The Influence Of Probiotics Used In Dairy On Intestinal Barrier Function By Caco-2Cells

Probiotics are widely used in food industry during the past two decades, They areidentified to have many good efforts to their host，although the mechanisms involvedare not fully understood. Most probiotics have a long history of safe use and fall intothe category of organisms classified as “generally regarded as safe”. Some reportsabout negative probiotic effects have surfaced, which make people pay particularattention to the safety of probiotics.[Objective] Lactobacillus bulgaricus, Streptococcus thermophilus andLactobacillus acidophilus are commonly used as probiotics in fermented dairy milkssuch as yogurt in China. The aim of this study was to examine the influence of threeprobiotics strains on intestinal barrier function by Caco-2cells and discuss whetherthe potential safety problems emerge or not.[Methods] Caco-2cell viability was detected by MTT assay when exposed tothese three strains either alone or in combination. ELISA was used to determinewhether differentiated Caco-2cells produce cytokines IL-1β, IL-10,TNF-α and IFN-γin response to three strains. The Caco-2monolayer was established and phenol redpermeability was also examined after the monolayer exposing to Lactobacillusbulgaricus, Streptococcus thermophilus and Lactobacillus acidophilus at theconcentration of108cfu/mL. Transmission electron microscopy was used to observethe morphology of tight junction. The cytoskeletal protein β-tublin responses ofdifferentiated Caco-2to three strains were also detected by Laser scanning confocalmicroscope. Western blot analysis was used for Tight Junction membraneprotein-occludin expression.[Results] Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillusacidophilus and mixed group had a significant inhibition on proliferation ofundifferentiated Caco-2cells at the final concentration (106-108cfu/mL), thepercentage of this inhibition had a dosage dependent. There was no significantinhibition lower than104cfu/mL. From the data, half inhibition concentration（IC50） was obtained. Lactobacillus bulgaricus IC50=1.19108cfu/mL, Lactobacillusacidophilus IC50=3.93108cfu/mL, Streptococcus thermophilus IC50=2.02108cfu/mL, mixed IC50=3.93108cfu/mL. The combination effects of three probiotics onCaco-2cells showed no significant difference compared with single action. It showedthat there was no increased secretion of IL-1β, IL-10, TNF-α and IFN-γ detected byELISA from differentiated Caco-2cells to the three strains after48hours. The dataclearly showed that phenol red diffusion was significantly increased after exposing toStreptococcus thermophilus72hours. Lactobacillus bulgaricus and Lactobacillusacidophilus have showed no influence on phenol red diffusion of Caco-2cellmonolayer. The morphology of tight junction was not changed after the three strainstreatment. The cytoskeletal protein β-tublin was not influenced after the three strainstreatment. Western blot analysis demonstrated that the quantity of occludin expressionwas not changed even at the dose of108cfu/mL probiotics for24hours.[Conclusion] It suggested that adverse effects didn’t occur even exposing tothese strains at high dose for24hours in human intestinal tract by a vitro study.