# AIP Bioluminescence for Rapid and Selective Detection of Bacteria and Yeasts in Wine
## 4 Discussion
The sensibility of the test was confirmed on a second trial, when wine samples were contaminated with the maximum tolerable microbial concentration to consider those wines shelf-stable (50 CFU/L of yeasts and 1000 CFU/L of bacteria), according to producers' indications. Through the developed method, both bacteria and yeasts were detected in all the tested wine samples despite the very low concentration. The filtration steps involved in the protocol are in fact able to collect all the cells present in the total filtered volume of sample. Due to the very low number of yeasts on the first filter, pore fouling is unlikely, resulting in the passage of all the bacteria through the filter [21], which were detected after resuscitation. On the other hand, the analysis of artificially inoculated vermouth samples detected only yeasts. This could be due to the bacteria's lower tolerance to alcohol [13],which is highly concentrated in vermouth, or to the presence of antimicrobials. In fact, vermouth is described as a fortified sweetened wine with added herbs and spices that are known to have anti-microbial characteristics, ascribed to essential oils [22]. These conditions could have caused bacteria death or damage, resulting in the absence of ATP produced by those cells. On the contrary, yeasts can survive for more than 30 h at 20% (\(v/v\)) ethanol concentration [23], and in the presence of high sugar and essential oil concentrations [24]. Thus, ATP from yeast cells could still be measured in vermouth. Finally, some inhibitory substances may be present in the vermouth samples, influencing the luciferase activity [18]. However, filters were rinsed to flush the compounds that would affect the luminescence and interfere with microbial resuscitation [19]. Better results were obtained in the third trial, when both bacteria and yeasts were detected in one vermouth sample. This could be ascribed to the use of a different resuscitation medium (REScy instead of MRScy) with an increased recovering capacity, which could have been able to favor the repair of cell damage, resulting in the production of measurable ATP. This is in agreement with other authors who reported that the culture medium for bacterial resuscitation could affect the luciferase reaction in terms of signal level and signal stability [19]. All the conducted trials allowed the detection of microbial contamination in wine samples in 24 h (including incubation for cell resuscitation). This was in accordance with [PERSON] and colleagues [6], who detected the slowest growing strains in wine after 24 h incubation on a selective medium, instead of 6 days with the standard filtration method applied in wine quality control. Compared with other techniques, ATP has been indicated as the best choice in food and beverage analyses when decisions must be taken in the shortest time, as it is simple, easy to use by food microbiology laboratory staff, and has little requirement for laboratory facilities, allowing the rapid estimation of very low levels of cells [25]. Furthermore, with the presently proposed protocol, one of the major drawbacks of the bioluminescence assay, i.e., the lack of specificity, was overcome [7]. In conclusion, ATP bioluminescence, coupled with serial filtration and the use of selective media for cell resuscitation, can be an efficient system for the rapid and simultaneous detection of bacteria and yeasts, in different types of wines. This would be of great interest for industries, for which an early detection of both yeasts and bacteria would help in the decision-making process. Conceptualization, J.R. and B.B. ; Data curation, S.M., E.B., V.C., S.W. and A.J. ; Formal analysis, S.M. and A.J. ; Funding acquisition, J.R.; Investigation, S.M. and A.J. ; Methodology, S.M., A.J. and B.B. ; Project administration, J.R. and B.B. ; Resources, J.R. and S.W. ; Supervision, J.R. and B.B. ; Validation, S.W. and A.J. ; Visualization, S.M., E.B. and B.B. ; Writing--original draft, S.M., E.B., V.C. and B.B. ; Writing--review and editing, S.M., E.B., V.C., J.R., S.W., A.J. and B.B.