# AIP Bioluminescence for Rapid and Selective Detection of Bacteria and Yeasts in Wine
## 2 Materials and Methods
### Bacterial Strains and Deliberate Wine Contamination  
For the contamination of samples, two strains of _Saccharomyces cerevisiae_ were used, one (SC1) belonging to the microbial collection of the Department of Food and Drug of the University of Parma and one (Premium Prosecco, Vason\({}^{\circledR}\), Verona, Italy) belonging to the microbial collection of G.I.V. (Gruppo Italiano Vini, Calmasino di Bardolino, Verona, Italy). These two strains were used together in a 1:1 mix of the respective overnight cultures to simulate yeast contamination in wine samples. On the other hand, to simulate contamination by LAB, of relevance for malolactic fermentation, one _Pediococcus pentosaceus_ (Pp 2089) strain, belonging to the microbial collection of the Department of Food and Drug of the University of Parma, was used.  
Bacteria and yeast were stored at \(-80\)\({}^{\circ}\)C, in MRS (De Man Rogosa Sharpe, Oxoid, Milan) with 12.5% of glycerol for bacteria, and YPD (AMRESCO LLC, Ohio) with 12.5% of glycerol for yeasts. Cultures were then revitalized inoculating 200 uL of thawed cultures in 6 mL of sterile MRS (De Man Rogosa Sharpe, Oxoid, Milan, Italy) for LAB, and YPD for yeasts. Both media were incubated for 24 h at 30 \({}^{\circ}\)C in aerobic conditions. Overnight cultures were microscopically counted on glass counters to verify the microbial cell load (data not shown). Overnight growth showed an average concentration of \(10^{7}\) CFU/mL for yeasts and \(10^{9}\) CFU/mL for Pp 2089.  
Decimal dilutions from the overnight cultures were prepared in Ringer's solution (Oxoid, Milan) for each strain, to achieve the concentration to be used for the inoculum.  
In a first trial, twelve wines (R1-6; W1-6) were inoculated with 2 different cell concentrations: (i) \(10^{4}\) CFU/mL yeasts and \(10^{7}\) CFU/mL LAB and (ii) \(10^{4}\) CFU/mL yeasts and \(10^{3}\) CFU/mL LAB. These inocula were chosen to mimic microbial populations possibly present in wines [10; 14; 15].  
In a second trial, 3 wines (W7-8, R7) and 2 vermouths (V1-2) were inoculated with 1000 CFU/L LAB and 50 CFU/L yeasts, indicated by the producers as the maximum microbial concentration tolerated to consider the wines stable during shelf life. The wines for the second trial were chosen as representative cases among the product indicated by the producers as the potentially most susceptible to microbial spoilage.  
The same samples, 3 wines (W7-8, R7) and 2 vermouths (V1-2), were inoculated with the same microbial concentration of the second trial and analyzed in a third trial, with modified cells in resuscitation conditions.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Wine Samples** & **Sparkling** & **Alcohol (\%)** & **Sugar (g/L)** \\ \hline R1 & No & 13.00 & 3.7 \\ R2 & No & 13.00 & 1 \\ R3 & No & 13.00 & 3.1 \\ R4 & No & 6.00 & 125 \\ R5 & Yes & 6.00 & 110 \\ R6 & Yes & 12.00 & 15 \\ W1 & No & 12.00 & 3.5 \\ W2 & No & 13.00 & 23.9 \\ W3 & Yes & 5.00 & 125 \\ W4 & Yes & 11.00 & 13 \\ W5 & Yes & 6.00 & 110 \\ W6 & Yes & 12.00 & 5 \\ W7 & Yes & 8.00 & 80 \\ W8 & Yes & 12.00 & 10 \\ R7 & Yes & 12.00 & 10 \\ V1 & No & 15.00 & 152 \\ V2 & No & 15.00 & 168 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Characteristics of analyzed wines; “W” is for white wines, “R” for the red wines and “V” for vermouth.