Magdalena Olszewska

Patterns of survival and volatile metabolites of selected Lactobacillus strains during long-term incubation in milk

The focus of this study was to monitor the survival of populations and the volatile compound profiles of selected Lactobacillus strains during long-term incubation in milk. The enumeration of cells was determined by both the Direct Epifluorescent Filter Technique using carboxyfluorescein diacetate (CFDA) staining and the plate method. Volatile compounds were analysed by the gas-chromatography technique. All strains exhibited good survival in cultured milks, but Lactobacillus crispatus L800 was the only strain with comparable growth and viability in milk, assessed by plate and epifluorescence methods. The significant differences in cell numbers between plate and microscopic counts were obtained for L. acidophilus strains. The investigated strains exhibited different metabolic profiles. Depending on the strain used, 3 to 8 compounds were produced. The strains produced significantly higher concentrations of acetic acid, compared to other volatiles. Lactobacillus strains differed from one another in number and contents of the volatile compounds.

Utilization of physiological and taxonomic fluorescent probes to study Lactobacilli cells and response to pH challenge

pH stress is recognized as an important feature for Lactobacillus in relation to lifestyle and commercial utility. Hence, this study aims to investigate the cell function of Lactobacilli cells subjected to pHs between 7.0 and 2.0. For this purpose, the Lactobacilli isolates of vegetable origin were first hybridized with fluorescent oligonucleotide rRNA probes for detecting Lactobacillus species. Then, cells were exposed to pH stress and labelled with fluorescent probes, carboxyfluorescein diacetate (CFDA) and propidium iodine (PI), which provided the insight into esterase activity and membrane integrity of cells. Among isolates, fluorescence in situ hybridization (FISH) enabled us to specifically detect L. plantarum and L. brevis. Interestingly, FCM analysis revealed that at pHs between 7.0 and 4.0 the cell membrane was intact, while after the exposure at pH 3.0, and 2.0 became perturbed or impaired. Finally, L. brevis and L. plantarum differed from each other in fluorescence labeling behaviour and culturability. However, the results showed that the same standard protocol for labeling enables discrimination of subpopulations of tested species. Depending on the species, the substantial culturability loss was observed at pH 3.0 and 2.0. These results suggest that the taxonomic and physiological fluorescent probes could be suitable for in situ detection of specific bacteria and rapid assessment of the physiological status of cells.

Biofilm formation by Pseudomonas aeruginosa and disinfectant susceptibility of planktonic and biofilm cells

Olszewska M.A., Kocot A.M., Stanowicka A., Laniewska-Trokenheim Ł. (2016): Biofilm formation by Pseudomonas aeruginosa and disinfectant susceptibility of planktonic and biofilm cells. Czech J. Food Sci., 34. Epifluorescence microscopy (EFM) was used to study the biofilm formation of Pseudomonas aeruginosa after 6, 24, 30, 48, 54, 72, 78, and 96 h growth in a chamber slide system. For this purpose, the biofilm was stained with the Live/Dead BacLight, wherein live and dead cells were visualised based on the cell membrane integrity. With the use of EFM we described 8of 9-stage biofilm characteristics after 78 h of growth, since the majority of microscopic fields were fully covered with attached cells. However, the 96-h growth resulted in the cell detachment and revealed 30% of dead cells of all those cells that remained on the surface. The susceptibility testing of planktonic and biofilm cells to two disinfectants, chlorine-based and quaternary ammonium compound-based, revealed that biofilm cells were more tolerant to a chlorine-based sanitiser than planktonic counterparts. P. aeruginosa was inhibited by lower concentrations of the quaternary ammonium compound-based sanitiser than the chlorine-based sanitiser, which on the other hand was more effective in cell inactivation, as both the MIC/MBC (inhibitory/bactericidal) measurement and the CFDA/PI (carboxyfluorescein diacetate/propidium iodide) staining indicated.

Physiological functions at single-cell level of Lactobacillus spp. isolated from traditionally fermented cabbage in response to different pH conditions

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.

Seasonal variances in bacterial microbiota and volatile organic compounds in raw milk

The aim of this study was to define the composition of microbiota and the volatile organic compounds (VOCs) in samples of raw milk collected for 22 months between 2012 and 2014 originated from north-eastern region of Poland. The results revealed that the VOCs profile changed with respect to the season of milk collection, and milk collected in autumn was characterized by a higher content of acetic acid (C2), propionic acid (C3) and valeric acid (C5), whereas spring was characterized by a frequent presence of acetone (Ac), ethanol (Et) and ethyl acetate (EtAc). Bacterial species composition changed considerably within the tested period and some bacterial species/groups occurred seasonally, e.g. L. helveticus (summer), L. casei (winter). The results show usefulness of the applied techniques (PCR-DGGE and HS-GC) and data analysis (PCA, correlation coefficients) methods in characterizing the raw milk quality intended for dairy production.