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Exopolysaccharides Produced by Lactobacillus rhamnosus KL 53A and Lactobacillus casei Fyos Affect Their Adhesion to Enterocytes

CORINNA KONIECZNA
CORINNA KONIECZNA
, 
MICHAŁ SŁODZIŃSKI
MICHAŁ SŁODZIŃSKI
 and 
MARCIN T. SCHMIDT
MARCIN T. SCHMIDT
   | Sep 04, 2018
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Article Category: original-paper
Published Online: Sep 04, 2018
Page range: 273 - 281
Received: Feb 01, 2017
Accepted: Apr 20, 2018
DOI: https://doi.org/10.21307/pjm-2018-032
Keywords
© 2018 Corinna Konieczna et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Adhesion efficiency of the fixed bacteria before degycosylation and after deglycosylation. LKL 53A, L. rhamnosus KL 53A, LcF, L. casei Fyos. The bars represent average percentages of bacterial cells that adhered to differentiated Caco-2 enterocytes, the whiskers represent average adhesion ± standard error.
Adhesion efficiency of the fixed bacteria before degycosylation and after deglycosylation. LKL 53A, L. rhamnosus KL 53A, LcF, L. casei Fyos. The bars represent average percentages of bacterial cells that adhered to differentiated Caco-2 enterocytes, the whiskers represent average adhesion ± standard error.

Fig. 2.

Normalized to 100% adhesion efficiency of L. casei Fyos grown in the presence of glycosidase inhibitors: castanospermine (Cas), deoxymannojirimycin hydrochloride (dMM), swainsonine (Swa), kifunensine (Kif), deoxynojirimycin hydrochloride (dNM) and without inhibitor (contr). The bars represent average percentages of bacterial cells that adhered to differentiated Caco-2 enterocytes, the whiskers represent average adhesion ± standard error. Statistically significant changes (p < 0.05) are marked with asterisks (*).
Normalized to 100% adhesion efficiency of L. casei Fyos grown in the presence of glycosidase inhibitors: castanospermine (Cas), deoxymannojirimycin hydrochloride (dMM), swainsonine (Swa), kifunensine (Kif), deoxynojirimycin hydrochloride (dNM) and without inhibitor (contr). The bars represent average percentages of bacterial cells that adhered to differentiated Caco-2 enterocytes, the whiskers represent average adhesion ± standard error. Statistically significant changes (p < 0.05) are marked with asterisks (*).

Fig. 3.

A comparison of relative protein number in the L. casei Fyos proteomic mass spectrometry data of the control cells (contr) and of the cells grown in the presence of swainsonine (Swa).
A comparison of relative protein number in the L. casei Fyos proteomic mass spectrometry data of the control cells (contr) and of the cells grown in the presence of swainsonine (Swa).

Fig. 4.

Changes in adhesion efficiency to Caco-2 cells of two lactic acid bacteria strains co-cultured with anaerobic gut bacteria. Adhesion efficiency was normalized to 100%. Statistically significant changes (p < 0.05) are marked with asterisks (*). C – control: either L. casei Fyos or L. rhamnosus KL 53A; LcF/FP, L. casei Fyos co-cultured with F. prausnitzii DSM 17677; LcF/BL, L. casei Fyos co-cultured with B. luti DSM 14534; LKL 53A/FP, L. rhamnosus KL 53A co-cultured with F. prausnitzii DSM 17677; LKL 53A/BL, L. rhamnosus KL 53A co-cultured with B. luti DSM 14534.
Changes in adhesion efficiency to Caco-2 cells of two lactic acid bacteria strains co-cultured with anaerobic gut bacteria. Adhesion efficiency was normalized to 100%. Statistically significant changes (p < 0.05) are marked with asterisks (*). C – control: either L. casei Fyos or L. rhamnosus KL 53A; LcF/FP, L. casei Fyos co-cultured with F. prausnitzii DSM 17677; LcF/BL, L. casei Fyos co-cultured with B. luti DSM 14534; LKL 53A/FP, L. rhamnosus KL 53A co-cultured with F. prausnitzii DSM 17677; LKL 53A/BL, L. rhamnosus KL 53A co-cultured with B. luti DSM 14534.

Composition of the Protein Deglycosylation Mix.

EnzymeHydrolyzed boundsNumber of enzymatic units added per 100 μl of reaction
O-GlycosidaseCatalyzes the removal of core 1 and core 3 O-linked disaccharides from glycoproteins.400000 U
PNGase FCleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins unless α(1-3) core fucosylated  5000 U
NeuraminidaseCatalyzes the hydrolysis of terminal, non-reducing α2,3, α2,6, and α2,8 linked N-acetylneuraminic acid residues from glycoproteins and oligosaccharides.    500 U
β1-4 GalactosidaseCatalyzes the hydrolysis of terminal, non-reducing β1-4 linked D-galactopyranosyl residues from oligosaccharides and glycoproteins.      80 U
β-N-AcetylglucosaminidaseCatalyzes the hydrolysis of terminal, non-reducing β-N-Acetylglucosamine residues from oligosaccharides and glycoproteins.      40 U

Function of glycosidase inhibitors used to inhibit EPS synthesis in L. casei Fyos.

GT inhibitorThe enzyme inhibited
Castanospermineβ-glucosidaseβ-xylosidaseα-mannosidaseβ-mannosidaseMaltase-glucoamylase
Deoxymannojirimycinα-(1→2)-mannosidase
Kifunensine
Swainsonineα-(1→3)-mannosidaseα-(1→6)-mannosidase
Deoxynojirimycin hydrochlorideα-glucosidaseMaltase-glucoamylase

Monosaccharides composition of the EPS produced by L. rhamnosus KL 53A and L. casei Fyos when grown on MRS agar (anaerobic conditions, 37°C, 18 h).

Monomer% of a monomer in EPS isolated from the strain tested
Lactobacillus rhamnosus KL 53ALactobacillus casei Fyos
Maltose0.710
Glucose30.915.7
Galactose53.731.4
Arabinose14.651.6
Ribose01.4
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