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The goals of this secondment were achieved with the collaboration of Gina Tavares (University of Coimbra), Maria Guerrero (Biosearch), Stephanie Passot (AgroParisTech) and Fernanda Fonseca (INRAE).

Probiotic production requires going through different processes before being marketed and consumed or added to any food, such as freezing, lyophilization, spray drying. These processes cause various types of stress, affecting both the viability and the functional properties of bacteria. One of the goals of PREMIUM project is to develop preservation processes for lactic acid bacteria involving Layer-by-Layer (LbL) encapsulation. This method takes advantage of the sequential deposition of oppositely charged polymers driven by electrostatic interactions to form nanoscale thin films on planar or colloidal surfaces like bacteria. However, LbL is typically time-consuming, and automation of the polymer deposition steps is difficult to scale up. In this regard, this study aims to optimize the scale-up of LbL microencapsulation of Lactiplantibacillus plantarum WCFS1. Chitosan and alginate were used as cationic and anionic polyelectrolytes, respectively. Processing and formulation parameters like biomass production, washing steps, number of polymer layers and biomass/polymer ratio during the encapsulation were evaluated.

The principal achievements of this secondment are:

1. Improvement in the chitosan dissolution process led to a significant reduction of the processing time from 16 to 2 hours and the acetic acid concentration needed for the dissolution from 1 to 0.034%v/v.
2. The bacterial biomass produced at constant pH (pH 5.8) and harvested at the early stationary phase showed a lower decrease of cultivability after LbL encapsulation (0.5 to 1 Log CFU/mL) than those produced without pH control (4 Log CFU/mL decrease).
3. The elimination of washing steps during the encapsulation resulted in a reduction of total processing time (120 min saved) and the consumed resources (e.g., energy, water, etc.).
4. The increment of polymer:biomass ratio during encapsulation enhanced the biomass processing concentration until 10 times, enabling the processing of 10 L of culture in 1L of polymers solutions.

As whole the results obtained support the feasibility of scaling-up the LbL encapsulation process for the delivery of sensitive lactic acid bacteria strains while ensuring their safe arrival to the gut.

General work scheme:

Photos of the secondment: