采用改良的微孔板法培养纳豆芽孢杆菌(Bacillus subtilis natto),通过结晶紫染色定量分析产生的生物膜。结果表明:培养条件为pH 7、温度37 ℃、培养72 h且在5 g/100 mL葡萄糖-5 g/100 mL NaCl条件下成膜能力最佳。柠檬酸二铵和聚乙二醇-200(polyethylene glycol-200,PEG-200)随着质量浓度的增加对生物膜的形成量呈现先下降后上升的趋势,十二烷基磺酸钠(sodium dodecyl sulfate,SDS)抑制生物膜的形成,十六烷基三甲基溴化铵(cetyl trimethyl ammonium bromide,CTAB)促进生物膜的形成。所以低温抑制生物膜的形成,一定浓度的NaCl和葡萄糖促进生物膜形成,不同的表面活性剂对其生物膜的影响机制不同,为纳豆芽孢杆菌生物膜的研究提供理论基础。
冯静静
,
杨自名
,
吴静
,
李伟
,
Sokhna Mbacke Gningue
,
赵礼军
,
周文豪
,
薛正莲
,
王洲
,
刘艳
. 培养条件及表面活性剂的添加对纳豆芽孢杆菌生物膜形成的影响[J]. 食品与发酵工业, 2020
, 46(3)
: 90
-96
.
DOI: 10.13995/j.cnki.11-1802/ts.022148
The formation of biofilms by Bacillus subtilis natto can increase the production of vitamin K2. However, there is no report on the factors that can significantly influenced the formation of biofilm of Bacillus subtilis natto. Therefore, culture conditions and surfactants were used in this study to investigate their effect. Bacillus subtilis natto was cultivated by modified microplate method, and the concentration of biofilm was quantitatively analyzed by crystal violet staining. The results showed that the ability of biofilm formation is strongest under the conditions of 5 g/100 mL glucose-5 g/100 mL NaCl (percentage indicates mass concentration),pH 7 and cultivated at 37 ℃ for 72 h. Different surfactants had different effect on biofilm formation of Bacillus subtilis natto. The production of biofilm decreased when the concentration of diammonium citrate or polyethylene glycol-200 (PEG-200) between 0-0.4 g/L. While, when the concentration of diammonium citrate between 0.4-0.8 g/L and PEG-200 between 0.4-1.0 g/L, the production of biofilm increased. The formation of biofilm was inhibited when Sodium dodecyl sulfate (SDS) presented and it was increased when cetyl trimethyl ammonium bromide (CTAB) was added. Low temperature inhibits the formation of biofilm, while at proper concentration of NaCl and glucose can promote the formation of biofilms. The research provided a theoretical basis on the biofilm formation of Bacillus subtilis natto.
[1] 王伟. 乳酸菌抗菌肽Lac-B23对荧光假单胞菌生物膜的消减作用研究[D].哈尔滨:哈尔滨工业大学,2017.
[2] Massimiliano Marvasi,Pieter T. Visscher,brunellaPerito,giorgio mastromei,lilliam casillas-martínez. physiological requirements for carbonate precipitation during biofilm development of Bacillus subtilis etfA mutant[J]. FEMS Microbiology Ecology,2010,71(3):341-350.
[3] LAURA (KAATZ) W,JASON R M,JAMES P D,et al. Production and analysis of a Bacillus subtilis biofilm comprised of vegetative cells and spores using a modified colony biofilm model[J]. Journal of Microbiological Methods,2018,148.
[4] DE L F C, REFFUVEILLE F, FERNÁNDEZ L, et al. Bacterial biofilm development as a multicellular adaptation: antibiotic resistance and new therapeutic strategies[J]. CurrOpin Microbiol, 2013, 16:580-589.
[5] DEJEA C M, FATHI P, CRAIG J M, et al. Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria[J]. Science, 2018, 359(6375): 592-597.
[6] LIU J, MARTINEZ-CORRAL R, PRINDLE A, et al. Coupling between distant biofilms and emergence of nutrient time-sharing[J]. Science, 2017, 356(6338):638-642.
