应用Illumina高通量测序技术分析3种酒曲中微生物多样性
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摘要
为了解贵州花溪(酒曲A)、盘州(酒曲B)、安顺(酒曲C)3个地区的酒曲中微生物群落组成和多样性,运用高通量测序技术对酒曲中的细菌16S rDNA V3-V4区和真菌ITS1区进行测序,比较不同地区酒曲中微生物群落结构差异。测定不同地区酒曲的基本理化指标,与酒曲主要细菌种群进行相关性分析。结果显示,细菌获得129 510条有效序列,1 030个OTU;真菌获得60 945条有效序列,19个OTU。细菌的多样性分析表明,酒曲B中Shannon指数明显低于酒曲A和酒曲C,酒曲A和酒曲C的细菌群落结构更为接近。真菌的多样性分析表明,酒曲C中Shannon指数明显低于酒曲A和酒曲B,酒曲A和酒曲B的真菌群落结构更为接近。在门水平上,酒曲A的优势细菌门为变形菌门(Proteobacteria),优势真菌门为接合菌门(Zygomycota);酒曲B的优势细菌门为厚壁菌门(Firmicutes),优势真菌门为接合菌门;酒曲C的优势细菌门为变形菌门(Proteobacteria),优势真菌门为子囊菌门(Ascomycota)。在属水平上,酒曲A优势细菌属为红球菌属(Rhodococcus),酒曲C和酒曲B的优势细菌属均为芽孢杆菌属(Bacillus);酒曲A和酒曲B的优势真菌属为米根霉属(Rhizopus),而酒曲C的优势真菌属为酵母属(Saccharomyces)。不同地方来源的酒曲中的微生物组成及多样性在属和门水平上有明显差异;酒曲理化指标与酒曲细菌种群结构具有一定相关性。
This study aimed to understand the microbial diversity of rice wine koji in Guizhou and to compare the bacterial and fungal community structures of rice wine kojis from different regions of this province. The composition and structure of bacterial and fungal communities in Huaxi(A), Panzhou(B) and Anshun(C) kojis were analyzed by high-throughput sequencing of the bacterial 16 S rDNA V3-V4 region and of the fungal ITS1 region. Then the basic physicochemical indicators of kojis were determined and correlated with the diversity of major bacterial populations. The results showed that a total of 129 510 bacterial effective sequences and 1 030 operational taxonomic units(OTUs) were obtained, together with60 945 fungal effective sequences and 19 OTUs. The bacterial diversity analysis showed that the Shannon index in koji B was significantly lower than those in kojis A and C, while the bacterial community structures of kojis A and C were more similar. The fungal diversity analysis showed that the Shannon index in koji C was significantly lower than those in kojis A and B, and the fungal community structures of kojis A and B were more similar to each other. At the level of phylum,Proteobacteria was the dominant bacteria and Zygomycota was the dominant fungi in koji A; Firmicutes was the dominant bacteria and Zygomycota was the dominant fungi in koji B; Proteobacteria was the dominant bacteria and Ascomycota was the dominant fungi in koji C. At the genus level, the dominant bacteria of koji A was Rhodococcus, and the dominant genus of kojis C and B was Bacillus. The dominant fungal genus of both kojis A and B was Rhizopus, while the dominant fungal genus of koji C was Saccharomyces. There was a significant regional variation in the microbial composition and diversity in rice wine koji at both the genus and phylum levels. The physicochemical indicators exhibited a correlation with the bacterial community structure.
This study aimed to understand the microbial diversity of rice wine koji in Guizhou and to compare the bacterial and fungal community structures of rice wine kojis from different regions of this province. The composition and structure of bacterial and fungal communities in Huaxi(A), Panzhou(B) and Anshun(C) kojis were analyzed by high-throughput sequencing of the bacterial 16 S rDNA V3-V4 region and of the fungal ITS1 region. Then the basic physicochemical indicators of kojis were determined and correlated with the diversity of major bacterial populations. The results showed that a total of 129 510 bacterial effective sequences and 1 030 operational taxonomic units(OTUs) were obtained, together with60 945 fungal effective sequences and 19 OTUs. The bacterial diversity analysis showed that the Shannon index in koji B was significantly lower than those in kojis A and C, while the bacterial community structures of kojis A and C were more similar. The fungal diversity analysis showed that the Shannon index in koji C was significantly lower than those in kojis A and B, and the fungal community structures of kojis A and B were more similar to each other. At the level of phylum,Proteobacteria was the dominant bacteria and Zygomycota was the dominant fungi in koji A; Firmicutes was the dominant bacteria and Zygomycota was the dominant fungi in koji B; Proteobacteria was the dominant bacteria and Ascomycota was the dominant fungi in koji C. At the genus level, the dominant bacteria of koji A was Rhodococcus, and the dominant genus of kojis C and B was Bacillus. The dominant fungal genus of both kojis A and B was Rhizopus, while the dominant fungal genus of koji C was Saccharomyces. There was a significant regional variation in the microbial composition and diversity in rice wine koji at both the genus and phylum levels. The physicochemical indicators exhibited a correlation with the bacterial community structure.
