酱香高温大曲、酒醅和窖泥的真菌多样性分析
|
摘要
基于HiSeq 2500高通量测序技术,利用r DNA-ITS1序列解析酱香高温大曲、酒醅和窖泥中真菌微生物的组成及其多样性。结果表明,本研究收集的高温大曲、酒醅和窖泥样品共产出236,402条clean tags,有效序列的平均长度分别为304 bp、258 bp和280 bp;在分类操作单元(OTU)水平上得到各样品的OTU个数分别为83、70和308,其中三者共有的OTU个数为13;窖泥中真核微生物具有最高的丰富度和多样性,酒醅的多样性高于高温大曲。研究表明,酱香高温大曲、酒醅和窖泥之间的真菌菌群组成和多样性差异明显。酱香高温大曲主要由优势菌属嗜热丝孢菌属(Thermomyces)和嗜热子囊菌属(Thermoascus)组成。酒醅中优势菌属包括假丝酵母属(Candida)、拟青霉属(Paecilomyces)和嗜热丝孢菌属(Thermomyces)。上述研究是首次对郎酒生产过程中的主要真菌微生物的组成进行鉴定和多样性分析,为酱香型高温制曲、高温堆积发酵、厌氧发酵等工艺的改进和完善提供一定的理论基础。
To investigate the composition and the diversity of fungal microflora in high-temperature Jiangxiang Daqu, fermented grains and pit mud, high-throughput sequencing technology was applied to analyze their rDNA-ITS1 sequences. The results showed that, 236,402 clean tags were produced from the collected samples, and the average length of the effective sequences were 304 bp, 258 bp and 280 bp respectively. At the operational taxonomic unit(OTU) level, the OTU numbers of each sample were 83, 70 and 308 respectively, and the number of OTUs shared by the three samples was 13. The eukaryotic microflora in pit mud displayed the highest diversity, and the microflora diversity in fermented grains was higher than that in Daqu. The research also demonstrated a significant difference in the composition and the diversity of fungal microflora among high-temperature Jiangxiang Daqu, fermented grains and pit mud. Thermomyces and Thermoascus were the dominant bacteria species in Daqu, and Candida, Paecilomyces and Thermomyces were the dominant bacteria species in fermented grains. The study laid the foundation and theoretical basis for elucidating the special mechanism of high-temperature Daqu making, high-temperature stacking fermentation, and anaerobic fermentation etc.
To investigate the composition and the diversity of fungal microflora in high-temperature Jiangxiang Daqu, fermented grains and pit mud, high-throughput sequencing technology was applied to analyze their rDNA-ITS1 sequences. The results showed that, 236,402 clean tags were produced from the collected samples, and the average length of the effective sequences were 304 bp, 258 bp and 280 bp respectively. At the operational taxonomic unit(OTU) level, the OTU numbers of each sample were 83, 70 and 308 respectively, and the number of OTUs shared by the three samples was 13. The eukaryotic microflora in pit mud displayed the highest diversity, and the microflora diversity in fermented grains was higher than that in Daqu. The research also demonstrated a significant difference in the composition and the diversity of fungal microflora among high-temperature Jiangxiang Daqu, fermented grains and pit mud. Thermomyces and Thermoascus were the dominant bacteria species in Daqu, and Candida, Paecilomyces and Thermomyces were the dominant bacteria species in fermented grains. The study laid the foundation and theoretical basis for elucidating the special mechanism of high-temperature Daqu making, high-temperature stacking fermentation, and anaerobic fermentation etc.
引文
[1]杨国华,邱树毅,黄永光.酱香白酒生产中产香微生物研究[J].中国酿造,2011(4):24-27.
[2]云敏,邓杰,李佳勇,等.酱香型郎酒酱味来源之分析推测[J].酿酒科技,2009(1):69-71.
[3]范文来,徐岩.应用液液萃取结合正相色谱技术鉴定汾酒与郎酒挥发性成分(下)[J].酿酒科技,2013(3):17-27.
