Lyophilized alginate-based microspheres containing Lactobacillus fermentum D12, an exopolysaccharides producer, contribute to the strain's functionality in vitro

Abstract

Lactobacillus (Limosilactobacillus) fermentum D12 is an exopolysaccharide (EPS) producing strain whose genome contains a putative eps operon. Whole-genome analysis of D12 was performed to disclose the essential genes correlated with activation of precursor molecules, elongation and export of the polysaccharide chain, and regulation of EPS synthesis. These included the genes required for EPS biosynthesis such as epsA, B, C, D and E, also gt, wzx, and wzy and those involved in the activation of the precursor molecules galE, galT and galU. Both the biosynthesis and export mechanism of EPS were proposed based on functional annotation. When grown on MRS broth with an additional 2% w/v glucose, L. fermentum D12 secreted up to 200 mg/L of a mixture of EPSs, whose porous structure was visualized by scanning electron microscopy (SEM). Structural information obtained by ¹HNMR spectroscopy together with composition and linkage analyses, suggested the presence of at least two different EPSs, a branched heteropolysaccharide containing t-Glcp and 2,6-linked Galf, and glycogen. Since recent reports showed that polysaccharides facilitate the probiotic-host interactions, we at first sought to evaluate the functional potential of L. fermentum D12. Strain D12 survived simulated gastrointestinal tract (GIT) conditions, exhibited antibacterial activity against enteropathogenic bacteria, adhered to Caco-2 cells in vitro, and as such showed potential for in vivo functionality. The EPS crude extract positively influenced D12 strain capacity to survive during freeze-drying and to adhere to extracellular matrix (ECM) proteins but did not interfere Caco-2 and mucin adherence when added at concentrations of 0.2, 0.5, and 1.0 mg/mL. Since the viable bacterial count of free D12 cells was 3 logarithmic units lower after the exposure to simulated GIT conditions than the initial count, the bacterial cells had been loaded into alginate for viability improvement. Microspheres of D12 cells, which were previously analyzed at SEM, significantly influenced their survival during freeze-drying and in simulated GIT conditions. Furthermore, the addition of the prebiotic substrates mannitol and lactulose improved the viability of L. fermentum D12 in freeze-dried alginate microspheres during 1-year storage at 4 °C compared to the control.

Keywords: Eps cluster; Exopolysaccharides; Limosilactobacillus (Lactobacillus) fermentum; Prebiotic; Probiotic.

Fig. 1

a Sequencing data were analyzed with DNA Plotter to construct the genomic atlas. Circles illustrate the following from outermost to innermost rings: (1) the entire chromosome; (2) the location of the contigs; (3) the local % GC plot and innermost, the GC-skew. b Subsystem distribution of L. fermentum D12 based on the RAST. The underlined text indicates the subsystem features involved in exopolysaccharide (EPS) biosynthesis

Fig. 2

a Putative exopolysaccharide gene cluster (eps) located in contig NZ_RHMA01000026.1 and other genes involved in exopolysaccharide production located in contig NZ_RHMA01000008.1 by L. fermentum D12 strain. epsA: transcriptional regulator; epsC: tyrosine kinase modulator; epsD: tyrosine kinase; epsB: manganese-dependent tyrosine phosphatase; wzx: flippase; glf: UDP-galactopyranose mutase; epsC´: tyrosine kinase modulator/chain length determination; wzy: polysaccharide polymerase; gt: glycosyltransferase; epsE: Undecaprenyl galactose phosphotransferase; C: cytoplasmic membrane; P: periplasm. Transcribing directions, gene functions and module size are respectively shown by oriented arrows, different colors and arrow length. b Putative genes encoding the enzymes involved in the activation of the precursor molecules: glucose pyranose and galactose furanose. c Proposal of the putative biosynthetic model of the EPS cluster of L. fermentum D12. This image was created with Biorender.com

Fig. 3

Size-exclusion chromatography elution profile of EPS-r separated on a Sephacryl S-400 column. Fractions were pooled as shown

Fig. 4.

¹H NMR spectra of EPS-r1 (a) and EPS-r2 (b) in D₂O recorded at 500 MHz and 50 °C. a α-1,4 Glcp and α-1,6 Glcp = anomeric protons of 1,4- and 1,6-linked glucopyranose; b in the EPS-r2 spectrum the stars indicate the two most intense anomeric proton resonances attributed to t-Glcp and 2,6-Galf

Fig. 5

Adhesion ability of L. fermentum D12 to major EMC proteins, mucin and Caco-2 cells in the presence of increasing concentrations of the EPS fractions. *Significantly different (P < 0.01) from other samples

Fig. 6

Viability of freeze-dried alginate-microspheres of cells (gray filled circle), and freeze-dried free cells (black filled circle) of L. fermentum D12, exposed to simulated GIT conditions (arrow indicates changed conditions: after 2 h incubation in simulated gastric fluid at pH = 2.0; 4 h incubation in the simulated intestinal fluid at pH = 8.3 was performed). Results are expressed as mean values of three independent experiments and error bars represent standard deviations

Fig. 7

Survival of alginate microencapsulated L. fermentum D12 cells with prebiotics, during 1-year storage at 4 °C with the addition of different prebiotics: inulin with fructooligosaccharides (FOS), mannitol, lactulose; and without prebiotics. The results are expressed as mean values of three independent experiments and error bars represent standard deviations