引文
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[19]李越鲲,孙燕飞,雷勇辉,等.枸杞根际土壤真菌群落多样性的高通量测序[J].微生物学报,2017,57(7):1049-1059.DOI:10.13343/j.cnki.wsxb.20160410.
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[21]BEAUMONT M.Flavouring composition prepared by fermentation with Bacillus spp.[J].International Journal of Food Microbiology,2002,75(3):189-196.DOI:10.1016/S0168-1605(01)00706-1.
[22]MITTER E K,DE FREITAS J R,GERMIDA J J.Bacterial root microbiome of plants growing in oil sands reclamation covers[J].Frontiers in Microbiology,2017,8:1-14.DOI:10.3389/fmicb.2017.00849.
[23]张双燕,廖永红,纪南,等.基于高通量测序技术分析北京清香型大曲微生物多样性[J].中国酿造,2016,35(11):49-53.DOI:10.11882/j.issn.0254-5071.2016.11.010.
[24]庄名扬.中国白酒香味物质形成机理及酿酒工艺的调控[J].食品与发酵科技,2007,43(2):109-113.DOI:10.3969/j.issn.1674-506X.2007.02.001.
[25]ZHAO S,ZHOU N,ZHAO Z Y,et al.High-throughput sequencing analysis of the endophytic bacterial diversity and dynamics in roots of the halophyte Salicornia europaea[J].Current Microbiology,2016,72(5):557-562.DOI:10.1007/s00284-016-0990-3.
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[27]BORA S S,KEOT J,DAS S,et al.Metagenomics analysis of microbial communities associated with a traditional rice wine starter culture(Xaj-pitha)of Assam,India[J].3 Biotech,2016,6(2):153.DOI:10.1007/s13205-016-0471-1.
[28]宋北,王继华,杜丛,等.大米酒酒曲中优势菌群的探究[J].哈尔滨师范大学自然科学学报,2010,26(2):69-73.DOI:10.3969/j.issn.1000-5617.2010.02.021.
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[30]PORTILLO M D C,MAS A.Analysis of microbial diversity and dynamics during wine fermentation of Grenache grape variety by high-throughput barcoding sequencing[J].LWT-Food Science and Technology,2016,72:317-321.DOI:101016/j.lwt.2016.05.009.
[31]YOUSSEF N,SHEIK C S,KRUMHOLZ L R,et al.Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNAgene-based environmental surveys[J].Applied&Environmental Microbiology,2009,75(16):5227.DOI:10.1128/AEM.00592-09.
[32]纪令士,邢咏梅.双歧杆菌与肠道微生态平衡[J].青岛医药卫生,2005,37(5):355-356.DOI:10.3969/j.issn.1006-5571.2005.05.028.
[33]陈玲,袁玉菊,曾丽云,等.16S rDNA克隆文库法与高通量测序法在浓香型大曲微生物群落结构分析中的对比研究[J].酿酒科技,2015(12):33-36.DOI:10.13746/j.njkj.2015145.
[34]王丹丹,沈馨,董蕴,等.孝感凤窝酒曲真菌多样性评价[J].中国酿造,2017,36(11):38-42.DOI:10.11882/j.issn.0254-5071.2017.11.009.
[35]乔晓梅,赵景龙,杜小威,等.高通量测序法对清香大曲真菌群落结构的分析[J].酿酒科技,2015(4):28-31.DOI:10.13746/j.njkj.2015076.
[36]罗惠波,王彩虹,甄攀,等.基于ITS基因文库法研究清香型大曲真菌群落结构[J].酿酒科技,2014(5):31-35.DOI:10.13746/j.njkj.2014.0006.
