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Application of FT-IR and flow cytometry to evaluate the effect of sodium chloride on probiotic bacteria
The aim of the study was to investigate the effect of varying sodium chloride concentrations on cell membrane, viability and proteolytic activity of probiotic bacteria. Reconstituted skim milk was inoculated with Lactobacillus acidophilus at varying salt concentrations (0 - 10% NaCl) and pH levels (4.0, 5.0 and 6.0) and ACE-inhibitory activity and proteolytic activity were determined. Additionally, the effects of NaCl reduction and its substitution with KCl on cell membrane of certain probiotic bacteria (Lb. acidophilus, Lb. casei and B. longum) and a pathogenic bacterium, Escherichia coli were investigated using Fourier transform infrared spectroscopy (FT-IR). A critical NaCl concentration that inhibited the growth of E. coli without significantly affecting the growth of probiotic bacteria was determined by monitoring cell growth and FT-IR spectra. To evaluate the effect of substitution of NaCl with KCl, substitution was carried out at critical total salt concentration at varying concentrations (0%, 25%, 50%, 75% and 100% KCl). Furthermore, the effects of varying NaCl concentrations on viability, membrane integrity and metabolic activity of these probiotic bacteria were studied using conventional technique and flow cytometry. The findings revealed that in Lb. acidophilus degree of proteolysis increased with higher salt concentration at pH 5.0 and 6.0 and ACE-inhibitory activity was highest at pH 5.0 at all salt concentrations. Fourier transform infrared spectroscopy results demonstrated significant shifts occurring in amide-I and amide-III regions when Lb. acidophilus was subjected to varying salt concentrations. Further, the conventional technique revealed that 2.5% was the critical level of NaCl to inhibit the growth of E. coli without significantly affecting the growth of most probiotic bacteria. The FT-IR analysis also highlighted the changes that occurred mainly in amide regions on increasing NaCl concentration from 2.5 to 3% in most bacteria. The findings suggest that 50% substitution of NaCl with KCl at 2.5% total salt could inhibit E. coli, without affecting the probiotic bacteria. Lastly, the observations from conventional culture technique were compared with the findings from flow cytometric analysis on metabolic activities of the cells and it was revealed that there was a correlation between culturability and dye extrusion ability of Lb. casei and B. longum. However, a certain population of Lb. acidophilus was viable as per the plate count method but the efflux activity was compromised. The metabolic activity of Lb. casei was found to be highest among the three probiotic bacteria.
Keywords: FTIR, Flow cytometry, probiotic bacteria