| Summary: | While brewing probiotic beer using <i>Saccharomyces cerevisiae</i> var. <i>boulardii</i>, we noticed the yeast potentially makes biofilm in glass bottles as the bottles get hazy. In this study, <i>S. cerevisiae</i> var. <i>boulardii</i> CNCM I-745 was used as a starter culture to produce probiotic beer. We studied the biofilm parameters combined with <i>FLO11</i> mRNA expression and used light and scanning electron microscopy to document biofilm formation and structure. Our results revealed that ageing the beer and maturing from a sugar-rich to a sugar-limited beer, along with the stress factors from the brewing process (pH reduction and produced metabolites), led to an increase in biofilm mass; however, the viable count remained relatively stable (approximately 7.1 log<sub>10</sub> cells/mL). Biofilm <i>S. boulardii</i> cells showed significantly higher <i>FLO11</i> mRNA expression in the exponential and stationary phase compared to the planktonic cells. This study, therefore, provides evidence that <i>S. cerevisiae</i> var. <i>boulardii</i> makes biofilm on glass surfaces during beer bottle ageing. The impact of complications caused by formed biofilms on returnable bottles emphasizes the significance of this study.
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