FTIR-Based Study of Starch Retrogradation and Protein Structure in Chickpea-Enriched Gluten-Free Bread During Storage

This study investigated the effect of chickpea flour (CF) on the staling behavior of gluten-free bread (GFB) by comparing a formulation containing 50% rice flour (RF) and 50% CF (CFB) with a control bread based on rice flour supplemented with whey protein (RFB). Bread samples were stored at room temperature for up to 7 days. Changes in color, reflectance, starch and protein structure, specific volume, crumb structure, texture, and staling kinetics were monitored. CFB exhibited a darker and more yellow crumb and crust, with lower reflectance intensity, and showed greater color stability during storage. Fourier-transform infrared (FTIR) spectroscopy revealed higher overall starch crystallinity and more stable relative crystallinity degree (RCD) values in CFB (58.74-59.05%) compared to RFB (46.19-40.52%) throughout storage, indicating early amylose-driven ordering and a more stable molecular organization of starch. Protein secondary structure analysis showed that CFB had a higher β-sheet content (35.05-37.49) than the RFB formulation (30.37-31.16), indicating stronger protein aggregation. In contrast, macroscopic quality parameters showed that CFB had lower specific volume (1.65 vs. 1.93) and porosity (17.17 vs. 21.01 cm³/g) than RFB, resulting in higher hardness (15.92 vs. 6.15 N) and accelerated staling kinetics (k_corr) (0.28 vs. 0.14 day^-1), indicating faster crumb firming despite the observed molecular-level stability. Overall, the results demonstrated that CF contributes to enhanced molecular organization of starch and increased nutritional value of GFB, while its technological performance at the macroscopic level remains formulation-dependent. These findings highlight the need for targeted formulation and process optimization to balance molecular stability with desirable textural properties in CFB.