[37]FANG R S,DONG Y C,CHEN F,et al.Bacterial diversity analysis during the fermentation processing of traditional Chinese yellow rice wine revealed by 16S rDNA 454 pyrosequencing[J].Journal of Food Science,2015,80(10):M2265-M2271.DOI:10.1111/1750-3841.13018.
[2]李健容,蔡爱群.民间传统酒曲主要微生物的分离及鉴定[J].酿酒科技,2007(5):111-115;121.DOI:10.3969/j.issn.1001-9286.2007.05.030.
[3]程荣瑞金,李祖明,王德良,等.中国酒曲微生物研究进展[J].中国酿造,2009,28(6):5-8.DOI:10.3969/j.issn.0254-5071.2009.06.002.
[4]HUI W,HOU Q,CAO C,et al.Identification of microbial profile of koji using single molecule,real-time sequencing technology[J].Journal of Food Science,2017,82(5):1193-1199.DOI:10.1111/1750-3841.13699.
[5]沈馨,尚雪娇,董蕴,等.基于MiSeq高通量测序技术对3个孝感凤窝酒曲细菌多样性的评价[J].中国微生态学杂志,2018(5):527-529.DOI:10.13381/j,cnki cjm 201805007.
[6]相飞.甜酒曲中微生物群落结构及辣蓼甜酒曲的制曲工艺研究[D].上海:上海海洋大学,2015.
[7]刘孟华,唐清兰,徐姿静,等.基于宏基因组学的剑南春酒曲细菌多样性分析[J].酿酒,2016,43(5):30-33.DOI:10.3969/j.issn.1002-8110.2016.05.009.
[8]李伟程,侯强川,于洁,等.传统发酵乳制品中微生物多样性研究[J].食品工业科技,2018,39(1):131-136.DOI:10.13386/j.issn1002-0306.2018.01.025.
[9]程秦楠,栗东芳,杨瑞馥.高通量测序技术及其在微生物学研究中的应用[J].微生物学报,2011,51(4):445-457.DOI:10.13343/j.cnki.wsxb.2011.04.013.
[10]ZHU Q L,LIU S,GAO P,et al.High-throughput sequencing technology and its application[J].Journal of Northeast Agricultural University(English Edition),2014,21(3):84-96.DOI:10.1016/S1006-8104(14)60073-8.
[11]YANG S,LEE J,KWAK J,et al.Fungi associated with the traditional starter cultures used for rice wine in Korea[J].Journal of the Korean Society for Applied Biological Chemistry,2011,54(6):933-943.DOI:10.1007/BF03253183.
[12]JING T,TANG X,MING T,et al.Analysis of the bacterial communities in two liquors of soy sauce aroma as revealed by high-throughput sequencing of the 16S rRNA V4 hypervariable region[J].BioMed Research International,2017,2017:6271358.DOI:10.1155/2017/6271358.
[13]王福荣.酿酒分析与检测[M].北京:化学工业出版社,2012:11-18.
[14]ERCOLINI D.High-throughput sequencing and metagenomics:moving forward in the culture-independent analysis of food microbial ecology[J].Applied&Environmental Microbiology,2013,79(10):3148-3155.DOI:10.1128/AEM.00256-13.
[15]谭映月,胡萍,谢和.应用PCR-DGGE技术分析酱香型白酒酒曲细菌多样性[J].酿酒科技,2012(10):107-111.DOI:10.13746/j.njkj.2012.10.024.
[16]曹荣,刘淇,赵玲,等.基于高通量测序的牡蛎冷藏过程中微生物群落分析[J].农业工程学报,2016,32(20):275-280.DOI:10.11975/j.issn.1002-6819.2016.20.036.
[17]张敏,张艳,黄丽丽,等.基于16S rDNA高通量测序方法比较新疆西北部地区乳品中微生物的多样性[J].食品科学,2017,38(20):27-33.DOI:10.7506/spkx1002-6630-201720005.
[18]唐凯,贾丽娟,高晓丹,等.浑善达克沙地生物土壤结皮及其下层土壤中好氧不产氧光营养细菌群落结构及多样性[J].微生物学报,2018,58(2):228-237.DOI:10.13343/j.cnki.wsxb.20170097.
[19]李越鲲,孙燕飞,雷勇辉,等.枸杞根际土壤真菌群落多样性的高通量测序[J].微生物学报,2017,57(7):1049-1059.DOI:10.13343/j.cnki.wsxb.20160410.
