Date of Award

Summer 8-30-2022

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Food Science and Human Nutrition

Advisor

Brian Perkins

Second Committee Member

Jennifer Perry

Third Committee Member

Jason Bolton

Abstract

Mixed cultures are prevalent in the food industry. From sour beer to sourdough bread, mix culture popularity is growing. A number of microbiological mixtures are used in these unique fermenting processes to create distinctive flavor profiles of consumers’ favorite foods. Although mixed cultures seem ubiquitous, they are often not well enumerated. The aim of this thesis was to create a novel rapid image-based cytometry method to enumerate mixed cultures in beer. Imaged-based cytometry can be used to rapidly enumerate mixed microbial cultures, as opposed to traditional plate counting methods that can take days to grow and count. A novel method was developed using the Nexcelom Cellometer X2 (X2) using fluorescent dyes and size exclusion automated counting on a mixed culture containing the microorganisms, lactic acid bacteria and yeast (L. plantarum and S. cerevisiae). Traditional spread plating using MRS and ADPA selective plates were used to validate this new methodology. The enumeration results were not found to be significantly different (P>0.05) when analyzed by ANOVA comparing X2 to traditional plating for log(CFU/mL). The difference between the log(CFU/mL) of the X2 and traditional plating was also within a ±0.5 log(CFU/mL). The difference was noted as having a iv negligible impact on commercial brewing applications. The X2 counts were most precise when the log(CFU/mL) for yeast were between approximately 5 to 7 log(CFU/mL) and 6 to 8 log(CFU/mL) for lactic acid bacteria cells. These ranges are important as they encompass sour beer fermentation pitch rates and can be actively used by brewers today. Overall, the novel image-based cytometry method accomplished the goal of precisely and consistently counting a mixed culture (L. plantarum and S. cerevisiae) in beer media within the identified ranges and correlated well with the standard process of traditional plating methods. This technique enables brewers to make proactive decisions during fermentation, saves brewers time, and can be a cost effective alternative to other enumeration methods.

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