growth kinetic models of R. arrhizus were diverse, and the curves significantly differ, with less relatedness to morphological and phylogenetic varieties. Lo wer restraining growth, optimum growth, higher restraining growth, and fatal temperatures were 4-9, 30-37, 40-49, and 40-52 °C, respectively. Strains XY00454 and XY00469 grew rapidly and adapted well to higher temperatures and therefore were potential for industrial production. [Conclusion] R. arrhizus is still evolving violently and diverging actively in morphology, molecular and physiology, while not developing any independent populations. It is feasible to screen fermentation potential isolates based on thermal adaptability.
[Background] Strains of Rhizopus arrhizus vary in physiological and biochemical indicators, but relative genetic background is unclear, hindering their further applications in fermentation. [Objective] This study explored temperature-growth kinetic models among strains of R. arrhizus in order to lay a foundation for investigating population genetics and for screening materials potential in production. [Methods] R. arrhizus isolated from Asia and Europe were first identified by morphology and then by molecular phylogeny reconstructed with ITS and IGS rDNA. Finally, their temperature-growth kinetics was analyzed by directly measuring colonial diameters on medium plates. [Results] The temperature-growth kinetic models of R. arrhizus were diverse, and the curves significantly differ, with less relatedness to morphological and phylogenetic varieties. Lo wer restraining growth, optimum growth, higher restraining growth, and fatal temperatures were 4-9, 30-37, 40-49, and 40-52 °C, respectively. Strains XY00454 and XY00469 grew rapidly and adapted well to higher temperatures and therefore were potential for industrial production. [Conclusion] R. arrhizus is still evolving violently and diverging actively in morphology, molecular and physiology, while not developing any independent populations. It is feasible to screen fermentation potential isolates based on thermal adaptability.
引文
[1]Zheng RY,Chen GQ,Huang H,et al.A monograph of Rhizopus[J].Sydowia,2007,59(2):273-372
[2]Coban HB,Demirci A.Enhancement and modeling of microparticle-added Rhizopus oryzae lactic acid production[J].Bioprocess and Biosystems Engineering,2016,39(2):323-330
[3]Thitiprasert S,Songserm P,Boonkong W,et al.Manipulating pyruvate decarboxylase by addition of enzyme regulators during fermentation of Rhizopus oryzae to enhance lactic acid production[J].Applied Biochemistry and Biotechnology,2014,174(5):1795-1809
[4]Naude A,Nicol W.Fumaric acid fermentation with immobilised Rhizopus oryzae:quantifying time-dependent variations in catabolic flux[J].Process Biochemistry,2017,56:8-20
[5]Pimtong V,Ounaeb S,Thitiprasert S,et al.Enhanced effectiveness of Rhizopus oryzae by immobilization in a static bed fermentor for L-lactic acid production[J].Process Biochemistry,2017,52:44-52
[6]Liu Y,Yang Y,Sun T,et al.Optimization of medium components and fermentation conditions for L-malic acid production by Rhizopus oryzae[J].Food Science,2015,36(11):100-109(in Chinese)刘亚,杨英,孙婷,等.米根霉发酵产L-苹果酸的工艺优化[J].食品科学,2015,36(11):100-109
[7]Wang WW,Ren PK.Selective breeding of gamma linolenic acid high-yielding strain of Rhizopus arrhizus and studies on solid state fermentation conditions[J].Mycosystema,2002,21(1):92-97(in Chinese)王卫卫,任鹏康.少根根霉γ-亚麻酸高产菌株选育及发酵条件优化[J].菌物系统,2002,21(1):92-97
[8]Wang WW,Ren PK,Yan M,et al.Studies on solid state fermentation conditions of GLA lipid from Rhizopus arrhizus[J].Food Science,2002,23(6):94-98(in Chinese)王卫卫,任鹏康,闫明,等.少根根霉γ-亚麻酸固态发酵条件的研究[J].食品科学,2002,23(6):94-98