[20]TAMAKI H,WRIGHT C L,LI X,et al.Analysis of 16S rRNAamplicon sequencing options on the Roche/454 next-generation titanium sequencing platform[J].PLoS ONE,2011,6(9):e25263.DOI:10.1371/journal.pone.0025263.
[21]BEAUMONT M.Flavouring composition prepared by fermentation with Bacillus spp.[J].International Journal of Food Microbiology,2002,75(3):189-196.DOI:10.1016/S0168-1605(01)00706-1.
[22]MITTER E K,DE FREITAS J R,GERMIDA J J.Bacterial root microbiome of plants growing in oil sands reclamation covers[J].Frontiers in Microbiology,2017,8:1-14.DOI:10.3389/fmicb.2017.00849.
[23]张双燕,廖永红,纪南,等.基于高通量测序技术分析北京清香型大曲微生物多样性[J].中国酿造,2016,35(11):49-53.DOI:10.11882/j.issn.0254-5071.2016.11.010.
[24]庄名扬.中国白酒香味物质形成机理及酿酒工艺的调控[J].食品与发酵科技,2007,43(2):109-113.DOI:10.3969/j.issn.1674-506X.2007.02.001.
[25]ZHAO S,ZHOU N,ZHAO Z Y,et al.High-throughput sequencing analysis of the endophytic bacterial diversity and dynamics in roots of the halophyte Salicornia europaea[J].Current Microbiology,2016,72(5):557-562.DOI:10.1007/s00284-016-0990-3.
[26]李艳,董振玲,牟德华.羊羔美酒大曲中酵母菌多样性及分子鉴定[J].食品科学,2014,35(13):144-149.DOI:10.7506/spkx1002-6630-201405029.
[27]BORA S S,KEOT J,DAS S,et al.Metagenomics analysis of microbial communities associated with a traditional rice wine starter culture(Xaj-pitha)of Assam,India[J].3 Biotech,2016,6(2):153.DOI:10.1007/s13205-016-0471-1.
[28]宋北,王继华,杜丛,等.大米酒酒曲中优势菌群的探究[J].哈尔滨师范大学自然科学学报,2010,26(2):69-73.DOI:10.3969/j.issn.1000-5617.2010.02.021.
[29]ZHANG Y,PING Y,ZHOU R,et al.High throughput sequencingbased analysis of microbial diversity in dental unit waterlines supports the importance of providing safe water for clinical use[J].Journal of Infection&Public Health,2017.DOI:10.1016/j.jiph.2017.09.017.
[30]PORTILLO M D C,MAS A.Analysis of microbial diversity and dynamics during wine fermentation of Grenache grape variety by high-throughput barcoding sequencing[J].LWT-Food Science and Technology,2016,72:317-321.DOI:101016/j.lwt.2016.05.009.
[31]YOUSSEF N,SHEIK C S,KRUMHOLZ L R,et al.Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNAgene-based environmental surveys[J].Applied&Environmental Microbiology,2009,75(16):5227.DOI:10.1128/AEM.00592-09.
[32]纪令士,邢咏梅.双歧杆菌与肠道微生态平衡[J].青岛医药卫生,2005,37(5):355-356.DOI:10.3969/j.issn.1006-5571.2005.05.028.
[33]陈玲,袁玉菊,曾丽云,等.16S rDNA克隆文库法与高通量测序法在浓香型大曲微生物群落结构分析中的对比研究[J].酿酒科技,2015(12):33-36.DOI:10.13746/j.njkj.2015145.
[34]王丹丹,沈馨,董蕴,等.孝感凤窝酒曲真菌多样性评价[J].中国酿造,2017,36(11):38-42.DOI:10.11882/j.issn.0254-5071.2017.11.009.
[35]乔晓梅,赵景龙,杜小威,等.高通量测序法对清香大曲真菌群落结构的分析[J].酿酒科技,2015(4):28-31.DOI:10.13746/j.njkj.2015076.
[36]罗惠波,王彩虹,甄攀,等.基于ITS基因文库法研究清香型大曲真菌群落结构[J].酿酒科技,2014(5):31-35.DOI:10.13746/j.njkj.2014.0006.
[37]FANG R S,DONG Y C,CHEN F,et al.Bacterial diversity analysis during the fermentation processing of traditional Chinese yellow rice wine revealed by 16S rDNA 454 pyrosequencing[J].Journal of Food Science,2015,80(10):M2265-M2271.DOI:10.1111/1750-3841.13018.