Molecular mechanisms of plant polyphenols inhibiting the formation of heterocyclic amines in roasted meat

Harmful substances such as heterocyclic amines (HAs) generated during the roasting process seriously affect the food safety of meat products. Polyphenols, as natural antioxidants, have been shown to have significant effects in inhibiting the generation of HAs. However, due to the wide variety of polyphenols and HAs, the inhibition patterns between polyphenols of different structures and specific HAs and their mechanisms of action are still unclear, and in addition, there is a lack of systematic studies on the mechanisms of polyphenols' effects on HAs generated by proteins involved in the production of HAs. This study systematically investigated the inhibitory effects and molecular mechanisms of polyphenols with different structural characteristics on the formation of free and protein-bound HAs by establishing model systems from three perspectives: the grilling process, protein interactions, and the structural features of polyphenols. Firstly, seven polyphenols with different classes and structural features, including m-hydroxyl and o-hydroxyl groups, were selected and added to lamb patties for roasting to investigate their inhibitory effects on various types of HAs. The inhibitory efficiency of different types of polyphenols on total HAs formation in roasted meat followed the order: stilbenes ≈ coumarins ≈ chalcones > flavonoids > phenolic acids. Cluster analysis and validation using model systems further demonstrated that polyphenols with m-hydroxyl structures have a stronger inhibition of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 1-methyl-9H-pyrido[3,4-b]-indole (Harman) and 9H-pyrido[3,4-b]indole (Norharman) by capturing the reaction intermediate carbonyl compounds, while those with the o-hydroxyl group had a stronger inhibitory effect on 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) by scavenging free radicals. Secondly, the effects of resveratrol and HAs on the conformational changes of sarcoplasmic protein (SP) and myofibrillar protein (MP) were evaluated to investigate the inhibitory patterns and mechanisms of polyphenols on protein-bound HAs. The results showed that resveratrol effectively inhibited both free and bound HAs formation mediated by MP, while for SP, it only suppressed the formation of free HAs. Moreover, the addition of polyphenols promoted the conversion of bound HAs into their free forms. The observed changes in free amino acid content detected only in the SP system suggest that polyphenols mainly inhibit the formation of protein-bound HAs by preventing MP from directly replacing amino acids in the reaction. Moreover, polyphenols not only suppress the generation of free HAs but may also reduce overall HAs content by facilitating the binding of free HAs with SP. Furthermore, analysis of resveratrol’s effects on the secondary structure, fluorescence intensity, and molecular docking with MP revealed strong interactions between resveratrol and MP, primarily through hydrogen bonding and hydrophobic interactions. These interactions reduce the exposure of amino acids involved in HAs formation and increase the exposure of non-precursor amino acids, thereby regulating the formation of MP-bound HAs. Additionally, the effects of resveratrol and free HAs on the particle size, surface hydrophobicity, and intermolecular forces of SP suggest that free HAs promote the formation of bound HAs by altering the surface hydrophobicity of the SP solution. Polyphenols may influence the formation of SP-bound HAs by modulating ionic and disulfide bond interactions. Finally, a combination of experimental analysis and theoretical calculations was used to investigate the molecular characteristics of various polyphenols containing m-hydroxyl groups and their correlation with the ability to trap reactive carbonyl compounds or inhibit PhIP formation. The results showed that the effectiveness of polyphenols in inhibiting PhIP formation was closely related to the concentration of reactive carbonyl intermediates during the reaction but was not directly associated with the carbonyl-trapping capacity of the polyphenols. Furthermore, analysis of the effects of polyphenols on the thermal degradation of phenylalanine indicated that their inhibitory effect on PhIP formation involves not only the trapping of carbonyl compounds but also the suppression of phenylalanine pyrolysis. In addition, the HOMO–LUMO gap of the polyphenols showed a significant positive correlation with their inhibition rate of major carbonyl compounds derived from phenylalanine degradation, while exhibiting a highly significant negative correlation with the binding energy of polyphenol–phenylacetaldehyde adducts. These findings suggest that the HOMO–LUMO gap of a polyphenol molecule can serve as a predictive indicator of its effectiveness in inhibiting PhIP formation. In summary, this study systematically investigated the inhibitory patterns and underlying mechanisms of HAs formation in roasted meat by polyphenols with different structural features. The inhibitory effect of polyphenols on HAs formation is closely related to their molecular structure. As exogenous additives, polyphenols can competitively bind to key precursors (such as amino acids or proteins) or intermediates (such as free radicals or carbonyl compounds) involved in HAs formation, thereby effectively suppressing their formation. This study provides a theoretical basis for screening efficient HAs inhibitors and offers scientific support for the development of healthier and safer meat products.