[7] WILKING J N, ZABURDAEV V, DE VOLDER M, et al. Liquid transport facilitated by channels in Bacillus subtilis biofilms[J]. Proc Natl Acad Sci USA, 2013, 110(3): 848-852.
[8] HOBLEY L, KIM S H, MAEZATO Y, et al. Norspermidine is not a self-produced trigger for biofilm disassembly[J]. Cell, 2014, 156:844-854.
[9] KOLODKIN-GAL I, ROMERO D, CAO S, et al. D-amino acids trigger biofilm disassembly[J]. Science, 2010, 238:627-629.
[10] Kolodkin-Gal I, Cao S, Chai L, B?ttcher T, Kolter R, Clardy J, Losick R. A self-produced trigger for biofilm disassembly that targets exopolysaccharide[J]. Cell, 2012, 149:684-692.
[11] BLAIR K M, TURNER L, WINKELMAN J T, et al. A molecular clutch disables lagella in the Bacillus subtilis biofilm[J]. Science, 2008, 320:1 636-1 638.
[12] XAVIER J B, FOSTER K R. Cooperation and conflict in microbial biofilms[J]. Proc Natl Acad Sci USA, 2007, 104:876-881.
[13] 刘珏玲,杨伟峰,王毅.细菌生物膜与表面活性剂抗菌的研究进展[J].中国病原生物学杂志,2016,11(9):858-860;867.
[14] 查飞,王洲,薛正莲,等.副溶血性弧菌生物膜形成及表面活性剂的影响[J].食品与发酵工业,2019,45(9):49-54.
[15] 葛芸,汤斌,李松.纳豆芽孢杆菌产纳豆激酶发酵工艺控制及纤溶酶分析[J].食品与发酵工业,2019.45(16):176-180;186.
[16] ANNON.Technology-food technology; investigators from nihon university have reported new data on food technology (the japanese fermented food natto inhibits sucrose-dependent biofilm formation by Cariogenic streptococci)[J]. Food Weekly News,2018.
[17] Juliana O. Moraes,Ellen A. Cruz,ítaloPinheiro,Tereza C.M. Oliveira,Ver nicaAlvarenga,Anderson S. Sant’Ana,Marciane Magnani. An ordinal logistic regression approach to predict the variability on biofilm formation stages by five Salmonella enterica strains on polypropylene and glass surfaces as affected by pH, temperature and NaCl[J]. Food Microbiology,2019,83:95-103.
[18] 徐寒莉. 盐分对自养与异养条件下短程硝化工艺和生物膜特性的影响研究[D].杭州:浙江大学,2017.
[19] 靳嘉巍,张力,查锡良,等.葡萄糖对表皮葡萄球菌生物被膜形成的影响及调节机制的研究[J].微生物学报,2005(3):431-436.
[20] TONE M R,SOLVEIGLANGSRUD,ASKILDHOLCK,et al. Different patterns of biofilm formation in Staphylococcus aureus under food-related stress conditions[J]. International Journal of Food Microbiology,2007,116(3):372-383.
[21] 佘鹏飞,陈丽华,许欢,等.D-氨基酸在生物膜分散中的作用研究进展[J].中国病原生物学杂志,2015,10(04):377-380.
[22] Desrosiers Martin,MynttiMatthew,James Garth. Methods for removing bacterial biofilms: in vitro study using clinical chronic rhinosinusitis specimens.[J]. American Journal of Rhinology,2007,21(5):527-532.
[23] BOO S T,WEIZHANG,JOE J H,et al. The extracellular matrix protects P seudomonas aeruginosa biofilms by limiting the penetration of tobramycin[J]. Environmental Microbiology,2013,15(10):2 865-2 878.
[24] MULCAHY H,CHARRON-MAZENODLAETITIA,LEWENZA S. Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms[J]. PLoS Pathogens (Online),2008,4(11).
[25] XIAO Chen,PHILIP S S. Biofilm removal caused by chemical treatments[J]. Water Research,2000,34(17):4 229-4 233.
[26] EDGAR R C. Search and clustering orders of magnitude faster than BLAST.[J]. Bioinformatics (Online),2010,26(19).