富硒长双歧杆菌DD98菌株的黏附特性及黏附机制初步研究
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摘要
利用Caco-2细胞作为体外黏附模型,通过细菌黏附率评价一株新的长双歧杆菌菌株DD98(Bifidobacterium longum DD98,DD98)富硒前后的黏附能力,测定细菌自动聚集能力与表面疏水性,采用荧光定量PCR技术检测细菌表面Tuf蛋白mRNA表达量差异。结果表明,与DD98相比,富硒DD98(Selenium-enriched Bifidobacterium longum DD98,Se-DD98)黏附于Caco-2细胞的数量从(39.12±17.57)CFU/100 cells上升到(581.17±62.79) CFU/100 cells,Se-DD98对Caco-2细胞的黏附能力极显著升高(p<0.01)。相比DD98,Se-DD98自动聚集能力略有上升,且其表面疏水性从5.12%±0.16%上升到20.25%±2.14%,极显著性升高(p<0.01);荧光定量PCR结果表明,与Caco-2细胞共孵育2 h后,Se-DD98菌体表面Tuf黏附蛋白表达显著上调(p<0.05)。表明DD98经富硒培养后,黏附能力显著提升,其黏附能力的增强可能与细菌表面Tuf黏附蛋白的改变有关。
In order to study whether there was a difference in the adhesion ability of a new Bifidobacterium strain DD98(Bifidobacterium longum DD98,DD98)before and after selenium enrichment,bacterial adhesion rate was evaluated by in vitro adhesion model of Caco-2 cells,the auto-aggregation ability and surface hydrophobicity of the bacteria were also determined,Tuf mRNA,as the quantitative expression of bacterial surface protein was detected by real-time fluorescent quantitative PCR. The results showed that selenium-enriched Bifidobacterium longum DD98(Se-DD98)presented extremely higher adhesive ability to Caco-2 cells than DD98(p<0.01).The average number of DD98 bacteria adhering to Caco-2 cells was(39.12±17.57)CFU/100 cells,while the number of Se-DD98 was(581.17±62.79)CFU/100 cells. Compared with the DD98,the auto-aggregation ability of Se-DD98 increased slightly,and the surface hydrophobicity increased significantly from 5.12%±0.16% to 20.25%±2.14%(p<0.01).The results of real-time PCR showed that the expression of Tuf protein mRNA on Se-DD98 cells significantly up-regulated after 2 h incubation with Caco-2 cells(p<0.05). It was indicated that the adhesion ability of DD98 after selenium-enriched culture was significantly improved,and the enhancement of adhesion might be related to the change of Tuf protein.
In order to study whether there was a difference in the adhesion ability of a new Bifidobacterium strain DD98(Bifidobacterium longum DD98,DD98)before and after selenium enrichment,bacterial adhesion rate was evaluated by in vitro adhesion model of Caco-2 cells,the auto-aggregation ability and surface hydrophobicity of the bacteria were also determined,Tuf mRNA,as the quantitative expression of bacterial surface protein was detected by real-time fluorescent quantitative PCR. The results showed that selenium-enriched Bifidobacterium longum DD98(Se-DD98)presented extremely higher adhesive ability to Caco-2 cells than DD98(p<0.01).The average number of DD98 bacteria adhering to Caco-2 cells was(39.12±17.57)CFU/100 cells,while the number of Se-DD98 was(581.17±62.79)CFU/100 cells. Compared with the DD98,the auto-aggregation ability of Se-DD98 increased slightly,and the surface hydrophobicity increased significantly from 5.12%±0.16% to 20.25%±2.14%(p<0.01).The results of real-time PCR showed that the expression of Tuf protein mRNA on Se-DD98 cells significantly up-regulated after 2 h incubation with Caco-2 cells(p<0.05). It was indicated that the adhesion ability of DD98 after selenium-enriched culture was significantly improved,and the enhancement of adhesion might be related to the change of Tuf protein.
引文
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[2]Short S P,Pilat J M,Williams C S.Roles for selenium and selenoprotein P in the development,progression,and prevention of intestinal disease[J].Free Radical Biology and Medicine,2018,127:26-35.
[3]Mrvcic J,Stanzer D,Solic E,et al.Interaction of lactic acid bacteria with metal ions:Opportunities for improving food safety and quality[J].World J Microbiol Biotechnol,2012,28(9):2771-2782.
