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ABSTRACT: Thermoduric bacteria are known to affect the quality of Cheddar cheese, with manifested defects including slits, weak body, and blowing. Thermoduric bacteria are likely to increase in numbers during cheese-making, as in-process conditions are conducive to proliferation. The present study was conducted to track thermoduric bacterial progression during an 18- to 20-h Cheddar cheese production run and during ripening when the pasteurizer was washed at midway through the production day. This study also correlated a broad range of chemical changes to the growth of thermoduric bacteria during ripening. Three independent cheese trials were performed at 3.5- ± 0.5-mo intervals. Samples were drawn in duplicates at 4 different times of the day: at the start of the run (vat 1), prior to a midday wash of the pasteurizer (vat 20), after the midday wash of the pasteurizer (vat 21), and at the end of the run (vat 42) for raw milk, pasteurized milk, and cheese. Cheeses were also tested during ripening for 6 mo. Results showed that raw milk total bacterial counts comprised 0.24% thermoduric mesophiles (TM) and 0.12% thermoduric thermophiles (TT). The thermoduric thermophilic bacterial counts increased by log10 1.23 during the pasteurizer run of 9 to 10 h, indicating a buildup of thermoduric thermophilic bacteria during the pasteurization process itself. Midday washing reduced thermophilic counts by log10 1.36, as evident by pre- and post-midday wash counts. However, a thermophilic buildup during post-midday wash was again noticed near the end of the 20-h run. We found that TT bacteria decreased in the first 60 d of ripening, whereas TM bacteria increased during the same period. However, TT bacteria increased later during 60 to 180 d of ripening. Bacillus licheniformis was the most frequently isolated bacteria in this study and was recovered at all production stages sampled during the cheese-making and ripening. We observed a significant increase in the level of orotic and uric acids in the vat made at the end of the day. No significant difference in the overall chemical composition, proteolysis, sugar, or other organic acids was observed in cheese made at the start versus the end of the production run.