Download L. helveticus

The 2nd International Symposium „VERA JOHANIDES”
BIOTECHNOLOGY IN CROATIA by 2020
Zagreb, May 10-11, 2013
Role of S-layer proteins in probiotic activity
of Lactobacillus strains
Jasna Beganović, Ksenija Uroić, Andreja Leboš Pavunc, Blaženka Kos, Jagoda Šušković
Laboratory for antibiotic, enzyme, probiotic and starter cultures technology
Department of Biochemical Engineering
Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia
Strategy for the selection of probiotic strains in
Laboratory for antibiotics, enzymes, probiotics and starter cultures technology
(Šušković et al., 1992.)
Accurate
taxonomic
identifications
General
Biosafety
BCCM confirmed taxonomic nomenclature of our 9 selected strains:
Lactobacillus helveticus M92, L. plantarum L4, L. brevis D6, L. brevis ZG1,
L. brevis SF9B, L. paraplantarum SF15B, L. fermentum A8, Enterococcus
faecium L3, E. faecium A7…
GRAS (Generally Regarded As Safe, according US FDA) status of selected strains
Resistance to pH, gastric juice
bile, pancreatic juice
(Antibiotic susceptibility)
Technological
Activity and viability
(production/processing)
Antimicrobial activity
Antagonism to pathogens
Functional
aspects
Adherence to intestinal
epithelium/tissue
Influencing
metabolic activities
Stimulation immune response
PhD Thesis (Šušković, 1996)
PhD Thesis (Šušković, 1996)
Master Thesis (Kos, 1995)
PhD Thesis (Uroić, in progress)
PhD Thesis (Šušković, 1996)
Master Thesis (Frece, 2003)
PhD Thesis (Leboš Pavunc, 2012)
PhD Thesis (Kos, 2001)
PhDThesis (Uroić, in progress )
PhD Thesis (Beganović, 2008)
PhD Thesis (Šušković, 1996)
PhD Thesis (Kos, 2001)
PhD Thesis (Leboš Pavunc, 2012)
PhD Thesis (Frece, 2007)
PhD Thesis (Beganović, 2008)
PhD Thesis (Uroić, in progress)
Characteristics of Lactobacillus S-layer proteins
•
•
•
•
monomolecular crystalline arrays composed of (glyco)proteins
located on external side of cell envelope
identified in different microorganisms from the domains of Bacteria and Archaea
detected in just a few strains among 117 know Lactobacillus species:
– lower MW : 25-71 kDa
– highly basic proteins (pI = 9.35-10.4)
– mostly non-glycosylated
– signal peptide (N- terminal secretion signal ) typical for Sec pathway (25-30 AA)
cell membrane
cell wall
S-layer
S-layer present on the L. brevis D6 cell surface
performed by transmission electron microscopy (PhD in progress, Ksenija Uroić)
Detection of S-layer proteins of Lactobacillus strains
from Laboratory of antibiotics, enzymes, probiotics and starter cultures technology
SDS-PAGE surface protein profiles
S-layer proteins:
1. L. paraplantarum SF15B
2. L. brevis D6
3. L. brevis ZG1
4. L. brevis SF9B
PCR analysis with the specific primers
ATGAAGAAAAATTTAAGAAT and
CACCGATCTTGTAGTA.
1
2
S
1. L. helveticus M92
2. L. plantarum L4
S- DNA standard
slpA gene (GenBank acession number HM140425)
L. helveticus M92 S-layer protein identified by SDS-PAGE coupled to LC-MS/MS
S
1
(LC-MS/MS)
SlpA
protein
nESI linear ion trap-MS
Nano HPLC
Peptide separation
S – low MW protein standard;
1 – S-layer, purified by dialysis
Peptide sequences assigned to SlpA protein by Bioworks 3.2.
Beganović et al., (2010) Journal of Proteomic Research, 9 (2): 677-688
Beganović et al., (2011) Antonie van Leeuwenhoek, 100 (1): 43-53
Role of S-layer proteins in probiotic activity
of Lactobacillus strains
1. Adhesion of Lactobacillus strains to IPEC-1 cell line
(porcine intestinal epithelial cells)
16%
14%
12%
% adhesion
10%
A600=0,25
8%
A600=0,5
A600=1
6%
4%
2%
0%
1110
1112
1115
1118
M92
D6
Lactobacillus strains are labeled by thymidine and the radioactivity of the samples was
measured by liquid scintillation (L. helveticus M92 as reference strain)
Role of S-layer proteins in probiotic activity
of Lactobacillus strains
2. Inhibition of adhesion of enterotoxigenic Echerichia coli
to IPEC-1 cell line by Lactobacillus strains
- COMPETITION simultaneous addition of
Lactobacillus and
E. coli ERL 2055
- DISPLACMENT addition of Lactobacillus
after incubation
of E. coli ERL 2055
100%
% inhibition
80%
60%
40%
20%
0%
1110
1112
1115
1118
M92
D6
- EXCLUSION addition of Lactobacillus
before incubation
of E. coli ERL 2055
Role of S-layer proteins in probiotic activity
of Lactobacillus strains
3. Imunomodulation mediated by Lactobacillus purified S- layer proteins
• Extraction of S-layers from Lactobacillus cell surface
1
2
3
4
5
6
1
1 - L. brevis GRL1
2 - L. amylovorus GRL 1110
3 - L. amylovorus GRL 1111
4 - L. amylovorus GRL 1112
5 - L. helveticus M92
6 – L. plantarum D6
2
3
4
5
6
Induction of IL-1, IL-6, IL-10, IL-12, TNF cytokines production
in human monocyte-derived dendritic cells with Lactobacillus strains and purified
S-layer proteins - determined by cytokine specific ELISA
Stimulation of HEK Blue cell lines -TLR2, TLR4, TLR5 and NOD2with Lactobacillus strains and purified S-layer proteins
Maturation of dendritic cells in response to Lactobacillus bacterial cells and purified
S-layer proteins analysed by Flow Cytometric Analysis (FACS)
120
120
CD86
100
% positive cells
100
80
60
40
80
60
40
20
20
0
0
% positive cells
% positive cells
HLA-DR II
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
CD83
Bacteria/S-layer proteins
induced expression of
moDC maturation markers
HLA class II, CD86 and
CD83
Biotechnological protocol in
Laboratory for antibiotics, enzymes, probiotics and starter cultures technology,
for probiotic and starter culture production technology
Collection of lactic acid bacteria (ZBMK)
over than 300 characterised LAB strains
Inoculation
Growth medium
Phenotypic characterisation
probiotic strain / starter culture
Inoculum
Growth
Sterilisation
Production process control:
- microbiological control
- genetic control
Nutrient medium removal
Wet biomass of probiotic / starter
culture
lyoprotectant addition
MICROENCAPSULATION
LYOPHILIZATION
microbiological control
genetic control
MIKROENCAPSULATION
microbiological control
genetic control
Probiotic bacterium/
starter culture
functionality control
SCIENTIFIC PROJECTS & COLLABORATIONS:
o National scientific project
“Probiotics, prebiotics and functional starter cultures” No. 058- 1990-2007
o International project
SEE-ERA.NET PLUS: PSALAB No. 195/1
Project leader: PhD Jagoda Šušković, full prof.
o Collaborative project
with research team of PhD Airi Palva, prof., University of Helsinki, Finland