Tailoring the properties of rhamnose-rich exopolysaccharides from cheese whey via continuous fed-batch fermentation for active packaging applications

• Cheese whey was valorized into a microbial exopolysaccharide via fed-batch fermentation. • Continuous feeding enabled control of EPS molecular weight and dispersity. • Continuous feeding yielded a 4-fold increase in EPS molecular weight compared to batch mode. • The EPS showed excellent film-forming ability and compatibility with geraniol. • EPS-based films exhibited sustained antimicrobial activity for active packaging. This study explores the sustainable bioconversion of cheese whey (CW) into high-value exopolysaccharides (EPS) by Halomonas caseinilytica K1 through the optimization of bioreactor operation modes. Batch, fed-batch with pulse feeding, and fed-batch with continuous feeding were compared to evaluate their impact on cell growth, EPS production, and polymer characteristics. The continuous fed-batch strategy markedly outperformed the other modes, achieving the highest biomass concentration (89.95 g/L) and EPS titer (11.67 g/L), corresponding to 2.12- and 5.16-fold increases relative to pulse-fed and batch cultures, respectively. This cultivation approach significantly influenced EPS structure, yielding a rhamnose-enriched polymer with a high molecular weight of 2.25 × 10⁵ Da, corresponding to a fourfold increase. The optimized EPS (EPS-K1-B3) was subsequently applied to fabricate biodegradable antimicrobial films incorporating geraniol (10%). These films displayed good structural integrity and demonstrated sustained inhibitory activity against Escherichia coli and Listeria innocua over a 22-day release period. Overall, this work establishes an integrated bioprocess–structure–function framework, demonstrating how controlled fermentation conditions can tailor EPS properties for targeted applications in active food packaging while contributing to the valorization of agro-industrial waste streams.