Yeast mitochondrial adaptation to freeze thaw stress depends on pre freeze fermentation state.

During frozen dough production, transportation, and processing, temperature fluctuations expose yeast to freeze-thaw (FT) stress, causing yeast cellular damage. This study investigated the effects of FT cycles on mitochondrial structure and function in yeast under different fermentation processes. As FT cycles increased, cell mortality, mitochondrial membrane potential, and superoxide anion (O2.-) generation rate significantly increased, with the highest values observed in the FT6 group. Cytochrome c content and cytochrome c oxidase activity varied among groups, while isocitrate dehydrogenase activity was highest in FT-0. Acetyl-CoA synthetase activity increased with FT cycles, and α-ketoglutarate dehydrogenase activity was higher in FT6 than in FT3. Gene expression of ACS1 and KGD increased with cycles, whereas YNO1, AGT32, and MDM34 were lower in FT3 than in FT0 and FT6. Compared with the control, expression of IDH2, CYC7, NDI1, AIF1, DNM1, AGT11, AGT8, and MDM12 was upregulated under FT stress. Collectively, FT stress induces yeast cell death and impairs mitochondrial function by modulating key enzyme activities, promoting O2.- production, altering membrane potential, and upregulating genes involved in mitochondrial division and autophagy.