EVALUATION OF – AMINOBUTYRIC ACID (GABA) – PRODUCING LACTOBACILLI AS A POTENTIAL PROBIOTICS
Tolinacki M., Sokovic S., Djokic J., Zivkovic M., Popovic D., Mihajlovic S.
Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade, Serbia
G-aminobutyric acid (GABA), a nonprotein amino acid, possesses several physiological functions such as neurotransmission, induction of hypotension, and diuretic and tranquilizer effects. GABA is synthesized from glutamate by the activity of glutamic acid decarboxylase (GAD). GABA-producing lactobacilli are promising candidates as starters for manufacture of GABA-rich foods and to synthesize GRAS (generally recognized as safe)-grade GABA. Given that LMM-IMGGE possesses a unique collection of natural lactic acid bacteria (LAB) isolates we screened the collection for the best GABA-producing candidates with probiotic potential.
In silico analysis, conserved CoreF/CoreR primers as well as a degenerate primer GAD typing method were used to screen the presence of a glutamate decarboxylase gene, gadB in the LMM collection of autochthonous LAB isolates. Qualitative and quantitative assessment of GABA in the supernatants of selected LAB isolates was performed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Susceptibility to various antibiotics was determined according to EFSA recommendations. Antimicrobial activity was determined by deferred antagonism method. The survival of the selected LAB during the passage through the gastrointestinal tract (GIT) was studied in an in vitro model. The adhesion and pathogens’ exclusion abilities of the GABA producing LAB were assessed with the epithelial intestinal cell line Caco-2 and HT-29 MTX.
The 81% of lactococci, 64% of leuconostocs, 52% of lactobacilli, 33% of streptococci and 17% of enterococci from LMM collection were positive for the presence of the gadB gene. Preliminary screening of LAB carrying the gadB gene by TLC methodology demonstrated that the best GABA producers are among lactobacilli. The highest GABA producers were determined by HPLC among Lactobacillus brevis isolates. In order to evaluate probiotic potential of selected L. brevis isolates numerous in vitro tests were carried out. All tested lactobacilli were sensitive to all tested antibiotics. L. brevis BGLMM10 strain inhibited 12 indicator microorganisms. All tested lactobacilli strains succesfully survived in the simulated GIT conditions and showed decreases lower than 0.5 log cycle with respect to their initial cell density (8.6 to 8.9 log CFU/ml). L. brevis BGZLS10-17 showed the best adhesion ability to epithelial cell lines (15%). The exclusion assay showed that the highest reduction of the growth of Escherichia coli ATCC 25922 and Salmonella Enteritidis C2 9039 was observed in the presence of L. brevis BGZLS10-17 (80%) and L. brevis BGZLS30-2 (60%), respectively.
The selection of a microbial strain to be used as a probiotic is a rather complex process. A degenerate primer GAD prescreening typing method combined with TLC and reversed-phase HPLC confirmation was an efficient and cost-effective method to identify high-GABA-producing LAB. Numerous in vitro tests stressed five L. brevis isolates as good probiotic candidates that could be eventually used in formulation of functional starter cultures for production of the innovative foods.
Keywords: GABA, Lactobacillus brevis , Probiotic, TLC, gadB
Tolinacki M., et al. (2016). Evaluation of - aminobutyric acid (GABA) - producing lactobacilli as a potential probiotics. Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 120.). IPC2016