Optimization of a brown rice protein bar supplemented with Spirulina and phycocyanin: FTIR-based analysis of protein secondary structures and multivariate quality assessment

• Spirulina & phycocyanin reduce protein bar hardening during storage. • Optimized formula: 2% Spirulina & 0.2% phycocyanin enhance texture & appeal. • FTIR analysis links reduced hardness to flexible protein secondary structures. • Antioxidant capacity significantly improved with Spirulina & phycocyanin. • PCA & PLSR models reveal relationships between protein structure & quality. Protein bars are popular functional foods, but they often experience undesirable physicochemical changes during storage, such as hardening and sensory deterioration, which can limit their consumer acceptance. This study aimed to develop and optimize a novel protein bar formulation fortified with brown rice flour, Spirulina platensis, and phycocyanin to improve nutritional value, texture stability, and antioxidant capacity. A face-centered composite design was employed to systematically investigate the effects of Spirulina (0–2%) and phycocyanin (0–0.4%) levels on physicochemical properties, microbial safety, antioxidant activity, and sensory quality. Antioxidant capacity was evaluated using DPPH, FRAP, total polyphenol, and flavonoid assays. The DPPH scavenging activity increased by 26.81% compared to the control, while texture profile analysis showed a 5.29% reduction in hardness in the optimized samples. FTIR spectroscopy revealed structural modifications in protein secondary structures, including decreased α-helix and intermolecular β-sheet content, and increased intramolecular β-sheets, β-turns, and random coils, which corresponded with improved textural and sensory properties. The optimized formulation, containing 2% Spirulina and 0.4% phycocyanin, showed the most desirable combination of nutritional, structural, and sensory attributes. Furthermore, multivariate statistical analyses (Principal Component Analysis and Partial Least Squares Regression) confirmed strong correlations between protein structural changes and quality parameters, emphasizing the critical role of molecular-level analysis in product development. These findings suggest that Spirulina and phycocyanin can serve as effective functional ingredients for producing nutritionally enhanced and shelf-stable protein bars with improved structural and sensory qualities.