Optimized Rapid Detection Conditions Using Carboxyfluorescein Diacetate Staining for Beer-Spoilage Lactic Acid Bacteria and Wild Yeast in Product Quality Control

In microbiological quality control of final beer products, traditional culture methods are widely used for their high sensitivity and ability to distinguish viable from dead cells. However, they require long cultivation periods. The microcolony method overcomes this limitation by retaining the advantages of culture methods while shortening the cultivation time. In this approach, microorganisms are captured on a membrane filter, briefly incubated on agar media for microcolony formation, and detected using fluorescence-based technology. This study aimed to establish a rapid and sensitive microcolony detection method for beer-spoilage lactic acid bacteria (LAB) and wild yeasts using the EZ-Fluo Rapid Detection System (EZ-Fluo system), which offers a cost advantage among available fluorescence-based detection systems. Because its detection sensitivity was initially insufficient, we optimized the experimental conditions and membrane filter selection. Detection of the slow-growing species Fructilactobacillus lindneri was improved using the membrane filter A045R047Z-P (Advantec Toyo). Furthermore, fluorescence reactions at 37 °C (LAB) and 30 °C (yeast) enabled sustained microcolony growth after detection, supporting visible colony formation and species identification. Under these optimized conditions, major beer-spoilage species were detected with recovery rates exceeding 100% after 72 h of incubation, compared with the 120 h required by traditional methods. In brewery trials, no false positives were observed in 30 beer products. Thus, the EZ-Fluo system, combined with optimized filter selection, can provide results within 3 days, compared with the standard 5-day culture methods, thereby accelerating fresh beer management and reducing warehousing costs.