Pilot-scale production, purification, and structural characterization of a glycosaminogalactan exopolysaccharide from Lactiplantibacillus pentosus

Exopolysaccharides (EPS) produced by lactic acid bacteria are increasingly recognized for their structural diversity and functional properties, contributing to food texture and offering potential applications in health, pharmaceuticals, and biotechnology. In this study, we report the multi-gram scale production and characterization of a novel EPS from Lactiplantibacillus pentosus KW1. EPS was produced in a 100 L fed-batch bioreactor using an ultrafiltered semi-defined medium. Proteomic analysis confirmed consistent expression of proteins within the proposed EPS biosynthetic cluster throughout fermentation. The culture underwent heat treatment, two-phase separation, depth filtration, and ultrafiltration for EPS recovery. Size-exclusion chromatography and NMR analysis identified a high-molecular-weight EPS (EPS A, >800 kDa) and two wall teichoic acids with distinct structures. EPS A was obtained at 92 mg/L and characterized as a repeating tetramer composed of pyruvylated galactose (Pyr-Gal), GlcNAc, Gal, and GalNAc. Monosaccharide composition was determined by high-performance anion-exchange chromatography, and structural elucidation was achieved via two-dimensional NMR spectroscopy. All monosaccharides were confirmed as ᴅ-sugars by reversed-phase HPLC. This work provides a foundation for understanding EPS from L. pentosus and its potential role in improving texture and stability in fermented foods.