Linking process variables to exopolysaccharide yield, molecular size and viscosity in Leuconostoc citreum CNTA 860 fermentation

The demand for clean label thickening agents follows the trend in the food and biotechnology industries away from chemically derived hydrocolloids. Exopolysaccharides (EPS) from lactic acid bacteria (LAB) have the potential to meet this demand, as they are produced during fermentation and exhibit texturising functionality, making them promising candidates for applications where natural origin is valued. This study examined EPS fermentation process from Leuconostoc citreum CNTA 860 using a two-level fractional factorial design (2⁶⁻²) to assess interaction effects among six operational factors. Initial sucrose concentration was the main driver of EPS synthesis, although agitation and inoculum size also significantly influenced EPS productivity and viscosity development. Regression models reached high predictive performance (R² = 0.989; Predicted R² = 0.925 at 12 h), confirming model adequacy during early fermentation. Under optimized conditions (600 rpm, 150 g/L sucrose, C/N = 100, inoculum 10⁸ CFU/mL, 25 °C, no sucrose pulsing), EPS concentration reached 70 g/L and broth viscosity 8200 cP after 24 h, exceptionally high values for L. citreum fermentations. SEC-HPLC revealed that the presence of a 3.26–6.50 kDa glucan fraction (20–40 glucose units) strongly correlated with viscosity (r = 0.70, p<0.001). This approach provides a robust framework for scale-up and positions EPS from L. citreum CNTA 860 as promising biothickeners for food and biotechnology applications. • Time-resolved DoE linked process key variables to EPS structure and rheology • Initial sucrose and agitation jointly drove EPS concentration and viscosity • A 3.3–6.5 kDa glucan fraction (20–40 glucose units) governed viscosity development • Leuconostoc citreum CNTA 860 excelled in EPS production with thickening capacity • Optimal conditions yielded 70 g/L EPS and 8200 cP viscosity, highest for L. citreum