"Single-Cell Encapsulation of Lactiplantibacillus Plantarum Via Layer-by-Layer Technique for Enhanced Viability and Shelf Life: Insights on Multilayer Assembly on Bacteria".

The efficacy of probiotics depends on their ability to survive gastrointestinal transit; however, conventional formulations often experience losses of 2-3 log units before reaching the intestine. Here, we report the single-cell encapsulation of Lactiplantibacillus plantarum C8 using the layer-by-layer (LbL) assembly of poly-L-lysine (PLL) and alginate (Alg), resulting in a protective two-layer coating. Coating assembly was monitored by flow cytometry and validated by quartz crystal microbalance measurements. Flow cytometry revealed an unusually high degree of polymer incorporation during the first deposition step. Transmission electron microscopy, atomic force microscopy, and confocal laser scanning microscopy confirmed the formation of continuous and dense polymer coatings around individual cells. The bilayer coating enhanced bacterial survival following freeze-drying while preserving growth capacity, reaching approximately 9.3 log CFU/mg after 24 h, compared with 8.72, 8.84, and 8.26 log CFU/mg for uncoated bacteria and bacteria coated with four and six layers, respectively. Under simulated gastric conditions, coated bacteria exhibited higher viability (4.6 ± 0.3 log CFU/mg) than uncoated cells (3.5 ± 0.2 log CFU/mg). Moreover, coated bacteria retained approximately 5.7 log CFU/mg after 60 days at 4°C, extending storage stability by approximately 10 days.