Encapsulation and subsequent freeze-drying of beneficial lactobacilli for their potential inclusion in pharmabiotic formulations for vaginal applications

Abstract

One of the main challenges during the design of pharmabiotic products is that the select microorganisms resist to the processes of biomass production, combination in the final product and storage during the shelf life period. In this work, the resistance of biofilm-forming vaginal Lactobacillus reuteri CRL 1324 to encapsulation, freeze-drying and storage were assayed. L. reuteri was encapsulated applying the extrusion-ionic gelation technique, using microbial polymers (1% xanthan gum-0.75% gellan gum). Capsule aliquots were freeze-dried with or without lyoprotectors (12% lactose-6% skim milk). Capsules were stored at room and refrigeration conditions for 150 days. The following evaluations were performed: viability of bioactive ingredient (i.e. viable L. reuteri) before and after encapsulation, freeze-drying and storage; microorganism release and resistance to the genital tract conditions in a medium simulating the vaginal fluid (pH = 4.2), and maintenance of the beneficial properties (biofilm formation and Streptococcus agalactiae NH17 inhibition). High yield of capsules, high encapsulation efficiency and viable entrapped lactobacilli were obtained. The resistance of encapsulated L. reuteri to lyophilization was higher with lactose/milk. At 150 days of storage, viable cells from freeze-dried capsules (with or without lyoprotectors) stored at refrigeration temperature were only recovered. From the optimal system (lyophilized capsules with lyoprotectors), L. reuteri was released in a culture medium simulating vaginal fluid, maintaining its viability during 24 h at 37°C and the capability to form biofilm and to inhibit S. agalactiae NH17 growth. In conclusion, the combination of encapsulation (by extrusion-ionic gelation) and freeze-drying processes in presence of lyoprotectors, and the subsequent storage at refrigeration conditions favored the maintenance of L. reuteri CRL 1324 viability and functionality. Encapsulated and freeze-dried beneficial lactobacilli can then be included in a suitable pharmaceutical form for vaginal application, to prevent or treat female urogenital infections.