In Situ Cross-Linked Porous Starch Microencapsulation Enhances the Colonization of Lactobacillus In Vivo

Abstract

In this study, we developed novel porous starch (PS)/Lactobacillus (LS) microcapsules via in situ cross-linking with sodium trimetaphosphate (STMP), using Lactobacillus johnsonii (LJ), Lactobacillus acidophilus (LA), and Lactobacillus rhamnosus GG (LGG) as representative strains. Scanning electron microscopy (SEM) revealed that the cross-linked microcapsules (designated as PS/LS-CL: PS/LJ-CL, PS/LA-CL, PS/LGG-CL) formed aggregated structures with denser microarchitecture compared to uncross-linked porous starch/Lactobacillus microcapsules (designated as PS/LS: PS/LJ, PS/LA, PS/LGG). The encapsulation efficiencies of PS/LJ-CL, PS/LA-CL, and PS/LGG-CL (79.56%, 78.49%, and 55.96%, respectively) significantly surpassed those of their uncross-linked counterparts (67.92%, 58.68%, and 47.71%, p < 0.05). In addition, the cross-linked porous starch microcapsules improved the survival rate of Lactobacillus during simulated gastrointestinal digestion and long-time storage. Importantly, the oral gavage of PS/LS-CL, PS/LA-CL, and PS/LGG-CL significantly increased the amount of Lactobacillus. The colonization efficiency of all the tested Lactobacillus in mice was detected by both gradient dilution plate counting and quantitative real-time PCR (qRT-PCR). These findings indicate the potential function of the in situ cross-linked porous starch microcapsules as a robust delivery system to enhance the colonization of probiotics in vivo.

Keywords: delivery system; encapsulation efficiency; oral gavage; simulated digestion; survival rate.

Figure 1

Schematic diagrams for the preparation of PS/LS and PS/LS-CL.

Figure 2

Electron micrographs of (A) porous starch (PS), (B) Lactobacillus johnsonii (LJ), (C) Lactobacillus acidophilus (LA), (D) Lactobacillus rhamnosus GG (LGG), (E) LJ encapsulated in PS (PS/LJ), (F) LA encapsulated in PS (PS/LA), (G) LGG encapsulated in PS (PS/LGG), (H) LJ encapsulated in PS via in situ cross-linking (PS/LJ-CL), (I) LA encapsulated in PS via in situ cross-linking (PS/LA-CL), (J) LGG encapsulated in PS via in situ cross-linking (PS/LGG-CL). The regions marked by red circles indicate Lactobacillus adhesion on the surface of porous starch or penetration into its micropores.

Figure 3

Survival Lactobacillus in PS/LS-CL and PS/LS during storage time (4 °C, 60% RH).

Figure 4

In vivo colonization detection in mouse colon after 7-day oral gavage. (A) BALB/c mice (6–8 weeks old) received daily oral gavage for 7 consecutive days, and their fresh fecal samples were aseptically collected 48 h after the final administration, immediately snap-frozen in liquid nitrogen, and stored at −80 °C until analysis. The control group: daily oral gavage of 200 μL 0.01 M phosphate-buffered saline (PBS, pH 7.4); LS groups (LJ, LA, LGG) daily oral gavage of 200 μL PBS containing free Lactobacillus cells (1 × 10⁸ CFU); PS/LS-CL groups (PS/LJ-CL, PS/LA-CL, PS/LGG-CL): daily oral gavage of 200 μL PBS containing Lactobacillus cells (1 × 10⁸ CFU). (B–D) the abundance of LJ, LA, and LGG in fecal samples via gradient dilution plate counting. (E–G) the abundance of LJ, LA, and LGG in fecal samples via qRT-PCR. ns: not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.