烟叶陈化过程细菌群落演替特征
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摘要
仓储生态因子和烟叶化学成分的改变直接影响烟叶微生物群落结构和功能。以储存在贵阳库(GY)、坛厂库(TC)及紫云库(ZY)的云南保山C3F烟叶为研究对象,对不同陈化时间(0、6、12、18、24个月)的烟叶样品提取微生物总DNA,利用Illumina Hi Seq平台对细菌的16S rRNA V4区进行高通量测序,并结合主要化学成分分析,以期揭示烟叶陈化过程中细菌群落演替规律及其与烟叶化学成分间的作用关系。研究结果表明,烟叶细菌群落以假单胞菌属、鞘氨醇单胞菌属、寡养单胞菌属、芽孢杆菌属为优势属;随陈化时间的增加,细菌优势群落呈现出变形菌门和厚壁菌门间消长变化的趋势,陈化后期芽孢杆菌(厚壁菌门)优势度明显增强;烟叶陈化过程中,细菌优势功能群变化与化学成分逐级降解间呈显著的相关关系,主要表现为由降解糖类菌群向降解淀粉类菌群,再向降解纤维素类菌群变化的趋势;其中,影响菌群演替的关键因素是水溶性总糖和纤维素。研究结果揭示了在烟叶陈化过程中存在显著的细菌群落演替特征,加深了对烟叶陈化机制的理解,为微生物调控烟叶陈化过程提供了科学依据。
In this study,we investigated the relationship between the community succession and chemical composition of the bacteria isolated from tobacco. The tobacco sample C3F( 2013) obtained from Baoshan City,Yunnan Province,was stored in Guiyang( GY),Tanchang( TC),and Ziyun( ZY) storerooms. The 16 S rRNA genes of the bacteria isolated from the samples aged 0,6,12,18,and 24 months were sequenced using the Illumina HiSeq2500 high-throughput sequencing platform. The results showed that the Pseudomonas,Sphingomonas,Stenotrophomonas,and Bacillus genera were dominant in the bacterial community. With an increase in aging time,a growth-decline trend was observed between Proteobacteria and Firmicutes in the dominant bacterial communities, and the genus Bacillus was significantly dominant in the later successional stage. In the process of tobacco aging,a significant correlation was found between the dominant functional groups of bacteria and the progressive decomposition of their chemical components. The dynamics of the dominant functional groups of bacteria changed from carbohydrate-like to starch-like and then to cellulose-like. The water-soluble sugar and cellulose were the key factors involved in succession. This study revealed a significant characteristic of the bacterial community succession during aging in tobacco,thereby elucidating the mechanism of tobacco aging and providing a scientific basis for using microbes to control tobacco aging.
In this study,we investigated the relationship between the community succession and chemical composition of the bacteria isolated from tobacco. The tobacco sample C3F( 2013) obtained from Baoshan City,Yunnan Province,was stored in Guiyang( GY),Tanchang( TC),and Ziyun( ZY) storerooms. The 16 S rRNA genes of the bacteria isolated from the samples aged 0,6,12,18,and 24 months were sequenced using the Illumina HiSeq2500 high-throughput sequencing platform. The results showed that the Pseudomonas,Sphingomonas,Stenotrophomonas,and Bacillus genera were dominant in the bacterial community. With an increase in aging time,a growth-decline trend was observed between Proteobacteria and Firmicutes in the dominant bacterial communities, and the genus Bacillus was significantly dominant in the later successional stage. In the process of tobacco aging,a significant correlation was found between the dominant functional groups of bacteria and the progressive decomposition of their chemical components. The dynamics of the dominant functional groups of bacteria changed from carbohydrate-like to starch-like and then to cellulose-like. The water-soluble sugar and cellulose were the key factors involved in succession. This study revealed a significant characteristic of the bacterial community succession during aging in tobacco,thereby elucidating the mechanism of tobacco aging and providing a scientific basis for using microbes to control tobacco aging.
引文
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[8]贾蓓蕾,魏涛,黄申,贾春晓,杨靖,张改红,白冰,毛多斌.α-胡萝卜素降解产香菌株的分离、鉴定及发酵条件优化.食品与发酵工业,2015,41(1):34-39.
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[15]李永忠,丁金玲.烟草化学成分分析实验指导书[D].昆明:云南农业大学烟草学院,2007.
