嗜热链球菌的电转化条件
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摘要
为了获得高产活性共轭亚油酸(conjugated linoleic acid,CLA)的嗜热链球菌并开发功能性发酵乳,研究甘氨酸浓度、菌体生物量、电极缓冲液、电压、质粒浓度对电转化效率的影响。结果表明,嗜热链球菌菌体生长与甘氨酸质量浓度呈现负相关,质量浓度为10 g/L时电转化效率最高。电转化效率的影响呈现先上升后下降的趋势;随着菌体生物量、转化电压和质粒浓度的增加,OD600=0. 8、电压1. 8 k V、质粒质量浓度为1. 0 g/L时电转化效率最高;电转缓冲液bufferⅡ(0.5 mol/L蔗糖,1 mmol/L柠檬酸铵; p H 6.0)表现出较好的转化效率。优化嗜热链球菌的电转化条件,提高其转化率,为乳酸菌的高效转化提供一定的理论基础,并为生产具有CLA生理功能的发酵乳提供新的思路。
In order to obtain Streptococcus thermophilus with high yield of conjugated linoleic acid( CLA) to develop functional fermented milk,effects of glycine concentration,bacterial density,buffer solution,voltage and plasmid concentration on electroporation efficiency of S. thermophilus were investigated. The growth of S. thermophiles was negatively correlated with glycine concentration,and its electroporation efficiency was the highest when the cells grew with 10 g/L glycine. The efficiency increased and then decreased with increasing cell density,transformation voltage,and plasmid concentration. The optimal conditions for electroporation were: cell density of 0. 8( OD600),voltage of 1. 8 kV,and plasmid concentration of 1. 0 g/L. Moreover,buffer II( pH = 6. 0) composed of 0. 5 mol/L sucrose and 1 mmol/L ammonium citrate showed good transformation efficiency. By optimizing electrotransformation conditions,the transformation rate of S. thermophiles increased,which provides a theoretical basis for transforming lactic acid bacteria efficiently and also gives a new idea for producing fermented milk with CLA.
In order to obtain Streptococcus thermophilus with high yield of conjugated linoleic acid( CLA) to develop functional fermented milk,effects of glycine concentration,bacterial density,buffer solution,voltage and plasmid concentration on electroporation efficiency of S. thermophilus were investigated. The growth of S. thermophiles was negatively correlated with glycine concentration,and its electroporation efficiency was the highest when the cells grew with 10 g/L glycine. The efficiency increased and then decreased with increasing cell density,transformation voltage,and plasmid concentration. The optimal conditions for electroporation were: cell density of 0. 8( OD600),voltage of 1. 8 kV,and plasmid concentration of 1. 0 g/L. Moreover,buffer II( pH = 6. 0) composed of 0. 5 mol/L sucrose and 1 mmol/L ammonium citrate showed good transformation efficiency. By optimizing electrotransformation conditions,the transformation rate of S. thermophiles increased,which provides a theoretical basis for transforming lactic acid bacteria efficiently and also gives a new idea for producing fermented milk with CLA.
引文
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[26]张旭,崔艳华,张兰威,等.乳酸菌电转化条件研究[J].兰州大学学报(自科版),2009,45(6):26-33.
[27]范璟,席雪冬,黄彦,等.植物乳杆菌g63电转化方法的优化[J].食品科学,2016,37(3):180-185.
[28]朱森康,黄磊,李燕飞,等.制备高效大肠杆菌电转化感受态细胞和电转化条件的研究[J].生物技术通报,2011(10):206-209.
[29]于建宁,宋小敬,王公金,等.乳酸乳球菌电转化方法的优化[J].江苏农业科学,2013,41(8):34-36.
[30]高玲,邓阳,陆兆新,等.多黏类芽孢杆菌jsa-9电转化方法的优化[J].食品科学,2014,35(11):89-94.
[2] NIKFARDJAM M P,DIETRICH H. Biology of microorganisms on grapes,in must and in wine[J]. 2009:259-271.
[3]韩希妍,孙大庆,相丽,等.乳酸菌风味代谢物质的基因调控[J].微生物学报,2007,47(6):1 105-1 109.
[4]梅林,王志耕.酸奶、乳酸菌饮料风味及其部分理化特性的研究[J].乳业科学与技术,2004,27(1):17-19.
[5] TETTELIN H,MASIGNANI V,CIESLEWICZ M J,et al.Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae:Implications for the microbial “Pan-Genome”[J]. Proceedings of the national academy of sciences of the United States of America,2005,102(39):13 950-13 955.
