Medium Complexity Modulates Kefiran Yield and Thermal Stability in Whey-Based Fermentations: Insights from Systematic Supplementation and Comprehensive Physicochemical Characterization

Kefiran is a bioactive exopolysaccharide produced by kefir grains, whose synthesis is strongly influenced by culture medium composition. In this study, cheese whey was evaluated as an alternative fermentation substrate for kefiran production, and the effect of supplementation with fermentable sugars (glucose, galactose, and lactose) and casein was assessed under controlled conditions. Kefir grains were cultivated in whey- and milk-based media, and kefiran production was quantified using an anthrone-based method, while grain growth and carbohydrate consumption were monitored. Supplementation with sugars and casein reduced kefiran production by up to 34.6% and did not improve yield, whereas unsupplemented whey supported the highest kefiran concentration (86.9 ± 3.7 mg/L), comparable to that obtained in semi-skimmed milk (84.0 ± 3.0 mg/L). The recovered polysaccharide was characterized by Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy (1H NMR), X-ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), showing structural and physicochemical properties comparable to kefiran obtained from semi-skimmed milk. These results indicate that whey constitutes a feasible and simple fermentation medium for kefiran production, and that increased medium complexity does not necessarily improve process performance.