[9]Thanh VN,Thuy NT,Chi NT,et al.New insight into microbial diversity and functions in traditional Vietnamese alcoholic fermentation[J].International Journal of Food Microbiology,2016,232:15-21
[10]Guillén M,Benaiges MD,Valero F.Improved ethyl butyrate synthesis catalyzed by an immobilized recombinant Rhizopus oryzae lipase:a comprehensive statistical study by production,reaction rate and yield analysis[J].Journal of Molecular Catalysis B:Enzymatic,2016,133(S1):S371-S376
[11]Ortega S,Máximo MF,Montiel MC,et al.Esterification of polyglycerol with polycondensed ricinoleic acid catalysed by immobilised Rhizopus oryzae lipase[J].Bioprocess and Biosystems Engineering,2013,36(9):1291-1302
[12]Zheng YX,Wang YL,Pan J,et al.Semi-continuous production of high-activity pectinases by immobilized Rhizopus oryzae using tobacco wastewater as substrate and their utilization in the hydrolysis of pectin-containing lignocellulosic biomass at high solid content[J].Bioresource Technology,2017,241:1138-1144
[13]He Y,Zheng YX,Pan J,et al.Pectinase production with Rhizopus oryzae mycelium immobilized on cotton matrix and treatment of tobacco stem[J].Chemical Industry and Engineering Progress,2016,35(5):1494-1501(in Chinese)何源,郑羽西,潘君,等.棉布支架固定化米根霉联产果胶酶及用于处理烟梗[J].化工迚展,2016,35(5):1494-1501
[14]Zhang LQ,Gu S,Shao H,et al.Isolation,determination and aroma product characterization of fungus producing irnoe[J].Mycosystema,1999,18(1):49-54(in Chinese)张玱琪,谷甦,邵华,等.发酵产鸢尾酮真菌的分离鉴定及生香特性的初步研究[J].菌物系统,1999,18(1):49-54
[15]Li CT,Tian FH,Li JN,et al.Diosgenin biotranformed by Rhizopus arrhizus var.arrhizus[J].Mycosystema,2012,31(5):754-761(in Chinese)李长田,田风华,李江楠,等.少根根霉原变种发酵生产薯蓣皂苷元[J].菌物学报,2012,31(5):754-761
[16]Li P,Sun CP,Wang Y,et al.Efficiently regioselective glucosylation of estrogen analogues mediated by fungus Rhizopus oryzae AS 3.2380[J].Catalysis Communications,2017,97:106-110
[17]Singh P,Paul S,Shivaprakash MR,et al.Stress response in medically important Mucorales[J].Mycoses,2016,59(10):628-635
[18]Kuang Q,Sun M,Shi DL,et al.Study on the L-lactic acid production by ammonia resistant strain R.oryzae JS-N02-02[J].Biotechnology,2005,15(4):65-67(in Chinese)匡群,孙梅,施大林,等.耐氨米根霉发酵生产L-乳酸的研究[J].生物技术,2005,15(4):65-67
[19]Lyu CW,Xu Q,Chen J,et al.Physiological characteristics of a high-glucose resistant Rhizopus oryzae[J].Chinese Journal of Bioprocess Engineering,2015,13(3):36-40,54(in Chinese)吕春微,徐晴,陈姣,等.耐高糖米根霉菌株的生理特性[J].生物加工过程,2015,13(3):36-40,54
[20]Liu YX.Research between pathogens identification and key control techniques of two Jujube Fruit Fungal Diseases in Xinjiang[D].Urumqi:Master’s Thesis of Xinjiang Agricultural University,2016(in Chinese)刘艳祥.新疆红枣果实两种真菌病害病原鉴定及关键防治技术研究[D].乌鲁木齐:新疆农业大学硕士学位论文,2016
[21]Ge HF,Chen GZ.Rhinocerebral mucormycosis caused by Rhizopus oryzae:a case report[J].Journal of Clinical Dermatology,2015,44(8):505-507(in Chinese)葛红芬,陈官芝.米根霉致鼻脑毛霉病[J].临床皮肤科杂志,2015,44(8):505-507
[22]Xiu YH.Carbon utilization and growth kinetics of Shewanella putrefaciens isolated from fish[D].Shanghai:Master’s Thesis of Shanghai Ocean University,2017(in Chinese)修艳辉.鱼源腐败希瓦氏菌生长动力学及碳源代谢研究[D].上海:上海海洋大学硕士学位论文,2017
[23]White TJ,Bruns T,Lee S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenties[A]//Innis MA,Gelfand DH,Gelfand DH,et al.PCR Protocols:A Guide to Methods and Applications[M].San Diego:Academic Press,1990:315-322
[24]Liu XY.Preliminary studies on the molecular systematics of Mucorales[D].Beijing:Doctoral Dissertation of Chinese Academy of Sciences Institute of Microbiology,2008(in Chinese)刘小勇.毛霉目分子系统学初步研究[D].北京:中国科学院微生物研究所博士学位论文,2008
