G. Potter (1), C. Stanton (1), R. A. SPEERS (1); (1) ICBD-HWU, Edinburgh, U.K.

Poster

Zymolectins, glycoprotein receptors on the yeast cell surface, are believed to be one of the predominant factors governing brewing yeast flocculation due to binding that occurs with carbohydrate ligands on adjacent cells. Questions still remain about the regulation of zymolectin activity over the course of beer fermentations. Past researchers have used numerous methods to monitor zymolectin activity, including spectrofluorometry/Langmuir analysis, immunofluorescence staining, and molecular biological techniques. Based on these studies, some brewing scientists have concluded zymolectin activity is genetically induced, while others believe it is constitutive. This study sought to address this lack of consensus using two new approaches: 1) an assay that measures zymolectin concentrations during lab-scale fermentations with a flow cytometer and a FITC-avidin fluorescent probe; and 2) a statistical technique to detect sub-populations within histograms prepared from flow cytometry data. It was hypothesized that flow cytometer techniques would generate data that would facilitate the detection of sub-populations of cells with different levels of zymolectins, thus supporting the finding that zymolectin activity is genetically induced. During the lab-scale fermentations, Langmuir analysis of the flow cytometer fluorescence measurements revealed that mean zymolectin levels initially decreased then later increased over the course of the fermentations. Additionally, histograms constructed from the fluorescence data suggested the flow cytometer was able to detect sub-populations of cells with more and less zymolectins. Large amounts of data were also gathered that gave information on cell size (measured as forward scatter) and cellular complexity (noted as side scatter) because of the capacity of flow cytometry for multiparametric analysis. Statistical analyses of the forward scatter measurements using deconvolution software identified three sub-populations of cells present during the fermentations. After corroboration with conventional microscopy, these sub-populations were presumed to represent daughter cells, mother cells, and mother cells with buds. Over the course of the fermentations, presumed sub-populations of daughter cells increased and presumed sub-populations of mother cells with buds decreased, implying cell size tended toward uniformity. Side scatter measurements (which reflect cell granularity) were not able to detect these cell sub-populations. These initial results suggest this new flow cytometric assay has more specificity and sensitivity then past methods that attempted to study the presence of zymolectin on the yeast cell. Repeated trials are required to verify some of the assumptions made herein, but it is believed this assay has the potential to answer many questions about the finer details of zymolectin activity regulation. Future work plans to extract more pertinent information from the large data sets with improved gating methods based on cell size and age.

Alex Speers is a professor and the director of the International Centre of Brewing and Distilling at Heriot-Watt University, Edinburgh, Scotland. Previously he was a professor in the Food Science program at Dalhousie University, Halifax, NS, Canada. Born in Creston, BC, Canada, he gained B.S. (Agr.), M.S., and Ph.D. degrees at UBC in Vancouver, BC, Canada. In the past, Alex has been employed in the Quality Assurance Departments of both Labatt and Molson Breweries. His current research interests include various aspects of the brewing and distilling process, including fermentability, yeast flocculation, fermentation modeling, extract calculations, and the properties of (and problems created by) barley malt. He has organized, presented, or participated in brewing events in the United States, Australia, Canada, China, and Ireland. Alex has spent sabbaticals at CUB/Fosters in Melbourne, Australia, and the Columbia Brewing Company in Creston. He is a past chair of the editorial board of the MBAA Technical Quarterly. Alex belongs to several professional societies and is a member of the editorial boards of the ASBC Journal, JIB, and TQ. He has published or presented more than 150 papers, is a Fellow of the Institute of Brewing and Distilling and a chartered scientist. In 2011 he received the W. J. Eva award from the Canadian Institute of Food Science and Technology. Alex has served on numerous ASBC Technical Subcommittees.

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