Bench scale Layer-by-Layer microencapasulation of Lactiplantibacillus plantarum WCFS1

Abstract

Layer-by-Layer (LbL) self-assembly encapsulation is a promising technology for the protection and delivery of lactic acid bacteria. However, laboratory-scale encapsulation is often time-consuming, involves intensive protocols tailored for small-scale operations, requires substantial amounts of energy and water, and results in a low yield of encapsulated biomass. Scaling-up this process to a bench-bioreactor scale is not simply a matter of increasing culture volume as different key parameters (not particularly relevant at lab scale) become critical, including biomass production, the number of polymer layers, and the biomass-to-polymer mass ratio. To our knowledge, this work is the first to address the optimization of each stage of the encapsulation process for Lactiplantibacillus plantarum WCFS1. These stages include biomass production, handling of encapsulation polymers [chitosan (Chi) and alginate (Alg)], critical LbL parameters (e.g., biomass concentration, washing steps). The encapsulation efficiency was assessed by plate-counting microorganisms before and after coating with the polymers layers, followed by spray- and freeze-drying dehydration using fructo-oligosaccharides (FOS) and maltodextrin as carriers. Once dehydrated, microorganisms were either exposed to gastrointestinal conditions or stored for 30 days at 25 and 30 °C. Supplementing culture media with glucose, controlling pH, and harvesting at the early stationary phase during biomass production increased the bacterial recovery after LbL encapsulation (decrease < 1 log unit) compared to bacteria grown under non-controlled conditions (decrease of 4 log units). Coating bacteria (B) with up to two polymer layers (B|Chi or B|Chi|Alg) did not significantly affect bacterial culturability, unlike adding further layers. Zeta-potential measurements enabled the determination of the optimal biomass-to-polymer mass ratio. Using up to a 10:1 bacterial-to-polymer ratio did not change the z-potential for B|Chi or B|Chi|Alg samples. After drying, a synergistic effect between the LbL coating and carrier compounds (FOS and maltodextrin) was observed in terms of culturability. LbL encapsulation mitigated thermal and acidic stresses during spray-drying and gastrointestinal exposure. These findings support scaling-up LbL encapsulation for delivering sensitive lactic acid bacteria strains to the gut.

Keywords: Carrier agent; Encapsulation; Freeze- & spray-drying; Fructo-oligosaccharides; Lactic acid bacteria; Upscaling.