Effects of Trehalose and Galacto-Oligosaccharides on Freeze-Drying Survival and Storage Stability of Bifidobacterium longum B18

Maintaining the viability of Bifidobacterium longum B18 throughout lyophilization and storage remains a key challenge for its application as a probiotic ingredient in functional foods. This study screened 18 lyoprotectants, optimized trehalose (TRE) and galacto-oligosaccharide (GOS) concentrations, and evaluated post-drying survival, enzyme activities, membrane integrity, morphology, and 90-day storage stability. The 8% (w/v) TRE + 3% (w/v) GOS formulation yielded the highest post-freeze-drying survival (90.6%). It also showed higher post-drying Na+/K+-ATPase, Ca2+/Mg2+-ATPase, lactate dehydrogenase, and β-galactosidase activities than the PBS control, although responses relative to the individual protectants varied by enzyme. Qualitative fluorescence and scanning electron microscopy observations indicated limited propidium iodide-positive staining and less apparent surface damage. During storage, viability decreased in all formulations. Based on descriptive comparisons, viability retention was numerically greater at 4 and −20 °C than at 25 °C. After 90 days, the combined formulation retained 49.4% and 45.7% survival at −20 and 4 °C, respectively, compared with 38.8% and 35.8% for TRE and 30.2% and 20.3% for GOS. At 25 °C, survival decreased to 0.1%. However, because the formulations differed in total added lyoprotectant concentration, the observed differences do not establish complementary or synergistic protection by TRE and GOS. These findings support further evaluation of 8% TRE + 3% GOS for refrigerated or frozen functional foods. However, this preliminary 90-day study does not establish commercial shelf-life suitability, which requires validation in representative food matrices under commercial processing, packaging, and longer-term storage conditions.