Extrusion of Dissolved Oxygen by Exopolysaccharide From Leuconostoc mesenteroides and Its Implications in Relief of the Oxygen Stress

Abstract

Strains of Leuconostoc are generally facultatively anaerobic and exposure to oxygen might be detrimental; therefore, strategies to combat the oxygen stress are essential for these bacteria to survive and flourish in the oxygenic atmosphere. Despite the extensive applications in industry, the fundamental issues concerning the aerobic life of Leuconostocs remain to be addressed. In this study, we have demonstrated that Leuconostoc mesenteroides CGMCC10064 cultivated in sucrose medium would acquire a growth advantage over that in glucose medium under oxygenic conditions, as reflected by more viable cells and less accumulation of reactive oxygen species. Further analysis showed that the growth advantage was dependent on exopolysaccharide (EPS) synthesized by a secreted glucansucrase. Determination of the dissolved oxygen in the culture suggested that the growth improvement was mediated by extrusion of dissolved oxygen from the aqueous circumstances. Growth experiments performed with the purified EPS showed that supplementation of 5 g/L EPS in the medium could improve the aerobic growth of L. mesenteroides by ∼10-fold. Moreover, the purified EPS was also effective in promoting the aerobic growth of oxygen-sensitive Lactobacillus and Bifidobacterium. These results demonstrate that EPS of L. mesenteroides plays a critical role in relief of the oxygen stress, and suggest the potential of the EPS in manufacture as well as preservation of oxygen-sensitive probiotics.

Keywords: Leuconostoc mesenteroides; dissolved oxygen; exopolysaccharide; oxygen tolerance; oxygen-sensitive probiotics.

FIGURE 1

The aerobic growth of L. mesenteroides CGMCC10064. (A) Aerobic growth of CGMCC10064 in TYC medium with glucose (Glc) or sucrose (Suc) as reflected by changes in the number of viable cells. (B) Amount of EPS at different time points during the aerobic growth of CGMCC10064. In order to test the requirement of EPS for aerobic growth, 1% sucralose was supplemented for inhibition of EPS synthesis. The effect of EPS inhibition on the aerobic growth of CGMCC10064 can be seen in (A). All the experiments were performed in triplicate and the average results as well as the standard deviations are included in the graphs.

FIGURE 2

EPS synthesis by a secreted GS enzyme. (A) Total proteins from the supernatant (the secreted proteins) of both culture (Glc and Suc) were extracted by precipitation with (NH₄)₂SO₄ as described in Section “Materials and Methods.” Equivalent amount of total protein was separated on 8% SDS-PAGE and subjected to silver staining. A differential expressed protein band of ∼180 kDa was indicated by an arrowhead. (B) In situ detection of glucansucrase activity. A parallel SDS-PAGE was incubated in 50 mM sodium acetate buffer containing 50 g/L sucrose buffered at pH5.6. An active band was detected by the appearance of opaque polymer.

FIGURE 3

Accumulation of ROS in cells of CGMCC10064 after exposure to environmental oxygen. The cultures were exposed to environment oxygen by agitation at 180 rpm under aerobic circumstances. Samples were taken at the indicated time points and the amount of ROS within the cells were determined using DCFH-DA as a probe and the results were normalized to total protein content (relative ROS expressed as DCF/mg pro). The average values of three measurements and the standard deviations are shown in the graph.

FIGURE 4

(A) Changes of the concentration of dissolved oxygen in the medium during the aerobic growth of CGMCC10064. Cultures of CGMCC10064 were incubated with agitation at 180 rpm. Samples were taken every 24 h and the concentration of dissolved oxygen was determined. (B) Extrusion of dissolved oxygen by purified EPS in aqueous solutions. The concentration of dissolved oxygen in EPS solutions with different content of purified EPS was detected as described in Section “Materials and Methods.” A trend line was drawn by exponential fitting and shown as gray curve. All the detections were performed in triplicate, and the average values are shown in the graphs.

FIGURE 5

The effect of purified EPS on the aerobic growth of strains of Leuconostoc, Lactobacillus, and Bifidobacterium. Growth of the indicated strains (L. mesenteroides CGMCC10064 and ATCC10830a, Lactobacillus plantarum ST-III, and Bifidobacterium longum NCC2705) in TYC media in the absence or presence of the purified EPS from CGMCC10064 was analyzed through quantifying the number of viable cells. The logarithms were calculated and shown in the graph. The average values of three determinations and the standard deviations are shown in the graph. A reference line was drawn to indicate the approximate initial value (i.e., the logarithm of the number of viable cells as estimated) of the cultures before exposure to oxygen.