Such evidence of therapeutic effects
Such evidence of therapeutic effects suggested that LAB and bifidobacteria could be used for inhibiting the growth as well as eradicating the biofilm of dermatological pathogen S. aureus. Thus, the aim of this study is to evaluate the antimicrobial activity of LAB and bifidobacteria, as well as the antimicrobial compounds produced in the extracellular extracts of these bacteria, against S. aureus.
Discussion LAB has been receiving significant attention lately due to their ability to exhibit an antagonistic effect on closely related bacteria and many other food-borne and dermatological pathogens. In this study, the extracellular extracts (CFS) of 90 strains of LAB and bifidobacteria were used for antimicrobial activity evaluation, as it was proven in a previous study that the extracellular fraction contained more inhibitives than the intracellular extracts. Five LAB strains, which showed a higher percentage of growth inhibition in screening, namely, L. bulgaricus FTDC 8611 (A30), W. cibaria BD 1514h (B7), L. fermentum BD 1512n (C5), L. fermentum BD 8913f (D10), and L. casei BD 1511a (E10), were selected for detailed studies. The primary antimicrobial effect exerted by LAB is due to the production of organic acids, which reduces the pH of the immediate environment, rendering it unsuitable for the growth of a broad range of Gram-positive bacteria. From the results (Figure 1) obtained, the deleterious effect of LAB on S. aureus was mainly due to the production of organic acids, as neutralization of CFS resulted in more than 50% drop in the percentage of inhibition from the initial level. Upon fermentation, the pH of the CFS was reduced to almost 4.0 for all strains (Table 2), which was unfavorable for the growth of S. aureus, as its survival requires the pH to be in the range 4.5–9.3. In addition, the lipophilic and undissociated nature of lactic and acetic acids allow the molecules to exhibit antibacterial action through the penetration of bacterial membrane. The higher pH environment in the bacterial cytoplasm will cause the acids to dissociate and interrupt the transport process in the nk1 receptor antagonist by disrupting the proton motive force. Bioactive peptides and/or bacteriocins are important antimicrobial metabolites produced by LAB, which are proteinaceous in nature. Such compounds inhibit specific microorganisms, particularly Gram-positive bacteria. In order to investigate whether the antimicrobial compounds that inhibited the growth of S. aureus were proteinaceous, the CFS of selected LAB was neutralized to eliminate the effects of acids and treated with protease. Proteolytic enzymes that degrade proteins present in the CFS would therefore render the proteinaceous antimicrobial compounds ineffective in exerting their bactericidal effect. Among the selected strains, the CFS of two strains, W. cibaria BD 1514h (B7) and L. fermentum BD 8913f (D10), showed a significant reduction (p < 0.05) in the inhibitive action compared to the neutralized CFS (Figure 1). The presence of proteinaceous antimicrobial compounds in these two strains was confirmed. In addition, the inhibitive action of the precipitated protein fractions of all selected strains was postulated to be a result of the concentration of bioactive peptide-like compounds through the salting out method. Many bioactive peptides of LAB were produced in small amounts; therefore, to effectively evaluate the antimicrobial activities of such peptides, one crucial step was to concentrate the CCFS with the ammonium sulfate precipitation method, which was employed in this study. Bioactive peptides including bacteriocins can easily form pores on the cytoplasmic membrane of sensitive cells and disrupt nucleic acids, subsequently leading to ion leakage, loss of proton motive force, and ultimately cell death. The characterization of CFS showed that the antimicrobial compounds produced by LAB, namely organic acids, hydrogen peroxide, and diacetyl, were strain dependent (Tables 2 and 3). L. bulgaricus FTDC 8611 (A30), the strongest of all these strains, produced a large amount (p < 0.05) of acetic acid and total acids compared to other strains (Table 2). Such data correlated well with the high percentage of inhibition exerted by this strain (Figure 1). The effect of acetic acid (pKa 4.74), although present in a small amount, is more lethal than that of lactic acid because the concentration of undissociated acetic acid is two to four times that of lactic acid at pH 4.0–4.6. The higher percentage (p < 0.05) of inhibition exhibited by L. bulgaricus FTDC 8611 (A30) was postulated to be a result of the synergistic effect between the overall antimicrobial compounds produced. Two or more inhibitory factors would result in an inhibitory action greater than either of the different factors (synergism). The antagonistic activity of diacetyl occurs through the blocking of enzyme\'s catalytic site responsible for arginine utilization, rendering the cells incapable of synthesizing essential proteins. Hydrogen peroxide oxidizes targeted bacterial membrane via peroxidation, leaving the cells with increased membrane permeability and denatured metabolic enzymes. The inhibition effect of bacteriocins was also found to increase efficiently via synergism between organic acids and bacteriocins.