[4]Bom A,Nemcová R,Guba D M P.The possibilities of potentiating the efficacy of probiotics[J].Trends in Food Science and Technology,2002(4):121-126.
[5]Roman M,Jitaru P,Barbante C.Selenium biochemistry and its role for human health[J].Metallomics,2014,6(1):25-54.
[6]Ip C,Birringer M,Block E,et al.Chemical speciation influences comparative activity of selenium-enriched garlic and yeast in mammary cancer prevention[J].J Agric Food Chem,2000,48(6):2062-2070.
[7]Arena M P,Capozzi V,Spano G,et al.The potential of lactic acid bacteria to colonize biotic and abiotic surfaces and the investigation of their interactions and mechanisms[J].Appl Microbiol Biotechnol,2017,101(7):2641-2657.
[8]Bentley-Hewitt K L,Narbad A,Majsak-Newman G,et al.Lactobacilli survival and adhesion to colonic epithelial cell lines is dependent on long chain fatty acid exposure[J].European Journal of Lipid Science and Technology,2017:1700062.
[9]Ferrando V F V,Quiberoni A Q A,Reinheimer J R J,et al.Functional properties of Lactobacillus plantarum strains:A study in vitro of heat stress influence[J].Food Microbiology,2016:154-161.
[10]Ren D,Li C,Qin Y,et al.Inhibition of Staphylococcus aureus adherence to Caco-2 cells by lactobacilli and cell surface properties that influence attachment[J].Anaerobe,2012,18(5):508-515.
[11]Valeriano V D,Parungao-Balolong M M,Kang D K.In vitro evaluation of the mucin-adhesion ability and probiotic potential of Lactobacillus mucosae LM1[J].J Appl Microbiol,2014,117(2):485-497.
[12]Rosenberg M,Judes H,Weiss E.Cell surface hydrophobicity of dental plaque microorganisms in situ[J].Infect Immun,1983,42(2):831-834.
[13]Dill K A.The meaning of hydrophobicity[J].Science,1990,250(4978):297-298.
[14]赵维俊.益生菌表面疏水性与自动聚集能力的研究[D].西安:陕西科技大学,2012.
[15]李清,刘小莉,王英,等.植物乳杆菌表面性质及对Caco-2细胞的黏附[J].食品科学,2015,36(9):97-101.
[16]Del R B,Sgorbati B,Miglioli M,et al.Adhesion,autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum[J].Lett Appl Microbiol,2000,31(6):438-442.
[17]Wei X,Yan X,Chen X,et al.Proteomic analysis of the interaction of Bifidobacterium longum NCC2705 with the intestine cells Caco-2 and identification of plasminogen receptors[J].J Proteomics,2014,108:89-98.
[18]宗方方,谭俊,邵雷,等.长双歧杆菌优势菌株的高通量筛选[J].食品工业科技,2016,37(15):150-153.
[19]Castellano P,Perez I M,Longo B L,et al.Lactobacillus spp.impair the ability of Listeria monocytogenes FBUNT to adhere to and invade Caco-2 cells[J].Biotechnol Lett,2018,40(8):1237-1244.
[20]唐文君,范鑫鹏,倪兵,等.腹毛目纤毛虫颗粒尖毛虫射出胞器的研究[J].生物学杂志,2016,33(4):38-42.
[21]王丽群,张佰荣,王彦,等.双歧杆菌体外对Caco-2的黏附及其表面性质分析[J].微生物学报,2010,50(5):606-613.
[22]Collado M C,Meriluoto J,Salminen S.Adhesion and aggregation properties of probiotic and pathogen strains[J].European Food Research and Technology,2008(5):1065-1073.
[23]Tuo Y,Yu H,Ai L,et al.Aggregation and adhesion properties of 22 Lactobacillus strains[J].J Dairy Sci,2013,96(7):4252-4257.
[24]Rong J,Zheng H,Liu M,et al.Probiotic and anti-inflammatory attributes of an isolate Lactobacillus helveticus NS8 from Mongolian fermented koumiss[J].BMC Microbiol,2015,15:196.
[25]陈宣男.长双歧杆菌NCC2705与宿主细胞Caco2相互作用的研究[D].长春:吉林大学,2010.