The Impact of Formulation and Freeze Drying on the Properties and Performance of Freeze-Dried Limosilactobacillus reuteri R2LC

Freeze drying is a commonly used method for preserving probiotic bacteria and live biotherapeutic products. Before drying, the bacterial cells are formulated with a lyoprotectant, and the design of these two process steps are crucial to achieve a high-quality product. There are several factors that may affect the biological and physicochemical properties of the freeze-dried cells and we have used a Design of Experiment approach to investigate the effects of formulation and freeze-drying parameters on properties and performance of Limosilactobacillus reuteri R2LC. The biological characteristics of the dried bacteria were evaluated by measuring cell survival, metabolic activity and stability, and physicochemical characteristics were studied using visual inspection, differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and analysis of residual moisture content and bacterial aggregation. A comparison between the lyoprotectants trehalose and sucrose showed that the latter gave better freeze-drying survival, metabolic activity, and storage stability. We also want to highlight that there was a correlation between bacterial concentration, metabolic activity, and aggregation of bacteria, where a higher concentration (1010 CFU/mL) resulted in both higher metabolic activity and aggregation. Several other process and formulation factors affected both the biological and physicochemical properties of freeze-dried L. reuteri R2LC and it could be concluded that care must be taken to develop a production method that generates a product with high and consistent quality. These results may, or may not, be strain specific.