一株产菌核青霉的分离鉴定与生物学特性研究
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摘要
分别采取湖北神农架自然保护区、山西关帝山国家自然保护区、山西北岳恒山林区、山西五台山林区以及山西长治太行山大峡谷区域中的针叶林、混交林根部表层20cm处土壤,进行青霉菌的分离。经过菌落和个体形态观察,分离得到73株青霉,其中从山西五台山油松土壤中分离得到1株能产菌核的青霉,命名为Q1菌株。本实验按照Pitt的分类方法对Q1菌株在4种培养基上进行了菌落特征和个体形态特征观察。结果表明：Q1菌株在MEA和CYA培养基表面生长比较好,菌核内有一定色素积累,在CA培养基表面菌核颜色为黑色；菌核多为不规则型,帚状枝为单轮生,瓶梗5-8轮生,经检索,Q1菌株属于汤姆青霉系。
研究了无机盐和碳氮源对青霉Q1菌株菌核生物量的影响作用,结果表明：K2HPO4的单因子效果最好；K2HPO4+KCl+MgSO4表现出的正协同效应最好。FeSO4对菌核内色素的积累有一定的影响,FeSO4存在时菌核内不能积累色素,菌核生物量下降。5种碳源均能被Q1菌株利用,当以麦芽糖为唯一碳源时,得到的菌核生物量明显高于其他糖；3种有机氮源都能够使Q1菌株形成菌核,其中酵母膏最为有利,形成的菌核量最高。培养基的含碳量保持在20 g/L左右,含氮量保持在0.24-0.48 g/L时,对Q1菌株菌核形成较有利。
通过紫外光谱全波扫描和薄层层析,结果显示,Q1菌核内色素为类胡萝卜素。同时对色素的稳定性进行了初步研究,Q1菌株的色素对光照比较敏感,见光易分解；温度对Q1菌株的色素稳定性影响较小,但过高的温度会破坏其结构,色素保存率大大降低；色素对金属离子不稳定,应尽量避免与金属器物的接触,以便使色素得到最佳保存。
首次采用液体培养法对本实验室已有的两株产菌核青霉PT95、Q1菌株进行了培养,结果筛选出MEA液体培养基为最佳培养基,但需要在静置条件下。液体培养基能够使菌核成熟的时间缩短1-2d,菌核内积累的色素量也比固体培养基高。
Seventy-three strains of Penicillium were isolated from the soil samples collected from respectively coniferous forest and mixed forest of Shennongjia Nature Reserve in Hubei Province, Shanxi Guandi Mountain National Nature Reserve, Shanxi Beiyue Hengshan, Shanxi Wutai Mount, and Taihang Grand Canyon in Shanxi Changzhi. By observing colonies and individual morphology, one strain isolated from the pine trees soil in Shanxi Wutai Mountain was found to can produce sclerotia, and it was named as Q1 strain. In this study, the colonies and individual morphology characteristics of Q1 strain in four medium according to Pitt were observed. The results showed that Q1 strain could grow better in the surface of MEA and CYA medium and accumulate some pigment in sclerotia. The sclerotia color in CA medium was black, irregular, pinicilli monorerticilati strictly,5-8 whorls. Based the characters above,Q1 strain was identified to P.thomii series.
This study examined the respective effect of various inorganic salts, carbon and nitrogen sources on sclerotia biomass of Q1 strain. The results showed that K2HP04 was more essential to the sclerotia formation for Q1 strain. Positive cooperative effect of K2HPO4+KCl+MgSO4 was the best. FeSO4 had some effect on the accumulation of pigment in Sclerotia, and had negative effect on the formation of sclerotia. All of five carbon sources tested could be utilized by the strain Q1. The maltose was the best carbon source and could result in higher significantly sclerotia biomass than other carbon sources. Three kinds of organic nitrogen sources favored the formation of sclerotia of strain Q1.The yeast extract was the best nitrogen source. The highest sclerotia biomass was obtained when the carbon and nitrogen content of the medium was maintained respectively at 20 g/L and 0.24-0.48 g/L.
