胆盐对植物乳杆菌NCU116应激基因和关键生理指标的影响
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摘要
植物乳杆菌NCU116是1株分离自四川传统泡菜的优良益生菌菌株,具有降血糖、调节肠道菌群、缓解便秘等功能。乳酸菌在进入人体胃肠道时,胆盐、胃酸等不利条件会降低其生理活性。为研究胆盐胁迫对NCU116的影响,文章分别研究了在质量浓度0、0. 3、0. 5、0. 7、1 g/100 mL的胆盐胁迫下NCU116转录水平及关键生理指标的变化。结果表明:NCU116受到0. 3 g/100 mL质量浓度的胆盐胁迫后,分子伴侣蛋白基因dnaK、groES、热休克蛋白基因hsp、6-磷酸果糖激酶基因pfk、ATP依赖型DNA解旋酶基因uvrD1等的相对转录水平显著上调;通过生理分析,发现细胞表面疏水性和自凝聚特性降低、细胞膜出现破损、葡萄糖代谢受到抑制;但随着胆盐质量浓度继续上升至0. 7、1. 0 g/100 mL时,NCU116受到的影响减弱。胞内氨基酸总含量受胆盐胁迫后增加,表明NCU116有良好的胆盐耐受能力,具有在胃肠道发挥益生功能的潜在能力。
Lactobacillus plantarum NCU116 is an excellent probiotic strain isolated from Sichuan traditional sauerkraut. When lactic acid bacteria enter the human gastrointestinal tract,they often encounter adverse conditions,such as bile salts and gastric acid,which may reduce their physiological activities. In order to study the effects of bile salts on L. plantarum NCU116,changes in transcription levels of associated genes as well as key physiological indicators of L. plantarum NCU116 under the stress of 0,0. 3,0. 5,0. 7,and 1 g/100 mL bile salts were analyzed. The results showed that the transcription levels of dnaK and groES,hsp,pfk,and uvr D1,which encode for molecular chaperone protein,heat shock protein,6-phosphofructose kinase,and ATP-dependent DNA helicase,respectively,were significantly up-regulated by 0. 3 g/100 mL bile salts. Moreover,the cellular surface hydrophobicity and self-aggregation characteristics of L. plantarum NCU116 reduced. Besides,its cell membrane integrity was destroyed,and its glucose metabolism was inhibited. However,the impacts of bile salts on L. plantarum NCU116 weakened as the concentration of bile salts increased to 0. 7 and 1 g/100 mL. In addition,bile salts increased the contents of intracellular total amino acids contents. This study revealed that L. plantarum NCU116 has good ability to resist bile salts and therefore has the potential to exert its probiotic functions in the gastrointestinal tract.
Lactobacillus plantarum NCU116 is an excellent probiotic strain isolated from Sichuan traditional sauerkraut. When lactic acid bacteria enter the human gastrointestinal tract,they often encounter adverse conditions,such as bile salts and gastric acid,which may reduce their physiological activities. In order to study the effects of bile salts on L. plantarum NCU116,changes in transcription levels of associated genes as well as key physiological indicators of L. plantarum NCU116 under the stress of 0,0. 3,0. 5,0. 7,and 1 g/100 mL bile salts were analyzed. The results showed that the transcription levels of dnaK and groES,hsp,pfk,and uvr D1,which encode for molecular chaperone protein,heat shock protein,6-phosphofructose kinase,and ATP-dependent DNA helicase,respectively,were significantly up-regulated by 0. 3 g/100 mL bile salts. Moreover,the cellular surface hydrophobicity and self-aggregation characteristics of L. plantarum NCU116 reduced. Besides,its cell membrane integrity was destroyed,and its glucose metabolism was inhibited. However,the impacts of bile salts on L. plantarum NCU116 weakened as the concentration of bile salts increased to 0. 7 and 1 g/100 mL. In addition,bile salts increased the contents of intracellular total amino acids contents. This study revealed that L. plantarum NCU116 has good ability to resist bile salts and therefore has the potential to exert its probiotic functions in the gastrointestinal tract.