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[17]Caporaso J G,Kuczynski J,Stombaugh J,Bittinger K,Bushman F D,Costello E K,Fierer N,Pe1a A G,Goodrich J K,Gordon J I,Huttley GA,Kelley S T,Knights D,Koenig J E,Ley R E,Lozupone C A,Mc Donald D,Muegge B D,Pirrung M,Reeder J,Sevinsky J R,Turnbaugh PJ,Walters W A,Widmann J,Yatsunenko T,Zaneveld J,Knight R.QIIME allows analysis of high-throughput community sequencing data.Nature Methods,2010,7(5):335-336.
[18]毛伟华,吴三玲,张旭.土壤微生物16S r DNA的Ion Torrent PGM高通量检测方法构建与应用.浙江农业学报,2015,27(12):2165-2170.
[19]李晓强.陈化烤烟烟叶表面优势菌株的定量检测与诱变增效研究[D].郑州:河南农业大学,2008.
[20]张晓娟.雪茄外包皮烟人工发酵工艺及叶表微生物区系研究[D].郑州:河南农业大学,2006.
[21]陈小敏,林影,梁书利.烟叶表面微生物群落结构鉴定及其产酶分析.基因组学与应用生物学,2016,35(7):1760-1766.
[22]Gurusamy R,Sakthivel N.Current status on biochemistry and molecular biology of microbial degradation of nicotine.The Scientific World Journal,2013,2013:125385.
[23]苟敏,曲媛媛,杨桦,周集体,李昂,关晓燕,艾芳芳.鞘氨醇单胞菌:降解芳香化合物的新型微生物资源.应用与环境生物学报,2008,14(2):276-282.
[24]杨金奎,段焰青,陈春梅,李庆华,黄静文,张克勤.醇化烟叶表面可培养微生物的鉴定和系统发育分析.烟草科技,2008,(11):51-55.
[25]伍雪莹,梁书利,韩双艳,陈小敏,林影.不同陈化期烤烟叶表细菌的多样性及发育分析.广东农业科学,2014,41(18):28-33,38-38.
[26]Chopyk J,Chattopadhyay S,Kulkarni P,Smyth E M,Hittle L E,Paulson J N,Pop M,Buehler S S,Clark P I,Mongodin E F,Sapkota A R.Temporal variations in cigarette tobacco bacterial community composition and tobacco-specific nitrosamine content are influenced by brand and storage conditions.Frontiers in Microbiology,2017,8:358.
[27]叶建斌,闫记,刘向真,杨宗灿,申洪涛,刘茂林,杨雪鹏,王根发,彭玉富,卢昶彤.原烟复烤前后细菌种群变化研究.河南农业科学,2017,46(1):154-159.
[2]汪长国,李宁,寇明钰,冯广林,杨文敏,吴艳,刘林,戴亚,张燕.复烤烟叶异地醇化过程中生物活性的变化.中国农业大学学报,2013,18(2):105-109.
[3]浦绍占,马永凯,刘敏,宋鹏飞,王毅,苏杨,季秀玲,魏云林.玉溪地区自然陈化烟叶表面可培养细菌多样性研究.中国微生态学杂志,2016,28(8):920-925.
[4]Di Giacomo M,Paolino M,Silvestro D,Vigliotta G,Imperi F,Visca P,Alifano P,Parente D.Microbial community structure and dynamics of dark fire-cured tobacco fermentation.Applied and Environmental Microbiology,2007,73(3):825-837.
[5]Vigliotta G,Di Giacomo M,Carata E,Massardo D R,Tredici S M,Silvestro D,Paolino M,Pontieri P,Del Giudice L,Parente D,Alifano P.Nitrite metabolism in Debaryomyces hansenii TOB-Y7,a yeast strain involved in tobacco fermentation.Applied Microbiology and Biotechnology,2007,75(3):633-645.
[6]李浩宇,崔尹赡,季秀玲,魏云林,余会喜,马永凯,王毅,苏杨,唐兴宏.醇化烟叶产蛋白酶菌株的分离鉴定及其对烟叶内在品质影响.中国微生态学杂志,2015,27(12):1376-1380.
[7]Maldonado-Robledo G,Rodriguez-Bustamante E,Sanchez-Contreras A,Rodriguez-Sanoja R,Sanchez S.Production of tobacco aroma from lutein.Specific role of the microorganisms involved in the process.Applied Microbiology and Biotechnology,2003,62(5/6):484-488.
[8]贾蓓蕾,魏涛,黄申,贾春晓,杨靖,张改红,白冰,毛多斌.α-胡萝卜素降解产香菌株的分离、鉴定及发酵条件优化.食品与发酵工业,2015,41(1):34-39.
