Characterization of bacteriocin-producing lactic asid bacteria (LAB) isolated from gastrointestine of clarias macrocephalus (broadhead catfish)

Abstract

The increase in demand of aquaculture products has led to intensive aquaculture; unfortunately, aquatic animals are often exposed to stressful conditions. Problems related to diseases and deterioration of environmental conditions often occurs, resulting in serious economic losses. In this study, the LAB strains from gastrointestinal of Broadhead catfish were identified and characterized by Gram staining and by other biochemical tests such as catalase, lactose utilization and API 50CHL (BioMerieux). Out of 50 isolates, 15 isolates from sample 1 and 11 isolates from sample 2 were found to be positive towards lactose utilization test and they were also identified as Gram positive cocci. Only 22 isolates showed negative reactions towards catalase test. Out of 22 isolates, only isolate A5 demonstrated a clear bactericidal effect against Bacillus cereus, Staphylococcus aureus and Salmonella thyphimurium, thus, this isolate was chosen for further characterization. PCR amplification of 16S rRNA sequence (given NCBI accession number, KP 064393) positively identified isolate A5 as a member of Lactococcus lactis with 100% DNA homology. Cell free supernatant fluid collected from Lactococcus lactis strain A5, when evaluated against Gram positive and Gram negative pathogens, showed inhibitory action against indicator microorganisms by using disc diffusion method. Bacteriocin was extracted using chloroform extraction method and complete inactivation of antimicrobial activity after treatment with Proteinase K confirmed the proteinaceous nature of bacteriocin extracted. Stability of the bacteriocin in the presence of catalase enzyme ruled out the possibility of antagonistic activity due to either hydrogen peroxide or organic acids. The bacteriocin extract was heat stable and has a molecular weight of 3.4 kDa based on SDS-PAGE analysis. The antibacterial activity was significantly increased by the addition of SDS, Triton X-100, Tween 20 and Tween 80. This probiotic strain, exclusively isolated from Malaysian catfish, produced an antimicrobial protein or bacteriocin which can be used as probiotic to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms in aquaculture feeding. Keywords: LAB, probiotic, bacteriocin, SDS-PAGE, Lactococcus lactis