Background and Objectives The present study investigated the effects of different freezing temperatures and different freeze‐thaw cycles on the physical, chemical, functional, and structural properties of gluten protein. The water‐holding capacity, rheological properties, water distribution, microstructure, and secondary structure were measured. Findings With the same number of freeze‐thaw cycles, the water‐holding capacity of gluten was higher at low temperature, and the viscoelasticity and binding water content increased slightly with decreasing temperature. At −30°C and −24°C, the microstructure of gluten protein was more uniform and relatively intact. Lower temperatures significantly decreased the ratio of α‐helix and β‐turns and significantly increased the ratio of β‐sheets. At the same temperature, the water‐holding capacity, bound water content, and viscoelasticity of the freeze‐thaw cycle gradually decreased with the increase in the number of freeze‐thaw cycles. Compared to the first freeze‐thaw cycle (F1), the holes in the microstructure of gluten in the fifth freeze‐thaw cycle (F5) were larger and fractured. Conclusions Increasing the number of freeze‐thaw cycles damaged the properties of gluten protein, and lower temperatures were more conducive to maintaining the stability of gluten properties. Significance and Novelty This study revealed the changes in gluten protein quality during storage at −6°C, −12°C, −18°C, −24°C, and −30°C and different numbers of freeze‐thaw cycles. The results provide a theoretical basis for the quality management and control of frozen dough during storage and transportation.

In recent years, with the acceleration of the pace of people's lives and improved living standards, the demand and market opportunities for frozen flour products have grown steadily. Frozen dough technology is increasingly used to preserve fresh dough. Frozen dough technology can preserve the appearance, flavor, and color of the product to a certain extent and maintain its original nutrients and freshness (Akyüz & Mazı, 2020;Zhang et al., 2018), prolonging the shelf life and slowing the aging of dough products. Frozen dough technology has many advantages, such as safety, convenience, and rapidity; therefore, it has broad prospects for development (Omedi & Huang, 2018). However, the generation and recrystallization of ice crystals in the freezing process have a negative impact on the frozen dough, including the destruction of the gluten network structure and the decrease in yeast fermentation activity (Ban et al., 2016;Phimolsiripol et al., 2008). These effects lead to the cracking and collapse of the surfaces of frozen products, causing them to taste rough and hard.The change in gluten protein is one of the main factors leading to the decline in the quality of frozen flour products. Gluten protein is mainly composed of glutenin and gliadin. Glutenin determines the elasticity of dough, while gliadin is associated with the dough's viscosity (Elizabeth et al., 2011). The unique amino acid composition of

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