# AIP Bioluminescence for Rapid and Selective Detection of Bacteria and Yeasts in Wine
## 4 Discussion  
Differently from traditional methods used to detect microorganisms in wines, modern detection methods have focused on providing highly specific and rapid results capable of detecting microorganisms at very low populations [9]. Despite some successful applications of the ATP bioluminescence assay to wine samples [6; 18; 19], the method is not currently used in wineries mainly due to the lack of specificity. In the present study we developed a protocol, which coupled a two-filtration step with the bioluminescence assay, allowing for the simultaneous detection of yeasts and bacteria in wines. Filtration of liquid samples prior to the bioluminescence assay, formerly proposed by other authors [18; 19], to increase the concentration of microbial cells during analysis, eliminates compounds that quench light emission and stop bioluminescence inhibitors. However, one filtration step through a 0.45 um pore size filter would retain all microbial cells [18], hampering the possibility to discriminate between ATP from yeasts or bacteria. In the present study, double filtration (1.2 um first and 0.45 um afterward) was applied to artificially contaminated wine samples with different amounts of a mixture of yeast and bacteria. The effectiveness of filtration steps was confirmed by spread-plating the eluates crossing the 1.2 um filter on both YPDch and MRScy agar media selective for yeasts and bacteria, respectively. The absence of colonies on the YPDch agar plates confirmed that yeasts were retained on the 1.2 um filter, while the presence of colonies on the MRScy agar plates confirmed that bacteria crossed that filter. Spread-plating on selective media was used also to confirm the presence/absence of yeasts on 1.2 um filters and bacteria on 0.45 um filters as revealed by the ATP measurements. Samples that tested positive by the bioluminescence (>3 times blank values) gave colonies on the respective plates. Other authors [19] found good correlation between plate growth of cells and light output from the ATP bioluminescence assay. Two different microbial concentrations, varying the amount of LAB, were used to contaminate wines. In both cases (LAB 10\({}^{7}\) and 10\({}^{4}\)), ATP measurements were able to detect yeasts in all the tested wines, while bacterial contamination was detected in 8 samples out of 12. Several possible reasons may explain this result. After the first filtration step, 10\({}^{7}\) cells/mL of yeasts are retained on the 1.2 um filter. This could limit the number of bacterial cells able to cross the filter, due to internal pore fouling [20]. On the other hand, other authors have previously reported the effectiveness of serial filtration for separating yeast cells from bacteria in beer, observing that all bacteria filtered avoided entrapment on a 1.2 um filter and were successfully retained and cultivated on a 0.22 um filter [21]. More recently, it has been observed that some bacteria and yeast present in a viable but non-cultivable state could pass through a 0.45 um filter [11]. It is thus feasible that part of the bacteria inoculated in the wines could have entered in that state due to factors such as alcohol or filtration under vacuum [19], ending in the final eluate. On the other hand, stressful conditions could have led to cell death, hampering their detection both with ATP measurements and plating. RLU results were confirmed by plating the cultured resuscitation media on selective agar media for the samples, except one (R2), for which plating on MRScy did not reveal bacterial colonies at the lowest concentration. The limit of detection of the bioluminescence assay has been reported to be one cell per sample, similar to traditional plating [6]. However, it is known that culture-based assays may fail to detect viable but non-cultivable cells that still harbor measurable ATP [12].