[4]唐婧,苏迪,徐小蓉,等.基于宏基因组学的茅台酒酒曲细菌的多样性分析[J].贵州农业科学,2014,42(11):180-183.
[5]傅国城.中国白酒科技创新步入分子生物学时代[J].酿酒,2017(2):9-12.
[6]黄永光,黄旭,黄平.茅台酒酿酒极端环境与极端酿酒微生物[J].酿酒科技,2006(12):47-50.
[7]蒋红军.茅台酒制曲发酵过程中微生物演替及作用规律[J].酿酒科技,2004(3):39-40.
[8]许玲,张秋月.几株生香酵母的分离筛选及应用[J].酿酒科技,2010(12):48-49.
[9]胡晓龙,赵东,葛隐,等.五粮液大曲中产酯化酶红曲霉的分离及酯化酶的纯化[J].酿酒,2011,38(3):35-38.
[10]曾娟,施思,吴正云,等.浓香型白酒醅中高产酒精酵母菌的定向筛选及分子鉴定[J].中国酿造,2011(2):26-28.
[11]赵爽,杨春霞,窦屾,等.白酒生产中酿酒微生物研究进展[J].中国酿造,2012,31(4):5-8.
[12]侯小歌,王俊英,李学思,等.浓香型白酒糟醅及窖泥产香功能菌的研究进展[J].微生物学通报,2013,40(7):1257-1265.
[13] SHOKRALLA S, SPALL J L, GIBSON J F, et al.Next-generation sequencing technologies for environmental DNA research[J].Molecular ecology,2012,21(8):1794-1805.
[14]王兴春,杨致荣,王敏,等.高通量测序技术及其应用[J].中国生物工程杂志,2012,32(1):109-114.
[15] MAGOC T, SALZBERG S L. FLASH:fast length adjustment of short reads to improve genome assemblies[J].Bioinformatics,2011,27(21):2957-2963.
[16] BOLGER A M, LOHSE M, USADEL B. Trimmomatic:a flexible trimmer for Illumina sequence data[J].Bioinformatics,2014,30(15):2114-2120.
[17] EDGAR R C, HAAS B J, CLEMENTE J C, et al.UCHIME improves sensitivity and speed of chimera detection[J].Bioinformatics,2011,27(16):2194-2200.
[18] EDGAR R C. Search and clustering orders of magnitude faster than BLAST[J].Bioinformatics,2010,26(19):2460-2461.
[19] BOKULICH N A, SUBRAMANIAN S, FAITH J J, et al. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing[J].Nature methods,2013,10(1):57.
[20] K?LJALG U, NILSSON R H, ABARENKOV K, et al.Towards a unified paradigm for sequence-based identification of fungi[J].Molecular ecology,2013,22(21):5271-5277.
[21] WANG Q, GARRITY G M, TIEDJE J M, et al. Naive Bayesian classifier for rapid assignment of r RNA sequences into the new bacterial taxonomy[J].Applied and environmental microbiology,2007,73(16):5261-5267.
[22] LARKIN M A, BLACKSHIELDS G, BROWN N, et al.Clustal W and Clustal X version 2.0[J].bioinformatics,2007,23(21):2947-2948.
[23] SCHLOSS P D, WESTCOTT S L, RYABIN T, et al.Introducing mothur:open-source, platform-independent,community-supported software for describing and comparing microbial communities[J].Applied and environmental microbiology,2009,75(23):7537-7541.
[24]蒋英丽,邓皖玉,王亚军,等.酱香高温大曲微生物菌群演化规律研究[J].酿酒科技,2018(12):1-9.
[25]范光先,王和玉,崔同弼,等.茅台酒生产过程中的微生物研究进展[J].酿酒科技,2006(10):75-77.
[26]董瑞丽,罗惠波,叶光斌.浓香型大曲中的嗜热真菌[J].中国酿造,2011(4):75-77.
[27]苏畅,窦晓,叶新,等.基于ITS4/5 r RNA区序列对不同时期大曲中霉菌的分离与鉴定[J].现代食品科技,2018,34(3):54-58.
