耐酸乳酸杆菌物种特异性引物设计及其在古井贡酒窖泥酒醅质量评价中的初步应用
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摘要
古井贡酒的酿造过程中,窖泥和酒醅中发现大量耐酵乳杆菌(Lactobacillus acetotolerans),对该菌快速特异性检测非常重要。该文利用L. acetotolerans菌及大量Lactobacillus类似菌种的全基因组序列与PRIMERBLAST方法,设计了几对L. acetotolerans特异性引物,其中2对经过系列验证具有较好的特异性,可结合QPCR技术对不同质量和不同发酵阶段的窖泥进行L. acetotolerans含量的快速检测。结果表明,古井贡酒窖泥中L. acetotolerans含量总体明显低于酒醅,老窖泥中L. acetotolerans含量总体明显低于新窖池窖泥;而酒醅中老厂区和新厂区L. acetotolerans含量平均来看都很高,但样本之间存在明显波动。所用L. acetotolerans特异性引物可对浓香型白酒发酵样本中L. acetotolerans的相对含量测定并辅助评估窖泥酒醅质量。
When brewing Gujing Tribute liquor,a large number of Lactobacillus acetotolerans were found in both pitmud and Zaopei. Therefore,it is important to rapidly detect L. acetotolerans specifically. In this paper,whole genome sequences of Lactobacillus bacteria and PRIMER-BLAST method were used to design L. acetotolerans-specific primers. Two pairs of primers were confirmed with decent specificity and combined with quantitative polymerase chain reaction( q PCR) to rapidly measure the amount of L. acetotolerans with different qualities and at different fermentation stages in pitmud and Zaopei. The results showed that the population of L. acetotolerans in Gujing Tribute pitmud was apparently lower than that of Zaopei and was apparently lower in old pitmud than that in pitmud of new plant. Moreover,L. acetotolerans in Zaopei from both new and old plants were generally high with apparent fluctuations among samples. In general,QPCR mediated by L. acetotolerans-specific primers can be used for L. acetotolerans quantification and facilitate quality assessment of pitmud or Zaopei in strong-aroma liquor production.
When brewing Gujing Tribute liquor,a large number of Lactobacillus acetotolerans were found in both pitmud and Zaopei. Therefore,it is important to rapidly detect L. acetotolerans specifically. In this paper,whole genome sequences of Lactobacillus bacteria and PRIMER-BLAST method were used to design L. acetotolerans-specific primers. Two pairs of primers were confirmed with decent specificity and combined with quantitative polymerase chain reaction( q PCR) to rapidly measure the amount of L. acetotolerans with different qualities and at different fermentation stages in pitmud and Zaopei. The results showed that the population of L. acetotolerans in Gujing Tribute pitmud was apparently lower than that of Zaopei and was apparently lower in old pitmud than that in pitmud of new plant. Moreover,L. acetotolerans in Zaopei from both new and old plants were generally high with apparent fluctuations among samples. In general,QPCR mediated by L. acetotolerans-specific primers can be used for L. acetotolerans quantification and facilitate quality assessment of pitmud or Zaopei in strong-aroma liquor production.
引文
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[21] LUO Ximei,GAO Zhilei,ZHANG Huimin,et al. Phylum-specific primer design and implication in quantification of the microbial community structure in Gu Jing Gong pit mud[J]. Advanced Materials Research,2014,3 607(1 051):311-316.
[22] ZHANG Huimin,GUO Changlu,HE Hongkui,et al.QPCR quantification strategy of minor species in a complex microbial community using species-specific primers[J]. Journal of Pure&Applied Microbiology,2013,7:601-610.
[23]赵长青,杨秦欢,邓静,等.窖泥的评定指标检测[J].生物技术进展,2012,2(3):212-216.
[24]张强,沈才洪,刘清斌,等.窖泥质量评价研究进展[J].酿酒科技,2013(7):84-86.
[25]卫春会,黄治国,甄攀.窖泥微生物群落SSCP分析条件优化的研究[J].四川理工学院学报(自然科学版),2010,23(6):695-698.
[26]徐岩,吴群,王石垒.一种快速筛选、鉴定及定量发酵食品中耐酸乳杆菌的方法[P].江苏:CN106906302A,2017-06-30.
[2]余乾伟.传统白酒酿造技术[M].北京:中国轻工业出版社,2010.
[3]张会敏,古井贡酒微生物群落结构及其与主要风味物质的关联研究[D].哈尔滨:哈尔滨工业大学,2016.
[4] HU X,DU H,REN C,et al. Illuminating anaerobic microbial community and cooccurrence patterns across a quality gradient in Chinese liquor fermentation pit muds[J]. Applied and Environmental Microbiology,2016,82(8):2 506-2 515.