[25]Kearse M,Moir R,Wilson A,et al.Geneious basic:an integrated and extendable desktop software platform for the organization and analysis of sequence data[J].Bioinformatics,2012,28(12):1647-1649
[26]Liu XY,Huang H,Zheng RY.Delimitation of Rhizopus varieties based on IGS rDNA sequences[J].Sydowia,2008,60(1):93-112
[27]Liu XY,Huang H,Zheng RY.Molecular phylogenetic relationships within Rhizopus based on combined analyses of ITS rDNA and pyrG gene sequences[J].Sydowia,2007,59(2):235-253
[28]Yang JG,Zhao ZK,Ma YY.Preparation of pure-species Rhizopus oryzae starter and its technical optimization[J].Liquor-Making Science&Technology,2014(9):33-36,41(in Chinese)杨建刚,赵中开,马莹莹.纯种米根霉种曲的制备及工艺条件优化[J].酿酒科技,2014(9):33-36,41
[29]Wang P,Zhang FY.Study on the growth features and the fermenting properties of Rhizopus oryzae F6[J].Liquor-Making Science&Technology,2014(3):21-24(in Chinese)王鹏,张凤英.米根霉F6生长和发酵特性的研究[J].酿酒科技,2014(3):21-24
[30]Li JS,Zhu CL.Contrast of the properties of five quality Rhizopus strains and their application[J].Liquor-Making Science&Technology,2005(3):43-45(in Chinese)李釐生,朱春林.五株优良根霉的特性比较及其推广应用[J].酿酒科技,2005(3):43-45
[2]Coban HB,Demirci A.Enhancement and modeling of microparticle-added Rhizopus oryzae lactic acid production[J].Bioprocess and Biosystems Engineering,2016,39(2):323-330
[3]Thitiprasert S,Songserm P,Boonkong W,et al.Manipulating pyruvate decarboxylase by addition of enzyme regulators during fermentation of Rhizopus oryzae to enhance lactic acid production[J].Applied Biochemistry and Biotechnology,2014,174(5):1795-1809
[4]Naude A,Nicol W.Fumaric acid fermentation with immobilised Rhizopus oryzae:quantifying time-dependent variations in catabolic flux[J].Process Biochemistry,2017,56:8-20
[5]Pimtong V,Ounaeb S,Thitiprasert S,et al.Enhanced effectiveness of Rhizopus oryzae by immobilization in a static bed fermentor for L-lactic acid production[J].Process Biochemistry,2017,52:44-52
[6]Liu Y,Yang Y,Sun T,et al.Optimization of medium components and fermentation conditions for L-malic acid production by Rhizopus oryzae[J].Food Science,2015,36(11):100-109(in Chinese)刘亚,杨英,孙婷,等.米根霉发酵产L-苹果酸的工艺优化[J].食品科学,2015,36(11):100-109
[7]Wang WW,Ren PK.Selective breeding of gamma linolenic acid high-yielding strain of Rhizopus arrhizus and studies on solid state fermentation conditions[J].Mycosystema,2002,21(1):92-97(in Chinese)王卫卫,任鹏康.少根根霉γ-亚麻酸高产菌株选育及发酵条件优化[J].菌物系统,2002,21(1):92-97
[8]Wang WW,Ren PK,Yan M,et al.Studies on solid state fermentation conditions of GLA lipid from Rhizopus arrhizus[J].Food Science,2002,23(6):94-98(in Chinese)王卫卫,任鹏康,闫明,等.少根根霉γ-亚麻酸固态发酵条件的研究[J].食品科学,2002,23(6):94-98
[9]Thanh VN,Thuy NT,Chi NT,et al.New insight into microbial diversity and functions in traditional Vietnamese alcoholic fermentation[J].International Journal of Food Microbiology,2016,232:15-21
[10]Guillén M,Benaiges MD,Valero F.Improved ethyl butyrate synthesis catalyzed by an immobilized recombinant Rhizopus oryzae lipase:a comprehensive statistical study by production,reaction rate and yield analysis[J].Journal of Molecular Catalysis B:Enzymatic,2016,133(S1):S371-S376
[11]Ortega S,Máximo MF,Montiel MC,et al.Esterification of polyglycerol with polycondensed ricinoleic acid catalysed by immobilised Rhizopus oryzae lipase[J].Bioprocess and Biosystems Engineering,2013,36(9):1291-1302
[12]Zheng YX,Wang YL,Pan J,et al.Semi-continuous production of high-activity pectinases by immobilized Rhizopus oryzae using tobacco wastewater as substrate and their utilization in the hydrolysis of pectin-containing lignocellulosic biomass at high solid content[J].Bioresource Technology,2017,241:1138-1144
