Bianchi, Fernanda

Chlumsky, O

Hansen, K.M.

Jespersen, Lene

Larsen, Nadja

Leser, Thomas

Sivieri, Katia

Srimahaeak, Thanyaporn

English

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetIn vitro studies of the impact of pectins on adhesion of Lactobacillus spp. to humanepithelial cells and intestinal barrier integritySrimahaeak, Thanyaporn; Bianchi, Fernanda; Chlumsky, O; Sivieri, Katia ; Hansen, K.M.;Leser, Thomas; Larsen, Nadja; Jespersen, LenePublication date:2018Citation for published version (APA):Srimahaeak, T., Bianchi, F., Chlumsky, O., Sivieri, K., Hansen, K. M., Leser, T., ... Jespersen, L. (2018). In vitrostudies of the impact of pectins on adhesion of Lactobacillus spp. to human epithelial cells and intestinal barrierintegrity. Poster session presented at International Scientific Conference on Probiotics and Prebiotics 2018,Budapest, Hungary.Download date: 03. Feb. 2020In vitro studies of the impact of pectins on adhesion of Lactobacillus spp. to human epithelial cells and intestinal barrier integrity DEPARTMENT OF FOOD SCIENCEFACULTY OF SCIENCEUNIVERSITY OF COPENHAGENSrimahaeak T.a, Bianchi F.a,c, Chlumsky O.b, Sivieri K.c, Hansen K.M.d, Leser T.e, Larsen N.a and Jespersen L.aa University of Copenhagen, Department of Food Science - Frederiksberg, Denmarkb Masaryk University, Department of Experimental biology - Kamenice, Czech Republicc State University of São Paulo, Department of Food and Nutrition - Araraquara, Brazild CP Kelco ApS, Lille Skensved, Denmarke Chr. Hansen, Hørsholm, DenmarkIntroduction and objectivesPectins are complex polysaccharides extracted from plants and used in food industry as stabilizers and gelling agents. Pectins are commonly referred as emerging prebiotics which beneficial effects in the gut need to be confirmed. The aim of this study: To investigate the effect of pectins on adhesion of probiotic Lactobacillus species to human epithelial cells and integrity of intestinal cell monolayers.Materials and MethodsPectins: P1 (Harsh extracted from orange), P2 (Mild extracted from lemon), P3 (Harsh extracted chemically deesterified from lime) and P4 (Harsh extracted pectin from lemon) (provided by CP Kelco ApS, Denmark).Bacterial strains: Lactobacillus fermentum PCC and Lactobacillus reuteriRC-14 (provided by Chr. Hansen, Denmark).Cell culture: Human colon adenocarcinoma Caco-2 cell line. Assays conditions:  Caco-2 cells were maintained in DMEM, 37ºC, and 5% CO2 for 14 days to reach differentiation. Bacterial strains PCC and RC-14 (overnight culture): 108 CFU per well  Pectins P1 – P4: final concentration 0.2% (w/v) in test solution (PBS + DMEM,  pH 7.3). Adhesion assay TEER assay Gene expression analysisConclusion• Pectins have a potential to improve bacterial adhesion to intestinal cells and further enhance strengthening of epithelial barriers by probiotic Lactobacillus spp. in the gut.• The beneficial effects of bacterial-pectin combinations in this study were strain-specific and differed between the pectins, indicating involvement of specific structural factors in bacterial-pectin interactions.• The mechanisms behind these interactions and the interplay between the structural properties of pectins and their beneficial effects need to be further elucidated.ResultsContact informationThanyaporn Srimahaeak,PhD studentBioactive components from by-products of food processing used in asynbiotic approach for improvinghuman health and well-being.Food MicrobiologyDepartment of Food ScienceUniversity of CopenhagenRolighedsvej 26, 1958 Frederiksberg CThanya@food.ku.dkAcknowledgementsAdhesion (%)ControlPCC/P1PCC/P2PCC/P3PCC/P402 04 06 08 01 0 0****************Adhesion (%)ControlRC-14/P1RC-14/P2RC-14/P3RC-14/P402 04 06 08 01 0 0****T im e  (H rs )Ratio (TEERt/TEER0)0 4 8 1 2 1 6 2 0 2 40 .00 .51 .01 .52 .0C