Screening And Mechanism Of Lactobacillus For Promoting Absorption Of SCFAs By Intestinal Epithelial Cells

Short Chain Fatty Acids(SCFAs)can not only provide energy as nutrients,but also play an important role in the interaction between intestinal flora and host.SCFAs has a wide range of physiological activities and biological effects,such as regulating nutrient metabolism and inhibiting endogenous cholesterol synthesis.When the intestinal flora is disordered,it will hinder the absorption of SCFAs,and then affect the health of the body.Probiotic Lactic acid bacteria(LAB)can play its prebiotic role by improving the absorption of SCFAs,but the mechanism is not clear.Therefore,in this paper,by establishing the model of intestinal epithelial cells absorbing SCFAs in vitro,we screened the Lactobacillus which can promote the intestinal epithelial cells to absorb SCFAs,and studied its regulation on SCFAs transporter to explore its possible mechanism of action,which provides theoretical basis for the application of probiotic functional fermented milk and probiotic preparation.The main research contents are as follows:1.Through acid and bile salt resistance,mucin adhesion,Caco-2 cell adhesion test and drug resistance test,the lactic acid bacteria strains with high adhesion were screened from the long-lived population in Bama,Guangxi.The results showed that among the 40 strains of lactic acid bacteria,18 strains of lactic acid bacteria,such as P1,P67,P58,P128,after passing through artificial gastric juice and artificial intestinal fluid in turn,had a survival rate of more than 10%;among them,the adhesion rates of strains P123,P198,P97,P58,F146,P1,P11,P67,P54 to mucin and Caco-2 cells were higher than 30%;the drug resistance of strains P1,P67,P58,P54,P123,P198,P97 and F146 was relatively low.According to API and 16S rDNA identification,P1,P67,P58,P54,P123,P198 and P97 are all Lactobacillus plantarum,and strain F146 is Lactobacillus fermentum.2.The integrity and stability of Caco-2 monolayer model were evaluated by morphological characteristics,transmembrane electrical resistance(TEER),apparent permeability coefficients(Papp),cell ultrastructure and cell counting kit-8(CCK-8).The results showed that when 1.2 ×105 cells/mL Caco-2 cell suspension was inoculated into Transwell 12 well plate,the TEER of Caco-2 cell model was 1032.8 Ω·cm2 on the 7th day,1319.31 Ω·cm2 on the 15th day,and the Papp value was less than 1×10-6 cm/s.The tight junction and microvilli structure could be clearly observed under transmission electron microscope.The established Caco-2 cell model could reach 1.2×10-6 cm/s.The cell survival rate was more than 91%when the cells were stable in propionic acid and butyric acid solution for 3 h,which indicated that the cell model was successfully established and could be used for subsequent absorption test.3.Using quadrupole mass spectrometry to establish a method for the determination of SCFAs in cells by full scanning and sim scanning,lactic acid bacteria strains with high adhesion were selected and inoculated into Caco-2 cells with 1 mM propionic acid and 1 mM butyric acid to absorb SCFAs.The lactic acid bacteria which could promote the absorption of SCFAs by intestinal epithelial cells were screened out by culturing for 3 h with the uninoculated bacteria as the control.The results showed that the relative standard deviation(RSD)of SCFAs standard solution was small,and the linear range was good,the correlation coefficients were greater than 0.999,and the quality control samples of propionic acid and butyric acid were less than 30%.The experimental data were stable and reliable.The method was suitable for the detection of propionic acid and butyric acid in cells.The contents of propionic acid and butyric acid in Caco-2 cells in the control group were 0.156 and 0.087 μg/ml,respectively.The contents of propionic acid in Caco-2 monolayer model inoculated with Lactobacillus plantarum P54,P58,P67,P97,P123 and P198 were 0.290,0.335,0.282,0.305,0.306 and 0.311 μg/ml,respectively.The content of butyric acid in Caco-2 monolayer model inoculated with Lactobacillus fermentans F146 and Lactobacillus plantarum P1 was 0.092 and 0.104 μg/ml,respectively.The butyric acid content in Caco-2 monolayer model inoculated with Lactobacillus plantarum P54,P58,P67,P97,P123 and P198 was 0.022,0.024,0.018,0.022 and 0.021 μg/ml,respectively.4.The effect of lactic acid bacteria on SCFAs transporter gene expression was studied by real-time fluorescent quantitative PCR.The strains were inoculated into Caco-2 cells with 1 mM propionic acid and 1 mM butyric acid to absorb SCFAs.The cells were cultured for 3 h with the uninfected cells as the control.The results showed that compared with the control group,Lactobacillus plantarum P54,P58,P97,F146,P198,P1 significantly increased the relative expression of MCT1 in Caco-2 cells(P<0.05),while Lactobacillus plantarum P54,P58,P67,P97,P123,P198 significantly increased the relative expression of SMCT1(P<0.05).