[9]向文良,张文学,胡承,王忠彦,汤月琴,木田建次,重松亨.中国浓香型白酒窖池中原核微生物的特性及系统发育分析.四川大学学报:工程科学版,2005,37(1):39-42.
[10]Morin A,Porter A,Joly J,Ratavicius A.Evolution of tobacco-specific nitrosamines and microbial populations during flue-curing of tobacco under direct and indirect heating.Beitrge Zur Tabakforschung International/Contributions to Tobacco Research,2004,21(1):40-46.
[11]Su C,Lei L P,Duan Y Q,Zhang K Q,Yang J K.Culture-independent methods for studying environmental microorganisms:methods,application,and perspective.Applied Microbiology and Biotechnology,2012,93(3):993-1003.
[12]龚俊,刘玉配,李媛媛.烟叶表面微生物类群两种检测方法的比较研究.华东师范大学学报:自然科学版,2016,(3):92-101.
[13]Zhao M Q,Wang B X,Li F X,Qiu L Y,Li F F,Wang S M,Cui J K.Analysis of bacterial communities on aging flue-cured tobacco leaves by 16Sr DNA PCR-DGGE technology.Applied Microbiology and Biotechnology,2007,73(6):1435-1440.
[14]Su C,Gu W,Zhe W,Zhang K Q,Duan Y Q,Yang J K.Diversity and phylogeny of bacteria on Zimbabwe tobacco leaves estimated by 16S rRNAsequence analysis.Applied Microbiology and Biotechnology,2011,92(5):1033-1044.
[15]李永忠,丁金玲.烟草化学成分分析实验指导书[D].昆明:云南农业大学烟草学院,2007.
[16]Magoc T,Salzberg S L.FLASH:fast length adjustment of short reads to improve genome assemblies.Bioinformatics,2011,27(21):2957-2963.
[17]Caporaso J G,Kuczynski J,Stombaugh J,Bittinger K,Bushman F D,Costello E K,Fierer N,Pe1a A G,Goodrich J K,Gordon J I,Huttley GA,Kelley S T,Knights D,Koenig J E,Ley R E,Lozupone C A,Mc Donald D,Muegge B D,Pirrung M,Reeder J,Sevinsky J R,Turnbaugh PJ,Walters W A,Widmann J,Yatsunenko T,Zaneveld J,Knight R.QIIME allows analysis of high-throughput community sequencing data.Nature Methods,2010,7(5):335-336.
[18]毛伟华,吴三玲,张旭.土壤微生物16S r DNA的Ion Torrent PGM高通量检测方法构建与应用.浙江农业学报,2015,27(12):2165-2170.
[19]李晓强.陈化烤烟烟叶表面优势菌株的定量检测与诱变增效研究[D].郑州:河南农业大学,2008.
[20]张晓娟.雪茄外包皮烟人工发酵工艺及叶表微生物区系研究[D].郑州:河南农业大学,2006.
[21]陈小敏,林影,梁书利.烟叶表面微生物群落结构鉴定及其产酶分析.基因组学与应用生物学,2016,35(7):1760-1766.
[22]Gurusamy R,Sakthivel N.Current status on biochemistry and molecular biology of microbial degradation of nicotine.The Scientific World Journal,2013,2013:125385.
[23]苟敏,曲媛媛,杨桦,周集体,李昂,关晓燕,艾芳芳.鞘氨醇单胞菌:降解芳香化合物的新型微生物资源.应用与环境生物学报,2008,14(2):276-282.
[24]杨金奎,段焰青,陈春梅,李庆华,黄静文,张克勤.醇化烟叶表面可培养微生物的鉴定和系统发育分析.烟草科技,2008,(11):51-55.
[25]伍雪莹,梁书利,韩双艳,陈小敏,林影.不同陈化期烤烟叶表细菌的多样性及发育分析.广东农业科学,2014,41(18):28-33,38-38.
[26]Chopyk J,Chattopadhyay S,Kulkarni P,Smyth E M,Hittle L E,Paulson J N,Pop M,Buehler S S,Clark P I,Mongodin E F,Sapkota A R.Temporal variations in cigarette tobacco bacterial community composition and tobacco-specific nitrosamine content are influenced by brand and storage conditions.Frontiers in Microbiology,2017,8:358.
[27]叶建斌,闫记,刘向真,杨宗灿,申洪涛,刘茂林,杨雪鹏,王根发,彭玉富,卢昶彤.原烟复烤前后细菌种群变化研究.河南农业科学,2017,46(1):154-159.