[6] TETTELIN H,RILEY D,CATTUTO C,et al. Comparative genomics:The bacterial pan-genome[J]. Current Opinion in Microbiology,2008,11(5):472-477.
[7] DAI W,WANG Y,WANG J,et al. Application of recombined plasmid p MG36e-nisi-gfp in lactic acid bacteria[J].Journal of Tianjin University of Science&Technology,2012,20(3):179-184.
[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.
[9]官雪芳,郑怡,徐庆贤,等.一株酸奶发酵嗜热链球菌的筛选及特性研究[J].西北农业学报,2018,27(1):124-130.
[10]赵丽艳,杨柳,陈宇飞.酸豆乳益生菌发酵条件探析[J].食品安全导刊,2018(3):129-129.
[11]吴军林,柏建玲,莫树平,等.乳酸菌r8高密度培养的发酵工艺研究[J].现代食品科技,2018,34(2):164-170.
[12]韩庆功,崔艳红,王元元,等.植物乳杆菌的生理特性及体外益生效果研究[J].粮食与饲料工业,2018(3):42-46.
[13]孙敏,袁凤霞,曹晓虹,等.传统发酵食品中耐胃肠道环境乳酸菌的筛选及其在酸乳发酵中的应用[J].食品与发酵工业,2018,44(3):114-120.
[14]宋程飞,郝敬云,程蔚兰,等.杜氏盐藻电击转化体系的优化[J].山西农业大学学报(自然科学版),2018,38(3):36-42.
[15] ZHANG Y,KE X,GUO H,et al. Research of electroporation processing of Lactococcus lactis ML23 with the constitutive expressionvector p MG36e[J]. Journal of Agricultural University of Hebei,2010,37(4):121-130.
[16] GUO Y,GU H,ZHANG X,et al. Research of electrotransformation processing of Lactobacilli delbrueckii subsp. Bulgaricus with the constitutive expression vector p MG36e[J]. Journal of Chinese Institute of Food Science and Technology,2011,5(2):59-66.
[17]北婷婷,李晨,谷新晰,等.鼠李糖乳杆菌电转化条件的研究[J].中国食品学报,2017,17(4):65-71.
[18]李计来,崔文禹,刘敬,等. Cho Dg44细胞电转染条件的优化[J].中国生物制品学杂志,2017,30(11):1 207-1 210.
[19] PAPAGIANNI M,AVRAMIDIS N,FILIOUSSIS G. High efficiency electrotransformation of Lactococcus lactis Spp.Lactis cells pretreated with lithium acetate and dithiothreitol[J]. BMC biotechnology,2007,7(1):15-27.
[20]蒋华波,夏梓元,张琼阁,等.大肠埃希菌tg1电穿孔法转化条件优化研究[J].生物技术进展,2017,7(1):72-76.
[21]张放,周载鑫,张丹丹,等.电穿孔法转染人原代成纤维细胞条件的优化[J].第二军医大学学报,2017,38(10):1 311-1 314.
[22] LANDETE J M,ARQUS J L,PEIROTN,et al. An improved method for the electrotransformation of lactic acid bacteria:A comparative survey[J]. Journal of Microbiological Methods,2014,105(10):130-133.
[23] TAGAWA J,INOUE T,NAITO M,et al. Development of a novel plasmid vector p TIO-1 adapted for electrotransformation of Porphyromonas gingivalis[J]. Journal of Microbiological Methods,2014,105(12):174-179.
[24] HUANG Y,ZHANG D. Cloning and expression of the manganese superoxide dismutase gene of Escherichia Coli in Lactobacillus bulgaricus[J]. Food Science,2005,5(16):160-167.
[25]刘朝,乔建军,朱宏吉.乳酸菌肽聚糖的研究进展[J].微生物学通报,2016,43(1):188-197.
[26]张旭,崔艳华,张兰威,等.乳酸菌电转化条件研究[J].兰州大学学报(自科版),2009,45(6):26-33.
[27]范璟,席雪冬,黄彦,等.植物乳杆菌g63电转化方法的优化[J].食品科学,2016,37(3):180-185.
[28]朱森康,黄磊,李燕飞,等.制备高效大肠杆菌电转化感受态细胞和电转化条件的研究[J].生物技术通报,2011(10):206-209.
[29]于建宁,宋小敬,王公金,等.乳酸乳球菌电转化方法的优化[J].江苏农业科学,2013,41(8):34-36.
[30]高玲,邓阳,陆兆新,等.多黏类芽孢杆菌jsa-9电转化方法的优化[J].食品科学,2014,35(11):89-94.