Metabolism of fructooligosaccharides by Lactobacillus paracasei 1195

Abstract

Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [(3)H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) alpha-1,2 linked to two, three, or four fructose (F) units (GF(2), GF(3), and GF(4), respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF(2) and GF(3) was rapid, whereas little GF(4) uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.

FIG. 1.

FOS transport by L. paracasei 1195. Log-phase cells grown in MRS broth containing FOS (▪), sucrose (□), glucose (▴), or fructose (•) were harvested, washed twice, and resuspended in potassium phosphate buffer. The reactions were started by the addition of 1 mM [³H]FOS.

FIG. 2.

Inhibition of FOS transport by competitive sugars in L. paracasei 1195. Experiments were performed as described in the text. The control FOS uptake rate was 101 nmol per min per mg (dry weight) of cells.

FIG. 3.

Effect of vanadate on FOS uptake by arginine-energized cells of L. paracasei 1195. Cells were grown in MRS broth supplemented with 50 mM l-arginine to induce enzymes of the arginine deiminase pathway. Cells were harvested, washed, and de-energized by incubation with 2-deoxyglucose for 40 min at 37°C (▪). A portion of the cells were then energized with 50 mM arginine either with (•) or without (▴) 5 mM vanadate. Uptake assays were started by the addition of 1 mM [³H]FOS.