Synergistic effects of bee pollen and propolis extract on protein bar properties: a multivariate chemometric analysis

Protein bars are increasingly popular as functional snacks, yet they often suffer from texture hardening and microbial spoilage during storage. This study aimed to enhance their stability and sensory acceptability by incorporating two natural polyphenol-rich ingredients—bee pollen and propolis extract. A face-centered central composite design (CCD) was used to investigate the effects of these additives (0–2%) on the physicochemical, antioxidant, microbial, sensory, and FTIR-analyzed structural properties of brown rice-based protein bars. The optimized formulation, containing 2% bee pollen and 2% propolis extract, showed the most nutritional composition, reduced microbial growth, better texture retention, and enhanced sensory scores. Peroxide value significantly dropped from 4.61 to 2.62 meq O₂/kg, and total microbial count decreased by 43%, suggesting improved oxidative and microbial stability. Water activity reduced from 0.626 to 0.356, suggesting improved microbial safety. FTIR analysis revealed structural shifts, including decreased α-helix and intermolecular β-sheet content, and increased intramolecular β-sheets, β-turns, and random coils, associated with improved mouthfeel. Principal component analysis (PCA) and partial least squares regression (PLSR) confirmed strong correlations between protein secondary structures and quality parameters. In conclusion, the synergistic addition of propolis extract and bee pollen enhanced the nutritional, microbial, structural, and sensory characteristics of protein bars, supporting their potential use as clean-label functional ingredients in industrial applications.