引文
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[31]曾东,倪学勤,胡晓玲,等.玉米、豆粕和麦麸提取物对4株乳酸杆菌胆盐耐受力的影响[J].浙江大学学报(农业与生命科学版),2009,35(6):659-664.
[32] KOSTER K L,LEI Y P,ANDERSON M,et al. Effects of vitrified and nonvitrified sugars on phosphatidylcholine fluid-to-gel phase transitions[J]. Biophysical Journal,2000,78(4):1 932-1 946.
[33] BEGLEY M,GAHAN C G,HILL C. The interaction between bacteria and bile[J]. Fems Microbiology Reviews,2010,29(4):625-651.
[34]黄珊.基于转录组学与代谢组学的Streptococcus thermophilus TF96氨基酸代谢机制研究[D].哈尔滨:东北农业大学,2017.
[35]刘岩,王慧,史吉平,等.微生物法生产L-丝氨酸代谢工程研究进展[J].生物技术通报,2015,31(8):44-49.
[36] CHRISTENSEN J E,DUDLEY E G,PEDERSON J A,et al. Peptidases and amino acid catabolism in lactic acid bacteria[J]. Antonie van Leeuwenhoek,1999,76(1-4):217-246.
[2] VRIES M C D,VAUGHAN E E,KLEEREBEZEM M,et al. Lactobacillus plantarum—survival,functional and potential probiotic properties in the human intestinal tract[J]. International Dairy Journal,2006,16(9):1 018-1 028.
[3]熊涛.直投式发酵泡菜益生乳酸菌的选育与菌剂制备及其益生特性研究[D].南昌:南昌大学,2012.
[4]谢俊华.植物乳杆菌NCU116对肠道健康的影响[D].南昌:南昌大学,2016.
[5]李川.植物乳杆菌NCU116的益生功能及其作用机制[D].南昌:南昌大学,2015.
[6] RUIZ L,MARGOLLES A,SANCHEZ B. Bile resistance mechanisms in Lactobacillus and Bifidobacterium[J].Frontiers in Microbiology,2013,4(396):396.
[7] LAUBER W M,CARROLL J A,DUFIELD D R,et al.Mass spectrometry compatibility of two-dimensional gel protein stains[J]. Electrophoresis,2001,22(5):906-918.
[8]赵小茜,魏旭丹,陈戴玲,等.乳酸菌耐酸耐胆盐机制研究进展[J].乳业科学与技术,2017,40(3):33-36.
[9] FLAHAUT S,HARTKE A,GIARD J C,et al. Alkaline stress response in Enterococcus faecalis:Adaptation,crossprotection,and changes in protein synthesis[J]. Applied&Environmental Microbiology,1997,63(2):812-814.
[10] BRONP A,MOLENAAR D,VOS W M D,et al. DNA micro-array-based identification of bile-responsive genes in Lactobacillus plantarum[J]. Journal of Applied Microbiology,2010,100(4):728-738.
[11]毕洁.胆盐水解酶提高乳酸菌胆盐耐受能力的酶学与生理学机制研究[D].无锡:江南大学,2016.
[12] ROSENBERG M,GUTNICK D,ROSENBERG E. Adherence of bacteria to hydrocarbons:A simple method for measuring cell-surface hydrophobicity[J]. FEMS Microbiology Letters,1980,9(1):29-33.
[13]胡斌.植物乳杆菌胆盐耐受评价及内在影响因素分析[D].无锡:江南大学,2015.
[14] BAO Y,ZHANG Y,LI H,et al. In vitro screen of Lactobacillus plantarum,as probiotic bacteria and their fermented characteristics in soymilk[J]. Annals of Microbiology,2012,62(3):1 311-1 320.
[15] HEUMAN D M,BAJAJ R S,LIN Q. Adsorption of mixtures of bile salt taurine conjugates to lecithin-cholesterol membranes:implications for bile salt toxicity and cytoprotection[J]. Journal of Lipid Research,1996,37(3):562-573.
[16]陈欣,潘素华,胡黎黎,等.人源乳酸杆菌耐酸耐胆盐能力的测定和鉴定[J].现代预防医学,2006,33(3):278-279.
