| Popis: |
The human microbiome has recently garnered the interest of scientists and biopharma industries as studies have revealed the potential use of live bacteria known as probiotics as potential therapeutics for restoring and maintaining human health. These probiotic-biopharma formulations must contain the right strain(s) in sufficient numbers when administered to confer the desired health benefit. Cell dehydration is used to keep the probiotic microbes in an inactivated form during storage, thereby ensuring that there are enough viable cells still present when the probiotic is taken. However, the drying process itself is detrimental to the probiotic cells and can result in reduced viability and stability of cells over storage. In this study, various cryoprotectants were assessed for their ability to maintain cell integrity and improve yield during the freeze drying dehydration of Lactobacillus plantarum towards a potential topical pharmabiotic formulation. Inulin, sucrose, maltodextrin, and skimmed milk at 10% m/v concentration of the drying media were tested for their ability to protect bacterial cells during freeze drying and over a storage period of 12 weeks at 4oC and room temperature. Furthermore glucose, inulin, sucrose, and maltodextrin as sole carbon substrate were investigated as prebiotics in concentrations of 0.5% m/v, 2% m/v, and 4% m/v of the fermentation media by in vitro fermentation of L. plantarum in glucose-free MRS-free media. The influence of these cryoprotectants and prebiotics on L. plantarum was measured against cell viability, growth kinetic parameters (growth rate, lag phase, and maximum cell density), and pH reduction potential of L. plantarum. Improved survival of L. plantarum during freeze drying and over 12-weeks of storage was observed with all cryoprotectants. Skimmed milk demonstrated the highest protection after freeze drying, with a survival rate of 91% and viable cell counts of 9.1 × 108 ( CFU ml ) from an initial cell count prior to drying of 1.0 × 109 ( CFU ml ). Inulin demonstrated high protective efficiency, with 85% viability maintained during freeze drying which resulted in final cell counts of 1.1 × 109 ( CFU ml ) from an initial cell count of 1.3 × 109 ( CFU ml ). However, inulin provided the least protection over the 12 week storage period compared to cells dried in the presence of maltodextrin, sucrose, and skimmed milk, with cell counts of only 1.2 × 106 ( CFU ml ) at 4oC and 6.3 × 103 ( CFU ml ) at room temperature recorded at the end of the period. Following skimmed milk, which also demonstrated the highest stability of cells over storage, sucrose performed second best in maintaining the stability of cells at 4 oC at the end of the 12 weeks storage, with viability of 33% which resulted in final cell counts of 3.4 × 108 ( CFU ml ). Overall, the presence of cryoprotectants and prebiotics demonstrated a significant influence on propagation and viability. The presence of each of the various prebiotics as the sole carbon substrate in the fermentation media promoted proliferation of L. plantarum. An increase in cryoprotectant concentrations led to increased biomass yield but with no significant change in the growth rate and lag phase. Cells showed improved stability when stored at 4oC compared to room temperature. A delay in propagation up to 10 hours was observed upon rehydration of stored probiotic cells across all cases except for skimmed milk that resulted in a maximum delay in propagation of 2 hours at both storage temperatures. |
