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November 2018 WINES&VINES 81 PRACTICAL WINERY & VINEYARD WINEMAKING 2. Determine if conventional measure- ments of molecular AO SO 2 are fit for practical purposes. Experiment A red wine was created by adding anthocy- anin extract to a sterile-filtered white wine. This way, a comparison could be made be- tween a red and a white wine with varying anthocyanin content but otherwise identical composition. A range of SO 2 concentrations were added to 1-liter bottles of each wine, from 0 mg/L (the control) up to 128 mg/L. To test their antimicrobial activity, the wines were inoculated with a commonly used S. cerevisiae strain, EC1118. Samples were taken from the wines at intervals over 10 days to assess the survival and viability of the yeast, and to measure the free GDT , free AO , molecu- lar GDT and molecular AO SO 2 concentrations using HS-GDT and AO methods. Choice of measurement method is critical SO 2 analyses of wines agreed with previous results: molecular AO SO 2 concentrations in the red wine were significantly higher when measured by AO than by HS-GDT, but mea- surements in the white wine were similar between the two methods. (See Figure 3: Comparison of molecular SO 2 measurement methods.) For example, an addition of 49 mg/L SO 2 to the red wine resulted in a seem- ingly acceptable molecular AO SO 2 of close to 0.7 mg/L. However, based on the HS-GDT method, the molecular GDT SO 2 of this red wine was less than 0.05 mg/L. These results sup- port the hypothesis that anthocyanin-bound SO 2 complexes in red wines are responsible for the overestimation of true molecular SO 2 using conventional methods. Anthocyanin-bound SO 2 does not have antimicrobial activity Pairs of red and white wine with the same level of molecular GDT /molecular AO SO 2 as measured by each method were tested for yeast survivability (Figure 4). At low levels of molecular GDT /molecu- lar AO SO 2 (~0.2 mg/L), no effect on survivability was observed in either wine. In the white wine, a greater than 2-log reduction in survivability was observed when SO 2 measured by molecu- lar GDT and molecular AO was 0.6-0.7 mg/L. How- ever, in the red wine, a 2-log reduction in yeast survivability was observed for 0.69 mg/L mo- lecular GDT SO 2 , but no effect on survivability was observed for the red wine with a similar molecu- lar AO SO 2 value. These results indicate that an- thocyanin-bound SO 2 has little antimicrobial activity, because the only difference between molecular GDT and molecular AO values is that the latter includes anthocyanin-bound SO 2 . Conventional measurements overestimate free and molecular SO 2 in red wines and are not fit for practical purposes. All conventional approaches to SO 2 determina- tion (such as manual or automated Ripper ti- tration, AO or FIA) rely on an initial acidification step that will result in release of SO 2 from anthocyanin-bound SO 2 complexes. This will result in an overestimation of free SO 2 in red wines, and eventually molecular SO 2 , and thus an overestimation of antimicrobial activity against S. cerevisiae. Further work will be needed to determine if anthocyanin-bisul- fite complexes also lack activity against other micro-organisms, such as Brettanomyces yeast or spoilage bacteria like Pediococcus. The extent of overestimation will likely depend on the anthocyanin concentration, the presence of other binders, the pH and amount of SO 2 , and the calculation used to convert "Free AO" to molecular. As a result, there is not a straightforward linear relationship between the commonly measured molecular AO SO 2 and the molecular GDT SO 2 values. Practical considerations This work confirms previous studies that show standard approaches to SO 2 measurement can badly overestimate the true amount of free and molecular SO 2 in red wines. A reasonable re- sponse is, "Now what? If we have approxi- mately three-fold less molecular SO 2 than we think we do, does that mean we should be adding approximately three times more SO 2 ?" KEY POINTS In red wine, molecular SO 2 is greatly over- estimated by conventional measurement methods such as aeration-oxidation, the Ripper method and flow-injection analysis. Anthocyanin-bound SO 2 , which is incor- rectly measured as part of free and mo- lecular SO 2 with conventional methods, does not have antimicrobial activity. Head-space gas detection tubes can pro- vide a more accurate measurement of mo- lecular SO 2 and, hence, are a better predictor of antimicrobial activity. Results from head-space gas detection tube measurements may be most useful in indicating to a winemaker when to be cau- tious with a wine that is prone to microbial spoilage. Adding more SO 2 may not be practical when considering legal limits, color stability, aroma, etc. Figure 2: Head-Space Gas Detection Tubes apparatuses for measurement of molecular GDT SO 2 . A syringe is half-filled with wine, half with air. Following an equilibration period, the SO 2 in the head space is expelled through a colorimetric gas detection tube (GDT). Molecular AO or molecular GDT SO 2 (mg/L) Representative values of molecular GDT SO 2 and mo- lecular AO SO 2 (y-axis) following varying SO 2 additions to a white or red wine (x-axis). FIGURE 3: COMPARISON OF MOLECULAR SO 2 MEASUREMENT METHODS 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 White, 36 mg/L White, 62 mg/L Red, 24 mg/L Red, 49 mg/L n A-O n HS-GDT