2. Validation, Advantages and Limitations of the ‘Direct’ and ‘Indirect’ Impedance
Five red wine samples of a different vintage, with concentrations ranging from 103 and 107 cfu/mL, were analyzed. The enumeration of Brettanomyces in these samples was conducted using plate counting as a reference. Equations (2) and (3) were then used to calculate the ‘direct’ and ‘indirect’ impedance yeast concentration, respectively. A comparison between the three B. bruxellensis enumeration methods is presented in Figure 1. The ‘direct’ method was slightly more accurate with an average percentage error of 15.9%, compared to that of the ‘indirect’ method, which was 18.6% (Equation (1)). For samples with a Brettanomyces concentration above 104 cfu/mL, the ‘indirect’ method generally proved to be more accurate, with an average percentage error of 8.2% compared to that of the ‘direct’ method (13.2%). Contrastingly, the ‘direct’ method was more accurate at concentrations below 104 cfu/mL, with an average percentage error of 19.8% compared to 34.2% for the ‘indirect’ method. As expected, samples with a low yeast concentration are more difficult to quantify and thus presented results with higher variability. These results are in agreement with the results described earlier.
Figure 1. Enumeration of Brettanomyces bruxellensis contamination in wine: a comparison between plate counting and impedance technology; N is the concentration of B. bruxellensis in red wine (cfu/mL); different letters indicate that the average Log N (logarithm of yeast concentration) are significantly different according to Tukey’s test (p < 0.05).
One drawback of plate counting is the prolonged incubation periods of 120 h (5 days) required for observing Brettanomyces colonies on the plates. Using the ‘indirect’ impedance, incubation times are reduced to 0.8 to 58 h. The reduced incubation time could be explained by the accelerated yeast proliferation rate in liquid broth compared to semi-solid agar medium, conventionally used for plate counting. Moreover, the principle of detection is different in the semi-solid agar medium contained in plates, where every cell is counted by itself, and in the liquid broth, where all cells grow together. Avoiding prolonged incubation times also decreases the chance of sample contamination during the enumeration process. Accelerated detection and enumeration times are especially important for Brettanomyces in connection with the wine industry. This would enable faster implementation of preventative actions in the wine industry, such as the addition of more SO2 and cleaning of equipment, to reduce the negative effects of Brettanomyces contamination and hence wine quality degradation. The risk of large-scale wine dumping due to Brettanomyces growth would also be reduced, in turn reducing economic losses.
One other important aspect is that standard plate counting is more labor intensive and possibly more time consuming, due to the multiple operations required for sample preparation. For the ‘direct’ and ‘indirect’ impedance methods, the sample preparation time can be significantly reduced from approximately 1 h (plate counting) to less than 5 min (impedance methods) per sample. Therefore, impedance technology has the potential to increase the throughput of enumeration analyses thereby providing faster responses to wine producers. Lastly, even though the initial capital and material costs for the impedance technology is higher, this is offset by the reduction of economic risk, labor and turnaround time of samples analyzed.
It was focused on wine samples inoculated only with
B. bruxellensis. In addition, only exclusively fresh cultures of
B. bruxellensis were used in the experiments. Since the age and viability of yeasts (and other microorganisms) are different in real wines, future research on the enumeration of
B. bruxellensis in unfiltered wines should be conducted, including the identification of the impact of the age/condition of the yeast cells. Unfiltered wines contain the main fermenting organism and contaminant microorganisms (for example, other yeasts and lactic acid bacteria) that can interfere with impedance detection. This could involve pre-treating the wine samples before analysis or adding antimicrobial agents to the detection media directly. Cycloheximide, to which
Brettanomyces is known to be highly resistant, has been used to isolate this yeast from real wine samples, as
S. cerevisiae’s (the predominant fermenting yeast) is susceptible to this antimicrobial additive
[19].