Zinc protoporphyrin IX formation from myoglobin by Leuconostoc mesenteroides subsp. mesenteroides: Multi-step mechanism elucidation in a model system

A model system was established for zinc protoporphyrin IX (ZnPP) formation. • The system is based on pepsin-digested myoglobin as the sole heme substrate. • Acidification and proteolysis synergistically release heme from myoglobin. • Intracellular enzymes convert released heme into ZnPP and protoporphyrin IX (PPIX). • A reducing microenvironment protects porphyrins and sustains ZnPP formation. Zinc protoporphyrin IX (ZnPP) is a natural red pigment formed in fermented meat products, in which lactic acid bacteria are thought to play a key role; however, the mechanism remains unclear. This study aimed to investigate ZnPP formation by Leuconostoc mesenteroides subsp. mesenteroides (LM). A model system based on pepsin-digested myoglobin was established, and the conversion process was characterized by fluorescence spectroscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, and biochemical analyses. After optimizing the key parameters, the LM strain increased ZnPP production by 89.08% compared with the uninoculated control under the optimal conditions of 60 min pepsin digestion and 200 μM Zn 2+ . During fermentation, the strain acidified the system from pH 6.5 to 4.7–5.0 and secreted extracellular proteases, which together disrupted the myoglobin aggregates and promoted heme release. The mobilized heme was then converted into ZnPP and protoporphyrin IX (PPIX) through intracellular enzymatic activity, accompanied by an increase in non-heme iron and a decrease in free Zn 2+ . Additionally, the LM strain enhanced the total antioxidant capacity and reduced protein carbonyl content, indicating the formation of a reducing microenvironment that may protect porphyrin intermediates and enzyme activities. Collectively, these findings provide physiological and biochemical evidence for the key steps involved in ZnPP formation by LM and support a multi-step cooperative model for the conversion of myoglobin heme into ZnPP. Download: Download high-res image (248KB) Download: Download full-size image