Natural curing strategies for nitrite-free dry-fermented sausages: Effects of NOS-positive Staphylococcus spp., Zn-protoporphyrin and polyphenol-rich extracts on untargeted metabolome and sensory quality

Abstract

The reduction of permitted nitrite levels in meat products by the European Union, driven by concerns related to nitrosamine formation and consumer health, has intensified the need for effective and safe nitrite replacement strategies. In this context, the present study investigated the impact of selected natural curing approaches on the untargeted metabolome, lipid oxidation, and olfactory profile of dry-fermented model sausages. Nine formulations originating from a meat batter control without nitrite; control with nitrite; inoculated with Staphylococcus equorum (L33); inoculated with Staphylococcus saprophyticus (L49); formulated with a porcine liver extract rich in zinc protoporphyrin IX (ZnPPEx); formulated with polyphenol-rich extract (NATPRE) alone (CTRL-/NATPRE); or in combination with the other strategies (L33/NATPRE, L49/NATPRE, ZnPPEx/NATPRE, respectively). Untargeted UHPLC-HRMS metabolomics, TBARS assay, and descriptive olfactory analysis were applied before and after ripening (21 days), and multivariate modelling was used to assess treatment- and time-related effects. Both treatment and ripening time shaped the sausage metabolome, whereas their interaction was not significant, indicating stable treatment-specific metabolic trajectories throughout ripening. ZnPPEx-containing formulations displayed distinct metabolic signatures after preparation, suggesting that this extract obtention process contributed to the presence of fermentation-related compounds. Among microbial strategies, L33 most closely replicated the metabolic and sensory characteristics, combining low lipid oxidation with a typical dry-cured meat odour. NATPRE effectively limited lipid oxidation and modulated the metabolomic profile, contributing to the preservation of bioactive compounds. Correlation analysis between discriminant metabolites and sensory attributes highlighted links between amino acid- and lipid-derived metabolites and both desirable and undesirable odour descriptors. Overall, the combination of starter cultures and polyphenol-based antioxidants emerged as a promising strategy for nitrite replacement, whereas ZnPPEx require further optimisation to control off-odours and biogenic amine accumulation.