Effect of calcium ascorbate and maltitol on calcium signaling and genes of cell wall and membrane synthesis in yeast under freeze-thaw cycle.

Yeast in frozen fermented dough often faces freeze-thaw (FT) stress due to temperature fluctuations. This study investigated the synergistic protective effects of calcium ascorbate (CA) and maltitol (M) on yeast adaptation to FT stress. The composite modifier (CA + M) exhibited superior efficacy compared to individual additives in preserving yeast cell integrity and metabolic homeostasis. The composite modifier enhanced the expression of key calcium signaling proteins (MID1, CMD1) while maintaining calcineurin (CaN) activity at optimal levels, thereby preventing excessive stress responses. Crucially, CA + M comprehensively reconstructed yeast cell wall architecture by coordinately regulating glucan and chitin biosynthesis pathways (upregulating RHO1, CHS3/5/6, PIR1 while downregulating emergency synthesis genes FKS1/2, MNN9), and restored membrane lipid metabolism by resolving the uncoupling of ergosterol biosynthesis (ERG1/ERG26) and fatty acid pathways (OLE1/FAA1). Transcriptomic analysis revealed that the composite modifier effectively downregulated FT-induced overexpression of calcium signaling genes (MID1, CCH1, CNA1, CMD1, PMC1, PMR1), indicating a balanced stress adaptation mechanism. Consequently, yeast treated with CA + M maintained superior fermentation capacity and cellular integrity after FT cycles, demonstrating that the synergistic regulation of calcium signaling and cell wall/membrane biosynthesis pathways represents a promising strategy for enhancing yeast freeze-thaw tolerance in frozen fermented dough applications.