Application of flow citomety in the monitoring populations of viable yeasts during alcoholic fermentations

LERENA M.C.; VARGAS A.S.; MERCADO L.; STURM M.E.; COMBINA M.

Bento Gonzalves

Congreso; 39º Congresso Mundial de la Viña y el Vino; 2016

Organizacion Internacional de la Vid y el Vino (OIV)

Alcoholic fermentation (AF) is a critical process for wine production and it is based on the conversion of the main grape sugars (glucose and fructose) in alcohol and carbon dioxide; a process carried on by yeasts belonging to the gender Saccharomyces, mainly S. cerevisiae. Specifically the knowledge of metabolic and physiological parameters of yeasts along the fermentative process provides information of great importance to predict the normal development of the process, avoiding possible problematic fermentations. Traditionally the monitoring of the physiological state of yeasts has been performed using methylene blue staining and manually counting live/dead yeast on a Neubauer chamber. However, even when this method has been widely used it can be time consuming and susceptible to operator variations. That is the reason why in this study we aimed to assess the viability of yeasts along AF through staining with propidium iodide (PI) following analysis with flow cytometry. We also aimed to apply this methodology in order to understand the effect of an abrupt downshift of the temperature applied in different moments of AF on yeasts viability. We performed fermentations at laboratory scale using synthetic must SM300 inoculated with S. cerevisiae T73. Cold shock induction was performed by placing Erlenmeyers in cold chamber at 8-10°C for 16 hours in different moments of AF (day 2, day 4, day 6, day 8 and day 10). Samples were obtained periodically in which yeast viability was analyzed through flow citometry, previously staining the samples with PI; and also with optic microscopy in a Neubauer chamber, previously staining the samples with methylene blue. We observed that using flow citometry to monitor AF proved to be very useful. Within minutes an accurate count was obtained, providing information such as cell size and complexity, and physiological state of cells that enabled a thorough evaluation of cell state at any time of fermentation. The total populations during fermentation increased until a stable platelet count with an increased percentage of non-viable cells at the end of the process. Counts number of yeast obtained in Neubauer chamber with methylene blue staining showed oscillation in time, with differences of up to 40 million cells/mL were observed between the counts obtained on successive days. This fact was not observed in the counts obtained by flow cytometry. Generally the counts obtained in Neubauer chamber were higher than those obtained by flow cytometry. Regarding the estimation of viability, both techniques showed similar percentages of non-viable cells at different stages of fermentation. Fluctuations in the evolution of counts observed in the data obtained with Neubauer chamber could be attributed to technical limitations such as the small volume of sample analyzed and consequently low number of cells evaluated. Furthermore, this technique presents a high error attributable to the operator since it has many stages of sample handling, and counting depends on subjectivity of the operator when differentiating stained and unstained cells. In contrast, flow cytometry allows the analysis a greater number of cells and an objective classification of living and dead cells according to the fluorescence detection. Thus, flow cytometry allows the evaluation of hundreds of thousands of cells in a few minutes, providing accurate and reliable information with respect to cell number and viability, after staining with IP.