[28] SINGH S, MADLALA A M, PRIOR B A.Thermomyces lanuginosus:properties of strains and their hemicellulases[J].FEMS microbiology reviews,2003,27(1):3-16.
[29] DEACON J. Decomposition of filter paper cellulose by thermophilic fungi acting singly, in combination, and in sequence[J]. Transactions of the British Mycological Society,1985,85(4):663-669.
[30]姚粟,于学健,白飞荣,等.中国传统发酵食品用微生物菌种名单的研究[J].食品与发酵工业,2017,43(9):238.
[31]周恒刚,吴呜.话说河内白曲[J].酿酒,1993(2):9-14.
[32]张春林.泸州老窖大曲的质量、微生物与香气成分关系[D].无锡:江南大学,2012.
[33]芦文娟.新疆塔城地区原料乳及奶酪中酵母菌多样性的研究[D].石河子:石河子大学,2016.
[34]孙丽.腌制冬瓜酵母菌多样性及其品质分析[D].宁波:宁波大学,2014.
[35] ZHAO J S, ZHENG J, ZHOU R Q, et al. Microbial community structure of pit mud in a Chinese strong aromatic liquor fermentation pit[J].Journal of the Institute of Brewing,2012,118(4):356-360.
[36]王福桢.北方浓香型白酒发酵微生物多样性分布模式解析[J].呼和浩特:内蒙古大学,2014.
[37]张宿义,李宗珍,张良,等.泸州老窖国窖酿造环境空气真菌四季分布研究[J].酿酒科技,2011(2):36-39.
[38]刘茂柯,唐玉明,赵珂,等.浓香型白酒窖泥微生物群落结构及其选育应用研究进展[J].微生物学通报,2017,44(5):1222-1229.
[2]云敏,邓杰,李佳勇,等.酱香型郎酒酱味来源之分析推测[J].酿酒科技,2009(1):69-71.
[3]范文来,徐岩.应用液液萃取结合正相色谱技术鉴定汾酒与郎酒挥发性成分(下)[J].酿酒科技,2013(3):17-27.
[4]唐婧,苏迪,徐小蓉,等.基于宏基因组学的茅台酒酒曲细菌的多样性分析[J].贵州农业科学,2014,42(11):180-183.
[5]傅国城.中国白酒科技创新步入分子生物学时代[J].酿酒,2017(2):9-12.
[6]黄永光,黄旭,黄平.茅台酒酿酒极端环境与极端酿酒微生物[J].酿酒科技,2006(12):47-50.
[7]蒋红军.茅台酒制曲发酵过程中微生物演替及作用规律[J].酿酒科技,2004(3):39-40.
[8]许玲,张秋月.几株生香酵母的分离筛选及应用[J].酿酒科技,2010(12):48-49.
[9]胡晓龙,赵东,葛隐,等.五粮液大曲中产酯化酶红曲霉的分离及酯化酶的纯化[J].酿酒,2011,38(3):35-38.
[10]曾娟,施思,吴正云,等.浓香型白酒醅中高产酒精酵母菌的定向筛选及分子鉴定[J].中国酿造,2011(2):26-28.
[11]赵爽,杨春霞,窦屾,等.白酒生产中酿酒微生物研究进展[J].中国酿造,2012,31(4):5-8.
[12]侯小歌,王俊英,李学思,等.浓香型白酒糟醅及窖泥产香功能菌的研究进展[J].微生物学通报,2013,40(7):1257-1265.
[13] SHOKRALLA S, SPALL J L, GIBSON J F, et al.Next-generation sequencing technologies for environmental DNA research[J].Molecular ecology,2012,21(8):1794-1805.
[14]王兴春,杨致荣,王敏,等.高通量测序技术及其应用[J].中国生物工程杂志,2012,32(1):109-114.
[15] MAGOC T, SALZBERG S L. FLASH:fast length adjustment of short reads to improve genome assemblies[J].Bioinformatics,2011,27(21):2957-2963.