[5] LI Junwei,HE Hongkui,CAO Runjie,et al. Significant difference of the culturable anaerobic microbial species in Gujing Tribute pitmud with different ages[C]//IOP Conference Series:Materials Science and Engineering,2018 5th International Conference on Advanced Composite Materials and Manufacturing Engineering,2018-08-08.
[6] HONG X,CHEN J,LIU L,et al. Metagenomic sequencing reveals the relationship between microbiota composition and quality of Chinese rice wine[J]. Scientific Reports,2016,6:26 621.
[7]胡佳.利用免培养法对浓香型白酒曲药细菌菌群的解析研究[D].成都:四川大学,2007.
[8]李维青.浓香型白酒与乳酸菌、乳酸、乳酸乙酯[J].酿酒,2010,37(3):90-93.
[9] WANG H Y,ZHANG X J,ZHAO L P,et al. Analysis and comparison of the bacterial community in fermented grains during the fermentation for two different styles of Chinese liquor[J]. Journal of Industrial Microbiology&Biotechnology,2008,35(6):603-609.
[10] HARUTA S,UENO S,EGAWA I,et al. Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis[J]. International Journal of Food Microbiology,2006,109(1):79-87.
[11] LIU Junyan,DENG Yang,PETERS B M,et al. Transcriptomic analysis on the formation of the viable putative non-culturable state of beer-spoilage Lactobacillus acetotolerans[J]. Scientific Reports,2016,6:36 753.
[12]刘森,李林光,李可,等.中国浓香型白酒窖池窖泥中原核微生物群落的空间异质性[J].食品科学,2013,34(21):221-226.
[13] TOH H,MORITA H,TSUJI H,et al. Complete genome sequence of Lactobacillus acetotolerans RIB 9124(NBRC13120)isolated from putrefied(hiochi)Japanese Sake.[J]. Journal of Biotechnology,2015,214:214-215.
[14]张会敏,束莹,周庆伍,等.利用非培养技术初步研究古井贡酒大曲中的细菌群落结构[J].现代食品科技,2014,30(4):44-49.
[15]张会敏,李天婵,孙美青,等.利用非培养技术初步研究古井贡酒窖泥细菌群落结构[J].食品工业科技,2014,35(13):200-206;228.
[16]张会敏,王杰,周庆伍,等.利用非培养技术初步解析古井贡酒大曲真核微生物群落结构[J].食品工业,2014,35(6):158-162.
[17] ZHANG Huimin,HE Hongkui,YU Xiujuan,et al. Employment of near full-length ribosome gene TA-cloning and Primer-Blast to detect multiple species in a natural complex microbial community using species-specific primers designed with their genome sequences[J]. Molecular Biotechnology,2016,58:729–737.
[18] LIU Zuolin,HAN Ying,LI Junwei,et al. Analysis of microbial community structure of pit mud for Chinese strong-flavor liquor fermentation using next generation DNA sequencing of full-length 16S rRNA[J]. bioRxiv,2018. 31. DOI:http://dx. doi. 02g/10. 1101/380949.
[19] YE J,COULOURIS G,ZARETSKAYA I,et al. PrimerBLAST:A tool to design target-specific primers for polymerase chain reaction[J]. BMC Bioinformatics,2012,18(13):134.
[20] LI K Q,ZHANG H M,LI A J,et al. Differential microbial community complexity levels between different locations in the Gu Jing Gong pits[J]. International Conference on Medicine Sciences and Bioengineering,ICMSB 2014,2015:641-646.
[21] LUO Ximei,GAO Zhilei,ZHANG Huimin,et al. Phylum-specific primer design and implication in quantification of the microbial community structure in Gu Jing Gong pit mud[J]. Advanced Materials Research,2014,3 607(1 051):311-316.
[22] ZHANG Huimin,GUO Changlu,HE Hongkui,et al.QPCR quantification strategy of minor species in a complex microbial community using species-specific primers[J]. Journal of Pure&Applied Microbiology,2013,7:601-610.
[23]赵长青,杨秦欢,邓静,等.窖泥的评定指标检测[J].生物技术进展,2012,2(3):212-216.
[24]张强,沈才洪,刘清斌,等.窖泥质量评价研究进展[J].酿酒科技,2013(7):84-86.
[25]卫春会,黄治国,甄攀.窖泥微生物群落SSCP分析条件优化的研究[J].四川理工学院学报(自然科学版),2010,23(6):695-698.
[26]徐岩,吴群,王石垒.一种快速筛选、鉴定及定量发酵食品中耐酸乳杆菌的方法[P].江苏:CN106906302A,2017-06-30.