Influence of the pili of Lacticaseibacillus rhamnosus GG on its encapsulation and survival in mixed protein-starch gels assembled by in situ fermentation

Abstract

Preserving the viability of probiotics during storage and gastrointestinal digestion poses a significant challenge in the development of effective probiotic formulations. Thus, this study developed an in situ fermentation approach to encapsulate the probiotic Lacticaseibacillus rhamnosus GG (LGG) in a mixed whey protein/modified starch gel and evaluated (i) the role of the pili on gel formation and on the effectiveness of the gel to maintain cell viability during simulated digestion, and (ii) the storage stability of the encapsulated probiotics. Kinetic data of gels made with the wild-type (WT) or a pilus-depleted mutant (ΔspaCBA) strain showed a rapid in situ gel formation (<30 min) at room temperature after inoculating the polymeric mixture, driven by in situ fermentation and independently of the piliation of the cells. After simulated gastrointestinal digestion, the viability of encapsulated WT cells was ~3 log higher than free WT cells (P value 7 × 10^-4) and ~0.6 times higher than encapsulated ΔspaCBA cells (P value 9 × 10^-3). A higher release of ΔspaCBA vs WT cells from the gels was quantified, and confocal microscopy revealed the aggregation of ΔspaCBA but not WT cells within the gel cavities. These findings suggest the pili-dependent retention of LGG within the gel contributes to its protective effect. Finally, the hydrated gels sustained counts of LGG of 7.76-6.69 log CFU/g (depending on the relative humidity) during 2 months of storage at room temperature. In summary, bacteria-to-matrix interactions might influence the survival of probiotics during delivery, and the protein/starch gels could represent a cost-effective alternative for unrefrigerated storage and delivery of probiotics.

Importance: Many probiotic formulations struggle to maintain the viability of microbial cells over time and during their passage through the gastrointestinal tract. This has led to the development of encapsulation strategies for probiotics, most of which are either costly to implement or damage the cells during the encapsulation process. To overcome these limitations, this work developed a rapid fermentation-based approach to encapsulate probiotics in protein/starch gels as a strategy to keep the cells alive during storage and digestion. Moreover, this work explored the role of interactions between bacterial cells and their encapsulation matrix on the formation of the gels and in the protection the gels provided in maintaining the viability of cells during simulated digestion. Developing this in situ fermentation approach for the encapsulation of probiotics and understanding the bacteria-matrix interactions will lead to the development of more effective probiotic products that can be easily deployed in low-resource settings.

Keywords: encapsulation; gel; pili; probiotic; protein; starch.

Fig 1

Changes in pH (A), optical density (B), and viscoelastic properties (C) of a mixture of WPI/modified starch after inoculation with L. rhamnosus GG strain WT or its pilus-depleted mutant strain ΔspaCBA. Rheological measurements were carried out at 10 Hz, 0.3% strain, 1 mm gap.

Fig 2

Confocal images of mixed WPI/modified starch gels assembled via in situ fermentation by L. rhamnosus GG strain WT or its pilus-depleted mutant strain ΔspaCBA. Scale bar, 50 µm. WPI stained with FITC (green), modified starch stained with Rhodamine B (red), and bacterial cells stained with Sybr Green (green).

Fig 3

Effect of the strain (WT or ΔspaCBA) and delivery mode (free or encapsulated in WPI/modified starch gel) in the survival of L. rhamnosus GG, reported in log CFU/g (A) and percentage (B). Effect of the strain (WT or ΔspaCBA) and pH on the release of L. rhamnosus GG cells from the bulk WPI/modified starch gels (C).

Fig 4

Effect of relative humidity (%) on the viability of L. rhamnosus GG WT encapsulated in WPI/modified gels during room temperature storage.