Characterization of the lactic acid bacteria producers of gama-aminobutyric acid and their role in the prevention and treatment of experimental autoimmune encephalomyelitis

γ-aminobuterna kiselina (GABA) je biogeni amin prisutan kod biljaka, životinja i mikroorganizama i ostvaruje brojne fiziološke funkcije: inhibitor je neurotransmisije u sinapsama, ima umirujući efekat i ublažava hipertenziju i važan je imunosupresivni molekul. γ- aminobuterna kiselina nastaje ireverzibilnom dekarboksilacijom L-glutamata aktivnošću glutamat dekarboksilaze (GAD). Ovo istraživanje imalo je za cilj ispitivanje potencijala sojeva mlečnokiselinskih bakterija (MKB) izolovanih iz mlečnih proizvoda Zapadnog Balkana kao i iz proizvoda sa Kavkaza i iz Azerbejdžana za produkciju γ-aminobuterne kiseline, selekciju sojeva sa navedenih područja koji produkuju najviše ovog metabolita i njihovu detaljnu probiotičku karakterizaciju. Nakon selekcije soja, koji produkuje najveću koncentraciju γ-aminobuterne kiseline ispitivan je efekat živog soja i supernatanata kultura ovog soja na modelu inflamacije in vitro i na poboljšanje kliničke slike na modelu eksperimentalnog autoimunskog encefalomijelitisa, životinjskog modela multiple skleroze. Korišćenjem standardnih prajmera (CoreR/CoreF) prisustvo gadB gena, gena koji kodira glutamat dekarboksilazu, detektovano je kod 124 soja i to: 83 (27,4 %) laktobacila, 15 (4,9 %) laktokoka, 11 (3,6 %) leukonostoka, 8 (2,64 %) streptokoka, 6 (1,98 %) enterokoka i 1 (0,33 %) pediokoka od testirana 303 soja. TLC metodom je produkcija ovog metabolita detektovana je kod 4,6 % sojeva, odnosno 14 sojeva, od 124 testirana soja, a koji pripadaju vrstama Lactobacillus brevis i Lb. plantarum, dok je HPLC metodom izmerena koncentracija produkovane γ-aminobuterne kiseline. Simulirane uslove gastrointestinalnog trakta od 7 testiranih sojeva uspešno su preživela 4 soja vrste Lb. brevis BGLMM10, BGLMM11, BGZLS10-17 i BGZLS30-2. Ispitivanje bezbednosnog statusa sojeva pokazalo je da je soj BGZLS10-17 je bio osetljiv na sve testirane antibiotike, BGLMM11 je bio rezistentan na kanamicin, BGLMM10 je bio rezistentan na kanamicin i tetraciklin, dok je soj BGZLS30-2 bio rezistentan na ampicilin, kanamicin i tetraciklin, dok ni jedan od 4 ispitivana soja nije ispoljio ni hemolitičku ni želatinoznu aktivnost...microorganisms. GABA functions as an inhibitory neurotransmitter, has a calming effect alleviates hypertension, and is an effective immunosuppressive molecule. GABA is synthesized by irreversible decarboxylation of L-glutamate by glutamate decarboxylase (GAD) activity. The research goal of this study was to examine the potential of lactic acid bacteria (LAB), isolated from dairy products from the Western Balkans and from the products from the Caucasus and Azerbaijan, for the production of GABA, selection of the best GABA-producing strains, and their detailed probiotic characterization. After selecting the best GABA-producing strain next objective was to examine the effects of live strain, as well as, its culture supernatants on an in vitro inflammation models and on the clinical picture on experimental autoimmune encephalomyelitis (EAE), an animal multiple sclerosis (MS) model. Using standard (CoreR/CoreF) primers, the presence of the gadB gene, which encodes glutamate decarboxylase, was detected at 83 (27,4 %) Lactobacillus spp., 15 (4,9 %) Lactococcus spp., 11 (3,6 %) Leuconostoc spp., 8 (2,64 %) Streptococcus spp., 6 (1,98 %) Enterococcus spp., and 1 (0,33 %) Pediococcus spp out of 303 tested strains. By TLC method, the production of GABA was detected in 4.6% of the strains, ie 14 strains, out of 124 tested strains, belonging to the species Lactobacillus brevis and Lb. plantarum, while the concentration of produced GABA was measured by HPLC. The simulated gastrointestinal conditions successfully survived 4 out of 7 tested Lb. brevis strains: BGLMM10, BGLMM11, BGZLS10-17 and BGZLS30-2. Examination of the four Lb. brevis strains safety status showed that strain BGZLS10-17 was sensitive to all tested antibiotics, BGLMM11 was resistant to kanamycin, BGLMM10 was resistant to kanamycin and tetracycline, strain BGZLS30-2 was resistant to ampicillin, kanamycin and tetracycline, while none of the 4 tested strains showed hemolytic or gelatinous activity. The BGLMM10 strain exhibited the most potent antimicrobial activity, on 11 out of 18 pathogens, while BGZLS10-17 strain did not exhibit antimicrobial activity..