Isolation, optimization and physicochemical characterization of exopolysaccharide: Volatile metabolite profiling and functional properties of Enterococcus lactis strain Pondicherry University - Kangayam cattle milk

Metabolites are the key compounds involved in growth, reproduction and defense. This study characterizes the structure and function of extracellular metabolites, specifically exopolysaccharide (EPS) and volatile organic compounds, derived from Enterococcus lactis PU - KCM (Pondicherry University - Kangayam Cattle Milk). The production conditions of EPS were optimized using lactose (6.3%), yeast extract (3.0%), and pH (6.25), resulting in a yield of 4.01 ± 0.01 g/L (gravimetric) and a total sugar content of 116 ± 0.15 g/L (on a glucose-equivalent basis). Structural characterization revealed that the EPS was a neutrally charged heteropolysaccharide with a bimodal molecular weight distribution (5.89 × 10⁴ Da and 1.05 × 10⁵ Da). Its composition consists mainly of galactose (47.38%), glucose (23.93%), and xylose (2.68%), with some proteins and uncharacterized components. EPS was identified as amorphous and hydrophilic. PU-KCM metabolites contain volatile and semi-volatile organic compounds like fatty acid esters, alkanes, diterpenes, alkane hydrocarbons, fatty alcohol, dipeptides, and phenol. Functionally, EPS showed significant oil holding capacity (721.70 ± 5.30%), water swelling capacity (210 ± 8.16%), solubility index (80.83 ± 0.62%) and low water holding capacity (11.54 ± 0.25%) with antioxidant activity against ABTS (76.13 ± 0.07%). PU-KCM metabolites exhibited antioxidant activities against DPPH (62.76 ± 1.51%), ABTS (79.93 ± 0.66%), hydroxyl (52.25 ± 0.71%), reducing power (0.38 ± 0.0 OD₇₀₀), and nitric oxide (50.09 ± 0.15%) as well as broad antimicrobial activity against pathogens. The safety of these metabolites was evaluated using the RBC hemolysis assay. This study identifies these metabolites as functional biopolymers and bioactive compounds.