Identification, Antioxidant and Immunomodulatory Activities of a Neutral Exopolysaccharide from Lactiplantibacillus plantarum DMDL 9010

Abstract

Objectives: This study investigated the properties of a neutral exopolysaccharide (EPS-LP1) with an average molecular weight of 55,637 Da, isolated from Lactiplantibacillus plantarum DMDL 9010 (LP9010).

Results: The composition of EPS-LP1 includes galactose (Gal), glucose (Glu) and mannose (Man) in a molar ratio of 5.35:86.25:8.40. Notably, EPS-LP1 exhibits a smooth and rod-like surface along with thermal stability. Methylation combined with nuclear magnetic resonance analysis revealed that EPS-LP1 structured as t-Galp(1→, →6)-Glcp(1→, 4)-Glcp(1→ and →4,6)-Galp(1→), with relative molar ratio of 1.016:9.874:4.355:78.693:6.062, respectively. In the concentration range of 50 to 400 mg/mL, we observed the absence of cytotoxic effects from EPS-LP1 on RAW264.7 cells. Furthermore, EPS-LP1 demonstrated protective effects on RAW264.7 cells against oxidative damage by reducing the production of reactive oxygen species (ROS), malondialdehyde (MDA), and lactate dehydrogenase (LDH) release. Conversely, an increase in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and concentrations of glutathione (GSH) was observed. Immunoreactivity assays indicated that EPS-LP1 can effectively reduce the production of nitric oxide (NO) and inhibit the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Additionally, it inhibited the activation of the mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B gene binding (NF-kB) signaling pathway.

Conclusions: This research provides a foundation basis for further investigations into the neutral exopolysaccharide derived from LP9010.

Keywords: Lactiplantibacillus plantarum DMDL 9010; antioxidant activity; extracellular polysaccharide; immunoregulatory activity; structural characterization.

Figure 1

Production, separation, purification, and molecular weight of EPS from LP9010. (A) Chromatograms of EPS purified by Sephadex G-75 column, (B) UV, (C) standard curve of polysaccharide molecular weight (5.0–670 kDa), and (D) HPGPC spectrum of EPS-LP1.

Figure 2

General properties analysis of EPS-LP1. (A) SEM (a) 100 μm, (b) 10 μm, and (c) 10 μm, (B) Thermogravimetric (TG) analysis curve of EPS-LP1, and (C) FT-IR spectra.

Figure 3

Monosaccharide composition analysis and NMR spectra of EPS-LP1. (A) HPLC analysis of monosaccharide composition of monosaccharide standard product (Fuc = fucose, Rha = rhamnose, Ara = arabinose, Gal = galactose, Glc = glucose, Xyl = xylose, Man = mannose, Fru = fructose, Rib = ribose); (B) HPLC analysis of monosaccharide composition of EPS-LP1; (C) ¹H NMR spectrum; (D) ¹³C NMR spectrum.

Figure 4

Effects of EPS-LP1 on (A) cell viability, the levels of, (B) reactive oxygen species (ROS), (C) malondialdehyde (MDA), the activities of (D) lactate dehydrogenase (LDH), (E) superoxide dismutase (SOD), (F) catalase (CAT), (G) glutathione peroxidase (GSH-Px), and (H) GSH in RAW264.7 cells induced by H₂O₂. Different letters represent different significance levels from the control group (p < 0.05).

Figure 5

Effects of EPS-LP1 on (A) cell viability, (B) nitric oxide (NO) production, expression of (C) interleukin 6 (IL-6), (D) tumor necrosis factor-α (TNF-α) in RAW264.7 cells induced by lipopolysaccharide (LPS). Different letters represent different significance from the control group (p < 0.05).

Figure 6

Effects of EPS-LP1 on the MAPK pathway (A–C) in RAW264.7 cells; effects of EPS-LP1 on the NF-κB pathway (D,E) in RAW264.7 cells. Different letters represent different significance levels from the control group (p < 0.05).

Figure 7

The possible mechanism of EPS-LP1 inhibiting inflammation in RAW264.7 cells.