Abstract: This study developed a composite freeze-dried biocontrol agent for controlling postharvest gray mold in strawberries by co-culturing three antagonistic yeasts (Pichia guilliermondii, Meyerozyma caribbica, and Pichia rarassimilans). The nutritional conditions for mixed culture and the formulation of cryoprotectants were optimized using response surface methodology to enhance the biocontrol efficacy and storage stability of the agent. The results showed that the optimal medium composition and culture conditions that provided maximum biomass production were determined as 15.49 g/L sucrose, 16.73 g/L yeast extract, 4.74 g/L MgSO4, and 3.34 g/L KH2PO4, pH 6.0, inoculum size of 2% (P. guilliermondii:M. caribbica:P. rarassimilans = 3:1:2 (V/V)), temperature of 24 ℃, and culture time of 60 h. The optimal cryoprotectant formulation consisted of 9.63% skim milk powder, 14.61% xylo-oligosaccharides, and 21.82% inulin, yielding a high survival rate of 64% after freeze-drying. Scanning electron microscopy (SEM) observations indicated that this formulation could protect against freeze-drying damage, effectively maintaining the integrity of yeast cell morphology. The antifungal agent maintained high biological activity (4.07 × 10 9 CFU/mL) after 5 months of storage at −20 ℃. It could rapidly colonize strawberry wounds, effectively suppress Botrytis cinerea infection, and significantly delay spore germination and mycelial growth of the pathogen. This study provides an efficient and stable green biocontrol strategy against postharvest gray mold in strawberries.

Key words: composite yeast biocontrol agent; strawberry gray mold; biocontrol; dual response surface optimization