Characterization of the growth and flavor potential of strains of Oenococcus oeni in the presence of Saccharomyces cerevisiae in Real Time during winemaking

AVF Proj. ID: 281 / /Cat.: ,
Primary Investigator(s): Roy J. Thornton, Susan B. Rodriguez, Kenneth C. Fugelsang, Barry H. Gumpby

The overarching objective for this study was the determination of viable cell
concentrations of strains of Oenococcus oeni as influenced by inoculation time and yeast
strain in fermenting must. The first step of this research is to conduct ?real-time?
quantification and viability determinations of Oenococcus oeni and wine yeasts in wine
during and after the alcoholic fermentation. These data will assist in examining the
relationship, e.g., synergism, antagonism, neutrality, between different strains of wine
yeast and Oenococcus oeni. Traditional quantification of Oenococcus oeni in wine by
plating takes approximately a week. If the malolactic fermentation (MLF) is stuck, reinoculation
may be needed. Delaying this re-inoculation by a week is detrimental to wine
quality.

The polyclonal antibody/rabbit serum used in the 2003 research was purified and labeled
with a fluorescent compound. The improved antibody has eliminated the need for the
secondary fluorescent antibody and, thus, decreased the expense and the sample
preparation time. A flow cytometric assay of wine samples treated with the new antibody
and propidium iodide, a viability stain, has allowed the determination of the
concentration and viability of O. oeni in the wine, even in the presence of > 108 yeast
cells/mL, in approximately 30 min.

O. oeni cells were separated from debris and yeast cells using light scatter plots. The
Oenococcus population was ?gated?. In a second plot of cell fluorescence, the ?gated?
population was separated into dead, live and ?intermediate? O. oeni cell populations. The
two plots were saved in a template, so that when the new sample was ready, it could be
run and analyzed on the flow cytometer in 1-2 min.

O. oeni cells were separated from debris and yeast cells using light scatter plots. The
Oenococcus population was ?gated?. In a second plot of cell fluorescence, the ?gated?
population was separated into dead, live and ?intermediate? O. oeni cell populations. The
two plots were saved in a template, so that when the new sample was ready, it could be
run and analyzed on the flow cytometer in 1-2 min.

Data analysis indicates that the two Oenococcus oeni strains used ferment malic acid at
different rates. Furthermore, with simultaneous inoculation of wine yeast and bacteria,
the malic acid fermentation rates are significantly influenced by the specific strain of
wine yeast present.