Evaluation of the antimicrobial activity of probiotic Lactobacillus casei cell-free supernatant extract against Escherichia coli ATCC 25922

Abstract

The antimicrobial activity of the members of genus Lactobacillus has been investigated over the years due to their ability to regulate the gastrointestinal tract microflora. The present study was conducted to evaluate the antimicrobial activity of this commercially utilized Lactobacillus strain. Toward this end, the study aimed to evaluate the antimicrobial activity of Lactobacillus casei against Escherichia coli ATCC 25922. Specifically, the effects of the cell-free supernatant extract to the hydrophobicity, growth and viability of E. coli in culture broth after exposure to different concentrations of the extract were investigated to determine whether the extract is bactericidal or bacteriostatic. The hydrogen peroxide and protein content was also measured to characterize and explain the antimicrobial activity of the cell-free extract.

The cell-free supernatant extract was obtained from L. casei Shirota strain in MRS broth using saturated ammonium sulphate precipitation and dialysis using cold phosphate buffer solution to obtain heat-stable peptides and proteins. The extract was prepared into different concentrations and mixed to a culture broth to evaluate the antimicrobial activity against E. coli using turbidimetric method with subsequent inoculation to agar media to check for microorganism viability. Protein determination and H₂O₂ quantification were performed using spectrophotometric methods. The change in hydrophobicity was determined using Microbial Adhesion to Hydrocarbon Assay (MATH assay). One-way Analysis of Variance with post hoc Tukey HSD test at α=0.01 was utilized for statistical analysis.

The cell-free supernatant extract produced by 5.62 x 10¹¹ CFU*mL^-1 contained 60.51µg*mL^-1 heat-stable proteins and 0.014 ± 0.002 µmol*mL^-1 H₂O₂. Generally, the addition of the extract to the culture broth inhibited the growth of E. coli in a dose-dependent fashion due to lower optical density but did not exhibit bactericidal effects, since the microorganisms are still viable after inoculation to a culture medium. A concentration of 100% (v/v) of the extract showed the highest reduction in the hydrophobicity and bacteriostatic activity against E. coli. Thus, the cell-free supernatant extract of L. casei Shirota strain exhibits bacteriostatic, not bactericidal activity against E. coli ATCC 25922 in vitro. The extract also reduces the hydrophobicity of E. coli in a dose-dependent manner, possibly through non-specific cell surface interactions. Hydrogen peroxide is unlikely to be a cause of the antimicrobial property of the cell-free supernatant extract due to its very low concentration in the extract.