Full-wave UV spectra and TLC scanning results showed that the pigment in sclerotia of Q1 strain was carotenoids. At the same time, the stability of the pigment was studied. The pigment was sensitive to light, and under strong light easily decomposed. Under lower temperature the pigment was stable, but under high temperature it's structure would be destroyed and the pigment survival rate was significantly lower. The pigment was unstable under metal ions. So the pigment should avoid contact with metal objects in order to preserve it better.
Penicillium PT95 strain was another strain which could form sclerotia. In this study, PT95 and Q1 strain were incubated in liquid culture media to observe the formation of sclerotia. The result showed that under standing culture, MEA liquid medium was the best medium. Liquid medium could shorten the time of sclerotia mature for 1-2 days as compared with the solid medium. The accumulation of pigment in sclerotia in liquid medium was also higher than that in solid medium.
研究了无机盐和碳氮源对青霉Q1菌株菌核生物量的影响作用,结果表明：K2HPO4的单因子效果最好；K2HPO4+KCl+MgSO4表现出的正协同效应最好。FeSO4对菌核内色素的积累有一定的影响,FeSO4存在时菌核内不能积累色素,菌核生物量下降。5种碳源均能被Q1菌株利用,当以麦芽糖为唯一碳源时,得到的菌核生物量明显高于其他糖；3种有机氮源都能够使Q1菌株形成菌核,其中酵母膏最为有利,形成的菌核量最高。培养基的含碳量保持在20 g/L左右,含氮量保持在0.24-0.48 g/L时,对Q1菌株菌核形成较有利。
通过紫外光谱全波扫描和薄层层析,结果显示,Q1菌核内色素为类胡萝卜素。同时对色素的稳定性进行了初步研究,Q1菌株的色素对光照比较敏感,见光易分解；温度对Q1菌株的色素稳定性影响较小,但过高的温度会破坏其结构,色素保存率大大降低；色素对金属离子不稳定,应尽量避免与金属器物的接触,以便使色素得到最佳保存。
首次采用液体培养法对本实验室已有的两株产菌核青霉PT95、Q1菌株进行了培养,结果筛选出MEA液体培养基为最佳培养基,但需要在静置条件下。液体培养基能够使菌核成熟的时间缩短1-2d,菌核内积累的色素量也比固体培养基高。
Seventy-three strains of Penicillium were isolated from the soil samples collected from respectively coniferous forest and mixed forest of Shennongjia Nature Reserve in Hubei Province, Shanxi Guandi Mountain National Nature Reserve, Shanxi Beiyue Hengshan, Shanxi Wutai Mount, and Taihang Grand Canyon in Shanxi Changzhi. By observing colonies and individual morphology, one strain isolated from the pine trees soil in Shanxi Wutai Mountain was found to can produce sclerotia, and it was named as Q1 strain. In this study, the colonies and individual morphology characteristics of Q1 strain in four medium according to Pitt were observed. The results showed that Q1 strain could grow better in the surface of MEA and CYA medium and accumulate some pigment in sclerotia. The sclerotia color in CA medium was black, irregular, pinicilli monorerticilati strictly,5-8 whorls. Based the characters above,Q1 strain was identified to P.thomii series.
This study examined the respective effect of various inorganic salts, carbon and nitrogen sources on sclerotia biomass of Q1 strain. The results showed that K2HP04 was more essential to the sclerotia formation for Q1 strain. Positive cooperative effect of K2HPO4+KCl+MgSO4 was the best. FeSO4 had some effect on the accumulation of pigment in Sclerotia, and had negative effect on the formation of sclerotia. All of five carbon sources tested could be utilized by the strain Q1. The maltose was the best carbon source and could result in higher significantly sclerotia biomass than other carbon sources. Three kinds of organic nitrogen sources favored the formation of sclerotia of strain Q1.The yeast extract was the best nitrogen source. The highest sclerotia biomass was obtained when the carbon and nitrogen content of the medium was maintained respectively at 20 g/L and 0.24-0.48 g/L.
