Characterization of Antimicrobial Compounds Producing Lactic Acid Bacteria from Domestic Mammals and their Application as Probiotic or Food Preservative

Abstract

Foodborne pathogens are a major threat to human health and the food industry. These bacteria cause spoilage of food and diseases on consumption. Moreover, the emergence of antibiotic resistance in clinically important strains of bacteria has led to the search for alternative solutions. Lactic Acid Bacteria (LAB) are well known for their antagonistic potential against closely related microbial species. The antagonistic activity is strain specific. LAB and their inhibitory compounds are gaining attention as they are declared GRAS status by FDA and have been utilized as probiotics and substitute of food preservatives. LAB are cosmopolitan and are found in dairy fermented products, soil and intestine. Previous studies were based on their isolation from dairy products but intestinal microbiota of domestic mammals is a less explored area. Our current study was focused on isolation and characterization of LAB strain from intestinal microbiota with antagonistic activity against important clinical and foodborne pathogens. The probiotic properties of LAB strains were also analyzed. We explored their metabolites as food preservative and determine physiochemical characteristics of the active component. Moreover, we also tried to partially purify the active component and determine its influence on the cell wall of pathogen and detection of the bacteriocin encoding gene in one of the potent strain. Finally prophylactic potential of most prominent strain using Listeria monocytogenes infection model in mouse. A total of sixty three (63) strains were isolated from the intestine of 7 domestic mammals. Out of which 48 trains presented acid production property and were considered lactic acid bacteria. Out of 48 only 22 strains could exhibit antimicrobial activity against both gram positive and negative bacteria. The indicator strains used in the study include Staphylococcus aureus, Clostridium perfringens, Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis. Six strains were selected on the basis of high antagonistic potential presented against at least one gram positive and one gram negative target strain used in the study. The selected strains were identified on the basis of 16S rRNA gene sequencing and accession number was obtained from NCBI Genebank. They are identified as Lactobacillus brevis MF179529, Enterococcus ratti MF183967, Enterococcus sp. MH734728, () Enterococcus sp. MH734729, Enterococcus durans MF179526, Pediococcus sp. MH734727. These strains were initially named as H, R, A, C, O, P respectively. Probiotic potential of the strains was evaluated on the basis of tolerance to NaCl (1-8%), bile salt (0.5-2%), pH (2-6), temperatures (35-50%), antibiotic resistance and non-hemolytic nature towards RBCs. Our results indicated that 6 strains had probiotic potential and were safe for utilization as food preservative. Among all strain Lactobacillus brevis MF179529 exhibited higher probiotic potential being resistant to grow at pH2, 50°C temperature, 3% bile salt and non-hemolytic nature. Furthermore, it exhibited 85% hydrophobicity and 69% antibiotic sensitivity making it the most promising probiotic candidate. To analyze the potential of the active component of CFS (Cell Free Supernatant) of selected strain against selected pathogen tests were performed. The comparison of growth kinetics of foodborne pathogens (L. monocytogenes, C. perfringens, S. enteritidis and E. coli) in the presence of 20% CFS of our six LAB strains showed inhibition of pathogen in food products. Our LAB strains produced active metabolites in growth medium and their CFS was effective against selected foodborne pathogens and is recommended for further studies for its use as food preservative. Inhibitory activity among strains varied but CFS of Enterococcus ratti MF183967 was appeared to be more active against L. monocytogenes. The oral administration of Lactobacillus brevis MF179529 provides the protective effect to the Listeria monocytogenes infected in mice. L. brevis MF179529 reduces L. monocytogenes load in liver, spleen and intestine and also maintain intestinal microbial equilibrium. Further studies are needed to best understand the mechanisms by which this microorganism promotes resistance against infectious diseases. Chemical nature of CFS was determined by treatment with enzymes, surfactant, pH and temperatures. The results indicate the proteinaceous nature of the active compounds as their activity diminished after treatment with protinase K and pepsin. CFS of all strain remained active at pH4-10, their inhibitory activity remained stable up to 100°C. SDS-PAGE analysis of the partially purified fraction of CFS of E. ratti MF183967 indicated the presence of two peptide bands of 20 and 30KDa, out of which 20KDa showed antimicrobial activity against Methicillin resistant Staphylococcus aureus (MRSA). Mode of action of the active component of E. ratti on the cell wall of MRSA revealed its rupturing that was observed under scanning electron microscope (SEM). Presence of enterocin coding genes entp and entL50A also strengthen the production of enterocin like inhibitory substance, by E. ratti MF183967 and that is applicable in food preservation. It is concluded that our six strains possess high inhibitory potential against spoilage and clinical pathogens. E. ratti MF183967 produce 20KDa peptide which is responsible for antimicrobial activity against MRSA. Presence of enterocin coding genes entp and entL50A also strengthen the production of enterocin like inhibitory substance, by E. ratti MF183967. In addition, L. brevis MF179529 ingestion has health promoting capacities and a good candidate to be employed as a probiotic against listeria infection.