RESUMO
Here we report the whole genome sequence of Lactobacillus fermentum HFD1 strain, the producer of antibacterial peptides. The genome consists of one circular chromosome with 2101878 bp in length and GC-content of 51.8%, and includes linear DNA with 5386 bp in length with 100% identity to bacteriophage phiX174. The analysis of the genome has revealed 2049 genes encoding for proteins including 867 proteins without known function and 70 genes encoding for RNAs (10 rRNAs, 59 tRNAs and 1 tmRNA). Putative genes responsible for the biosynthesis of 4 antimicrobial peptides were identified. The NCBI Bioproject has been deposited at NCBI under the accession number PRJNA615901 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA615901/) and consist of full annotated genome and raw sequence data.
RESUMO
Application of NO-producing lactobacilli to a rat jejunum segment induced muscle relaxation that was potentiated after activation of bacterial NO production with NO synthase substrate L-arginine. Similar changes in the intestinal contractile activity were observed in response to exogenous NO formed by sodium nitroprusside. These results indicated the involvement of NO synthesized by probiotic lactobacilli in the regulation of the intestinal motor function.
Assuntos
Lactobacillus/metabolismo , Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Animais , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Lactobacillus/fisiologia , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Nitroprussiato/farmacologia , RatosRESUMO
BACKGROUND: Probiotics are live microorganisms, generally either lactobacilli or bifidobacteria, which when administered in adequate amounts confer a health benefit to the host [1]. Due to the growing evidence of health benefits associated with their use, probiotics are of increasing interest and represent now a significant growth area in the functional foods industry [2]. However, to be effective, orally administered probiotics should survive preparation of dosage forms and passage through acidic environment of the gastrointestinal tract (GIT). Reaching the intestine, these microorganisms should be able to establish themselves, remain viable and perform their beneficial actions. In this context, oral formulations have to protect probiotic bacteria from gastric acidity and delay their release in the small intestine in order to allow their complete release in the colon. OBJECTIVE: To evaluate effects of starch formulations of lactobacilli on their survival in gastric environment and probiotic properties. METHODS: Nineteen Lactobacillus strains belonging to the species L. fermentum (14 strains), L. plantarum (4 strains), and L. rhamnosus (1 strain), were isolated from dairy products and probiotics, and were used in this study. Lactobacilli were cultured in de Man, Rogosa, Sharpe (MRS) broth (Merck, Germany) under microaerobic conditions at 37°C.Amylolytic activity of lactobacilli, cultured for 3-5 days on MRS agar supplemented with 1% soluble potato starch (SPS), was determined with iodine reagent (0.01 M I2-KI solution).Loading in starch was performed with L. plantarum 8PA3 bacteria ("Dry lactobacterin", Perm, Russia), which were resuspended to the concentration 1010 cells/mL in 10 mL of 0.85% NaCl solution and added to 90 mL of 2.5% SPS solution. Resulting mixture was frozen at -18°C and then lyophilized (Martin Christ Alpha 1-2 LDplus, Germany).Atomic force microscopy (AFM) images of formulated L. plantarum 8PA3 cells were acquired in air by a Solver P47H atomic force microscope (NT-MDT, Moscow, Russia).Starch swelling and dissolution was studied in simulated colonic fluid (SCF), prepared according to [3] and in distilled water (pH = 6.0) as control. Amylase from Aspergillus oryzae (A8220, Sigma) was added to the solutions to study the influence of amylase. The formulation form was examined visually during 14 h incubation time.Fluorescence microscopy images were obtained with a Leica DM6000B (Germany) fluorescent microscope using Leica FW4000 software.L. plantarum 8PA3 loaded in SPS were placed either in HCl solution (pH 2), or in 2% oxgall bile solution, or in 0.85% NaCl solution. Viability was tested after 2, 4 and 6 h incubation at 37°C by plating diluted aliquots onto MRS agar with subsequent counting of bacterial colony forming units (CFU). In addition, viability was determined using LIVE/DEAD BacLight bacterial viability kit L-7012 (Molecular Probes, Invitrogen) as described elsewhere [4]. Fluorescence in the stained samples was estimated with BD FACS Canto II (USA) flow cytometer or fluorescent microscope.Nitric oxide (NO) production was assessed with DAF-FM DA and DAA fluorescent dyes as described earlier [4]. Each experiment was performed in triplicate. RESULTS: In the present study we studied the probiotic composition comprising of SPS and bacteria L. plantarum 8PA3. We used AFM to confirm effective fixation of the cells to carbohydrate. The compositions were found to swell quickly (~5 min) in aqueous solutions either containing amylase, or not. Tested starch formulations disintegrated during the first 5-10 min of incubation in amylase solutions whereas in amylase-free probes dissolution was less intensive (after ~30 min). Amylolysis of starch excipients was less pronounced in aqueous amylase solution than in SCF, supplemented with amylase. None of 19 studied Lactobacillus strains hydrolyzed SPS when growing on MRS agar supplemented with it. The amount of viable L. plantarum 8PA3 cells formulated with SPS was high and did not change when stored for 6 months at 4°C. The bacterial viability tests also demonstrated that after 6 h treatment with 2% bile or HCl (pH 2) L. plantarum 8PA3 exhibited increased sensitivity (viability 14% and 0.4%, respectively). However, in similar conditions no significant differences were noticed between bacterial viability obtained for formulated with starch and non-formulated bacteria. Furthermore, we showed that loading into SPS had no effect on bacterial production of nitric oxide (NO) - a pluripotent regulatory molecule in human organism. CONCLUSIONS: Overall, the results strongly support that formulation with polymeric matrices on the basis of SPS represent an appealing technology of probiotics production. It provides slow release of bacteria in target environment and does not alter their viability and NO biosynthesis. However, SPS excipient does not preserve the bacteria from harsh conditions of upper GIT. Therefore, we conclude that for oral administration the composition should be loaded in acid-resistant capsules.
RESUMO
An actual problem of analysis of probiotic lactobacilli resistance to antibiotics and other drugs used in the treatment of gastro-intestinal disturbances has been for the first time solved. The levels of resistance of 19 strains of Lactobacillus (14 strains of L. fermentum, 4 strains of L.plantarum and 1 strain of L.rhamnosus) isolated from commercial probiotics and sour milk products to 14 antibiotics of various nature, i.e. ß-lactams, aminoglycosides, macrolides, clindamycin, vancomycin, rifampicin, ciprofloxacin, tetracycline and chloramphenicol were determined. All the isolates were practically susceptible to the drugs of the first line antihelicobacterial therapy, i.e. amoxicillin and clarithromycin, that makes inexpedient the parallel use of the probiotics containing the above lactobacilli in the treatment of gastritis and gastric ulcer, despite the lactobacilli antagonism with respect to Helicobacter pylory. Lactobacilli are as well resistant to mesalazin and can be used for correction of dysbiosis in inflammatory affections of the intestine.