Full-wave UV spectra and TLC scanning results showed that the pigment in sclerotia of Q1 strain was carotenoids. At the same time, the stability of the pigment was studied. The pigment was sensitive to light, and under strong light easily decomposed. Under lower temperature the pigment was stable, but under high temperature it's structure would be destroyed and the pigment survival rate was significantly lower. The pigment was unstable under metal ions. So the pigment should avoid contact with metal objects in order to preserve it better.
Penicillium PT95 strain was another strain which could form sclerotia. In this study, PT95 and Q1 strain were incubated in liquid culture media to observe the formation of sclerotia. The result showed that under standing culture, MEA liquid medium was the best medium. Liquid medium could shorten the time of sclerotia mature for 1-2 days as compared with the solid medium. The accumulation of pigment in sclerotia in liquid medium was also higher than that in solid medium.
引文
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[4]姚圣梅,黎坚.白绢病菌菌核培养方法的研究[J].仲恺农业技术学院学报,1998,11(1)：6～10.
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[7]李国庆,王道本,周启等,核盘菌菌核多样性的研究[J].植物保护学报,1997,24(1)：59～64.
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[20]Purdy L H.Facors affecting apothecial production by Sclerotinia sclerotiorum[J]. Phytopathology.1956,46:409～410.
[21]Moore D. Developmental genetics of Coprinus cinereus:evidence that carpophores and sclerotia share a common pathway of initiation[J]. Current Genetics,1981,3:145～150.
[22]Marukawa S, Funakawa S, Satomura Y. Role of sclerin on morphogenesis in Sclerotinia sclerotiorum[J]. Agricultural and Biological Chemistry Bulletin,1975,39:645～650.
[23]Huang H C and Kozub G C. A simple method for production of apothecia from sclerotia of Sclerotinia scleratiarum, Plant Prot[J]. Bull,1989, 31:333～345.
[24]Kybal J. Changes in N and P content during growth of ergot sclerotia (Claviceps purpurea) due to nutrition supplied by rye[J]. Phytopathology, 1964,54:244～245.
[25]Briton G. The biochemistry of natural pigments[M]. Cambridge:Cambridge University Press,1983.
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[27]姜文侯,单志萍,孟好.β-胡萝卜素的应用、市场和天然型产品的发酵法生 产[J].食品与发酵工业,1994,20(3)：65～71.
[28]Weedon BCL, Moss GP. Structure and nomenclature of carotenoids[J]. Pure and Applied Chemistry,1995,41:407～413.
[29]Nelis H J, De Leenheer A P. Microbial sources of carotenoid pigments used in foods and feeds [J]. Journal of Bacteriology.1991,70:180～191.
[30]Moss GP, Weedon BCL. Chemistry and Biochemistry of Plant Pigment[J]. London:Academic Press,1976,148～149.
[31]Margalith P Z.Pigment microbiology[M]. London Chapman&Hall,1992, 59～65.
[32]Zechmeister L. Cis-trans isomeric carotenoids vitamins A and arylpolyenes[M]. Vienna:Springer-Verlag,1962,155～160.
[33]Tee E S.The Analysis of Carotenoid and retinoids[J]. Food Chemistry. 1991, (41):147～149.
[34]李洋,徐雅琴.超声波法提取沙棘类胡萝卜素条件的优化[J].食品科技2008,(1)：137～139.
[35]薛伟明,张效林,亢茂德等.红花黄色素的酶法提取应用研究[J].化学工程,1999,27(1)：42～50.
[36]张春兰,冯作山.微法萃取构祀色素的研究[Z],信阳农业高等专科学校报,2004,(1)：10～11.
[37]刘敬业,丘玲,王亮.薄层色谱分离测定烤烟中的β-胡萝卜素[J].昆明师专学校(自然科学版),1998,13(1)：1～3.
[38]夏薇,陈新志.烟叶中β-胡萝卜素的分离纯化和测定[J].色谱,2004,22(1)：54～56.
[39]徐响,刘光敏,王琦等.反相HPLC法测定沙棘全果油中类胡萝卜素[J].食品工业科技,2007,28(12)：206～207.
