In vitro controlled release of the probiotic strain Bacillus licheniformis PPL2016 microencapsulated: Simulating the digestive system by age class and sex in the blue swimming crab Callinectes arcuatus.

Abstract

This work aims to encapsulate Bacillus licheniformis PPL2016 (12 × 10⁶ CFU/mL), a marine probiotic characterized at a biochemical and molecular level, in sodium alginate (2%) microparticles and to evaluate its controlled and directed release in a simulated digestive system (DS) of the swimming crab Callinectes arcuatus, considering the following age classes and sexes: Adult Female, Juvenile Female, Adult Male, and Juvenile. The encapsulation process was carried out using the ionic gelation technique. The microcapsules were characterized physiochemically by their size, morphology, number of encapsulated bacteria after the encapsulation process, as well as bacterial survival after 45 days of storage (4 °C). The in vitro release and survival studies of bacteria inside the organs that make up the DS of C. arcuatus were carried out using a protocol developed in our laboratory by applying extracts of dissected organs from the DS (stomach, hepatopancreas and intestine) of the swimming crab. A χ² test (α = 0.05) was performed at linearization (Log₁₀) of the percentages of the controlled releases of microencapsulated B. licheniformis PPL2016 at different times (0 h, 4 h, 8 h, 12 h), corresponding to the extracts of the organs which simulated the digestive system of C. arcuatus. After biochemical characterization B. licheniformis PPL2016 was considered probiotic bacteria. Microparticles with an average size of 602 to 639 µm were obtained after using the ionic gelation method. Bacterial survival and encapsulation efficacy showed high cell viability and performance above 77.94%. Stability studies showed that storage at a temperature of 4 °C, kept almost 100% of viable bacteria for 15 days; however, cell viability decreased to a survival of 90% after 30 days of storage at this temperature. Regardless of reduced cell viability after 30 days, there are enough viable bacterial cells. Release and survival studies showed that alginate particles had a protective effect on bacteria, these results suggest that microparticles can be produced by a low-cost method. In juvenile males, the percentage of release of probiotic bacteria was greater in TIV in the enzyme extract of the intestine (12 h) with 95 ± 0.45%. Juvenile males had the lowest in vitro release at the stomach stage (0 h) and thus marks the significance for their low release of microcapsules at the beginning of the in vitro release (χ² = 6.7509; χ²_{Calculated Pool} = 13.5188; χ²_{Calculated Critical (0.05, 21)} = 11.5919; p < 0.05), with the highest significance in the intestine (12 h) (χ² = 1.2602; χ²_{Calculated Pool} = 13.5188; χ²_{Calculated Critical (0.05, 21)} = 11.5919; p < 0.05). Significant differences in vitro bacterial release were recorded for age classes and sexes of C. arcuatus.