Enhanced exopolysaccharide production and biological activity of Lactobacillus rhamnosus ZY with calcium and hydrogen peroxide

Exopolysaccharides (EPS) are important food and drug additives with beneficial antioxidant, anticancer, and immune-related effects on human health. However, the EPS is limited by low yields and the need for complex culture conditions in fermentation. Here, we report that hydrogen peroxide and calcium stimulated probiotic activity and production of crude exopolysaccharide (c-EPS) by Lactobacillus rhamnosus ZY. Accordingly, supplementation with 3 mM H₂O₂ allowed c-EPS biosynthesis to reach 567 mg/L after 24 h. Addition of both CaCl₂ and H₂O₂ resulted in a c-EPS yield of 2498 mg/L after 12 h, over 9-fold higher than that of an anaerobic culture. We observed that exposure to calcium and hydrogen peroxide made the cells more hydrophobic and led to the over-expression of GroEL, NADH peroxidase, and glyceraldehyde 3-phosphate dehydrogenase, thus increasing energy storage and EPS production. Chromatographic analysis revealed c-EPS was composed mainly of mannose (5.1%), galactose (15.3%), glucose (20–30%), and rhamnose (50–60%). Preliminary in vitro tests revealed that H₂O₂ and CaCl₂ enhanced the 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radical scavenging capacities, resulting in a notable protective effect against oxidative damage in NIH/3T3 cells. Our study provides a simple and cost-effective approach for achieving high yields of good quality EPS using Lactobacillus rhamnosus.