Estimation of microbial phosphate-accumulation abilities

Abstract

Phosphate binders and dialysis can have harmful side-effects during the treatments of hyperphosphatemia. Therefore, we evaluated the capability of intestinal bacteria (lactic acid bacteria and bifidobacteria) as phosphate-accumulating organisms (PAOs) for phosphate accumulation, with the aim of determining whether PAO-formulated food can prevent hyperphosphatemia in the early stages. However, methods for estimating microbial phosphate-accumulation capacities require significant improvements regarding specificity, cost, and simplicity. The presented method analyzed cell-free broth to assess the phosphate accumulation capability of cells. Active cells and the constructed phosphate-deficient cells were incubated in assay salt media. After incubation, phosphate-deficient cell-free broth was taken as sample and the blank was the active cell-free broth. Therefore, effects of interfering agents and other metabolites were avoided and enhanced the specificity remarkably. Phosphate contents were assessed by reactions with toluidine blue O. In contrast to the case in previous studies, the shift in the first absorbance peak was found to be inversely proportional to the phosphate concentration. The minimum detectable phosphate concentrations for the 11th isolate of Lactobacillus casei JCM 1134 and 8th isolate of Bifidobacterium adolescentis JCM 1275 were determined to be 1.24 and 0.4 mg/L, respectively. Further, the validation results were found to be significant (p-value < 0.05).

Figure 1

Metabolic activities of cells incubated in assay salt solution (which evaluated the phosphate accumulation abilities of cells) and monitored over time: (a) phosphate contents of supernatants of cells incubated in assay salt broth [(+P): ◊→L11 of L. casei JCM 1134 and •→B8 of B. adolescentis JCM 1275]. Standard errors are less than 8%. (b) Pre- and post-incubation DAPI micrographs of probiotics; b.1, b.2, b.5, and b.6 were incubated in phosphate-containing assay salt solution while b.3, b.4, b.7, and b.8 were incubated in phosphate-free assay salt solution. b.1→ at 0 h phosphate-deficient L11 of L. casei  JCM1134, b.2→ at 6 h L11 of L. casei JCM1134 (phosphate metabolising), b.3→ at 0 h active L11 of L. casei JCM1134, b.4→ at 6 h L11 of B8 of B. adolescentis JCM1275 (phosphate storing), b.5→ at 0 h phosphate-deficient B8 of B. adolescentis JCM1275, b.6→ at 6 h B8 of B. adolescentis JCM1275 (phosphate metabolising), b.7→ at 0 h active B8 of B. adolescentis JCM1275, b.8→ at 6 h B8 of B. adolescentis JCM 1275 (phosphate storing). White bars represent 2 μm.

Figure 2

Absorbance scans of reaction product (a and b; TBO and phosphate in concentration of 2 mg/L in cell-free broth). Net absorbances and shifts in wavelengths of first peaks were used to determine calibration curves (c, d). (a) and (c) are for L. casei JCM 1134 while (b) and (d) are for B. adolescentis JCM 1275. In (a) and (b), white dots → sample, black dots → blank, and grey dots → net absorbance. In (c) and (d), black circles  → change in wavelength and black squares → net absorbance. Standard errors of observed values were found less than 2%.