Use of Flow Cytometry To Follow the Physiological States of Microorganisms in Cider Fermentation Processes

Abstract

Analysis of microbiological samples from fermentation processes in the beverage industry (beer, wine, and cider) by traditional indirect, culture-based standard methods is timeconsuming, and the methods do not produce direct information about the physiological state of the microorganisms. Moreover, the plate-counting method detects only cells able to form colonies under the conditions of the medium that is used, ignoring the presence of cells that do not form colonies but are nevertheless metabolically active (6). As is known, standard laboratory culture media rarely resemble natural environmental conditions (32). The application of flow cytometry (FC) with fluorescent dyes is faster and more direct and has made it possible to distinguish stages beyond the classical definition of viability generally demonstrated by culturing (24). FC can be used to quantitatively measure the optical characteristics of cells as they pass, in single file, into a focused beam of light (for a review, see reference 33). As particles pass through the beam, three parameters are measured: forward light scatter, side angle light scatter, and fluorescence at selected wavelengths. Light scatter is related to cell mass, structure, surface properties, and the optical density of the internal medium. A variety of fluorescent dyes may be used to detect structural or functional cellular properties. In this way, different cellular functions, such as reproductive growth, metabolic activity, and membrane integrity, can be detected, allowing a definition of the term viable-but-not-culturable cells (VBNC) (24) (other authors have called them active but nonculturable). Previous studies have reported that there are two major adaptations that cells undergo during the formation of VBNC states: cell wall toughening and DNA condensation (29). The detection of metabolic activity provides presumptive evidence of reproductive growth by the demonstration of enzyme activity, such as esterases, along with membrane integrity. In the absence of metabolic activity, it is still possible to determine membrane integrity by either dye retention or dye exclusion, the latter using propidium iodide (PI). Cells without an intact membrane cannot maintain or generate the electrochemical gradient that generates the membrane potential and can be considered dead cells (24). The use of FC techniques was reported for the "at-line" study of Escherichia coli fermentations (14), which detected a considerable drop in cell viability (about 20%) during the latter stages of small-scale (5-liter), well-mixed fedbatch fermentations.Flow-cytometric applications using different dyes have been compared to standard methods to assess yeast cultures in baking, wine making, cider making, and brewing (1,5,7,8,10,11,17,19). The technique has also been used to study the membrane integrity of ethanol-stressed Oenococcus oeni cells (9). Most studies of FC applications have focused mainly on microorganims as pure cultures, and only a small number have analyzed mixed populations over time (30).The purp...

Quantitative Approach to Determining the Contribution of Viable-but-Nonculturable Subpopulations to Malolactic Fermentation Processes

“…A subpopulation of VBNC cells (calculated as the difference between metabolically active and viable cells) was found in both controls (Fig. 1a and d), as was detected previously under stress conditions during cider MLF (11). The use of sodium bisulfite caused a drop in cell viability even to the extent of a total absence (Fig.…”

“…MLF was carried out under different SO 2 concentrations (0, 30, and 60 ppm total) for inducing VBNC states. The fermentation medium was sterile apple must or "green" cider (obtained just after alcoholic fermentation and containing 5.6% [vol/vol] ethanol), obtained as previously described (11). Sodium bisulfite was used for SO 2 treatments.…”

Application of Flow Cytometry to Segregated Kinetic Modeling Based on the Physiological States of Microorganisms

“…It should be highlighted that the principle and cell numbers considered by each technique are different. After acid challenge of Streptococcus macedonicus (41), a heterogeneity within the bacterial population was observed, which was otherwise undetermined by the (20). As shown in Fig.…”

“…Aliquots for the same cell suspensions were stained with DRAQ5. In previous work (20) sonication of malolactic bacterium samples was tested (15 and 30 s) to avoid problems associated with cell aggregation. Results obtained led us to consider than the standard two phosphate-buffered saline washes performed, including vortexing, were enough to avoid aggregation.…”

Growth Promotion of Pepper Plants by Pantoea ananatis B1-9 and its Efficient Endophytic Colonization Capacity in Plant Tissues

“…However, DRAQ5™ was used as a fluorescent dye to stain the nuclear DNA of root hairs in Arabidopsis plants and to examine the physiological conditions of microorganisms during fermentation processes (Da Costa et al, 2006;Herrero et al, 2006). As compared with other fluorescent DNA dyes, it has many advantages, such as a low level of photobleaching and a wide range of red excitation/emission ranges that is compatible with other fluorescent proteins including GFP, cyan fluorescent protein, and yellow fluorescent protein (Martin et al, 2005).…”