Isolation and characterization of potential probiotic lactic acid bacteria isolated from cow milk and milk products

Abstract

Probiotics refer to the living microorganisms that exhibit beneficial effect on the health of human by the intestinal microbial balance. Most widely used probiotics are lactic acid bacterial group found in milk and milk products. This study was aimed to characterize the probiotic properties of lactic acid bacterial (LAB) strains isolated from cheddar cheese, yoghurt and cow milk. Bacterial strains naturally grown in milk, cheddar cheese and yoghurt were isolated using De Mann Rogosa Sharpe (MRS) agar medium and incubated at 37oC for 48 h separately under aerobic and anaerobic conditions. There were eight strains grown under aerobic conditions and they were isolated, purified and characterization was done based on the morphological and biochemical analysis such as gram staining, catalase test and motility test. All the eight isolates were either rod or cocci shaped, gram positive, catalase negative, non-motile and non-spore formers. These eight isolated strains were identified as lactic acid bacteria. When screening of the 8 isolates was done to determine their antimicrobial activities against five human pathogenic strains such as E.coli, Klebsiella pneumoniae, Pseudomonas eaeruginosa, Salmonella sp and Staphylococcus aureus, three isolates (M6, C1 and Y1) showed wide spectrum antimicrobial activity. To determine the probiotic properties of these three isolates, different tests such as tolerance to acid, NaCl and bile, lactose utilization and antibiotic resistance were done. Though all the three isolates showed resistance to stomach pH (pH 3.0), the strain C1 showed significantly higher tolerance to stomach pH than the other strains. Though the three isolates grew well in the presence of NaCl and 0.3% bile, the isolate M6 showed significantly higher growth with NaCl and 0.3% bile than the others. Even though all the three isolates had the capacity of utilizing lactose, the isolate M6 showed prominent colour change in the lactose utilization test than the other two strains. When antibiotic susceptibility of the isolated LABs were evaluated using four antibiotics such as Ampicillin, Streptomycin, Bacitracin and Gentamycin, the strain M6 showed significantly higher resistance to Ampicillin and Bacitracin but sensitive to Streptomycin and Gentamycin. Other two isolates (C1 and Y1) were sensitive to all the four antibiotics used. Based on the antibiotic sensitivity tests and the analysis of probiotic properties, the isolated strain M6 was confirmed as a potential probiotic lactic acid bacterium. This strain was identified as Lactobacillus plantarum strain CIP 103151 through16S rDNA sequence analysis.