[40]Romns H S, Harry H S. Evaluation and validation of an LC method for the analysis of carotenoids in vegetables and fruit[J]. Food Chemistry. 1997,59:599～603.
[41]杜咏梅,张怀宝,王晓玲等.光度法测定烟草中总类胡萝卜素的方法研究[J].中国烟草科学,2003,(3)：28～29.
[42]彭光华,李忠,张声华等.薄层色谱法分离鉴定枸杞子中的类胡萝卜素[J].营 养学报,1998,20(1)：76～78.
[43]左天觉.烟草的生产生理和生物化学[M].朱尊权译.上海：上海远东出版社,1993：386～396.
[44]杨庆尧.食用菌生物学[M].上海：上海科学技术出版社,1981.
[45]贾新成.食用菌的深层发酵与液体菌种的生产[J].河南科技,1991,(11)：15～16.
[46]王荣菜.香菇液体菌种的培养与应用[J].杭州食品科技,2000,59(4)：32～37.
[47]张李阳,熊晓辉,沈昌等.灵芝子实体及其深层培养产物特性的研究[J].中国食用菌,1998,12(3)：35～38.
[48]韩建荣,王肖娟,原香娥.青霉PT95菌株菌核内产类红萝卜素的研究[J].微生物学通报,1998,25(6)：319～321
[49]Quintanilla J. Penicillium caerulescens sp. nov. isolated from soil [J]. Mycopathologia,1983,82:101～104.
[2]Pitt JI. A laboratory guide common Penicilliuw species[M]. North Ryde: Division Of Food Processing,1988.
[3]Raper K B, Thom C. Manual of the Penicillia[M]. Baltimore:William & Wilkins Company,1949.
[4]姚圣梅,黎坚.白绢病菌菌核培养方法的研究[J].仲恺农业技术学院学报,1998,11(1)：6～10.
[5]Backhouse D, Willetts H J. Development and structure of infection cushion of Botrytis cinerea[J]. Transactions of the British Mycological Society, 1987,89:89～95.
[6]Garrett S D. Pathogenic root-infecting fungi[M]. Cambridge:Campridge University Press,1970.
[7]李国庆,王道本,周启等,核盘菌菌核多样性的研究[J].植物保护学报,1997,24(1)：59～64.
[8]Cooke R C. Changes in soluble carbohydrates during sclerotium formation by Sclerotinia sclerotiorum and S. trifoliorum[J]. Transactions of the British Mycological Society,1969,53:77～86.
[9]Townsend B B, Willetts H J. The development of sclerotia of certain fungi[J]. Transactions of the British Mycological Society,1954,37: 213～221.
[10]贺新生,张玲.羊肚菌菌丝体的培养[J].资源开发技术,1994,10(1)：23～25.
[11]Garrett S D. Soil fungi and soil fertility[M]. Oxford:Pergamon Press, 1963.
[12]尚春树,李素玲.羊肚菌驯化栽培试验初报[J],山西农业科学,1997,25(1)：88～90.
[13]Willetts H J, Wong A L. Ontogenetic diversity of sclerotia of Sclerotinia sclerotiorum and related species[J]. Transactions of the British Mycological Society,1971,57:515～524.
[14]王秀云.羊肚菌的菌核形成初探[J],食用菌,1997,(6)：4～5.
[15]Chanter D O, Thornley JHM. Mycelial growth and the initiation and growth of sporophores in the mushroom crop:a mathematical model[J]. Journal of General Microbiology,1978,106:55～65.
[16]Cooke R C. Morphogenesis of sclerotia. In:Smith JE (Ed.), Fungal differentiation, a contemporary Synthesis [J]. New York:Marcel Dekker, 1983. pp.397～418.
[17]Coley-Smith J R, Cooke R C. Survival and germination of fungal sclerotia[J]. Annual Review of Phytopathology,1971,9:65～92.
[18]Willetts H J, Bullock S. Developmental biology of sclerotia[J]. Mycological Research,1992,96:801～816.
