Association of beta-glucan endogenous production with increased stress tolerance of intestinal lactobacilli

Abstract

The exopolysaccharide beta-glucan has been reported to be associated with many health-promoting and prebiotic properties. The membrane-associated glycosyltransferase enzyme (encoded by the gtf gene), responsible for microbial beta-glucan production, catalyzes the conversion of sugar nucleotides into beta-glucan. In this study, the gtf gene from Pediococcus parvulus 2.6 was heterologously expressed in Lactobacillus paracasei NFBC 338. When grown in the presence of glucose (7%, wt/vol), the recombinant strain (pNZ44-GTF(+)) displayed a "ropy" phenotype, while scanning electron microscopy (SEM) revealed strands of polysaccharide-linking neighboring cells. Beta-glucan biosynthesis was confirmed by agglutination tests carried out with Streptococcus pneumoniae type 37-specific antibodies, which specifically detect glucan-producing cells. Further analysis showed a approximately 2-fold increase in viscosity in broth media for the beta-glucan-producing strain over 24 h compared to the control strain, which did not show any significant increase in viscosity. In addition, we analyzed the ability of beta-glucan-producing Lactobacillus paracasei NFBC 338 to survive both technological and gastrointestinal stresses. Heat stress assays revealed that production of the polysaccharide was associated with significantly increased protection during heat stress (60-fold), acid stress (20-fold), and simulated gastric juice stress (15-fold). Bile stress assays revealed a more modest but significant 5.5-fold increase in survival for the beta-glucan-producing strain compared to that of the control strain. These results suggest that production of a beta-glucan exopolysaccharide by strains destined for use as probiotics may afford them greater performance/protection during cultivation, processing, and ingestion. As such, expression of the gtf gene may prove to be a straightforward approach to improve strains that might otherwise prove sensitive in such applications.

FIG. 1.

(A) Loop touch test of pNZ44-GTF⁺ Lb. paracasei NFBC 338, displaying the “ropy” strand formed by the cellular mass of EPS-producing Lb. paracasei NFBC 338 grown in MRS broth containing 7% glucose. (B) Scanning electron micrograph (5 μm) of Lb. paracasei NFBC 338. pNZ44 (control) strain (left) and pNZ44-GTF⁺ beta-glucan-producing strain (right) after growth in 7% glucose. Arrows indicate EPS production. (C) Agglutination tests using S. pneumoniae type 37-specific antisera detected using phase-contrast microscopy. pNZ44 (control) with antisera (left), pNZ44-GTF⁺ with antisera (middle), and pNZ44-GTF⁺ without antisera (right).

FIG. 2.

Viscosity analysis (bars) and growth curve (circles) of pNZ44 (control) and pNZ44-GTF⁺ (beta-glucan producer), following growth anaerobically at 37°C in MRS broth supplemented with 7% glucose. Error bars represent standard errors of the means from 10 readings for each time point (viscosity) and standard errors of the means from triplicate experiments (growth curves).

FIG. 3.

Survival of the pNZ44 control and pNZ44-GTF⁺ beta-glucan-producing Lb. paracasei NFBC 338 in an elevated temperature (58°C). Overnight cultures (12 h) were centrifuged and resuspended in MRS and incubated at 58°C. Viable cell counts were performed at intervals by serial dilution in maximum recovery diluent solution and enumeration on MRS agar, followed by incubation at 37°C anaerobically for ∼48 h. The error bars represent standard errors of the means from triplicate experiments.

FIG. 4.

Survival of the pNZ44 control and pNZ44-GTF⁺ beta-glucan-producing Lb. paracasei NFBC 338 in acid (HCl, pH 2). Overnight cultures (12 h) were centrifuged, resuspended in HCl at pH 2, and incubated at 37°C. Viable cell counts were performed at intervals by serial dilution in maximum recovery diluent solution and enumeration on MRS agar, followed by incubation at 37°C anaerobically for ∼48 h. The error bars represent standard errors of the means from triplicate experiments.

FIG. 5.

Survival of the pNZ44 control and pNZ44-GTF⁺ beta-glucan-producing Lb. paracasei NFBC 338 in simulated gastric juice (pH 2). Overnight cultures (12 h) were centrifuged, resuspended in simulated gastric juice, and incubated at 37°C. Viable cell counts were performed at intervals by serial dilution in maximum recovery diluent solution and enumeration on MRS agar, followed by incubation at 37°C anaerobically for ∼48 h. The error bars represent standard errors of the means from triplicate experiments.

FIG. 6.

Survival of the pNZ44 control and pNZ44-GTF⁺ beta-glucan-producing Lb. paracasei NFBC 338 in porcine bile (0.7%). Overnight cultures (12 h, 3%) were inoculated into MRS broth containing 0.7% porcine bile and incubated at 37°C. Viable cell counts were performed at intervals by serial dilution in maximum recovery diluent solution and enumeration on MRS agar, followed by incubation at 37°C anaerobically for ∼48 h. The error bars represent standard errors of the means from triplicate experiments.