[13]He Y,Zheng YX,Pan J,et al.Pectinase production with Rhizopus oryzae mycelium immobilized on cotton matrix and treatment of tobacco stem[J].Chemical Industry and Engineering Progress,2016,35(5):1494-1501(in Chinese)何源,郑羽西,潘君,等.棉布支架固定化米根霉联产果胶酶及用于处理烟梗[J].化工迚展,2016,35(5):1494-1501
[14]Zhang LQ,Gu S,Shao H,et al.Isolation,determination and aroma product characterization of fungus producing irnoe[J].Mycosystema,1999,18(1):49-54(in Chinese)张玱琪,谷甦,邵华,等.发酵产鸢尾酮真菌的分离鉴定及生香特性的初步研究[J].菌物系统,1999,18(1):49-54
[15]Li CT,Tian FH,Li JN,et al.Diosgenin biotranformed by Rhizopus arrhizus var.arrhizus[J].Mycosystema,2012,31(5):754-761(in Chinese)李长田,田风华,李江楠,等.少根根霉原变种发酵生产薯蓣皂苷元[J].菌物学报,2012,31(5):754-761
[16]Li P,Sun CP,Wang Y,et al.Efficiently regioselective glucosylation of estrogen analogues mediated by fungus Rhizopus oryzae AS 3.2380[J].Catalysis Communications,2017,97:106-110
[17]Singh P,Paul S,Shivaprakash MR,et al.Stress response in medically important Mucorales[J].Mycoses,2016,59(10):628-635
[18]Kuang Q,Sun M,Shi DL,et al.Study on the L-lactic acid production by ammonia resistant strain R.oryzae JS-N02-02[J].Biotechnology,2005,15(4):65-67(in Chinese)匡群,孙梅,施大林,等.耐氨米根霉发酵生产L-乳酸的研究[J].生物技术,2005,15(4):65-67
[19]Lyu CW,Xu Q,Chen J,et al.Physiological characteristics of a high-glucose resistant Rhizopus oryzae[J].Chinese Journal of Bioprocess Engineering,2015,13(3):36-40,54(in Chinese)吕春微,徐晴,陈姣,等.耐高糖米根霉菌株的生理特性[J].生物加工过程,2015,13(3):36-40,54
[20]Liu YX.Research between pathogens identification and key control techniques of two Jujube Fruit Fungal Diseases in Xinjiang[D].Urumqi:Master’s Thesis of Xinjiang Agricultural University,2016(in Chinese)刘艳祥.新疆红枣果实两种真菌病害病原鉴定及关键防治技术研究[D].乌鲁木齐:新疆农业大学硕士学位论文,2016
[21]Ge HF,Chen GZ.Rhinocerebral mucormycosis caused by Rhizopus oryzae:a case report[J].Journal of Clinical Dermatology,2015,44(8):505-507(in Chinese)葛红芬,陈官芝.米根霉致鼻脑毛霉病[J].临床皮肤科杂志,2015,44(8):505-507
[22]Xiu YH.Carbon utilization and growth kinetics of Shewanella putrefaciens isolated from fish[D].Shanghai:Master’s Thesis of Shanghai Ocean University,2017(in Chinese)修艳辉.鱼源腐败希瓦氏菌生长动力学及碳源代谢研究[D].上海:上海海洋大学硕士学位论文,2017
[23]White TJ,Bruns T,Lee S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenties[A]//Innis MA,Gelfand DH,Gelfand DH,et al.PCR Protocols:A Guide to Methods and Applications[M].San Diego:Academic Press,1990:315-322
[24]Liu XY.Preliminary studies on the molecular systematics of Mucorales[D].Beijing:Doctoral Dissertation of Chinese Academy of Sciences Institute of Microbiology,2008(in Chinese)刘小勇.毛霉目分子系统学初步研究[D].北京:中国科学院微生物研究所博士学位论文,2008
[25]Kearse M,Moir R,Wilson A,et al.Geneious basic:an integrated and extendable desktop software platform for the organization and analysis of sequence data[J].Bioinformatics,2012,28(12):1647-1649
[26]Liu XY,Huang H,Zheng RY.Delimitation of Rhizopus varieties based on IGS rDNA sequences[J].Sydowia,2008,60(1):93-112
[27]Liu XY,Huang H,Zheng RY.Molecular phylogenetic relationships within Rhizopus based on combined analyses of ITS rDNA and pyrG gene sequences[J].Sydowia,2007,59(2):235-253
[28]Yang JG,Zhao ZK,Ma YY.Preparation of pure-species Rhizopus oryzae starter and its technical optimization[J].Liquor-Making Science&Technology,2014(9):33-36,41(in Chinese)杨建刚,赵中开,马莹莹.纯种米根霉种曲的制备及工艺条件优化[J].酿酒科技,2014(9):33-36,41
[29]Wang P,Zhang FY.Study on the growth features and the fermenting properties of Rhizopus oryzae F6[J].Liquor-Making Science&Technology,2014(3):21-24(in Chinese)王鹏,张凤英.米根霉F6生长和发酵特性的研究[J].酿酒科技,2014(3):21-24
[30]Li JS,Zhu CL.Contrast of the properties of five quality Rhizopus strains and their application[J].Liquor-Making Science&Technology,2005(3):43-45(in Chinese)李釐生,朱春林.五株优良根霉的特性比较及其推广应用[J].酿酒科技,2005(3):43-45