[19]Chet I, Henis Y. Sclerotial morphogenesis in fungi[J]. Annual Review of Phytopathology,1975,13:169～192.
[20]Purdy L H.Facors affecting apothecial production by Sclerotinia sclerotiorum[J]. Phytopathology.1956,46:409～410.
[21]Moore D. Developmental genetics of Coprinus cinereus:evidence that carpophores and sclerotia share a common pathway of initiation[J]. Current Genetics,1981,3:145～150.
[22]Marukawa S, Funakawa S, Satomura Y. Role of sclerin on morphogenesis in Sclerotinia sclerotiorum[J]. Agricultural and Biological Chemistry Bulletin,1975,39:645～650.
[23]Huang H C and Kozub G C. A simple method for production of apothecia from sclerotia of Sclerotinia scleratiarum, Plant Prot[J]. Bull,1989, 31:333～345.
[24]Kybal J. Changes in N and P content during growth of ergot sclerotia (Claviceps purpurea) due to nutrition supplied by rye[J]. Phytopathology, 1964,54:244～245.
[25]Briton G. The biochemistry of natural pigments[M]. Cambridge:Cambridge University Press,1983.
[26]Sandmann G. Carotenoid biosynthesis in microorganisms and plants[J]. European Journal of Biochemistry,1994,223:17～24.
[27]姜文侯,单志萍,孟好.β-胡萝卜素的应用、市场和天然型产品的发酵法生 产[J].食品与发酵工业,1994,20(3)：65～71.
[28]Weedon BCL, Moss GP. Structure and nomenclature of carotenoids[J]. Pure and Applied Chemistry,1995,41:407～413.
[29]Nelis H J, De Leenheer A P. Microbial sources of carotenoid pigments used in foods and feeds [J]. Journal of Bacteriology.1991,70:180～191.
[30]Moss GP, Weedon BCL. Chemistry and Biochemistry of Plant Pigment[J]. London:Academic Press,1976,148～149.
[31]Margalith P Z.Pigment microbiology[M]. London Chapman&Hall,1992, 59～65.
[32]Zechmeister L. Cis-trans isomeric carotenoids vitamins A and arylpolyenes[M]. Vienna:Springer-Verlag,1962,155～160.
[33]Tee E S.The Analysis of Carotenoid and retinoids[J]. Food Chemistry. 1991, (41):147～149.
[34]李洋,徐雅琴.超声波法提取沙棘类胡萝卜素条件的优化[J].食品科技2008,(1)：137～139.
[35]薛伟明,张效林,亢茂德等.红花黄色素的酶法提取应用研究[J].化学工程,1999,27(1)：42～50.
[36]张春兰,冯作山.微法萃取构祀色素的研究[Z],信阳农业高等专科学校报,2004,(1)：10～11.
[37]刘敬业,丘玲,王亮.薄层色谱分离测定烤烟中的β-胡萝卜素[J].昆明师专学校(自然科学版),1998,13(1)：1～3.
[38]夏薇,陈新志.烟叶中β-胡萝卜素的分离纯化和测定[J].色谱,2004,22(1)：54～56.
[39]徐响,刘光敏,王琦等.反相HPLC法测定沙棘全果油中类胡萝卜素[J].食品工业科技,2007,28(12)：206～207.
[40]Romns H S, Harry H S. Evaluation and validation of an LC method for the analysis of carotenoids in vegetables and fruit[J]. Food Chemistry. 1997,59:599～603.
[41]杜咏梅,张怀宝,王晓玲等.光度法测定烟草中总类胡萝卜素的方法研究[J].中国烟草科学,2003,(3)：28～29.
[42]彭光华,李忠,张声华等.薄层色谱法分离鉴定枸杞子中的类胡萝卜素[J].营 养学报,1998,20(1)：76～78.
[43]左天觉.烟草的生产生理和生物化学[M].朱尊权译.上海：上海远东出版社,1993：386～396.
[44]杨庆尧.食用菌生物学[M].上海：上海科学技术出版社,1981.
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