[17]李洋,赵欣,张玉,等.牦牛酸乳中耐酸耐胆盐乳酸菌的分离筛选和鉴定[J].食品与机械,2018,34(7):23-28; 33.
[18] BERNSTEIN H,PAYNE C M,BERNSTEIN C,et al.Activation of the promoters of genes associated with DNA damage,oxidative stress,ER stress and protein malfolding by the bile salt,deoxycholate[J]. Toxicology Letters,1999,108(1):37-46.
[19] AKERFELDT,KARIN. Guidebook to molecular chaperones and protein-folding catalysts\r. mary-jane gething[J]. The Quarterly Review of Biology,2000,75(1):48-49.
[20]刘倩颖.基于RT-PCR技术对植物乳杆菌耐盐分子机理的研究[D].沈阳:沈阳农业大学,2014.
[21]乌日娜.益生菌Lactobacillus casei Zhang蛋白质组学研究[D].呼和浩特:内蒙古农业大学,2009.
[22] GEIS A,DEMERDASH H A M E,HELLER K J. Sequence analysis and characterization of plasmids from Streptococcus thermophilus[J]. Biochemistry,2006,50(1):53-69.
[23] CONNOLLY J P R,ROE A J. Intracellular D-serine accumulation promotes genetic diversity via modulated induction of Rec A in enterohemorrhagic Escherichia coli[J]. Journal of Bacteriology,2016,198(24):3 318-3 328.
[24] SINGH P,PATIL K N,KHANDUJA J S,et al. Mycobacterium tuberculosis Uvr D1 and Uvr A proteins suppress DNA strand exchange promoted by cognate and noncognate Rec A proteins[J]. Biochemistry,2010,49(23):4 872.
[25] RHODIUS V A,SUH W C,NONAKA G,et al. Conserved and variable functions of theσE stress response in related genomes[J]. Plos Biology,2006,4(1):e2.
[26]杨振泉,靳彩娟,张咪,等.高粘附性戊糖片球菌的筛选、标记及其表面疏水与自凝聚性特征[J].食品与生物技术学报,2015,34(9):926-934.
[27] DIAO M,NGUYEN T A,TARAN E,et al. Effect of energy source,salt concentration and loading force on colloidal interactions between Acidithiobacillus ferrooxidans cells and mineral surfaces[J]. Colloids&Surfaces B Biointerfaces,2015,132:271-280.
[28] NOH D O,GILLILAND S E. Influence of bile on cellular integrity andβ-galactosidase activity of Lactobacillus acidophilus[J]. Journal of Dairy Science,1993,76(5):1 253-1 259.
[29] FERNANDEZ M, Z'UNIGA M. Amino acid catabolic pathways of lactic acid bacteria[J]. Critical Reviews in Microbiology,2006,32(3):155.
[30] POOLMAN B. Energy transduction in lactic acid bacteria[J]. Fems Microbiology Reviews,1993,12(1-3):125-147.
[31]曾东,倪学勤,胡晓玲,等.玉米、豆粕和麦麸提取物对4株乳酸杆菌胆盐耐受力的影响[J].浙江大学学报(农业与生命科学版),2009,35(6):659-664.
[32] KOSTER K L,LEI Y P,ANDERSON M,et al. Effects of vitrified and nonvitrified sugars on phosphatidylcholine fluid-to-gel phase transitions[J]. Biophysical Journal,2000,78(4):1 932-1 946.
[33] BEGLEY M,GAHAN C G,HILL C. The interaction between bacteria and bile[J]. Fems Microbiology Reviews,2010,29(4):625-651.
[34]黄珊.基于转录组学与代谢组学的Streptococcus thermophilus TF96氨基酸代谢机制研究[D].哈尔滨:东北农业大学,2017.
[35]刘岩,王慧,史吉平,等.微生物法生产L-丝氨酸代谢工程研究进展[J].生物技术通报,2015,31(8):44-49.
[36] CHRISTENSEN J E,DUDLEY E G,PEDERSON J A,et al. Peptidases and amino acid catabolism in lactic acid bacteria[J]. Antonie van Leeuwenhoek,1999,76(1-4):217-246.