o n tro lRC14R C14/P1R C14/P2R C14/P3R C14/P4P1=P2=P3=P4T im e  (H rs )Ratio (TEERt/TEER0)0 4 8 1 2 1 6 2 0 2 40 .51 .01 .5C o n tro lPCC PCC/P1 PCC/P2PCC/P3 PCC/P4 P1=P2=P3=P4Ratio (TEER/TEER0)ControlRC14P1RC14/P1P2RC14/P2P3RC14/P3P4RC14/P40123*Ratio (TEER/TEER0)ControlPCC P1PCC/P1P2PCC/P2P3PCC/P3P4PCC/P40123***Gene Control P2 P3 PCC PCC/P2 PCC/P3CLDN1 1.00 1.11 0.42 0.50 0.56 0.26CLDN2 1.00 0.77 0.92 0.91 1.08 0.42CLDN4 1.00 1.03 0.56 1.10 1.18 0.32TJP1 1.00 0.94 0.57 0.86 1.02 0.49Gene Control P2 P3 PCC PCC/P2 PCC/P3CCL20 1.00 0.86 5.59 11.73 16.41 10.08CXCL1 1.00 0.72 26.17 79.75 64.61 43.68CXCL2 1.00 0.95 0.99 14.47 13.65 10.54CXCL10 1.00 2.86 9.68 13.97 18.40 11.48IL8 1.00 1.28 9.21 28.97 30.37 14.34TNF 1.00 1.15 2.54 101.06 77.37 81.58CLDN1 1.00 0.25 0.49 1.29 0.77 0.38CLDN2 1.00 4.09 1.13 0.43 0.34 0.19CLDN4 1.00 0.96 0.90 2.20 2.66 2.88TJP1 1.00 1.24 0.55 0.84 0.76 0.30Gene Control P2 P3 PCC PCC/P2 PCC/P3CLDN1 1.00 1.13 1.02 0.89 1.03 1.02CLDN2 1.00 1.00 3.22 0.16 0.17 0.31CLDN4 1.00 0.86 0.48 2.79 2.65 9.67TJP1 1.00 0.85 1.09 0.62 0.52 1.36Adhesion1 HoursAdhesion4 HoursImmune reponseAdhesion10 Hours(a) (b)Figure 1. Adhesion of Lactobacillus spp. to Caco-2 cell monolayers as affected by pectins after 1h incubation  ((a) PCC and (b) RC-14). Adhesion assay: PCC and RC-14 increased adhesion to Caco-2 cells of 30% and 35%, respectively. Pectins P1, P2, and P4 improved adhesion of PCC (2-fold); their effect on RC-14 adhesion was insignificant Pectin P3 reduced the binding of both strains to Caco-2 cells. Measurement of transepithelial electrical resistance (TEER): TEER was increased up to 30% by treatments with PCC and RC-14. PCC in combination with  P1, P2 and P4 increased TEER up to 16% compared to PCC alone.  RC-14 in combination with P2 increased TEER up to 37%.  Other pectin combinations with  PCC (P3) and RC-14 (P1, P3 and P4) did not have significant effect on TEER. PCC in combination with pectins P2 and P3 altered mRNA expression in Caco-2 cells:  Expression of genes CCL20, CXCL1, CXLC2, CXCL10, IL8 and TNF∝ involved in immune responses were increased after 4h in all treatments.  Highest by 70-100 fold in TNF∝ after treatment with PCC alone and combined with P2 and P3. CLDN4 was induced by both P2 and P3 combined with PCC after 4h. CLDN1, CLDN2 and TJP1 were decreased (0.2-0.8 fold) after 1h in all treatments.  TJP1 was slightly increased (1.36-fold) after 10h incubation (PCC with P3).Figure 2. Transepithelial electrical resistance (TEER) across Caco-2 cell monolayers treated with PCC and RC-14 alone and in combination with pectins P1, P2, P3 and P4. (a) and (b) TEER curves during 24 h treatment; (c) and (d) differences between treatments at maximum TEER (after 12h incubation). Figure 3. Expression of genes encoding adhesion and immune response proteins in Caco-2 cells treated with PCC and pectins P2 and P3. • Responses in adhesion genes were determined after 1, 4, and 10 hours treatment (SYBR Green assay) and immune genes - after 4 h treatment (TaqMan assay, Fluidigm).(a) (b)(c) (d)Extract RNA from Caco-2 cellsqRT-PCR(Fluidigm/SYBR Green)Counts of bacterial cells bound to Caco-2 cells  on MRS agarThe Danish Council for Strategic ResearchCellZscope2 (Nano Anatytics)12 well-plate1hTranswell filter insert (0.4 µm pore size)12 well-plateIncubation 1, 4 and 10h

<em>In vitro</em> studies of the impact of pectins on adhesion of <em>Lactobacillus spp.</em> to human epithelial cells and intestinal barrier integrity

https://curis.ku.dk/ws/files/209387763/Thanyaporn_S_IPC2018_.pdf

<em>In vitro</em> studies of the impact of pectins on adhesion of <em>Lactobacillus spp.</em> to human epithelial cells and intestinal barrier integrity

Abstract

Similar works

Full text

Available Versions

Copenhagen University Research Information System