Changes in pH are significant environmental stresses that may be encountered by lactobacilli during fermentation processes or passage through the gastrointestinal tract. Here, we report the cell response of Lactobacillus spp. isolated from traditionally fermented cabbage subjected to acid/alkaline treatments at pH 2.5, 7.4 and 8.1, which represented pH conditions of the gastrointestinal tract. Among six isolates, four species of Lactobacillus plantarum and two of Lactobacillus brevis were identified by fluorescence in situ hybridization (FISH). The fluorescence-based strategy of combining carboxyfluorescein diacetate (CFDA) and propidium iodine (PI) into a dual-staining assay was used together with epifluorescence microscopy (EFM) and flow cytometry (FCM) for viability assessment. The results showed that the cells maintained esterase activity and membrane integrity at pH 8.1 and 7.4. There was also no loss of culturability as shown by plate counts. In contrast, the majority of 2.5 pH-treated cells had a low extent of esterase activity, and experienced membrane perturbation. For these samples, an extensive loss of culturability was demonstrated. Comparison of the results of an in situ assessment with that of the conventional culturing method has revealed that although part of the stressed population was unable to grow on the growth media, it was deemed viable using a CFDA/PI assay. However, there was no significant change in the cell morphology among pH-treated lactobacilli populations. These analyses are expected to be useful in understanding the cell response of Lactobacillus strains to pH stress and may facilitate future investigation into functional and industrial aspects of this response.
Keywords: Epifluorescence microscopy; Flow cytometry; Fluorescence in situ hybridization; Lactobacillus spp.; Viability; pH conditions.
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