Evolved Saccharomyces cerevisiae strains exhibit superior freeze-thaw tolerance and frozen dough performance via...

Frozen dough technology enables industrial-scale production of baked foods, yet freeze-thaw damage -particular yeast activity loss - remains a critical bottleneck. This study evaluated an ultra-tolerant Saccharomyces cerevisiae strain developed via hybrid fusion evolution using a - 20 °C, 4-day frozen dough model. The evolved strain demonstrated superior industrial adaptability, achieving >80 % specific volume retention (vs. 52-54 % in parental strains) and limiting the increase in hardness to only 38 %-49 % (vs. 69 %-102 % in parental strains). Untargeted metabolomics revealed multiple tolerance mechanisms in the evolved strain. These included enhanced unsaturation of membrane lipids (e.g., phosphatidylcholine (PC) (18:3)), accumulation of intermediates from the mevalonate pathway, upregulation of key antioxidant compounds including dihydrobiopterin and spermidine analogs, and a shift in energy metabolism involving coenzyme-related pathways. These molecular adaptations were supported by increased antioxidant capacity, as shown by DPPH, ABTS, and FRAP assays, and by maintained membrane integrity, confirmed through field-emission scanning electron microscopy. Overall, this study provides ultra-freeze-thaw-tolerant yeast strains along with mechanistic insights that support improved frozen dough quality, higher production efficiency, and greater sustainable in baking applications.