采用体外实验探究了嗜酸乳杆菌KLDS1.0901的浓度、生长阶段、表层蛋白对其黏附Caco-2细胞能力的影响。将未处理、去除表层蛋白和热致死的KLDS1.0901采用荧光标记灌胃给小鼠,取不同部位的肠黏膜,进行流式细胞检测。通过透射电镜和SDS-PAGE对表层蛋白粗品进行分析鉴定。结果表明,随着菌体浓度的增加,KLDS1.0901黏附率增大直至浓度为10~8CFU/mL黏附率达到饱和。菌体处于对数生长末期时,黏附特性更优。Caco-2单细胞层与KLDS1.0901表层蛋白共同孵育1 h黏附量显著下降(p <0.05),且加入的表层蛋白浓度越高,对其黏附抑制作用越强。未处理的KLDS1.0901细胞壁外围包裹着表层结构,但除去表层蛋白后表层结构即消失。表层蛋白粗品中表层蛋白含量约为15.6%,分子量在43 kDa左右。综上,嗜酸乳杆菌KLDS1.0901含有丰富的表层蛋白,且能较好的黏附于小鼠肠道,具有益生菌的潜力。
In vitro experiments were conducted to investigate the effects of concentration,growth stage and surface layer protein of Lactobacillus acidophilus KLDS1.0901 on its ability to adhere to Caco-2 cells.The untreated KLDS1.0901,the remove surface layer protein and the heat-killing KLDS1.0901 were fluorescently labeled with cFDA-SE,then administered to mice,and the intestinal mucosa of different parts of the intestine was taken for flow cytometry. Surface layer protein of KLDS1.0901 was crudely extracted by 5 mol/L LiCl and identified by transmission electron microscopy and SDS-PAGE.The results showed that with the increase of bacterial concentration,the adhesion rate of KLDS1.0901 increased,but reached saturation when the concentration was 10~8 CFU/mL.At the end of the logarithmic growth phase,the bacteria had better adhesion properties.After the Caco-2 monolayer was co-incubated with the surface layer protein for 1 h,the adhesion amount of KLDS1.0901 to it was significantly decreased. And the higher the concentration of the surface layer protein added,the stronger the inhibition of adhesion.Under transmission electron microscopy,the untreated KLDS1.0901 cell wall was surrounded by a surface structure,which disappeared after LiCl treatment.The extracted crude protein had a surface layer protein content of approximately 15.6% and a molecular weight of approximately 43 kDa. The results showed that Lactobacillus acidophilus KLDS1.0901 was rich in surface layer protein and could adhere to the intestinal tract of mice well.It had the potential of probiotics.
Science and Technology of Food Industry
surface layer proteins