[16] BOLGER A M, LOHSE M, USADEL B. Trimmomatic:a flexible trimmer for Illumina sequence data[J].Bioinformatics,2014,30(15):2114-2120.
[17] EDGAR R C, HAAS B J, CLEMENTE J C, et al.UCHIME improves sensitivity and speed of chimera detection[J].Bioinformatics,2011,27(16):2194-2200.
[18] EDGAR R C. Search and clustering orders of magnitude faster than BLAST[J].Bioinformatics,2010,26(19):2460-2461.
[19] BOKULICH N A, SUBRAMANIAN S, FAITH J J, et al. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing[J].Nature methods,2013,10(1):57.
[20] K?LJALG U, NILSSON R H, ABARENKOV K, et al.Towards a unified paradigm for sequence-based identification of fungi[J].Molecular ecology,2013,22(21):5271-5277.
[21] WANG Q, GARRITY G M, TIEDJE J M, et al. Naive Bayesian classifier for rapid assignment of r RNA sequences into the new bacterial taxonomy[J].Applied and environmental microbiology,2007,73(16):5261-5267.
[22] LARKIN M A, BLACKSHIELDS G, BROWN N, et al.Clustal W and Clustal X version 2.0[J].bioinformatics,2007,23(21):2947-2948.
[23] SCHLOSS P D, WESTCOTT S L, RYABIN T, et al.Introducing mothur:open-source, platform-independent,community-supported software for describing and comparing microbial communities[J].Applied and environmental microbiology,2009,75(23):7537-7541.
[24]蒋英丽,邓皖玉,王亚军,等.酱香高温大曲微生物菌群演化规律研究[J].酿酒科技,2018(12):1-9.
[25]范光先,王和玉,崔同弼,等.茅台酒生产过程中的微生物研究进展[J].酿酒科技,2006(10):75-77.
[26]董瑞丽,罗惠波,叶光斌.浓香型大曲中的嗜热真菌[J].中国酿造,2011(4):75-77.
[27]苏畅,窦晓,叶新,等.基于ITS4/5 r RNA区序列对不同时期大曲中霉菌的分离与鉴定[J].现代食品科技,2018,34(3):54-58.
[28] SINGH S, MADLALA A M, PRIOR B A.Thermomyces lanuginosus:properties of strains and their hemicellulases[J].FEMS microbiology reviews,2003,27(1):3-16.
[29] DEACON J. Decomposition of filter paper cellulose by thermophilic fungi acting singly, in combination, and in sequence[J]. Transactions of the British Mycological Society,1985,85(4):663-669.
[30]姚粟,于学健,白飞荣,等.中国传统发酵食品用微生物菌种名单的研究[J].食品与发酵工业,2017,43(9):238.
[31]周恒刚,吴呜.话说河内白曲[J].酿酒,1993(2):9-14.
[32]张春林.泸州老窖大曲的质量、微生物与香气成分关系[D].无锡:江南大学,2012.
[33]芦文娟.新疆塔城地区原料乳及奶酪中酵母菌多样性的研究[D].石河子:石河子大学,2016.
[34]孙丽.腌制冬瓜酵母菌多样性及其品质分析[D].宁波:宁波大学,2014.
[35] ZHAO J S, ZHENG J, ZHOU R Q, et al. Microbial community structure of pit mud in a Chinese strong aromatic liquor fermentation pit[J].Journal of the Institute of Brewing,2012,118(4):356-360.
[36]王福桢.北方浓香型白酒发酵微生物多样性分布模式解析[J].呼和浩特:内蒙古大学,2014.
[37]张宿义,李宗珍,张良,等.泸州老窖国窖酿造环境空气真菌四季分布研究[J].酿酒科技,2011(2):36-39.
[38]刘茂柯,唐玉明,赵珂,等.浓香型白酒窖泥微生物群落结构及其选育应用研究进展[J].微生物学通报,2017,44(5):1222-1229.