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• Mannosylerythritol lipid-A improved the rheological property and water distribution of frozen dough. • Mannosylerythritol lipid-A strengthened the gluten network of frozen dough. • Mannosylerythritol lipid-A enhanced the textural property of bread. • Mannosylerythritol lipid-A effectively sterilized Bacillus cereus in frozen dough. Biosurfactants have been put into applications in breadmaking industry, while the effects of mannosylerythritol lipid-A (MEL-A) on gluten network of frozen dough, bread quality and microbial spoilage were firstly investigated in this study. Rheology and differential scanning calorimetry (DSC) analysis showed that MEL-A significantly improved the rheological properties of frozen dough and reduced the content of frozen water. Further experiments showed that MEL-A promoted the formation of aggregates by interacting with gluten protein, and strengthened the gluten network through molecular weight distribution measurement and microstructure observation, effectively avoiding the destruction of ice crystals. A series of bread assessments illustrated MEL-A improved the loaf volume, gas retention ability and textural property. In addition, MEL-A (1.5%) killed 99.97% of the vegetative cells of Bacillus cereus and 75.54% of the spores, and at the same time had a slight inactivation effect on yeast. These results indicate that MEL-A has broad application prospects in the baking industry and the storage stage of flour products. [ABSTRACT FROM AUTHOR]