Graduate Thesis Or Dissertation


Development of a High Throughput Method for Screening of Clean-Label Mold Inhibitors in Cheddar Cheese Public Deposited

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  • Mold growth on cheddar-style cheese, though not a food safety issue, causes significant loss to the industry due to consumer rejection. For this reason, it is common for at-risk products, such as cheese shreds, to be coated with the mold inhibitor natamycin. While highly effective at extending shelf-life, preferences for clean-labels and organic foods is driving renewed interest in alternative mold inhibitors for the dairy industry. The purpose of this study was to develop a high-throughput method and use this to quantify mold grow on a cheese-mimicking matrix as a means to screen candidate clean-label inhibitors. In order to evaluate mold-growth at this scale, we adopted image-analysis using freely available ImageJ software (NIH) and the “read plate” plug-in, wherein changes in mold growth are measured using the lightness/darkness of pixels as a proxy for growth. Sensitivity of mold-growth detection was enhanced by incorporating methylene-blue dye into the cheese-mimicking agar. In order to better apply results to the food science industry, the “Growthcurver” package from R statistical software was used to calculate lag time, the time-point at which the mold has reached 50% maximum growth. This metric allowed us to compare delays in growth to the control and quickly assess inhibitor efficacy. To demonstrate the power of this approach, using a cheese-based agar, over eighty 96-well micro-titer cheese agar plates were prepared for inhibitor screening across the three proof of concept studies. Final results yielded analysis of 7,200 samples against varying concentrations of 7 inhibitors and combinations thereof. The results confirm the efficacy of natamycin, while alternative inhibitors caused less pronounced growth delays even at relatively high rates of application. Thyme oil and chitosan showed the greatest potential as alternative mold inhibitors. Thyme oil caused a delay in growth beyond the length of the experiment (168 hrs) for 5 of the 10 mold isolates. Chitosan showed a similar delay for 7 of the molds tested. Future work should focus on analysis of additional inhibitor combinations and incorporate sensory analysis to evaluate their applicability for the dairy industry.
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