Introduction
Study design
Contact:
andrea.mancini@fmach.it
Andrea Mancini, Elena Franciosi, Ilaria Carafa,
Francesca
Fava, Roberto Viola, Kieran Tuohy
Nutrition and Nutrigenomic Group, Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach (FEM),
38010 San Michele all’Adige (TN), Italy
Conclusions
Pancreatic fluid tolerance.
Tolerance test perfomed in NaHCO3 with 1.9 mg/ml pancreatin for 3h. Cell counts compared to type strain grown in the same condition (mean ± sd, N = 3).
Effect of bile salts after low pH treatment. Bile acid
(Oxbile) treatment
perfomed in MRS over time after pH 2.5 exposure (3h) (mean ± sd, N = 3).
Growth rate with bile salts. Bile acid (Oxbile)
treatment perfomed in MRS over time (mean ± sd, N = 3).
Lactobacillus brevis FEM 1874 was confirmed as a high GABA producing strain with potential as a probiotic for human use, given
it’s ability to survive under simulated gastro-intestinal physicochemical conditions. However, its ability to produce GABA within
the gut and consequently mediate health benefits via the gut:brain axis and immune system, must further be confirmed in vivo.
Bile Salt Hydrolysis (BSH) assay. BSH activity perfomed on MRS agar plates supplemented with 0.5% of bile acids. (mean ± sd, N = 3).
Acid tolerance (pH 2, 2.5, 3.2). Acid treatment perfomed in PBS at 0 and 3h (T0 and T3 resp). Cell counts compared to type strain grown in the same condition (mean ± sd, N = 3).
1) FEM 1874 survives in gastrointestinal phisicochemical condition
Results
3) FEM 1874 GABA genes sequences analysis and knock-out generation strategy
Probiotics and their metabolites can act as brain modulators through the gut:brain
axis system. In this respect γ-aminobutyric acid (GABA) gut bacteria produced, has
been identified as a possible factor improving brain function, inhibiting inflammation
and regulating energy metabolism.
-0.20 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 ND ND ND 2.5 3.2 7.2 2 low GABA medium GABA
high GABA very high GABA (L. brevis FEM 1874)
«Wild» Alpine cheese isolates GABA production.
GABA production (mg/L) after 24h in experimental buffer + glutamate [1mg/ml]. Total isolate considered 110. Mean ± sd, N = 3.
GABA production. GABA production after 24h in experimental buffer + glutamate [1mg/ml]. Mean ± sd, N = 3 gadR 591bp gadC 1506bp gadA 1440bp 5’ gadB 3’ 1407bp operon ~ 1,7Mb
based on L. brevis ATCC 367
FEM 1874 GABA genes sequenced: 100% identity. Based on L. brevis ATCC 367 gene
sequencing, nine sets of primer were used to sequence all GABA genes. 100% of identity was found, only a SNP in antiporter (gad C) with synonimus mutation (V to V) were evidenced. HindIII BamHI gad genes KpnI KpnI erm from pE194 Ampr ori P LacZ HindIII BamHI pUC19 2686bp Ampr ori P gad gene pUC19-gad gene 3238 to 3710bp Ampr ori P erm pUC19-gad gene-erm 4430 to 4902bp
gad genes knock-out plasmids contruction.
Schematic representation of the strategy employed in the study for the cintruction of the pUC19-gad gene-erm vectors.
“Wild” Alpine cheese isolate Lactobacillus brevis FEM 1874 was characterized for
potential probiotics traits and GABA production. Acid, bile and pancreatic fluid
resistance was assessed and the genetic loci for GABA production and export
characterized for the subsequent knock-out vectors construction.
2) FEM 1874 produced high GABA level
Probiotic potential of a high GABA producing strain,
Lactobacillus brevis
FEM 1874, isolated from traditional
“wild” Alpine cheese.
Strain BSH activity L. brevis DSM 20054 -L. brevis FEM 1874 + Strain GABA (mg/L) sd VSL#3 9.39 0.31 L. brevis DSM 20054 78.2 18.6 L. brevis FEM 1874 262.0 15.4
