Investigation of chemical and processing hurdle impacts upon microbial control and physicochemical quality of manufactured pepperoni products

This study investigated the stage-dependent effects of salt, nitrite, heat treatment, and water activity (aW) on physicochemical and microbiological parameters during fermented meat manufacture. Eight formulations representing a fractional subset of a 2 × 2 × 3 × 2 factorial design were evaluated across four processing stages (pre-fermentation, post-fermentation, post-heat treatment, and post-drying). A stage-resolved multivariate analysis of variance (MANOVA) framework was applied, followed by univariate testing with Benjamini–Hochberg false discovery rate (FDR) correction. Results demonstrated that hurdle effects were strongly stage dependent. Nitrite and its interaction with salt were associated with early stage microbial variation (η² ≤ 0.72), while heat treatment was the dominant factor influencing microbial reduction at the post-processing stage. Notably, short high-temperature heat treatments were most effective at microbial reduction, followed by longer lower-temperature heat treatments, with the intermediate treatment being the least effective, indicating that equivalent time–temperature combinations did not yield equivalent microbiological outcomes. At the final stage, salt and nitrite were associated with compositional, microbial, and quality-related parameters, while aW influenced product structure and concentration effects but was not retained as an independent factor in the multivariate model. These findings indicate that hurdle effects evolve throughout processing and should be considered within a dynamic, stage-specific framework. The results provide a basis for the development of integrated reformulation strategies to reduce salt and nitrite levels while maintaining microbial stability and product quality.