A novel exopolysaccharide produced by Lactobacillus coryniformis NA-3 exhibits antioxidant and biofilm-inhibiting properties in vitro

Abstract

Background: Exopolysaccharides (EPSs) secreted from lactic acid bacteria are carbohydrate polymers with reported biological activities. In this study, we extracted and characterized the composition as well as antioxidant and biofilm-inhibitory properties of EPS from Lactobacillus coryniformis NA-3 isolated from northeast Chinese sauerkraut (Suan Cai).

Methods: Lactobacillus coryniformis NA-3 was identified with 16S rDNA amplification and Neighbor Joining (NJ) phylogenetic analysis. EPS derived from Lactobacillus coryniformis NA-3 (EPS-NA3) was analyzed, including compositions by high-performance liquid chromatography (HPLC), functional groups by Fourier-transform infrared spectroscopy (FT-IR) and glycosidic bond configuration by Hydrogen-1 Nuclear Magnetic Resonance (¹H NMR). Antioxidant activity of EPS was evaluated with hydroxyl and superoxide radical-scavenging. Anti-biofilm activities of EPS-NA3 were checked through inhibition and dispersion.

Results: The monosaccharide composition of EPS included α-rhamnose, α-mannose, α-galactose, and α-glucose in a ratio of 2.6:1.0:5.0:3.3. The free radical-scavenging abilities of EPS-NA3 were 37.77% ± 1.56% and 78.87% ± 3.07% on hydroxyl and superoxide reactive oxygen species respectively. Moreover, EPS-NA3 attenuated the formation of Bacillus cereus and Salmonella typhimurium biofilms by inhibition ratios of approximately 80% and 40% respectively. Additionally, treatment with EPS-NA3 dispersed established biofilms of B. cereus and S. typhimurium by approximately 90% and 20% respectively.

Conclusion: These results suggest that EPS-NA3 may be developed as antioxidant and anti-biofilm agents for industrial and clinical applications due to its capacity of scavenging free radicals, inhibition of bacterial biofilm formation, and dispersion of established biofilms.

Keywords: Lactobacillus coryniformis NA-3; anti-biofilm; antioxidant; dispersion; exopolysaccharide; inhibition.

Fig. 1

Neighbor joining-dendrogram showing the phylogenetic relationship between16S rDNA nucleotide sequences of Lactobacillus spp., obtained from the GenBank database, and Lactobacillus coryniformis NA-3 16S sequence, isolated in this study.

Fig. 2

HPLC chromatogram showed the molecular weight of the EPS produced by Lactobacillus coryniformis NA-3, calculated by linear regression with dextran standards.

Fig. 3

The FT-IR spectrum of exopolysaccharide (EPS) produced by Lactobacillus coryniformis NA-3 with the KBr pellet pressing method; x-axis is wave number (cm⁻¹) and y-axis is transmittance (%).

Fig. 4

The¹H NMR spectrum of EPS-NA3.

Fig. 5

Free radical-scavenging activities of EPS derived from Lactobacillus coryniformis NA-3 and ascorbic acid (positive control): hydroxyl radicals (a) and superoxide radicals (b). Data are represented as mean ± standard deviation (SD) of three replicates per experiment. Three independent experiments were conducted for each assay.

Fig. 6

Representative micrographs (40×) showing the inhibitory effects of EPS on biofilm formation by pathogenic bacteria: Bacillus cereus (a1, a2, a3) and Salmonella typhimurium (b1, b2, b3) and spectrophotometric analyses of EPS biofilm inhibition. (a1, b1): Negative controls (pathogen only, no EPS) for biofilm formation by fluorescence microscopy; (a2, b2): Biofilm formation in the presence of EPS observed by fluorescence microscopy; (a3, b3): Inhibition ratios of EPS against B. cereus and S. typhimurium biofilms respectively. ^a,b,cDifferent letters indicate significant differences between concentrations (P < 0.05).

Fig. 7

Representative micrographs (40×) showing the dispersion activity by EPS against established biofilms of the pathogenic bacteria Bacillus cereus (a1, a2, a3) and Salmonella typhimurium (b1, b2, b3), and spectrophotometric analyses of EPS-mediated dispersion of biofilms produced by these species. (a1, b1): Observation by fluorescence microscopy of negative controls (pathogens only, no EPS) for biofilm dispersion; (a2, b2): Observation by fluorescence microscopy of biofilm dispersion by Lactobacillus coryniformis NA-3-derived EPS; (a3, b3): Dispersion ratios of EPS on different bacterial biofilms. ^a,b,cDifferent letters indicate significant differences between treatments (P < 0.05).